WO2019173787A1 - Constructions d'administration dérivées de toxines pour administration orale - Google Patents

Constructions d'administration dérivées de toxines pour administration orale Download PDF

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Publication number
WO2019173787A1
WO2019173787A1 PCT/US2019/021474 US2019021474W WO2019173787A1 WO 2019173787 A1 WO2019173787 A1 WO 2019173787A1 US 2019021474 W US2019021474 W US 2019021474W WO 2019173787 A1 WO2019173787 A1 WO 2019173787A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
carrier
delivery construct
acid sequence
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PCT/US2019/021474
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English (en)
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WO2019173787A8 (fr
Inventor
Keyi LIU
Julia Dawn MACKAY
Weijun Feng
Thomas Carl HUNTER
Randall J. Mrsny
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Applied Molecular Transport Inc.
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Application filed by Applied Molecular Transport Inc. filed Critical Applied Molecular Transport Inc.
Priority to CN201980031164.1A priority Critical patent/CN112105376A/zh
Priority to EP23192492.9A priority patent/EP4316586A3/fr
Priority to ES19717013T priority patent/ES2920428T3/es
Priority to EA202092126A priority patent/EA202092126A1/ru
Priority to SI201930291T priority patent/SI3762009T1/sl
Priority to CA3093386A priority patent/CA3093386A1/fr
Priority to KR1020207028975A priority patent/KR20210076881A/ko
Priority to PL19717013.7T priority patent/PL3762009T3/pl
Priority to EP19717013.7A priority patent/EP3762009B1/fr
Priority to SG11202009925PA priority patent/SG11202009925PA/en
Priority to DK19717013.7T priority patent/DK3762009T3/da
Priority to AU2019230230A priority patent/AU2019230230A1/en
Priority to EP22165874.3A priority patent/EP4082558B1/fr
Priority to KR1020217017372A priority patent/KR20210110800A/ko
Priority to CN201980088247.4A priority patent/CN113347997A/zh
Priority to IL282986A priority patent/IL282986B2/en
Priority to CA3119179A priority patent/CA3119179A1/fr
Priority to PCT/US2019/050708 priority patent/WO2020096695A1/fr
Priority to AU2019374703A priority patent/AU2019374703A1/en
Priority to SG11202104734YA priority patent/SG11202104734YA/en
Priority to TW108132886A priority patent/TW202031297A/zh
Priority to MX2021005382A priority patent/MX2021005382A/es
Priority to JP2021525126A priority patent/JP2022512976A/ja
Priority to EP19881649.8A priority patent/EP3826682A4/fr
Priority to BR112021009001A priority patent/BR112021009001A8/pt
Publication of WO2019173787A1 publication Critical patent/WO2019173787A1/fr
Priority to EP22159495.5A priority patent/EP4083058A3/fr
Priority to BR112021009003-7A priority patent/BR112021009003A2/pt
Priority to MX2021005346A priority patent/MX2021005346A/es
Priority to DK19207825.1T priority patent/DK3650037T3/da
Priority to EP19207825.1A priority patent/EP3650037B1/fr
Priority to JP2021525169A priority patent/JP2022506990A/ja
Priority to AU2019377117A priority patent/AU2019377117A1/en
Priority to ES19207825T priority patent/ES2911075T3/es
Priority to PT192078251T priority patent/PT3650037T/pt
Priority to CA3119060A priority patent/CA3119060A1/fr
Priority to PCT/US2019/060356 priority patent/WO2020097394A1/fr
Priority to TW108140533A priority patent/TW202031678A/zh
Priority to KR1020217017210A priority patent/KR20210110294A/ko
Priority to CN201980088287.9A priority patent/CN113423722A/zh
Priority to SG11202104721RA priority patent/SG11202104721RA/en
Priority to PL19207825T priority patent/PL3650037T3/pl
Priority to US16/686,671 priority patent/US20200140511A1/en
Priority to IL277209A priority patent/IL277209A/en
Priority to US17/015,011 priority patent/US11426466B2/en
Priority to IL282987A priority patent/IL282987B2/en
Priority to CL2021001209A priority patent/CL2021001209A1/es
Priority to CONC2021/0007359A priority patent/CO2021007359A2/es
Priority to CONC2021/0007402A priority patent/CO2021007402A2/es
Publication of WO2019173787A8 publication Critical patent/WO2019173787A8/fr
Priority to US17/868,077 priority patent/US20230158163A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02036—NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36)
    • 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 instant application contains a Sequence Listing in the form of a“paper copy” (PDF File) and a file containing the referenced sequences (SEQ ID NO: 1 - SEQ ID NO: 221) in computer readable form (ST25 format text file) which is submitted herein.
  • the Sequence Listing is shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1.822. Said ASCII copy, created on March 8, 2019, is named 40566-71 l_60l_SL.txt and is 350,816 bytes in size.
  • the gut epithelium has thwarted efforts to orally administer large molecule biologies because proteins cannot diffuse across the barrier or sneak through the tight junctions. When they are taken up by endocytosis— the only route left to them— they are typically degraded in lysosomes rather than being transported into the body. This inability to be readily absorbed across the intestinal epithelium continues to be a limiting factor in developing commercially viable oral formulations of these agents. The most common solution is to use systemic administration, but that can often create considerable side effects and reduce patient convenience that negatively affects compliance.
  • the present disclosure provides methods and composition for transport and/or delivery of a cargo molecule to certain location(s) within a cell (e.g., a supranuclear location) or across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a rodent or a human).
  • a cargo molecule can be directed to a set of location(s) by coupling it to a carrier molecule.
  • carrier molecule can interact with unique receptors both on the cell surface and intracellularly for the targeted delivery of the cargo.
  • carrier, cargos, and uses thereof are described herein.
  • the disclosure provides an isolated delivery construct that can comprise: a carrier derived from a domain I of an exotoxin and lacking a domain II, a domain lb and a domain III of the exotoxin; coupled to a heterologous cargo.
  • the carrier can consist essentially of the domain I of the exotoxin.
  • the delivery construct can deliver the heterologous cargo according to one or more of the following: across an epithelial cell via transcytosis; to the basal side of the epithelial cell; to a supranuclear region within the epithelial cell; or to the interior of the epithelial cell via endocytosis.
  • the carrier is configured to deliver a heterologous cargo to the basal side of an epithelial cell.
  • the disclosure provides an isolated delivery construct that can comprise: a chimeric carrier comprising an intracellular epithelial targeting domain; coupled to a heterologous cargo.
  • the disclosure provides an isolated delivery construct that can comprise: a chimeric carrier comprising a supranuclear epithelial targeting domain; coupled to the heterologous cargo.
  • the disclosure provides an isolated delivery construct that can comprise: a carrier coupled to a heterologous cargo, wherein the carrier interacts with one or more of ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, and does not display interaction with one or more of a clathrin or GPR78, or a combination thereof.
  • the interaction can be a selective interaction.
  • the interaction can be a pH-dependent interaction.
  • the interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan can occur on a surface of the epithelial cell, in the interior of an epithelial cell, or a combination thereof.
  • the delivery of the heterologous cargo across the epithelial cell can occur in vitro from the apical surface of the epithelial cell to a basolateral compartment.
  • the delivery of the heterologous cargo can occur in vitro from the apical surface of the epithelial cell to the interior of the epithelial cell.
  • the delivery of the heterologous cargo can occur in vitro from the apical surface of the epithelial cell to the supranuclear region within the epithelial cell.
  • the interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-8,TMEMT32, GRP75, ERGIC-53, or perlecan, or the combination thereof can occur in vitro on the apical surface of the epithelial cell, in the interior of the epithelial cell, or a combination thereof.
  • the epithelial cell can be a polarized epithelial cell.
  • the polarized epithelial cell can be part of a monolayer of polarized epithelial cells.
  • the polarized epithelial cell can be from a rodent or a human.
  • the polarized epithelial cell can be from a human.
  • the human polarized epithelial cell can be a human polarized gut epithelial cell.
  • the human polarized gut epithelial cell can be a Caco-2 cell.
  • the delivery of the heterologous cargo across the epithelial cell can occur in vivo from a gut of a subject to a basolateral compartment of a subject.
  • the delivery of the heterologous cargo can occur in vivo from a gut of a subject to the interior of the epithelial cell of a subject.
  • the delivery of the heterologous cargo can occur in vivo from a gut of a subject to the supranuclear region within the epithelial cell of a subject.
  • the interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-8,TMEMl32, GRP75, ERGIC-53, and perlecan, or the combination thereof, can occur in vivo on the apical surface of the epithelial cell of a subject, in the interior of the epithelial cell of the subject, or a combination thereof.
  • the subject can be a rodent or a human.
  • the subject can be a human and affected by one or more of the following: inflammatory bowel disease, psoriasis, bacterial sepsis, systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave’s disease, Sjogren’s disease, dermatomyositis, Hashimoto’s disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, scleroderma, non- Hodgkin’s lymphomas, Hodgkin’s lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignant melanoma, r
  • the epithelial cell can be a polarized epithelial cell.
  • the polarized epithelial cell can be a polarized gut epithelial cell.
  • the carrier can be a small molecule, a polypeptide, an aptamer, or a combination thereof.
  • the carrier can be a small molecule.
  • the carrier can be a polypeptide.
  • the polypeptide can be an antibody or a functional fragment thereof.
  • the carrier can be an aptamer.
  • the carrier can be derived from an exotoxin.
  • the carrier can be derived from a domain I of the exotoxin and lacks a domain II, a domain lb and a domain III of the exotoxin
  • the carrier that can be derived from a domain I of an exotoxin comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of the domain I of the exotoxin, or at least 80% sequence identity to a functional fragment thereof, wherein the exotoxin is a Cholix toxin or a Pseudomonas exotoxin A.
  • the carrier comprises at least 110 amino acid residues of the domain I of the exotoxin.
  • the carrier comprises at least 50 contiguous amino acid residues of the domain I of the exotoxin.
  • the carrier that lacks the domain II, the domain lb and the domain III of the exotoxin can comprise a portion of the domain II, the domain lb or the domain III of the exotoxin, or a combination thereof.
  • the portion can comprise no more than 70% of the amino acid residues of the domain II, the domain lb or the domain III of the exotoxin.
  • the exotoxin can be a Cholix toxin.
  • the carrier can comprise: an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 80% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise a deletion or mutation in one or more of the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • the carrier can comprise: an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 90% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 95% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise: an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 99% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise: an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • the carrier can comprise a spatial structure in which one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in close proximity to one or more amino acid residues of SEQ ID NO: 151, and one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in close proximity to one or more amino acid residues of SEQ ID NO: 152.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-187 or 1-206 of SEQ ID NO: 11 or 1-186 or 1-205 of SEQ ID NO: 10.
  • the carrier can comprise residues 1-187 of SEQ ID NO: 30 or 1-186 of SEQ ID NO: 31 and no more than 206 contiguous amino acid residues of SEQ ID NO: 1.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 10 - SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-151 or 1-187 of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the carrier can lack any one or more of the amino acid residues 1-39 of SEQ ID NO: 5 or amino acid residues 1-38 of SEQ ID NO: 4.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69 or SEQ ID NO:
  • the carrier can comprise residues 1-151 of SEQ ID NO: 5 or residues 1-150 of SEQ ID NO: 4 and no more than 187 contiguous amino acid residues of SEQ ID NO: 1
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-150 of SEQ ID NO: 6 or in one or more of amino acid residues 1-151 of SEQ ID NO: 7.
  • the carrier can comprise residues 1-134 of SEQ ID NO: 5 or residues 1-133 of SEQ ID NO: 4 and no more than 151 contiguous amino acid residues of SEQ ID NO: 1.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 106 - SEQ ID NO: 125 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or a functional fragment thereof.
  • the carrier or isolated delivery construct can comprise at least one but no more than 20 beta strands.
  • the exotoxin can be a Pseudomonas exotoxin A.
  • the carrier can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 137 or at least 80% identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-252 of SEQ ID NO: 137.
  • the carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 90% sequence identity to a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 95% sequence identity to a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 99% sequence identity to a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having 100% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or 100% sequence identity to a functional fragment thereof.
  • the carrier can comprise no more than 252 contiguous amino acid residues from SEQ ID NO: 134.
  • the carrier comprises residues 1-252 of SEQ ID NO: 134.
  • the carrier can comprise at least one N- terminal methionine residue.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO: 125, or 80% sequence identity to a functional fragment thereof.
  • the delivery construct can form a multimer.
  • the multimer can be formed by multimerization of the heterologous cargo.
  • the multimer can be a heteromer or a homomer
  • the homomer can be a homodimer.
  • the homodimer can be formed by dimerization of the heterologous cargo.
  • the present disclosure provides an isolated delivery construct that can comprise: a carrier comprising a first portion and a second portion, wherein the first portion is derived from a first exotoxin and the second portion is derived from a second exotoxin; coupled to a
  • the first exotoxin can be Cholix.
  • the second exotoxin can be PE.
  • the first portion can be derived from a domain I, a domain II, a domain lb, or a domain III of Cholix, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 125 or SEQ ID NO: 133, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11, a functional fragment thereof, or any combination thereof.
  • the second portion can be derived from a domain I, a domain II, a domain lb, or a domain III of PE, or any combination thereof.
  • the second portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 137 - SEQ ID NO: 145, a functional fragment thereof, or any combination thereof.
  • the first portion can be chemically coupled or
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence SEQ ID NO: 146 or SEQ ID NO: 147.
  • the carrier can be chemically coupled or recombinantly coupled to the heterologous cargo.
  • the carrier can be covalently coupled to the heterologous cargo.
  • the heterologous cargo can be coupled to the C-terminus of the carrier.
  • the heterologous cargo can be coupled to the N- terminus of the carrier.
  • the carrier can be coupled directly to the heterologous cargo.
  • the carrier can be coupled indirectly to the heterologous cargo.
  • the carrier can be coupled to the
  • the spacer can comprise an amino acid spacer.
  • the amino acid spacer can be between 1 and 50 amino acid residues in length.
  • the amino acid spacer can comprise one or more glycine residues and one or more serine residues.
  • the spacer can be a cleavable spacer.
  • the cleavable spacer can comprise an amino acid sequence set forth in any one of SEQ ID NO: 174 - SEQ ID NO: 206.
  • the spacer can be a non-cleavable spacer.
  • the non- cleavable spacer can comprise one or more of the amino acid sequences GTGGS (SEQ ID NO: 207), GGGGS (SEQ ID NO: 208), GGGGSGGGGS (SEQ ID NO: 209),
  • the spacer can comprise one or more fragments of the domain II, the domain lb or the domain III of the exotoxin, or a combination thereof.
  • the spacer can comprise at most 80 amino acid residues of the domain II, 80 amino acid residues of the domain III, or a combination thereof.
  • the heterologous cargo can be a macromolecule, a small molecule, a polypeptide, a nucleic acid, a mRNA, a miRNA, a shRNA, a siRNA, an antisense molecule, an antibody, a DNA, a plasmid, a vaccine, a polymer a nanoparticle, or a catalytically-active material.
  • the heterologous cargo can be a biologically active cargo.
  • the biologically active cargo can be a cytokine, a hormone, a therapeutic antibody, a functional fragment thereof, or any combination thereof.
  • the cytokine can be IL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-l l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-l 9, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, or IL-30.
  • the cytokine can have the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 218.
  • the hormone can have the amino acid sequence set forth in SEQ ID NO: 215 or SEQ ID NO: 216.
  • the therapeutic antibody can be an anti-TNFa antibody.
  • the anti-TNFa antibody can be adalimumab or infliximab.
  • the heterologous cargo can be a detectable agent.
  • the detectable agent can be a fluorophore, a contrast agent, an X-ray contrast agent, a PET agent, a
  • the fluorophore can be a red fluorescent protein (RFP).
  • the RFP can have the amino acid sequence set forth in SEQ ID NO: 220.
  • a delivery construct of the present disclosure can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158 - SEQ ID NO: 165, or at least 80% sequence identity to a functional fragment thereof.
  • a delivery construct of the present disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158 - SEQ ID NO: 165, or at least 90% sequence identity to a functional fragment thereof.
  • a delivery construct of the present disclosure can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158 - SEQ ID NO: 165, or at least 95% sequence identity to a functional fragment thereof.
  • a delivery construct of the present disclosure can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158 - SEQ ID NO: 165, or at least 99% sequence identity to a functional fragment thereof.
  • a delivery construct of the present disclosure can comprise an amino acid sequence having 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158— SEQ ID NO: 165, or 100% sequence identity to a functional fragment thereof.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: an isolated delivery construct as described herein; and a pharmaceutically acceptable carrier.
  • the composition can be formulated for oral administration, topical administration, pulmonary administration, intra-nasal administration, buccal administration, sublingual administration or ocular administration.
  • the composition can be formulated for oral administration.
  • the composition can be formulated in a capsule or tablet.
  • the present disclosure provides a host cell that can comprise a vector that expresses a delivery construct, wherein the host cell comprises a vector comprising a polynucleotide encoding an isolated delivery construct as described herein.
  • the present disclosure provides a method of delivering a heterologous cargo across an epithelial cell, the method can comprise: applying a delivery construct to the apical surface of the epithelial cell; and delivering the delivery construct to the basal side of the epithelial cell at a rate greater than 10 6 cm/sec, wherein the delivery construct comprises: a carrier; coupled to the heterologous cargo.
  • the method further comprises releasing the delivery construct from the basal side of the epithelial cell following delivery across the epithelial cell.
  • the carrier is configured to deliver a heterologous cargo to the basal side of an epithelial cell.
  • the present disclosure provides a method of delivering a heterologous cargo to the interior of an epithelial cell via endocytosis, the method can comprise: applying a delivery construct to the apical surface of the epithelial cell; and delivering the delivery construct to the interior of the epithelial cell via endocytosis, wherein the delivery construct comprises: a carrier; coupled to the heterologous cargo.
  • the present disclosure provides a method of interacting with ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, or a combination thereof, the method can comprise: applying a delivery construct to the apical surface of the epithelial cell; and interacting the delivery construct with the ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, or the combination thereof, wherein the delivery construct comprises: a carrier;
  • the present disclosure provides a method of treating a disease in a subject in need thereof, the method can comprise administering to the subject a delivery construct comprising: a carrier derived from a domain I of an exotoxin and lacking a domain II, a domain lb and a domain III of the exotoxin; coupled to a heterologous cargo; wherein the delivery construct is capable of delivering the heterologous cargo via transcytosis across an epithelial cell.
  • the present disclosure provides a method of diagnosing a disease in a subject in need thereof, the method can comprise administering to the subject a delivery construct comprising: a carrier derived from a domain I of an exotoxin and lacking a domain II, a domain lb and a domain III of the exotoxin; coupled to a heterologous cargo; wherein the delivery construct is capable of delivering the heterologous cargo via transcytosis across an epithelial cell.
  • the delivery of the heterologous cargo across the epithelial cell can occur in vitro from the apical surface of the epithelial cell to a basolateral compartment.
  • the delivery of the heterologous cargo can occur in vitro from the apical surface of the epithelial cell to the interior of the epithelial cell.
  • the delivery of the heterologous cargo can occur in vitro from the apical surface of the epithelial cell to the supranuclear region within the epithelial cell.
  • the interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-8,TMEMT32, GRP75, ERGIC-53, or perlecan, or the combination thereof, can occur in vitro on the apical surface of the epithelial cell, in the interior of the epithelial cell, or a combination thereof.
  • the epithelial cell can be a polarized epithelial cell.
  • the polarized epithelial cell can be part of a monolayer of polarized epithelial cells.
  • the polarized epithelial cell can be from a rodent.
  • the polarized epithelial cell can be from a human.
  • the human polarized epithelial cell can be a human polarized gut epithelial cell.
  • the human polarized gut epithelial cell can be a Caco-2 cell.
  • the delivery of the heterologous cargo across the epithelial cell can occur in vivo from a gut of a subject to a basolateral compartment of the subject.
  • the delivery of the heterologous cargo can occur in vivo from a gut of a subject to the interior of the epithelial cell of the subject.
  • the delivery of the heterologous cargo can occur in vivo from a gut of a subject to the supranuclear region within the epithelial cell of the subject.
  • the interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-8,TMEMT32, GRP75, ERGIC-53, and perlecan, or the combination thereof, can occur in vivo on the apical surface of the epithelial cell of a subject, in the interior of the epithelial cell of the subject, or a combination thereof.
  • the subject can be a rodent or a human.
  • the epithelial cell can be a polarized epithelial cell.
  • the polarized epithelial cell can be a polarized gut epithelial cell.
  • the method further can comprise formulating the delivery construct for administration to the subject.
  • the formulation can comprise one or more pharmaceutically acceptable carriers.
  • the delivery construct can be formulated for oral administration, topical administration, pulmonary administration, intra-nasal administration, buccal administration, sublingual administration or ocular administration.
  • the composition can be formulated for oral administration.
  • the disease can be an inflammatory disease, an autoimmune disease, a cancer, a metabolic disease, a fatty liver disease, or a growth hormone deficient growth disorder.
  • the inflammatory disease can be an inflammatory bowel disease, psoriasis or bacterial sepsis.
  • the inflammatory bowel disease can be Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet’s syndrome or indeterminate colitis.
  • the autoimmune disease can be systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave’s disease, Sjogren’s disease, dermatomyositis, Hashimoto’s disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, or scleroderma.
  • SLE systemic lupus erythematosus
  • pemphigus vulgaris myasthenia gravis
  • hemolytic anemia thrombocytopenia purpura
  • Grave’s disease Sjogren’s disease
  • the cancer can be a non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignant melanoma, rituximab resistant NHL, or leukemia.
  • the metabolic disease can be diabetes, obesity, diabetes as a consequence of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, or hyperlipidemia.
  • the carrier can be a small molecule.
  • the carrier can be a polypeptide.
  • the polypeptide can be an antibody or a functional fragment thereof.
  • the carrier can be an aptamer.
  • the carrier can be derived from an exotoxin.
  • the carrier can be derived from a domain I of the exotoxin and lacks a domain II, a domain lb and a domain III of the exotoxin.
  • the carrier can be derived from a domain I of an exotoxin comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of the domain I of the exotoxin, or at least 80% sequence identity to a functional fragment thereof, wherein the exotoxin is a Cholix toxin or a Pseudomonas exotoxin A.
  • the carrier can comprise: an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 95% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise: an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 99% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise: an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional fragment thereof.
  • the carrier comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-187 or 1-206 of SEQ ID NO: 5 or one or more of amino acid residues 1-186 or 1-205 of SEQ ID NO: 4.
  • the carrier can comprise residues 1- 187 of SEQ ID NO: 5 or residues 1-186 of SEQ ID NO: 4 and no more than 206 contiguous amino acid residues of SEQ ID NO: 1.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or a functional fragment thereof.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-151 or 1-187 of SEQ ID NO: 5 or in one or more of amino acid residues 1-150 or 1-186 of SEQ ID NO: 4.
  • the carrier can lack any one or more of the amino acid residues 1-39 of SEQ ID NO: 5 or residues 1-38 of SEQ ID NO: 4.
  • the carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or at least 80% sequence identity to a functional fragment thereof.
  • the carrier can comprise a deletion or mutation in one or more of amino acid residues 1-151 of SEQ ID NO: 5 or in one or more of amino acid residues 1-150 of SEQ ID NO: 4.
  • the carrier can comprise residues 1-134 of SEQ ID NO: 5 or residues 1-133 of SEQ ID NO: 4 and no more than 151 contiguous amino acid residues of SEQ ID NO: 1.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 125, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11, a functional fragment thereof, or any combination thereof.
  • the carrier can be coupled indirectly to the heterologous cargo.
  • the carrier can be coupled to the heterologous cargo via a spacer.
  • the spacer can comprise an amino acid spacer.
  • the amino acid spacer can comprise one or more glycine residues and one or more serine residues.
  • the amino acid spacer can be between 1 and 50 amino acid residues in length.
  • the spacer can be a cleavable spacer.
  • the cleavable spacer can comprise an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 174 - SEQ ID NO: 206.
  • the spacer can be a non-cleavable spacer.
  • the non-cleavable spacer can comprise one or more of the amino acid sequences GTGGS (SEQ ID NO: 207), GGGGS (SEQ ID NO: 208), GGGGS GGGGS (SEQ ID NO: 209), GGGGS GGGGS GGGGS (SEQ ID NO:
  • the spacer can comprise one or more fragments of the domain II, a domain lb or the domain III of the exotoxin, or a combination thereof.
  • the spacer can comprise at most 82 amino acid residues of the domain II, 82 amino acid residues of the domain III, or a combination thereof.
  • the hormone can have the amino acid sequence set forth in SEQ ID NO: 215 or SEQ ID NO: 216.
  • the therapeutic antibody can be an anti-TNFa antibody.
  • the anti-TNFa antibody can be adalimumab or infliximab.
  • the heterologous cargo can be a detectable agent.
  • the detectable agent can be a fluorophore, a contrast agent, an X-ray contrast agent, a PET agent, a nanoparticle, or a radioisotope.
  • the fluorophore can be a red fluorescent protein (RFP).
  • the RFP can have the amino acid sequence set forth in SEQ ID NO: 220
  • the present disclosure relates to novel non-naturally occurring delivery constructs that can comprise a bacterial toxin-derived chimeric carrier coupled to a biologically active cargo; wherein the chimeric carrier is derived from a domain I but does not comprise a domain II, a domain lb, or a domain III of the bacterial toxin (e.g., an exotoxin); and wherein the delivery construct is capable of delivering a heterologous (e.g., a biologically active) cargo via transcytosis transport across an epithelial cell (e.g., an intestinal epithelial cell).
  • a heterologous e.g., a biologically active
  • the carrier that a delivery construct of the present disclosure is comprised of can bind to receptor(s) known to be present on the apical membrane of an epithelial cell by one of skill in the art without limitation.
  • the receptor binding domain of the delivery construct can bind to low density lipoprotein receptor-related protein 1 (LRP1) or TMEM132 receptor.
  • the carrier can be a polypeptide derived from Cholix and/or PE and having: at most 5 amino acid residues; at most 10 amino acid residues; at most 15 amino acid residues; at most 20 amino acid residues; at most 30 amino acid residues; at most 40 amino acid residues; at most
  • the carrier can be derived from a domain I of a Cholix exotoxin and can comprise an amino acid sequence selected from the group consisting of an amino acid sequence having greater than 50% homology to SEQ ID NO: 4, having greater than 60% homology to SEQ ID NO: 4, having greater than 70% homology to SEQ ID NO: 4, having greater than 80% homology to SEQ ID NO: 4, having greater than 85% homology to SEQ ID NO: 4, having greater than 90% homology to SEQ ID NO: 4, and having greater than 95% homology to SEQ ID NO: 4.
  • the delivery construct is derived from cholix exotoxin (Cholix) and comprises the receptor binding domain polypeptide having the amino acid sequence set forth in SEQ ID NO: 4.
  • a delivery construct can comprise a carrier, wherein the carrier comprises one or more amino acid residues of one exotoxin domain I (e.g., a Cholix or PE domain I) is replaced by one or more amino acid residues of a second exotoxin domain I (e.g., a Cholix or PE domain I), (also referred to hereinafter as a hybrid or chimeric carrier).
  • the carrier can comprise an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 is replaced by one or more amino acid residues of SEQ ID NO: 137.
  • the carrier can comprise an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 137 is replaced by one or more amino acid residues of SEQ ID NO: 4.
  • the carrier can comprise an amino acid sequence wherein amino acid residues 177- 228 of SEQ ID NO: 137 are replaced by amino acid residues of a second bacterial carrier receptor binding domain polypeptide.
  • a carrier of the present disclosure that can be derived from a domain I of an exotoxin and can further comprise a portion of a domain II, a portion of a domain lb, and/or a portion of a domain III of the same or another exotoxin.
  • a carrier can comprise a domain I of an exotoxin, or a truncated and/or modified version thereof, and one or more portions derived from a domain II, domain lb, and/or domain III of the same or a different exotoxin.
  • the domain II, or modified domain II, and domain III, or modified domain III can be derived from the same bacterial toxin.
  • the domain II, or modified domain II, and domain III, or modified domain III can be derived from a bacterial carrier selected from the group consisting of cholix carrier (Cholix) and Pseudomonas exotoxin A (PE), botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like toxin.
  • Toxicity of the bacterial carrier e.g., Cholix or PE
  • the delivery constructs of the present disclosure can comprise a carrier coupled to a heterologous cargo.
  • the heterologous cargo can be a biologically active cargo.
  • the heterologous cargo can be a detectable agent.
  • the carrier can be coupled to a biologically active cargo to produce a delivery construct that is capable of delivering the biologically active cargo via transcytosis transport across an intestinal epithelium.
  • the biologically active cargo can be selected from e.g., a macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, or catalytically-active material.
  • the biologically active cargo can be an enzyme selected from hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, or PGE- adenosine deaminase.
  • the biologically active cargo can comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219, or any combination thereof.
  • the delivery constructs can comprise a delivery construct coupled to a biologically active cargo by a cleavable spacer.
  • the spacer can be cleavable by an enzyme that is present at a basolateral membrane of a polarized epithelial cell.
  • the spacer can be cleavable by an enzyme that is present in the plasma.
  • the cleavable spacer can comprise the amino acid sequence set forth in any one of SEQ ID NO: 174 - SEQ ID NO: 206.
  • the cleavable spacer can be a spacer that comprises an amino acid sequence that can be a known substrate for the tobacco etch virus (TEV) protease.
  • TEV tobacco etch virus
  • the cleavable spacer comprises the amino acid sequence set in forth in SEQ ID NO: 193.
  • the spacer can be cleavable by an enzyme that is present at a basal-lateral membrane of a polarized epithelial cell.
  • the spacer can be cleav
  • the cleavable spacers can comprise a peptide sequence (or like domain), which serves to inhibit, interfere with, or block the ability of the biologically active cargo to bind to receptors at the surface of epithelial cells, but wherein the delivery construct retains the ability of the cargo to activate it’s receptor after the delivery construct is transported across the epithelial barrier and the cargo is released from the delivery construct and spacer components of the construct.
  • the cleavable spacer can comprise the amino acid sequence set forth in, e.g., SEQ ID NO: 194 - SEQ ID NO: 206.
  • the present disclosure provides a method of treating an inflammatory disease in a subject that can comprise administering a pharmaceutical composition of the present disclosure to the subject.
  • the inflammatory disease is selected from an
  • the inflammatory bowel disease is Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome or indeterminate colitis.
  • the present disclosure provides a method of treating an autoimmune disease in a subject that can comprise administering a pharmaceutical composition of the present disclosure to the subject.
  • the autoimmune disease is systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave's disease, Sjogren's disease, dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, or scleroderma.
  • SLE systemic lupus erythematosus
  • pemphigus vulgaris myasthenia gravis
  • hemolytic anemia thrombocytopenia purpura
  • Grave's disease Sjogren's disease
  • dermatomyositis Hashimoto's disease
  • polymyositis inflammatory bowel disease
  • the present disclosure provides a method of treating a cancer in a subject that can comprise administering a pharmaceutical composition of the present disclosure to the subject.
  • the cancer to be treated includes, but is not limited to, non-Hodgkin's lymphomas, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignant melanoma and rituximab resistant NHL and leukemia.
  • the present disclosure provides a method of treating a subject having a metabolic disorder, said method can comprise administering a pharmaceutical composition of the present disclosure in an amount sufficient to treat said disorder, wherein said metabolic disorder is diabetes, obesity, diabetes as a consequence of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, or hyperlipidemia.
  • diabetes is diabetes, obesity, diabetes as a consequence of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, or hyperlipidemia.
  • the present disclosure provides a method of treating a subject having a fatty liver disease (e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH)), a gastrointestinal disease, or a neurodegenerative disease, said method comprising orally administering a pharmaceutical composition of the present disclosure in an amount sufficient to treat said disease.
  • a fatty liver disease e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH)
  • NASH nonalcoholic steatohepatitis
  • the present disclosure provides a method of treating a subject having a GH deficient growth disorder, said method can comprise administering a pharmaceutical composition of the present disclosure in an amount sufficient to treat said disorder, wherein said disorder is growth hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome, Prader-Willi syndrome, short stature homeobox-containing gene (SHOX) deficiency, chronic renal insufficiency, and idiopathic short stature short bowel syndrome, GH deficiency due to rare pituitary tumors or their treatment, and muscle-wasting disease associated with HIV/AIDS.
  • GDD growth hormone deficiency
  • TS Turner syndrome
  • Noonan syndrome Noonan syndrome
  • Prader-Willi syndrome short stature homeobox-containing gene
  • SHOX short stature homeobox-containing gene
  • a delivery construct can comprise a carrier comprising an amino acid sequence having at least 80% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 133 or SEQ ID NO: 137 - SEQ ID NO: 147.
  • a delivery construct can comprise a carrier comprising an amino acid sequence having at least 90% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 133 or SEQ ID NO: 137 - SEQ ID NO: 147.
  • a delivery construct can comprise a carrier comprising an amino acid sequence having at least 95% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 133 or SEQ ID NO: 137 - SEQ ID NO: 147.
  • a delivery construct can comprise a carrier comprising an amino acid sequence having at least 99% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 133 or SEQ ID NO: 137 - SEQ ID NO: 147.
  • the carrier can be derived from a Cholix domain I and can comprise an amino acid sequence having at least 80% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125 and/or SEQ ID NO: 148 - SEQ ID NO: 152.
  • the carrier can be derived from a Cholix domain I and can comprise an amino acid sequence having at least 90% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125 and/or SEQ ID NO: 148 - SEQ ID NO: 152.
  • the carrier can be derived from a Cholix domain I and can comprise an amino acid sequence having at least 95% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125 and/or SEQ ID NO: 148 - SEQ ID NO: 152.
  • the carrier can be derived from a Cholix domain I and can comprise an amino acid sequence having at least 99% sequence identity to any one or more of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125 and/or SEQ ID NO: 148 - SEQ ID NO: 152.
  • the present disclosure provides isolated delivery constructs that can be capable of binding a receptor on the luminal surface of intestinal epithelial cells with sufficient affinity to allow endocytosis; wherein the domain is a polypeptide comprising an amino acid sequence wherein one or more amino acid residues of one bacterial toxin domain I polypeptide is replaced by one or more amino acid residues of a second bacterial toxin (e.g., an exotoxin) domain I polypeptide.
  • a second bacterial toxin e.g., an exotoxin
  • the domain I of a first exotoxin can comprise a polypeptide which comprises an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 can be replaced by one or more amino acid residues of a second bacterial toxin (e.g., an exotoxin) receptor binding domain polypeptide.
  • the receptor binding domain can be a polypeptide which comprises an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 is replaced by one or more amino acid residues of SEQ ID NO: 137.
  • the receptor binding domain can be a polypeptide which comprises an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 137 is replaced by one or more amino acid residues a second bacterial toxin receptor binding domain polypeptide.
  • the receptor binding domain can be a polypeptide which comprises an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 136 is replaced by one or more amino acid residues of SEQ ID NO: 4.
  • a chimeric carrier can comprise a biologically active cargo coupled to the polypeptide to produce a chimeric construct; wherein the chimeric construct is capable of delivering the biologically active cargo.
  • the present disclosure provides a chimeric construct comprising a bacterial toxin- derived delivery construct; and a biologically active cargo; wherein the delivery construct is capable of delivering the biologically active cargo into an epithelial cell; and wherein the delivery construct does not comprise a bacterial toxin-derived translocation domain or a bacterial toxin-derived catalytic (cytotoxic) domain.
  • the present disclosure provides a chimeric construct consisting of a receptor binding domain of a bacterial toxin; and a biologically active cargo; wherein the delivery construct is capable of delivering the biologically active cargo into an epithelial cell, and wherein the chimeric construct is capable of binding a receptor on the luminal surface of intestinal epithelial cells.
  • vectors comprising polynucleotides encoding the non-naturally occurring delivery constructs and/or delivery constructs of the present disclosure; optionally, operably-linked to control sequences recognized by a host cell transformed with the vector; host cells comprising vectors comprising polynucleotides encoding the non-naturally occurring delivery constructs and/or delivery constructs of the present disclosure; a process for producing the non-naturally occurring delivery constructs and/or delivery constructs of the present disclosure comprising culturing host cells comprising vectors comprising polynucleotides encoding the non-naturally occurring delivery constructs and/or delivery constructs of the present disclosure such that the polynucleotide is expressed; and, optionally, recovering the non-naturally occurring delivery constructs and/or delivery constructs from host cell culture medium.
  • Disclosed herein is a use of a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of an inflammatory disease in a subject in need thereof.
  • Disclosed herein is a use of a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of an autoimmune disease in a subject in need thereof.
  • Disclosed herein is a use of a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of a cancer in a subject in need thereof.
  • a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of a metabolic disorder in a subject in need thereof.
  • Disclosed herein is a use of a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of a fatty liver disease in a subject in need thereof.
  • Disclosed herein is a use of a non-naturally occurring delivery construct of the present disclosure for the preparation of a medicament for treatment, prophylaxis and/or prevention of GH deficient growth disorder in a subject in need thereof.
  • FIG. 2 depicts (top to bottom) Constructs 9, 8 and 7.
  • FIG. 1A shows localization (fluorescence image) of construct 12 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. IB shows localization (white light image) of construct 12 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. ID shows localization (fluorescence image) of construct 11 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. IE shows localization (white light image) of construct 11 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 1G shows localization (fluorescence image) of construct 10 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. II shows localization (merge image, with DAPI) of construct 10 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 2A shows localization (fluorescence image) of construct 9 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 2B shows localization (white light image) of construct 9 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 2D shows localization (fluorescence image) of construct 8 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 2G shows localization (fluorescence image) of construct 7 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 21 shows localization (merge image, with DAPI) of construct 7 observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 3 depicts fluorescence microscopic detection of Construct 6 (prepared as described in EXAMPLE 1 herein) observed 20 min after intra-luminal injection using a rat intra-luminal injection model.
  • Fluorescence image, dark field illumination, composite of fluorescence image and dark field illumination white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface.
  • FIG. 3A depicts fluorescence microscopic detection of Construct 6 (anti-Cho,
  • FIG. 3B depicts dark field illumination detection of Construct 6 (anti-Cho, 1/500).
  • FIG. 3B depicts a composite of fluorescence image and dark field illumination detection of Construct 6 (anti-Cho, 1/500).
  • FIG. 3D depicts fluorescence microscopic detection of Construct 6 (anti-RPF, 1/50).
  • FIG. 3E depicts dark field illumination detection of Construct 6 (anti-RPF, 1/50).
  • FIG. 4 and FIG. 5 depict fluorescence microscopic detection of Construct 13 (SEQ ID NO: 146, prepared as described in EXAMPLE 5 herein) observed after 1 min (FIG. 4) and 20 min (FIG. 5) after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 4A shows fluorescence microscopic detection of Construct 13 after 1 min.
  • FIG. 4B shows fluorescence microscopic detection of Construct 13 after 1 min.
  • FIG. 4C shows fluorescence microscopic detection of Construct 13 after 1 min.
  • FIG. 4D shows fluorescence microscopic detection of Construct 13 after 1 min.
  • FIG. 4E shows fluorescence microscopic detection of Construct 13 after 1 min
  • FIG. 5A shows fluorescence microscopic detection of Construct 13 after 20 min.
  • FIG. 5B shows fluorescence microscopic detection of Construct 13 after 20 min.
  • FIG. 5C shows fluorescence microscopic detection of Construct 13 after 20 min.
  • FIG. 5D shows fluorescence microscopic detection of Construct 13 after 20 min.
  • FIG. 5E shows fluorescence microscopic detection of Construct 13 after 20 min
  • FIG. 6 and FIG. 7 depict fluorescence microscopic detection of Construct 14 (SEQ ID NO: 147, prepared as described in EXAMPLE 5 herein) observed after 1 min (FIG. 6) and 20 min (FIG. 7) after intra-luminal injection using a rat intra-luminal injection model (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 6A shows fluorescence microscopic detection of Construct 14 after 1 min.
  • FIG. 6B shows fluorescence microscopic detection of Construct 14 after 1 min.
  • FIG. 6C shows fluorescence microscopic detection of Construct 14 after 1 min.
  • FIG. 6D shows fluorescence microscopic detection of Construct 14 after 1 min.
  • FIG. 6E shows fluorescence microscopic detection of Construct 14 after 1 min
  • FIG. 7A shows fluorescence microscopic detection of Construct 14 after 20 min.
  • FIG. 7B shows fluorescence microscopic detection of Construct 14 after 20 min.
  • FIG. 7C shows fluorescence microscopic detection of Construct 14 after 20 min.
  • FIG. 7D shows fluorescence microscopic detection of Construct 14 after 20 min.
  • FIG. 7E shows fluorescence microscopic detection of Construct 14 after 20 min
  • FIG. 8A depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix 39 186 -(spacer with SEQ ID NO: 2lO)-HGH) observed 5 min after intra-luminal injection using a rat intra-luminal injection model (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 8B depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix 39 186 -(spacer with SEQ ID NO: 2lO)-HGH) observed 10 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 8C depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix 39 186 -(spacer with SEQ ID NO: 2lO)-HGH) observed 15 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 9A depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 160 (Cholix 1 187 -(spacer with SEQ ID NO: 210)- HGH) observed 5 min after intra-luminal injection using a rat intra-luminal injection model (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 9B depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 160 (Cholix 1 187 -(spacer with SEQ ID NO: 210)- HGH) observed 10 min after intra-luminal injection using a rat intra-luminal injection model.
  • FIG. 9C depicts fluorescence microscopic detection of the construct comprising an amino acid sequence set forth in SEQ ID NO: 160 (Cholix 1 187 -(spacer with SEQ ID NO: 210)- HGH) observed 15 min after intra-luminal injection using a rat intra-luminal injection model (white arrow #2 highlights the basal surface).
  • FIG. 10A shows Non-toxic Cholix (ntChx) transcytosis across human polarized intestinal epithelium in vitro.
  • FIG. 10B depicts a Western blot analysis of basal compartment contents 2 h after an apical application of 2.5 -200 mg/mL ntChx that were concentrated approximately lO-fold prior to analysis showing that the ntChx that transported was not significantly altered (e.g., chemically altered) during transport.
  • FIG. 10C depicts basal quantities of ntChx detected over a time course of 2 h by ELISA.
  • the graph shows a delay of -20-25 min in detectable quantities and comparable rates of transport for apical applications of 5-20 mg/mL at 37 °C, and a significant reduction in transport rate at 4 °C.
  • FIG. 12A depicts transcytosis of non-toxic Cholix (ntChx, SEQ ID NO: 3) in vivo after 1 minutes following intraluminal injection (ILI) into rat jejunum visualized by immunofluorescence microscopy.
  • FIG. 12B depicts transcytosis of non-toxic Cholix (ntChx, SEQ ID NO: 3) in vivo after 5 minutes following ILI into rat jejunum visualized by immunofluorescence microscopy.
  • FIG. 13A shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of early endosomal antigen 1 (EEA1) (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • EAA1 early endosomal antigen 1
  • FIG. 13B shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of Ras-related protein Rab 1 la (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 13C shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of trans-Golgi network (TGN)-38 protein.
  • FIG. 13D shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of calnexin (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 13E shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of Ras-related protein Rab 7 (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 13F shows in vivo ntChx transcytosis at 15 min after ILI of ntChx (SEQ ID NO: 3) into rat jejunum with simultaneous staining of Golgi-associated 58kDa
  • FIG. 14A shows small amounts of RFP (negative control) reaching cells within the lamina intestinal ( l-p ) with no detectable RFP in the villus epithelium (solid arrows point to apical surface of epithelium) at 30 min post ILL
  • a polyclonal antibody to RFP demonstrates small amounts of RFP can reach cells within the lamina intestinal (l-p) with no detectable RFP in the villus epithelium ; (solid arrows point to apical surface of epithelium) at 30 min post ILL
  • FIG. 14B shows that a construct comprising an amino acid sequence set forth in SEQ ID NO: 157 comprising full-length of non-toxic Cholix (ntChx, SEQ ID NO: 3) genetically fused to or conjugated to red fluorescent protein (RFP, SEQ ID NO: 220) is capable of efficient apical-to-basal transcytosis.
  • Full-length of non-toxic Cholix (ntChx, SEQ ID NO: 3) was genetically conjoined to the red fluorescent protein (RFP, SEQ ID NO:
  • FIG. 14C shows that Cholix domain I is sufficient for apical to basal transcytosis after intraluminal injection (ILI) into rat jejunum in vivo.
  • FIG. 14C shows that a Cholix truncated at the termination of domain I (amino acid residue 265 of SEQ ID NO: 1, plus an N- terminal methionine residue resulting in SEQ ID NO: 5) and that is genetically fused to RFP (e.g., thus having an amino acid sequence set forth in SEQ ID NO: 156) is capable of efficient apical-to-basal transcytosis, suggesting that Cholix domain I may be sufficient for apical to basal transcytosis after intraluminal injection (ILI) into rat jejunum in vivo.
  • ILI intraluminal injection
  • Solid white arrows #1 indicate the apical epithelial surface, and white arrow #2 highlights the basal surface.
  • Cholix domain I (SEQ ID NO: 5) was genetically conjoined to the red fluorescent protein (RFP, SEQ ID NO: 220).
  • FIG. 15 depicts apical-to-basal transport of human growth hormone (HGH, SEQ ID NO: 214) compared to chimeras of Cholix domain I and truncated elements of this domain (designated by amino acids) that were genetically conjoined to HGH.
  • HGH human growth hormone
  • SEQ ID NO: 214 chimeras of Cholix domain I and truncated elements of this domain (designated by amino acids) that were genetically conjoined to HGH.
  • Western blotting for HGH qualitatively assessed the capacity of these proteins to undergo apical-to-basal transport across polarized monolayers of primary human small intestinal epithelial cells in vitro after 2 h. The amounts of apically-applied materials were equivalent on a molar basis for HGH content and basal collections were concentrated ⁇ l0-fold prior to analysis.
  • FIG. 15A shows that background apical-to-basal transport of HGH alone in this model was minimal compared to that observed for the delivery construct comprising the amino acid sequence set forth in SEQ ID NO: 164, comprising a Cholix domain I (SEQ ID NO: 5), a spacer (SEQ ID NO: 210), and HGH (SEQ ID NO: 214).
  • Cholix domain I (SEQ ID NO: 5) truncations at positions 134, 151, 187 or at 40-187 of SEQ ID NO: 5 were incapable of facilitating apical -to-basal transport of conjoined HGH.
  • FIG. 15B shows that truncations of Cholix domain I (SEQ ID NO: 5) at positions 206, 245, or 251 demonstrated apical-to-basal transport of conjoined HGH. While truncations as positions 245 and 251 resulted in apical-to-basal transport comparable to that of the construct comprising the carrier with SEQ ID NO: 5, the chimera where Cholix domain I is truncated at position 206 showed a significant enhancement of apical-to-basal transport.
  • FIG. 16A shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16A shows that the Cholix -HGH construct with SEQ ID NO: 211 (comprises Cholix 1 133 + N-term.
  • FIG. 16B shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16B shows that the Cholix -HGH construct with SEQ ID NO: 212 (comprises Cholix 1 150 + N-term.
  • epithelial cells did enter epithelial cells (as opposed to protein with SEQ ID NO: 211) but remained in apical and basal vesicular pools and did not enter the lamina intestinal , thus enabling delivery the interior of an epithelial cell (e.g., a compartment at the basal side of the epithelial cell).
  • FIG. 16C shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16C shows that the Cholix -HGH construct with SEQ ID NO: 213 (comprises Cholix 1 186 + N-term.
  • methionine entered epithelial cells, reached apical and basal compartments and, significantly, also a supra-nuclear region of the cell, yet still remained inside the epithelial cell, suggesting that the functional peptide fragment having an amino acid sequence set forth in SEQ ID NO: 151 may allow access and delivery to supranuclear regions, yet does not allow release of the construct into a basolateral compartment (e.g., lamina propria).
  • FIG. 16D shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16D shows that the Cholix -HGH construct with SEQ ID NO: 218 (comprises Cholix 39 186 + N-term.
  • FIG. 16E shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16E shows that the Cholix -HGH construct with SEQ ID NO: 161 (comprises Cholix 1 205 + N-term.
  • FIG. 16F shows the assessment of Cholix domain I truncation-human growth hormone (HGH) chimera transport across rat jejunum epithelia monolayers in vivo 15 min after intraluminal injection as demonstrated by immunofluorescence microscopy.
  • FIG. 16F shows that the Cholix -HGH construct with SEQ ID NO: 164 (comprises Cholix 1 265 + N-term.
  • Methionine SEQ ID NO: 5
  • SEQ ID NO: 5 completed the transcytosis process as indicated by delivery of the chimera to cells within the lamina intestinal similar to the Cholix -HGH construct with SEQ ID NO: 164.
  • FIG. 17 shows that selected amino acid fragments of Cholix domain I achieve apical to basal transcytosis in vitro and in vivo.
  • a polymer framework containing peptide sequences of amino acids from positions 1-39, 134-151, 151-178, and 178-206 of Cholix domain I with SEQ ID NO: 5 in various combinations were labeled with different forms of quantum dots (e.g., cadmium sulfide, lead sulfide, etc.).
  • FIG. 17B shows in vivo transcytosis at 15 min of the Cholix 39 186 -HGH construct (SEQ ID NO: 218) (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 17C shows in vivo transcytosis at 15 min of the (SEQ ID NO: 5)-(4N)-RFP construct labeled with quantum dots (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 18 shows the amino acid sequence set forth in SEQ ID NO: 221 of a Cholix domain 1 (incl. and N-terminal methionine) having a spacer with an amino acid sequence set forth in SEQ ID NO: 210 attached to its C-terminus.
  • This spacer can be used to attach cargo moieties (e.g., therapeutic agents) to the Cholix carrier for transport across epithelial layers (e.g., the gut epithelium).
  • cargo moieties e.g., therapeutic agents
  • the highlighted amino acid fragments can provide certain functionalities in relation to transcytosis across epithelial layers.
  • the fragment with the amino acid residues of positions 134-151 of the sequence set forth in SEQ ID NO: 5 can promote apical entry of Cholix constructs into epithelial cells.
  • the highlighted fragment with amino acid residues 151-187 can promote early endosomal sorting.
  • the highlighted fragment with amino acid residues 187-206 can promote complete transcytosis of a Chx construct as described herein.
  • FIG. 19 shows potential glycosylation sites of the asparagine residues located at positions N98, N154, N165, and N224 of Cholix domain I having an amino acid sequence set forth in SEQ ID NO: 221.
  • FIG. 20 shows a general 3D structure of Cholix domain I (SEQ ID NO: 5) with highlighted functional fragments.
  • FIG. 20A shows a general 3D structure of Cholix domain I (SEQ ID NO: 5) with the highlighted functional fragment having an amino acid sequence of SEQ ID NO: 148 (residues 134-151 of SEQ ID NO: 5).
  • FIG. 20B shows a general 3D structure of Cholix domain I (SEQ ID NO: 5) with the highlighted functional fragment having an amino acid sequence of SEQ ID NO: 149 (e.g., residues 151-187 of SEQ ID NO: 5).
  • FIG. 20C shows a general 3D structure of Cholix domain I (SEQ ID NO: 5) with the highlighted functional fragment having an amino acid sequence of SEQ ID NO: 152 (e.g., residues 187-206 of SEQ ID NO: 5).
  • FIG. 21 illustrates a trafficking pathway analysis for the Cholix derived delivery construct having the amino acid sequence set forth in SEQ ID NO: 154 (the delivery construct is M+Cholix 386 -GGGGSGGGGSGGGGS (SEQ ID NO: 2lO)-IL-lO, from N- to C-terminus).
  • the delivery construct comprising Cholix domain (SEQ ID NO: 5) and human growth hormone (HGH) as cargo could also be used to show similar results as shown for the construct comprising M+Cholix 386 .
  • FIG. 21A shows that M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct strongly co-localized with the EEA1 antigen in cellular locations consistent with trafficking at both the apical and basal domains of enterocytes, suggesting the presence of the Cholix derived delivery constructs in early endosome compartments (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 21B show that the M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct (top right) strongly co-localizes with the Rab7 (top left) predominantly in the apical
  • FIG. 21C shows that LAMP1 was identified in large, specific vesicles consistent mature lysosomes that were devoid of M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery constructs (white arrows).
  • M+Cholix 386 -IL-10 SEQ ID NO: 154
  • FIG. 21D shows that M+Cholix 386 -IL-10 (SEQ ID NO: 154) chimera also strongly co-localized with clathrin-coated vesicles, particularly in areas adjacent to the nucleus and in the Rabl 1 predominantly in the basal compartment of enterocytes as well as in selected cells within the lamina propria.
  • FIG. 21E shows that M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct co- localizes with the endoplasmic reticulum as demonstrated by calnexin in a pattern adjacent to the nucleus in enterocytes and in a large fraction of cells with in the lamina propria.
  • M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct strongly co-localizes with the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) and the LAMN1 antigen appeared to re-distribute in response to carrier endocytosis and transcytosis, as shown for 1
  • FIG. 21F 5 (FIG. 21G), 10 (FIG. 21H), and 15 minutes after injection (FIG. 211).
  • FIG. 21F shows that the M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct strongly co-localizes with the endoplasmatic reticulum Golgi intermediate compartment
  • FIG. 211 shows M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct co- localization with LAMN1 antigen 15 minutes after injection.
  • FIG. 21L shows that the M+Cholix 386 -IL-lO (SEQ ID NO: 154) delivery construct showed some level of co-localization with the TGN38 antigen (top right), which showed a cellular distribution that was restricted to the apical side of nuclei in enterocytes and adjacent to the nucleus in a few cells within the lamina intestinal (white light and merge images shown bottom left and bottom right, respectively).
  • FIG. 21M shows that the M+Cholix 386 -IL-lO (SEQ ID NO: 154) delivery construct (staining shown in green, top right) strongly co-localizes with Rabl 1 (top left) predominantly in the basal compartment of enterocytes and in selected cells within the lamina intestinal (white light and merge images shown bottom left and bottom right, respectively).
  • FIG. 22 illustrates a 1D SDS-PAGE showing that an efficient protocol using nano- sized magnetic beads (25 nm or 100 nm diameter) decorated with non-toxic Cholix derived carrier elements can be used for specific protein capture to analyze proteins that interact with Cholix or carriers derived therefrom.
  • FIG. 23 illustrates that, after multiple washings, the magnetic bead-enriched vesicles can be solubilized in lysis buffer and the protein components present can be separated by 2-D SDS-PAGE for analysis.
  • FIG. 24 shows that patterns of these proteins can be compared to the total protein content of the cells and that mass spectrometry can be used to identify specific elements associated with vesicular structures accessed by the Cholix derived delivery constructs.
  • FIG. 26 shows that incubation of the Cholix carrier (having the amino acid set forth in SEQ ID NO: l54)-coated beads with the pure proteins and subsequent Western Blots or ELISA can enable detection of Cholix -protein interaction.
  • this figure shows interaction of Cholix carrier with heparan sulfate proteoglycan (HSPG), Dickkopf-related protein 1 (DKK1), the chaperone glucose-regulated protein 75 (GRP75), and cytokeratin-8 (K8 or CK8).
  • HSPG heparan sulfate proteoglycan
  • DKK1 Dickkopf-related protein 1
  • GFP75 chaperone glucose-regulated protein 75
  • cytokeratin-8 K8 or CK8
  • FIG. 27 shows microscopic co-localization of candidate proteins and Cholix derived delivery construct in rat jejunum.
  • Co-localization of a delivery construct comprising a Cholix carrier protein coupled to IL-10 (SEQ ID NO: 154, M+Cholix 386 -GGGGSGGGGSGGGGS (SEQ ID NO: 210)-IL- 10) with CK8 was shown in vivo.
  • FIG. 27A shows co-localization after rat jejunum was treated with a luminal application of M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct for 1 minute (white arrow #1 highlights the apical surface, and white arrow #2 highlights the basal surface).
  • FIG. 27B shows co-localization after rat jejunum was treated with a luminal application of M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct for 5 minute.
  • FIG. 27C shows co-localization after rat jejunum was treated with a luminal application of M+Cholix 386 -IL-10 (SEQ ID NO: 154) delivery construct for 10 minutes.
  • M+Cholix 386 -IL-10 SEQ ID NO: 1544
  • FIG. 28 shows a comparison of results obtained in an IHC study with the human atlas to ensure that the receptor distribution is consistent between rat in vivo studies and human intestine.
  • two of the receptors identified by mass spectrometry and verified in rat jejunum are examined.
  • FIG. 28A shows that the intestinal localization of GRP75 is consistent between rat and human intestine.
  • FIG. 28B shows that the intestinal localization of HSPC is consistent between rat and human intestine.
  • FIG. 29 shows effects of HSPG knockout by CRISPR on transport function of the delivery construct (SEQ ID NO: 164) comprising Cholix domain I (SEQ ID NO: 5) coupled to HGH (SEQ ID NO: 214) via a polyglycine-serine spacer (SEQ ID NO: 210) and HGH alone as internal control of non-selective transport.
  • Cells were seeded at l .5xl0 5 cells/mL in transwells.
  • transepithelial/transendothelial electrical resistance (TEER) was measured and PBS containing 20 ug/mL of the delivery construct was added to the apical chambers. After 3 h, basolateral samples were collected and concentrated. The extent of protein transport was analyzed by Western blotting using anti -HGH antibody. The results shown in FIG. 29
  • FIG. 30 shows knockout effects of K8, HSPC, and GRP75 on the transcytosis function of Cholix domain I derived delivery constructs.
  • Stable cell lines of Caco-2 cells lacking the expression of specific candidate proteins were used as monolayers in vitro to verify their requirement for carrier transcytosis using active and selective endogenous transport mechanisms.
  • the specific transport of the HGH-containing delivery construct vs non-selective transport of HGH alone was reduced in HSPG and GRP75 knockouts, but not the K8 knockout.
  • FIG. 30 A shows knockout effects of K8.
  • FIG. 30B shows knockout effects of HSPC.
  • FIG. 30C shows knockout effects of GRP75.
  • FIG. 30D shows the control experiment.
  • FIG. 31 shows Biacore binding interactions used to examine the pH-dependency of Cholix carrier-GRP75 interactions. Cholix carrier proteins were attached to magnetic beads using the biotin-streptavidin bioconjugation and incubated with purified GRP75 protein in buffer solutions with pH 5.5, 6.5, and 7.5, respectively. Highest binding affinity was shown at pH 6.5.
  • FIG. 32 shows an exemplary surface model of Cholix domain I (SEQ ID NO: 5) was used to highlight selected areas of potential interest in this transcytosis process due to their projection from the protein surface. It is interesting to note that two amino acids regions between M 1 and G 40 are adjacent to surface exposed amino acids D 151 -A 187 and A 187 -L 206 . Specifically,
  • L -I (domain XI) and T -I (domain X2) coordinate to form a pocket surrounded by several negative charges.
  • K -H (domain X3) coordinates with I -E (domain X4) to form a continuous ridge structure
  • FIG. 32A shows the proximity of domains X3 and X4.
  • FIG. 32B shows the proximity of domains XI and X2, as well as X3 and X4.
  • FIG. 32C shows the proximity of domains XI and X2.
  • FIG. 32D shows the proximity of domains XI and X2, as well as X3 and X4.
  • the present disclosure provides methods and compositions for transport and/or delivery of a cargo molecule to certain location(s) within a cell (e.g., a supranuclear location) or across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a rodent or a human).
  • a cargo molecule can be directed to a set of location(s) by coupling it to a carrier molecule.
  • carrier molecule can interact with unique receptors both on the cell surface and intracellularly for the targeted delivery of the cargo.
  • carrier, cargos, and uses thereof are described herein.
  • an amino acid sequence can comprise one or more modification to the amino acid sequences at the N-terminus.
  • An amino acid sequence as disclosed herein can comprise an“N-cap.”
  • an N-cap as disclosed herein can refer to a modification of an N-terminus of a peptide or polypeptide in a variety of ways, and particularly can refer to (i) the addition of one or more amino acid sequences or other moieties (e.g., affinity handles, cell- penetrating peptide sequences, etc.), and (ii) a modification of one or more amino acid residues within the first 1-10 N-terminal amino acids of a peptide or polypeptide, wherein the amino acid modification is relative to a reference sequence or a consensus sequence (see e.g., comparison of the first 4 N-terminal amino acid residues of polypeptide sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 1 and SEQ ID NO: 2 as described herein).
  • An N-cap can comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 or 100 additional amino acid residues that are attached to (e.g., chemically coupled to) to the N-terminus of an amino acid sequence, such as a Cholix derived carrier molecule.
  • An N-cap can further comprise one or more variations in the amino acid sequence at the N-terminus.
  • a Cholix domain I derived carrier can comprise an N-cap.
  • the N-cap can comprise substituting one or more N-terminal amino acid residues with other amino acid residues.
  • An N-cap can further comprise an N-terminal methionine residue.
  • One or more of these modifications can be a result of producing the Cholix domain I amino acid sequence in a bacterial production system (e.g., E. coli).
  • Cholix domain I can comprise amino acid residues 1-265 of SEQ ID NO: 1 which is set forth in SEQ ID NO: 4.
  • a bacterially expressed Cholix domain can comprise an amino acid sequence set forth in SEQ ID NO: 5, which as SEQ ID NO: 4 plus an N-terminal methionine residues, which can also be referred to herein as M+Cholix 1 265 or M+Cholix 265 .
  • the term“lacks a domain” or“lacking a domain” generally refers to not comprising a complete domain, but optionally comprising a portion or fragment thereof.
  • a carrier that is derived from a domain I of an exotoxin but lacks a domain II, a domain lb, and a domain III of said exotoxin generally refers to a carrier that does not comprise the full amino acid sequences of (e.g., 100% sequence identity to) any one of the domains II, lb, and III, but which can optionally comprise portions or fragments thereof.
  • a carrier derived from a Cholix domain and lacking a Cholix domain II as described herein can comprise Cholix domain I having an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 and an additional 50-80 amino acid residues of Cholix domain II (e.g., amino acids 1-50 or 1-80 of SEQ ID NO: 126), and or an additional 50-80 amino acid residues of Cholix domain III (e.g., amino acids 1-50 or 1-80 of SEQ ID NO: 128).
  • first molecule e.g., a polypeptide
  • second molecule e.g., a polypeptide, small molecule, etc.
  • the association can be via a chemical linkage, wherein the chemical linkage can be covalently or non-covalently.
  • a covalent chemical linkage between a first polypeptide and a second polypeptide can be produced by synthetically coupling the first polypeptide to the second polypeptide, or it can be produced by recombinant fusion of the first polypeptide to the second polypeptide.
  • a first (e.g., a first polypeptide) molecule can be chemically (e.g.,
  • a second molecule e.g., a second polypeptide
  • polypeptide “peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms“toxin”,“carrier”,“delivery construct”,“chimeric construct”,“protein”, and“polypeptide” can be used interchangeably and generally refer to a molecule that can be coupled to a heterologous cargo.
  • “delivery constructs” and“chimeric constructs” are“peptides”,“polypeptides”, or“proteins”, are described herein as chains of amino acids whose alpha carbons are linked through peptide bonds.
  • amino terminal therefore has a free amino group
  • terminal amino acid at the other end of the chain has a free carboxyl group
  • amino acid at the other end of the chain has a free carboxyl group.
  • amino terminus refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
  • the term“carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide.
  • Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether bond as opposed to an amide bond.
  • peptides, polypeptide, and proteins as described herein can be recombinantly produced or chemically synthesized (e.g., using solid-phase synthesis), or a combination thereof.
  • the term“delivery” generally refers to the presence of a molecule (e.g., a heterologous cargo) at a location (e.g., an intracellular compartment or a supranuclear region) for a certain period of time.
  • the term“delivery” can refer to the presence of a molecule (e.g., a heterologous cargo) at a location (e.g., an intracellular compartment or a supranuclear region) for a time that is sufficient to elicit a certain biological effect, such as an interaction (e.g., binding) with a protein (e.g., an enzyme or a receptor) at that location.
  • the delivery of a molecule (e.g., a heterologous cargo) to a location e.g., an intracellular
  • compartment or a supranuclear region can refer to the retention of the molecule at that location.
  • Retention of a molecule at a certain intracellular or extracellular region or compartment can be for a certain amount of time, e.g., at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at 30 minutes, or at least 60 minutes.
  • Retention of a molecule can depend on various factors such as the location where the molecule is retained and/or the types of molecular interactions that occur between the molecule (e.g., a carrier, a delivery construct, and/or a heterologous cargo).
  • delivery of a heterologous cargo to a basolateral compartment via transcytosis across a polarized epithelial cell can comprise retaining the heterologous cargo at the basolateral location for a time sufficient to elicit a certain effect, such as a therapeutic effect in case of a therapeutic and/or biologically active cargo.
  • Polypeptides of the disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties.
  • single or multiple amino acid substitutions e.g., conservative amino acid substitutions
  • A“conservative amino acid substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:
  • A“non-conservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class.
  • the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge
  • the carrier of a delivery constructs is a chimeric carrier comprising a peptide, polypeptide, small molecule, aptamer, fragments thereof, or any combination thereof.
  • antigenicity i.e., with a biological property of the protein.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +/- .1); glutamate (+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 are within + 2 is included, in various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.
  • a skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques.
  • One skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity.
  • the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides. Areas of these materials that can be important for biological activity or for structure could be subjected to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, the skilled artisan can predict the importance of amino acid residues in a
  • polypeptide that correspond to amino acid residues important for activity or structure in similar polypeptides.
  • One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art can predict the alignment of amino acid residues of a polypeptide with respect to its three-dimensional structure. One skilled in the art can choose to not make radical changes to amino acid residues predicted to be on the surface of the polypeptide, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants.
  • polypeptide fragment and“truncated polypeptide” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length protein.
  • fragments can be, e.g., at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length.
  • fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length.
  • a fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial spacer sequence).
  • polypeptide variant and“polypeptide mutant” as used herein refer to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
  • the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length.
  • Variants of the present disclosure include fusion proteins.
  • A“derivative” of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • % sequence identity is used interchangeably herein with the term“% identity” and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program.
  • 80% identity means the same thing as 80% sequence identity determined by a defined algorithm, and means that a given sequence is at least 80% identical to another length of another sequence.
  • the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
  • % sequence homology is used interchangeably herein with the term“% homology” and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence.
  • the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
  • Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN,
  • BLASTX BLASTX
  • TBLASTX BLASTP
  • TBLASTN BLASTTN
  • Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases.
  • the BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See id.
  • 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, Proc. NatT. Acad. Sci. USA, 90:5873-5787 (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.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., at most 0.1, at most 0.01, or at most 0.001.
  • Polynucleotide refers to a polymer composed of nucleotide units.
  • Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs.
  • Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds.
  • nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O- methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • PNAs peptide-nucleic acids
  • Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer.
  • the term“nucleic acid” typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides.
  • nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C)
  • this also includes an RNA sequence (i.e., A, U, G, C) in which“U” replaces“T.”
  • the DNA strand having the same sequence as an mRNA is referred to as the“coding strand”; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5’ -end of the RNA transcript are referred to as“upstream sequences”; sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as“downstream sequences.”
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides.
  • the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.
  • hybridizing specifically to” or“specific hybridization” or“selectively hybridize to” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences.
  • “Stringent hybridization” and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence-dependent, and are different under different environmental parameters.
  • highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the Tm for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than about 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 x SSC wash at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A high stringency wash can be preceded by a low stringency wash to remove background probe signal.
  • An exemplary medium stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 1 x SSC at 45°C for 15 minutes.
  • An exemplary low stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6 x SSC at 40°C for 15 minutes.
  • a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific
  • Primer refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a
  • a primer is typically single-stranded, but can be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.
  • a primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • Probe when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In instances where a probe provides a point of initiation for synthesis of a
  • a probe can also be a primer.
  • A“vector” is a polynucleotide that can be used to introduce other nucleic acids linked to it into a cell.
  • a“plasmid” refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated.
  • a viral vector e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • An“expression vector” is a type of vector that can direct the expression of a chosen polynucleotide.
  • A“regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked.
  • the regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid).
  • Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • a nucleotide sequence is“operably linked” to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the nucleotide sequence.
  • a cell of the present disclosure can be a eukaryotic cell or a prokaryotic cell.
  • a cell can be an epithelial cell.
  • An epithelial cell can be a polarized epithelial cell (e.g., a Caco-2 cell or a Chinese Hamster Ovary (CHO) cell).
  • a cell can be an animal cell or a plant cell.
  • An animal cell can include a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal.
  • a mammalian cell can be obtained from a primate, ape, equine, bovine, porcine, canine, feline, or rodent.
  • a mammal can be a primate, ape, dog, cat, rabbit, ferret, or the like.
  • a rodent can be a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig.
  • a bird cell can be from a canary, parakeet or parrots.
  • a reptile cell can be from a turtles, lizard or snake.
  • a fish cell can be from a tropical fish.
  • the fish cell can be from a zebrafish (e.g., Danino rerio).
  • a worm cell can be from a nematode (e.g., C. elegans).
  • An amphibian cell can be from a frog.
  • An arthropod cell can be from a tarantula or hermit crab.
  • a mammalian cell can also include cells obtained from a primate (e.g., a human or a non-human primate).
  • a mammalian cell can include a blood cell, a stem cell, an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, or an immune system cell.
  • the methods and compositions of the present disclosure are used in combination with one or more mammalian blood cells.
  • a host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
  • a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding series of nucleic acids, which can then be expressed in the host cell.
  • the phrase“recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a polypeptide-encoding series of nucleic acids to be expressed.
  • a host cell also can be a cell that comprises series of nucleic acids but does not express these at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the terms“complete transcytosis”,“efficient transcytosis”, or “transcytosis”, or“transport” can be used interchangeably and can refer to the transport of toxin- derived delivery constructs across epithelial layers such as the gut epithelium. These terms can refer to a complete transport of these construct as determined in the respective experiment using various techniques to assess transcytosis efficiency, such as fluorescence microscopy.
  • the terms“comprising” and“having” can be used interchangeably.
  • the terms“a polypeptide comprising an amino acid sequence of SEQ ID NO: 1” and“a polypeptide having an amino acid sequence of SEQ ID NO: 1” can be used.
  • polyacrylamide gel electrophoresis of a protein sample followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art.
  • higher resolution separation techniques can be provided by using HPLC or other means well known in the art for purification.
  • a“spacer” refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5’ end and to another complementary sequence at the 3’ end, thus joining two non-complementary sequences.
  • a “cleavable spacer” refers to a spacer that can be degraded or otherwise severed to separate the two components connected by the cleavable spacer. Cleavable spacers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like.
  • Cleavable spacers can also be cleaved by environmental cues, such as, for example, specific enzymatic activities, changes in temperature, pH, salt concentration, etc. when there is such a change in environment following transcytosis of the delivery constructs across a polarized epithelial membrane.
  • environmental cues such as, for example, specific enzymatic activities, changes in temperature, pH, salt concentration, etc. when there is such a change in environment following transcytosis of the delivery constructs across a polarized epithelial membrane.
  • a heterologous cargo e.g., a biologically active cargo
  • “Pharmaceutical composition” refers to a composition suitable for pharmaceutical use in an animal.
  • a pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier.“Pharmacologically effective amount” refers to that amount of an agent effective to produce the intended
  • “Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington’s Pharmaceutical Sciences, 2lst Ed. 2005, Mack Publishing Co, Easton.
  • the term“subject,” generally refers to a human or to another animal.
  • a subject can be of any age, for example, a subject can be an infant, a toddler, a child, a pre- adolescent, an adolescent, an adult, or an elderly individual.
  • Ranges can be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from 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 embodiment. It will be further understood that the endpoints of each of the ranges are in relation to the other endpoint, and independently of the other endpoint.
  • the term“about” as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 can include a range from 8.5 to 11.5.
  • Carriers that can be used to deliver a cargo to a location within a cell (e.g., epithelial cell) or across a cell (e.g., epithelial cell).
  • Such carriers can be a small molecule, a polypeptide, an aptamer, an antibody, a nucleic acid a fragment of any of the above, or a combination of any of the above.
  • Examples of a polypeptide contemplated herein include any polypeptide that is derived from a domain I of an exotoxin and lacking a domain II, a domain lb and a domain III of the exotoxin.
  • Such domain Fs include but are not limited to amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 137.
  • Polypeptides that are derived from any of the above sequences include those that have a high sequence homology to the above sequences (e.g., greater than 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity as defined in more detail herein).
  • Polypeptides that are derived from any of the above sequences include those that are fragments of the above which function to deliver a cargo to a defined location within a cell or across a cell (e.g., epithelial cell).
  • Examples of small molecules contemplated herein include those that are rationally designed to interact with one or more of the following receptors ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan and/or to have a similar or the same 3D structure of a domain I of an exotoxin (e.g., Cholix or PE), or a functional fragment of a domain I of an exotoxin.
  • an exotoxin e.g., Cholix or PE
  • contemplated herein include those that are rationally designed to interact with one or more of the following receptors ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan and/or to have a similar or the same 3D structure of a domain I of an exotoxin (e.g., Cholix or PE), or a functional fragment of a domain I of an exotoxin.
  • an exotoxin e.g., Cholix or PE
  • nucleic acids that are contemplated herein include those that are rationally designed to interact with one or more of the following receptors ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan and/or to have a similar or the same 3D structure of a domain I of an exotoxin (e.g., Cholix or PE), or a functional fragment of a domain I of an exotoxin.
  • the nucleic acid can be a mRNA, a siRNA, shRNA, or a cDNA.
  • compositions of the present disclosure are based on the inventors’ surprising finding that a carrier capable of interacting with one or more endogenous receptors (e.g., ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan) can provide rapid and efficient delivery of cargo into and/or across a cell such an epithelial cell.
  • endogenous receptors e.g., ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan
  • the methods and compositions described herein allow rapid and efficient transport and/or delivery of cargo molecules across epithelial cells and/or to the interior (e.g., to the intracellular vesicle or compartment or the cytosol) of epithelial cells (e.g., polarized gut epithelial cells).
  • the present disclosure provides constructs (e.g., isolated delivery constructs) that can comprise a carrier coupled to a heterologous cargo.
  • a carrier as disclosed herein can vary in molecular size and composition as well as other physicochemical parameters such as isoelectric point, overall molecular net charge, etc.
  • a carrier can be a small molecule, a polypeptide, an aptamer, a nucleic acid, a fragment and/or any combination thereof.
  • a carrier can be derived from an exotoxin, e.g., any exotoxin described herein.
  • a carrier can be a non-naturally occurring form of Cholix exotoxin (Cholix) or Pseudomonas exotoxin A (PE) comprising only a domain I (i.e., lacking a domain II (sometimes referred to as translocation domain), a domain lb and a domain III (sometimes referred to as cytotoxic domain)) and can be capable of transporting and/or delivering a cargo (e.g., a heterologous cargo such as biological, therapeutic, or diagnostic molecules) across intact epithelial cells (e.g., polarized gut epithelial cells) and epithelial cell barriers (e.g., Caco-2 cell monolayers or the gut epithelium of a subject) via transcytosis and/or to the interior of an epithelial cell (e.g., via apical endo
  • the present disclosure provides methods and compositions comprising a carrier, wherein the carrier can be coupled to a cargo, and as such, can deliver the cargo into or across epithelial cells.
  • the carrier can be a polypeptide, wherein the polypeptide can be derived from an exotoxin.
  • the exotoxin can be Cholix or PE, or any combination thereof (e.g., a carrier comprising one or more domains Cholix and PE, or truncated versions thereof).
  • a carrier as described herein can comprise elements or portions derived from both Cholix and PE, which can be referred to a chimeric carrier.
  • a Cholix domain I or a PE domain I can be sufficient for rapid and efficient transport and delivery of cargo across an epithelial cell.
  • Such transport and delivery may even be superior to the transcytosis function of the respective full-length Cholix or PE exotoxins (e.g., SEQ ID NO: 1, SEQ ID NO:
  • the exotoxin-derived carrier polypeptides described herein can utilize endogenous trafficking pathways, including endogenous receptors and receptor complexes, to achieve apical-to-basal transcytosis and/or uptake into the interior of a cell, such as an epithelial cell (e.g., enterocytes).
  • the delivery carriers of the present disclosure can access a basolateral compartment (e.g., lamina basement) and/or the interior of an epithelial cell without damaging the cell or cell layer and without being altered, degraded or modified (e.g., chemically or enzymatically altered or modified).
  • the carrier constructs (e.g., isolated delivery constructs) of the present disclosure can further utilize specific intracellular compartments during
  • transcytosis and/or intracellular delivery to achieve the described transport efficiency.
  • the present disclosure provides methods and compositions that can comprise carriers that use (or interaction with) a set of endogenous proteins and receptors involved in the apical-to- basal transcytosis process across epithelial cells, such as polarized intestinal epithelial cells (e.g., enterocytes), to mediate transcytosis of a carrier coupled to a cargo from a lumen bordering the apical surface of a mucous membrane to the basolateral side of a mucous membrane.
  • epithelial cells such as polarized intestinal epithelial cells (e.g., enterocytes)
  • the delivery constructs disclosed herein can engage in interactions with such proteins and receptors to provide efficient transport and delivery of various cargo molecules to locations within an epithelial cell and/or across an epithelial cell to the basal side of an epithelium (e.g., a gut epithelium of a subject).
  • the constructs described herein, such as delivery constructs can comprise a carrier coupled to a heterologous cargo, wherein the carrier and/or the heterologous cargo can interact with proteins and/or receptors during intracellular delivery (e.g., to a supranuclear region or to a compartment located at the basal side within the epithelial cell) or during transcytosis (e.g., vesicular transcytosis).
  • the carrier can interact with one or more proteins (e.g., receptors or enzymes). These interactions can be dynamical and/or pH-dependent. It is pointed out that the herein described interactions are examples only and are not limiting the methods and compositions of this disclosure to other interactions (e.g., with other proteins or receptors).
  • proteins e.g., receptors or enzymes.
  • compositions and methods disclosed herein provide efficient delivery and transport of various cargo molecules (e.g., small molecules as well as macromolecules) across epithelial cells and/or into epithelial cells.
  • the carriers described herein achieve such efficient delivery of cargo in a manner that does not impair the epithelial cell barrier nor the delivery construct itself.
  • the functional properties of the delivery constructs e.g., those of the carrier as well as the functions of the cargo
  • the presently described carriers utilize endogenous trafficking pathways to deliver exogenous or endogenous cargo molecules to specific locations. Those locations can be inside an epithelial cell and/or in basolateral compartments outside epithelial cells on the basal side, e.g., the lamina limbal.
  • the carriers of the present disclosure comprise can be derived from an exotoxin.
  • Bacterial protein toxins are well known in the art, and are discussed in such sources as Bums, D., et ah, eds., BACTERIAL PROTEIN TOXINS, ASM Press, Herndon Va. (2003), Aktories, K. and Just, T, eds., BACTERIAL PROTEIN TOXINS (HANDBOOK OF EXPERIMENTAL PHARMACOLOGY), Springer-Verlag, Berlin, Germany (2000), and Alouf, J. and Popoff, M., eds., THE COMPREHENSIVE SOURCEBOOK OF BACTERIAL PROTEIN TOXINS, Academic Press, Inc., San Diego, Calif (3rd Ed., 2006).
  • an exotoxin can comprise one or more domains.
  • an exotoxin can be Cholix or PE.
  • Cholix the following nomenclature is used herein to describe its various domains (N- to C-terminus) and using the functional Cholix variant having the amino acid sequence set forth in SEQ ID NO: 1 as a reference sequence: (i) domain I (amino acid residues 1-265, SEQ ID NO: 4), (ii) domain II (amino acid residues 266- 386, SEQ ID NO: 126), (iii) domain lb (amino acid residues 387-425, SEQ ID NO: 127), and (iv) domain III (amino acid residues 426-634, SEQ ID NO: 128).
  • the ranges of amino acid residues defining these domains can be flexible and variations of about 5-10 amino acid residues may still fall within the scope of this disclosure, e.g., describing an amino acid sequence comprising the amino acid residues 5-265 or 5-270, or 1- 260, or 5-260 of full-length Cholix may still be understood as a Cholix domain I and so forth.
  • the terms“domain I” and“receptor binding domain” of an exotoxin can be used interchangeably.
  • the terms“domain II” and“translocation domain” of an exotoxin can be used interchangeably.
  • the terms“domain III”,“catalytic domain” and“cytotoxic domain” of an exotoxin can be used interchangeably.
  • Pseudomonas aeruginosa exotoxin A PE
  • Corynebacterium diphtheria Diphtheria carrier DT
  • Vibrio cholera Cholix make up a family of bacterial protein toxins that act as ADP-ribosyltransferases.
  • the carrier can be derived from a Cholix toxin.
  • the carrier can be derived from a PE.
  • a Cholix polypeptide as described herein may be rendered non-toxic by one or more amino acid substitutions.
  • a Cholix derived polypeptide or carrier as described herein may be rendered non-toxic by substituting a glutamic acid residue at position 581 of the amino acid sequence set forth in SEQ ID NO: 2 with alanine, resulting in a Cholix construct comprising an amino acid sequence set forth in SEQ ID NO: 3.
  • Cholix and PE are organized into distinct domains (I, II, lb, and III) that are denoted based upon their structural relationships. Domain I appears to facilitate exotoxin internalization and transcytosis, whereas domains II, lb, and III provide other functions as, for example, enzymatic activity in case of domain III that can ADP-ribosylate elongation factor 2 to induce cell apoptosis via blockade of protein synthesis. It has previously been unknown what components of PE and Cholix proteins are involved in the trans-epithelial transcytosis process.
  • Cholix is secreted by Vibrio cholera as a 70.7 kDa protein composed of three prominent globular domains (la, II, and III) and one small subdomain (lb) connecting domains II and III similar to the structure of PE (Jorgensen, R. et al., J Biol Chem 283(16): 10671-10678, 2008).
  • Mature Cholix comprises a genus of functional variants, wherein each variant can differ in one or more amino acid residues compared to another variant.
  • all Cholix variants disclosed herein and encompassed in this disclosure are functional Cholix variants.
  • Cholix is a 634-residue protein, and two functional variants are specifically included herein, which are those having the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 1.
  • a nucleic acid encoding the mature Cholix as used herein is set forth in SEQ ID NO: 134.
  • Pseudomonas exotoxin A or“PE” is secreted by Pseudomonas aeruginosa as a 67 kDa protein composed of three prominent globular domains (la, II, and III) and one small subdomain (lb) connecting domains II and III (see Allured et. al., Proc. Natl. Acad. Sci. 83: 1320 1324, 1986).
  • Mature PE as used herein is a 6l3-residue protein, whose sequence is set forth in SEQ ID NO: 134.
  • a nucleic acid encoding mature PE as used herein is set forth in SEQ ID NO: 135.
  • the amino acid sequence of the mature Cholix toxin is set forth in SEQ ID NO: 1 and is used as the reference sequence, unless specified otherwise.
  • the amino acid sequence set forth in SEQ ID NO: 4 contains the amino acid residues 1-265 of the amino acid sequence of mature Cholix toxin set forth in SEQ ID NO: 1 and is defined as Cholix domain I.
  • the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 can also be described as“Chx 1 265 ” (or“Cholix 1 265 ” or“Cholix 265 ” or“Cholix domain I”).
  • any other, functionally active, Cholix exotoxin variants are encompassed in the present disclosure, e.g., those that comprise a consensus sequence defining the functional activity of the Cholix exotoxins.
  • a consensus sequence defining the functional activity of the Cholix exotoxins See e.g., Awasthi et al. Novel Cholix toxin variants, ADP-ribosylating toxins in Vibrio Cholerae Non-Ol/Non- 0139 strains, and their pathogenicity, Infection and Immunity, 81(2), p. 531-541 (2013)).
  • the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is a functional variant of SEQ ID NO: 1.
  • a domain I derived from that Cholix exotoxin sequence, or a truncated version thereof, can be used as a carrier for the rapid and efficient delivery of cargo.
  • a domain I polypeptide of the Cholix exotoxin with SEQ ID NO: 2 can also be described as amino acid residues 1-4 of SEQ ID NO: 2 + Cholix 5 265 .
  • a first carrier and a second carrier are produced in a different expression system (e.g., a bacterial or a mammalian expression system).
  • Bacterial expression systems include E. coli, and mammalian expression systems include CHO cells, for example.
  • a bacterially produced polypeptide can comprise an N-cap, wherein the N- cap can comprise one more modifications at the N-terminal of the polypeptide.
  • An N-cap can comprise an N-terminal methionine residue.
  • Cholix domain I derived carrier polypeptides that can be bacterially produced and that comprise such N-terminal methionine include those comprising the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO: 125.
  • the present disclosure contemplates isolated non-naturally occurring and bacterial toxin derived carriers (e.g., an exotoxin derived) that can be coupled to a cargo (e.g., a biologically active); wherein the carrier is capable of delivering the cargo (e.g., a biologically active) via transcytosis transport across the intestinal epithelium.
  • a cargo e.g., a biologically active
  • the carrier can be derived from a domain I of an exotoxin (e.g., Cholix or PE).
  • a carrier that is derived form a domain I of an exotoxin can lack a domain II (e.g., SEQ ID NO: 126 or SEQ ID NO: 138), a domain lb (e.g., SEQ ID NO: 127 or SEQ ID NO: 139), or a domain III (e.g, SEQ ID NO: 128 or SEQ ID NO: 140) of an exotoxin (e.g., Cholix or PE).
  • a domain II e.g., SEQ ID NO: 126 or SEQ ID NO: 138
  • a domain lb e.g., SEQ ID NO: 127 or SEQ ID NO: 139
  • a domain III e.g, SEQ ID NO: 128 or SEQ ID NO: 140
  • a carrier that“lacks” a domain II, domain lb, and a domain III of an exotoxin can still comprise a portion of the domain II, a domain lb, or the domain III of the exotoxin, or a combination thereof.
  • the term“lacking” as referred to herein means that a carrier does not comprise a complete domain II, a complete domain lb, or a complete domain III.
  • a carrier can comprise no more than 70% of the amino acid residues of a domain II, a domain lb, or a domain III of an exotoxin.
  • a carrier can comprise a Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 5) or a truncated version thereof, and further comprise the amino acid residues 1-82 of Cholix domain II (SEQ ID NO: 126).
  • a carrier can comprise no more than 60% of the amino acid residues of a domain II, a domain lb, or a domain III of an exotoxin.
  • a carrier can comprise no more than 50% of the amino acid residues of a domain II, a domain lb, or a domain III of an exotoxin.
  • a carrier can comprise no more than 25% of the amino acid residues of a domain II, a domain lb, or a domain III of an exotoxin.
  • a carrier can comprise no more than 10% of the amino acid residues of a domain II, a domain lb, or a domain III of an exotoxin.
  • the present disclosure contemplates isolated non-naturally occurring and bacterial toxin derived carriers (e.g., an exotoxin derived) that can be coupled to a cargo (e.g., a biologically active); wherein the carrier is capable of delivering the cargo (e.g., a biologically active) to the interior of an epithelial cell, such as an intracellular vesicle or compartment or the cytosol.
  • a cargo e.g., a biologically active
  • an epithelial cell such as an intracellular vesicle or compartment or the cytosol.
  • Regions and/or compartments in the interior of an epithelial cell can include regions and/or compartments on the apical side of the interior of an epithelial cell, regions and/or compartments on the basal side of the interior of an epithelial cell, supranuclear regions of an epithelial cell, or any combination thereof.
  • the epithelial cell can be a polarized gut epithelial cell.
  • the polarized gut epithelial cell can be part of a polarized epithelial cell monolayer (e.g., comprising Caco-2 cells) or it can be part of a gut epithelium of a subject (e.g., a rodent or a human).
  • a carrier can be derived from a bacterial carrier such as an exotoxin (e.g., Cholix and/or PE) and can be derived from a domain I of said exotoxin and can lack a domain II (e.g., SEQ ID NO: 126 or SEQ ID NO: 138), a domain lb (e.g, SEQ ID NO: 127 or SEQ ID NO:
  • the carrier can comprise a receptor binding domain or binding fragment, which can be a domain, region, or fragment within the exotoxin derived domain I, and which allows binding of the delivery construct to one or more selective or non-selective receptors on the luminal surface of an epithelial cell.
  • a receptor can be a selective receptor or a non-selective receptor, such as a non-selective scavenger receptor on the luminal surface of intestinal epithelial cells.
  • the one or more receptors that a carrier can interact with on the surface of an epithelial cell and/or during endocytosis can include a low density lipoprotein receptor-related protein 1 (LRP1) or a transmembrane protein 132 (TMEM132).
  • LRP1 low density lipoprotein receptor-related protein 1
  • TMEM132 transmembrane protein 132
  • the delivery construct can bind to one or more cell surface receptor that can be present on the apical membrane of an epithelial cell with sufficient affinity to allow endocytosis.
  • the delivery construct can bind to any receptor known to be present on the apical membrane of an epithelial cell by one of skill in the art without limitation.
  • the carrier can bind to LRP1.
  • the carrier can bind to TMEM132.
  • the carrier can bind to LRP1 and TMEM132.
  • a carrier can be derived from a domain I of an exotoxin.
  • the exotoxin is selected from the group consisting of Cholix and PE.
  • a carrier as described herein is derived from a domain I of an exotoxin, wherein the exotoxin is Cholix.
  • a carrier as described herein can comprise an amino acid sequence that is derived from that of Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 5).
  • a Cholix Domain I (e.g., SEQ ID NO: 4) can comprise amino acids 1-265 of SEQ ID NO: 1 or it can comprise amino acid sequence set forth in SEQ ID NO: 5 (e.g., when bacterially produced comprising an N-terminal methionine residue) and can be described as a “receptor binding domain” that functions as a ligand for a cell surface receptor and mediates Cholix binding and endocytosis.
  • a carrier can comprise an amino acid sequence with greater than 50% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence with greater than 60% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence with greater than 70% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence with greater than 80% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence with greater than 90% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence with greater than 95% homology to any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • Conservative or non-conservative substitutions can be made to an amino acid sequence of any one of SEQ ID NO: 4 - SEQ ID NO: 125.
  • an amino acid residue substitution will be identified by reference to the particular amino acid substitution at a specific amino acid residue.
  • the term “S30A” indicates that the“S” (serine, in standard single letter code) residue at position 30 in SEQ ID NO: 4 has been substituted with an“A” (alanine, in standard single letter code), and the modified carrier will be identified as“Cholix S30A ”.
  • a carrier can be a truncated version of a Cholix domain I sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • a carrier comprising a truncated version of a Cholix domain I can comprise an amino acid sequence set forth in any one of SEQ ID NO: 6 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence of any one of SEQ ID NO: 4 - SEQ ID NO: 125, wherein one or more amino residues of such sequence is deleted.
  • a carrier can comprise an amino acid sequence of any one of SEQ ID NO: 4 - SEQ ID NO: 125, wherein one or more amino acid residues can be substituted with another amino acid.
  • a truncated carrier can be identified by reference to the amino acid residues comprising the truncated toxin, e.g., a truncated Cholix carrier consisting of amino acid residues 1-260 of SEQ ID NO: 4 will be identified as Cholix 260 and so forth, according to nomenclature described herein.
  • a carrier comprises any of the amino acid sequences shown in TABLE 2, or fragment, or a combination thereof.
  • a carrier can be a polypeptide that is derived from a Cholix exotoxin and having: at most 5 amino acid residues; at most 10 amino acid residues; at most 15 amino acid residues; at most 20 amino acid residues; at most 30 amino acid residues; at most 40 amino acid residues; at most 50 amino acid residues; at most 60 amino acid residues; at most 70 amino acid residues; at most 80 amino acid residues; at most 90 amino acid residues; at most 100 amino acid residues; at most 1 10 amino acid residues; at most 120 amino acid residues; at most 130 amino acid residues; at most 140 amino acid residues; at most 150 amino acid residues; at most 160 amino acid residues; at most 170 amino acid residues; at most 180 amino acid residues; at most 190 amino acid residues; at most 200 amino acid residues; at most 210 amino acid residues; at most 220 amino acid residues; at most 230 amino acid residues; at most 240 amino acid residues; at most 250 amino acid residues; at most
  • the bacterial carrier receptor binding domain can be a polypeptide derived from Cholix and having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more sequence homology with SEQ ID NO: 4 or SEQ ID NO: 5.
  • the carrier can be a polypeptide derived from Cholix and having at most 10%, 20%, 30%, 40%,
  • a carrier can be derived from a domain I of a Cholix exotoxin.
  • a carrier that is derived from a domain I of an exotoxin can comprise an amino acid having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125, or at least 80% sequence identity to a functional fragment thereof.
  • a carrier that is derived from a domain I of an exotoxin can comprise an amino acid having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125, or at least 90% sequence identity to a functional fragment thereof.
  • a carrier that is derived from a domain I of an exotoxin can comprise an amino acid having at least 95% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125, or at least 95% sequence identity to a functional fragment thereof.
  • a carrier that is derived from a domain I of an exotoxin can comprise an amino acid having at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125, or at least 99% sequence identity to a functional fragment thereof.
  • a carrier that is derived from a domain I of an exotoxin can comprise an amino acid having 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125, or 100% sequence identity to a functional fragment thereof.
  • a carrier can be artificially synthesized.
  • a carrier can be an artificially synthesized polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more amino acid sequence homology to a Cholix domain I (e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 125).
  • a carrier can be a synthetic polypeptide having at most 10%, 20%, 30%,
  • the polypeptide that a carrier can be comprises of can be synthesized using solid-phase synthesis.
  • a carrier can be a polypeptide derived from Cholix and having at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence homology with any one of SEQ ID NO: 4 - SEQ ID NO: 125. Certain fragments within the amino acid sequence of the carrier can have specific functions that can be related to one or more aspects of the transcytosis process.
  • These functions can comprise crossing a polarized monolayer of primary human small intestinal epithelial cells or an intact gut epithelium, enabling or promoting endocytosis into an epithelial cell, apical-to-basal transport, release of the delivery construct from the basal membrane into a basolateral compartment, delivery into an intracellular vesicle or compartment or the cytosol of an epithelial cell, and/or delivery to a supranuclear region of an epithelial cell (e.g., a polarized gut epithelial cell).
  • a polarized monolayer of primary human small intestinal epithelial cells or an intact gut epithelium enabling or promoting endocytosis into an epithelial cell, apical-to-basal transport, release of the delivery construct from the basal membrane into a basolateral compartment, delivery into an intracellular vesicle or compartment or the cytosol of an epithelial cell, and/or delivery to a supranuclear region of
  • the present disclosure provides carriers that can have various functions (e.g., one or more of endocytosis, transcytosis, intracellular delivery, etc.).
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 80% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • Such a carrier can comprise a deletion or mutation in one or more of amino acid residues of the amino acid sequence set forth in SEQ ID NO: 4 (e.g., a Cholix domain I expressed in a mammalian cell) or SEQ ID NO: 5 (e.g., a Cholix domain I expressed in a bacterial cell).
  • Such a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 90% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 95% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 99% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • a carrier can comprise an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity to a functional fragment thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO: 1.
  • a carrier disclosed herein can comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a functional fragment thereof.
  • the carrier can comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional fragment thereof.
  • the carrier can comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a functional fragment thereof.
  • a functional carrier of the present disclosure can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • a carrier can comprise comprises an amino acid sequence having at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • a carrier can comprise a spatial structure in which one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in close proximity to one or more amino acid residues of SEQ ID NO: 151, and one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in close proximity to one or more amino acid residues of SEQ ID NO: 152.
  • a carrier of the present disclosure can be capable of delivering a cargo across an epithelial cell (e.g., a polarized epithelial cell).
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 80% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can further comprise a deletion or mutation in one or more of amino acid residues 1-187 or 1-205 of SEQ ID NO: 10 or 1-186 or 1-206 of SEQ ID NO: 11.
  • a carrier can comprise residues 1-186 of SEQ ID NO: 30 or 1-187 of SEQ ID NO:
  • the methods and compositions of the present disclosure can comprise a carrier comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 10 - SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 10 - SEQ ID NO: 31 or at least 90% sequence identity to a functional fragment thereof.
  • the carrier comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 10 - SEQ ID NO: 31 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 10 - SEQ ID NO: 31 or at least 99% sequence identity to a functional fragment thereof.
  • the carrier can comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment thereof.
  • Such a carrier can be capable of delivering cargo to an intracellular location. Such an intracellular location may be a supranuclear region.
  • Such a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can comprise a deletion or mutation in one or more of amino acid residues 1-151 or 1-187 of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the methods and compositions of the present disclosure provide a carrier that can lack any one or more of the amino acid residues 1-39 of SEQ ID NO: 5 or amino acid residues 1- 38 of SEQ ID NO: 4.
  • a carrier can be capable of delivering cargo to an intracellular location via endocytosis. Such a location can be an apical region or compartment.
  • a carrier can lack all of the amino acid residues 1-39 of SEQ ID NO: 5 or amino acid residues 1-38 of SEQ ID NO: 4.
  • a carrier can comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 80% sequence identity to a functional fragment thereof.
  • a carrier can comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 90% sequence identity to a functional fragment thereof.
  • a carrier can comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 95% sequence identity to a functional fragment thereof.
  • a carrier can comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 99% sequence identity to a functional fragment thereof.
  • a carrier can comprises residues 1-151 of SEQ ID NO: 5 or residues 1-150 of SEQ ID NO: 4 and no more than 187 contiguous amino acid residues of SEQ ID NO: 1.
  • a carrier of the present disclosure can comprise a truncated version of a Cholix domain I.
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107 or at least 80% sequence identity to a functional fragment thereof.
  • Such a carrier can be capable of delivering cargo to an intracellular location via endocytosis. Such a location can be an apical and/or a basal region or compartment.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can comprise the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or a functional fragment thereof.
  • a carrier of the present disclosure can comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 80% sequence identity to a functional fragment thereof.
  • Such a carrier can be capable of delivering cargo to an intracellular location via endocytosis. Such a location can be an apical region or compartment.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can further comprise a deletion or mutation in one or more of amino acid residues 1-151 of SEQ ID NO: 6 or in one or more of amino acid residues 1-150 of SEQ ID NO: 7.
  • a carrier as described herein can comprise residues 1-134 of SEQ ID NO: 5 or residues 1-133 of SEQ ID NO: 4 and no more than 151 contiguous amino acid residues of SEQ ID NO: 1.
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 106 - SEQ ID NO: 125 or at least 80% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 106 - SEQ ID NO: 125 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 106 - SEQ ID NO: 125 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 106 - SEQ ID NO: 125 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can comprise the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or a functional fragment thereof.
  • a carrier of the present disclosure can be derived from a domain I of an exotoxin.
  • the exotoxin can be Cholix.
  • a carrier that is derived from a Cholix domain I can comprise at least one but no more than 20 beta strands.
  • a carrier that is derived from a Cholix domain I can comprise at least one but no more than 15 beta strands.
  • a carrier that is derived from a Cholix domain I can comprise between 10 and 15 beta strands.
  • a carrier that is derived from a Cholix domain I can comprise at least one but less than 10 a-helices.
  • a carrier that is derived from a Cholix domain I can comprise between 1 and 5 a-helices.
  • a carrier of the present disclosure can comprise an amino acid fragment of Cholix domain I that can enable, promote, and/or enhance apical entry of the Cholix derived carrier into epithelial cells such as polarized gut epithelial cells.
  • a fragment can comprise the amino acid sequence set forth in SEQ ID NO: 148 and can promote and/or enhance apical entry of the Cholix derived carrier into epithelial cells on the apical epithelial/luminal surface. This may enhance the delivery and/or transport function of the carrier and increase the amount cargo molecules delivered and/or transported into and/or across an epithelial cell.
  • a carrier of the present disclosure can comprise an amino acid fragment of Cholix domain I that can enable, promote, and/or enhance apical-to-basal transcytosis of a Cholix- derived carrier as described herein.
  • Such an amino acid fragment that enables, promotes, and/or enhances apical-to-basal transcytosis of the delivery construct can comprise an amino acid sequence set forth in SEQ ID NO: 149 or SEQ ID NO: 150. This may enhance the delivery and/or transport function of the carrier and increase the amount cargo molecules delivered and/or transported into and/or across an epithelial cell.
  • a carrier comprising such fragment with SEQ ID NO: 149 or SEQ ID NO: 150 can increase the amount cargo molecules delivered and/or transported to a basal compartment. This may further enhance basal release of the carrier.
  • a carrier of the present disclosure can comprise an amino acid fragment of Cholix domain I that can enable, promote, and/or enhance early and/or late endosomal sorting, thereby enabling, promoting, and/or enhancing transport of the Cholix-derived carrier to a supranuclear region within an epithelial cell.
  • a peptide fragment of Cholix domain I comprising the amino acid sequence set forth in SEQ ID NO: 151 and can enable, promote, and/or enhance early endosomal sorting of a Cholix-derived delivery construct as described herein.
  • Supranuclear regions that may be targeted using such a carrier can include the endoplasmatic reticulum, the Golgi apparatus, and/or endosomes.
  • a carrier capable of accessing such region can provide efficient delivery of cargo to such regions.
  • a carrier of the present disclosure can comprise an amino acid fragment of Cholix domain I that can enable, promote, and/or enhance complete transcytosis of the Cholix-derived delivery construct across an intact epithelial layer such as the gut epithelium.
  • a fragment comprising the amino acid sequence set forth in SEQ ID NO: 152 can enable, promote, and/or enhance complete transcytosis of the Cholix-derived delivery construct by enabling basal release of the carrier and/or the delivery construct from the epithelial cell.
  • Complete transcytosis of the Cholix-derived delivery construct can be determined, for example, by measuring the presence of the delivery construct in a basolateral compartment or the lamina propia. The ability of such carriers to delivery cargo across intact epithelial cell layers can be of high significance as it allows the oral administration of drugs that would not be able to cross such epithelial layers by themselves.
  • the present disclosure contemplates the surprising finding that a carrier that is derived from a Cholix domain I and that lacks a Cholix domain II, domain lb, and domain III, is sufficient for rapid and efficient apical-to-basal transcytosis (e.g., and sufficient for rapid and efficient apical-to-basal transport of cargo via transcytosis). Furthermore, it is shown that certain portions of the amino acid sequence of Cholix domain I can have specific functions related to apical-to-basal transcytosis across an epithelial cell, and/or the delivery into the cytosol or interior of an epithelial cell.
  • a carrier of the present disclosure can comprise any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125.
  • a carrier of the present disclosure can comprise one or more of the functional amino acid peptide fragments of a Cholix domain I set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier of the present disclosure can comprise a Cholix domain I with an amino acid sequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 5.
  • the present disclosure further contemplates carriers that comprise one or more functional fragments of a Cholix domain I.
  • a carrier can comprise one or more of the functional amino acid sequences derived a Cholix domain I and set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • Such amino acid sequences can be linked together to form a polymeric polypeptide comprising a plurality of Cholix domain I derived peptide fragments.
  • Such a carrier of the present disclosure can comprise one or more amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152, or any combination thereof, that form a polymeric polypeptide capable of efficient transcytosis across epithelial layers such as the gut epithelium.
  • a polymeric peptide can comprise a plurality of amino acid fragment derived from Cholix domain I and such polymeric polypeptide can be used instead of and/or in addition to a Cholix domain I polypeptide or a truncated version thereof.
  • Such a non-naturally occurring synthetic polymeric peptide can possess superior or inferior transcytosis capabilities when used as a delivery construct compared to Cholix domain I or a truncated version thereof.
  • the functionality of such synthetic polypeptides can depend on several factors such spatial structure and geometry, stability, and/or the cargo that may be coupled to such polypeptide.
  • a carrier of the present disclosure may not be significantly altered in a chemical, structural, and/or conformational manner during the transcytosis process across an epithelial cell.
  • the Cholix toxin-derived carrier as disclosed herein can be used as an efficient delivery vehicle for various cargo molecules (e.g., therapeutic cargo molecules) as described herein.
  • a Cholix-derived carrier as described herein does not contain the domains II and III, but instead is attached to one or more cargo moieties (e.g., therapeutic cargo molecules) without having reduced transport and/or transcytosis capabilities compared to mature ntChx.
  • Transport of a carrier as described herein across an epithelial layer can comprise multiple steps.
  • Transport of a delivery construct e.g., a Cholix-derived delivery construct
  • Transport or transcytosis can include apical endocytosis, vesicular trafficking involving apical, basal, and/or supra-nuclear regions of enterocytes, and release from the basal membrane to reach the lamina propria.
  • a Cholix-derived delivery construct as described herein can utilize a receptor-mediated-type endocytosis process.
  • Receptor-mediated endocytosis can involve an amino acid sequence having at least 80% sequence identity to the amino acid set forth in SEQ ID NO: 148 or a fragment or derivative thereof, which, can provide access to an early endosomal vesicular compartment in the apical portion of enterocytes, e.g., via endocytosis.
  • An amino acid sequence having at least 80% sequence identity to the amino acid set forth in SEQ ID NO: 151 or a fragment or derivative thereof can allow, promote, or enhance the movement of a Cholix-derived delivery construct to a supranuclear region consistent with a sorting site in the cell for secretory events.
  • Movement of a delivery construct comprising a Cholix-derived carrier to the basal compartment of the cells can be more efficient when the carrier comprises an amino acid sequence having at least 80% sequence identity to the amino acid set forth in SEQ ID NO: 149 or SEQ ID NO: 150, or a fragment or derivative thereof.
  • An amino acid sequence having at least 80% sequence identity to the amino acid set forth in SEQ ID NO: 149, a fragment or derivative thereof, can provide a mechanism for secretion from the basal membrane that releases an intact and functional delivery construct (e.g., including the cargo moiety) into a basolateral compartment or the lamina basement from where it can reach various other locations (e.g., cells, tissues or organs) within an organism (e.g., in a human or in a rodent).
  • an intact and functional delivery construct e.g., including the cargo moiety
  • transport of a delivery construct as disclosed herein across an epithelial barrier generally does not involve enterocyte intoxication or disruption.
  • a delivery construct as disclosed herein can comprise a Cholix domain I, a fragment or truncated version thereof (e.g., any one of SEQ ID NO: 6 - SEQ ID NO: 125), or a polymeric peptide comprising a plurality of amino acid fragments derived from a Cholix domain I (e.g., SEQ ID NO: 148 - SEQ ID NO: 152), and wherein the other domains (e.g., domains II, domain lb, and III) can be replaced by various other moieties, such as spacers, heterologous cargos (e.g., therapeutic and/or biologically active cargo), small molecules, nucleic acids (e.g., aptamers or interfering RNAs), or any combination thereof, as further described herein.
  • a Cholix domain I e.g., any one of SEQ ID NO: 6 - SEQ ID NO: 125
  • a polymeric peptide comprising a plurality of amino acid fragments
  • a carrier of the present disclosure can be used to deliver various cargo molecules into and/or across epithelial cells in an efficient manner, e.g., when comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 125).
  • a carrier of the present disclosure can enable efficient endocytosis on the apical site and transport into the interior of an epithelial cell (e.g., an enterocyte and/or a polarized gut epithelial cell) such as an intracellular vesicle or compartment or the cytosol and/or a supranuclear region.
  • Such a carrier can comprise an amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 125.
  • constructs for delivery of cargo molecules into epithelial cells may comprise a truncated Cholix domain I or fragment of a Cholix domain I that does not comprise the amino acid sequence set forth in SEQ ID NO: 151, and/or the amino acid amino acid sequence set forth in SEQ ID NO: 152, or any combination thereof.
  • a carrier of the present disclosure can comprise one or more potential glycosylation sites.
  • the one or more glycosylation sites can be located within a Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 5).
  • a carrier as described herein can comprise the amino acid sequence set forth in SEQ ID NO: 5, wherein the asparagine residues N98, N154, N165, N224, or any combination thereof, can be potential glycosylation sites. Variation or mutation of one or more of these amino acid residues that can act as glycosylation sites can affect or reduce a function related to transcytosis of a delivery construct.
  • TABLE 3 shows exemplary functional peptide fragments of Cholix domain I that were identified to provide one or more functions related to apical-to-basal transcytosis.
  • a carrier can comprise an amino acid sequence having at least 80% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise at least one of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise at least two of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise at least three of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise at least four of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise all of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152.
  • a carrier as described herein can comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, or a functional fragment thereof.
  • the PE exotoxin domain I (SEQ ID NO: 137) comprises amino acids 1-252 of SEQ ID NO: 135 and has been described as a“receptor binding domain” that functions as a ligand for a cell surface receptor and mediates binding of PE to a cell.
  • a carrier of the present disclosure can be derived from PE and can comprise the receptor binding domain polypeptide having the amino acid sequence set forth in SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 50% homology to SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 60% homology to SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 70% homology to SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 80% homology to SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 90% homology to SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence with greater than 95% homology to SEQ ID NO: 137.
  • conservative or non-conservative substitutions can be made to the amino acid sequence of SEQ ID NO: 7, so long as the ability to mediate binding of the delivery construct to a cell is not substantially eliminated.
  • a carrier can comprise a receptor binding domain that is a truncated version of SEQ ID NO: 137.
  • a carrier can comprise a receptor binding domain polypeptide wherein one or more amino residues of SEQ ID NO: 137 are deleted.
  • a carrier can comprise a receptor binding domain polypeptide wherein one or more amino residues of SEQ ID NO: 137 are substituted with another amino acid.
  • a carrier of the present disclosure that is derived from a PE domain I can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 137 or at least 80% identity to a functional fragment thereof.
  • a carrier can comprise a deletion or mutation in one or more of amino acid residues 1-252 of SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 90% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 95% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 99% sequence identity to a functional fragment thereof.
  • a carrier can comprise an amino acid sequence having 100% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or 100% sequence identity to a functional fragment thereof.
  • a carrier e.g., a bacterial carrier receptor binding domain
  • a carrier can be a polypeptide derived from PE and having: at most 5 amino acid residues; at most 10 amino acid residues; at most 15 amino acid residues; at most 20 amino acid residues; at most 30 amino acid residues; at most 40 amino acid residues; at most 50 amino acid residues; at most 60 amino acid residues; at most 70 amino acid residues; at most 80 amino acid residues; at most 90 amino acid residues; at most 100 amino acid residues; at most 110 amino acid residues; at most 120 amino acid residues; at most 130 amino acid residues; at most 140 amino acid residues; at most 150 amino acid residues; at most 160 amino acid residues; at most 170 amino acid residues; at most 180 amino acid residues; at most 190 amino acid residues; at most 200 amino acid residues; at most 210 amino acid residues; at most 220 amino acid residues; at most 230 amino acid residues; at most 240 amino acid residues; at most
  • the bacterial carrier receptor binding domain can be a polypeptide derived from PE and having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more sequence homology with SEQ ID NO: 137.
  • the bacterial carrier receptor binding domain can be a polypeptide derived from PE and having at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence homology with SEQ ID NO: 137.
  • the amino acid residues can be consecutive.
  • the amino acid residues can be non-consecutive.
  • a carrier of the present disclosure can comprise a binding domain that is an artificially synthesized polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more amino acid sequence homology to PE domain I.
  • the carrier comprising a receptor binding domain can be a synthetic polypeptide having at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence homology to PE domain I set forth in SEQ ID NO: 137.
  • the polypeptide can be synthesized using solid-phase synthesis or recombinant expression.
  • a carrier of the present disclosure capable of delivering cargo across epithelial cells can comprise a receptor binding domain polypeptide (e.g., a domain I or a derivative thereof) wherein one or more amino acid residues of one bacterial carrier receptor binding domain polypeptide is replaced by one or more amino acid residues of a second bacterial carrier receptor binding domain polypeptide (also referred to hereinafter as“a hybrid receptor binding domain polypeptide”).
  • a carrier can comprise an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 are replaced by one or more amino acid residues of SEQ ID NO: 137.
  • a carrier can comprise an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 137 are replaced by one or more amino acid residues of SEQ ID NO: 4.
  • a carrier can comprise an amino acid sequence wherein amino acid residues 77-87 of SEQ ID NO: 4 (Cholix) are replaced by amino acid residues of a second bacterial carrier receptor binding domain polypeptide (e.g., a PE domain I).
  • a carrier can comprise an amino acid sequence wherein amino acid residues 188- 236 of SEQ ID NO: 4 are replaced by amino acid residues of a second bacterial carrier receptor binding domain polypeptide.
  • a carrier can comprise an amino acid sequence wherein amino acid residues 69-71 of SEQ ID NO: 137 are replaced by amino acid residues of a second bacterial carrier receptor binding domain polypeptide.
  • a carrier can also comprise an amino acid sequence wherein amino acid residues 177-228 of SEQ ID NO: 137 are replaced by amino acid residues of a second bacterial carrier receptor binding domain polypeptide.
  • a carrier of the present disclosure can comprise an amino acid sequence having at least 10%, 20%, 30%, 40%, 50%,
  • a carrier of the present disclosure that is derived from a domain I of an exotoxin can further comprise a portion of an exotoxin translocation domain, or modified translocation domain elements.
  • a translocation domain can be a domain II of an exotoxin.
  • a carrier of the present disclosure that is derived from a domain I of an exotoxin can further comprise a portion of a non-toxic catalytic domain or modified non-toxic catalytic domain elements.
  • a non-toxic catalytic domain can be a modified domain III of an exotoxin, e.g., those that comprise one or more amino acid variations and/or a deletion of one or more amino acid residues rendering the domain III non-toxic (e.g., an E581A substitution (e.g., SEQ ID NO: 3) or a DE581 deletion).
  • a translocation domain, or a modified translocation domain, and a non-toxic catalytic domain, or a modified non-toxic catalytic domain can be derived from the same bacterial toxin.
  • a translocation domain, or a modified translocation domain, and a non-toxic catalytic domain, or a modified non-toxic catalytic domain can be derived from a bacterial carrier selected from the group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin (PE), botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like toxin.
  • a bacterial carrier selected from the group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin (PE), botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like toxin.
  • Cholix domain II (SEQ ID NO: 126) comprises amino acids 266-386 of SEQ ID NO: 1).
  • a carrier of the present disclosure can comprise a Cholix derived carrier comprising the entire amino acid sequence of SEQ ID NO: 126, or can comprise a portion(s) of SEQ ID NO: 126. Further, conservative or non-conservative substitutions can be made to SEQ ID NO: 126.
  • a representative assay that can routinely be used by one of skill in the art to determine whether a transcytosis domain has transcytosis activity is described herein.
  • a carrier can comprise at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid residues of the entire amino acid sequence of SEQ ID NO: 126.
  • a carrier of the present disclosure can comprise a truncated Cholix domain II, e.g., those identified as Cholix 425 (SEQ ID NO: 129), Cholix 415 (SEQ ID NO: 130), Cholix 397 (SEQ ID NO: 131), Cholix 386 (SEQ ID NO: 132), Cholix 291 (SEQ ID NO: 133), and Cholix 265 (SEQ ID NO: 4).
  • a PE domain II (SEQ ID NO: 138) comprises amino acids 253- 364 of SEQ ID NO: 135).
  • a carrier of the present disclosure can comprise a PE carrier comprising the entire amino acid sequence of SEQ ID NO: 137, or can comprise a portion(s) of SEQ ID NO: 137.
  • PE domain I can be sufficient for rapid and efficient apical-to-basal transcytosis.
  • portion(s) of PE domain II can be used as a spacer to attach further payload, such as a heterologous cargo.
  • conservative or non conservative substitutions can be made to SEQ ID NO: 137.
  • a representative assay that can routinely be used by one of skill in the art to determine whether a transcytosis domain has transcytosis activity is described herein.
  • the transcytosis activity is not substantially eliminated so long as the activity is, e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as compared to a PE carrier comprising the entire amino acid sequence of SEQ ID NO: 137.
  • a carrier of the present disclosure can comprise a truncated PE domain II, e.g., those identified as PE 404 (SEQ ID NO: 141), PE 395 (SEQ ID NO: 142), PE 376 (SEQ ID NO: 143), PE 364 (SEQ ID NO: 144), PE 277 (SEQ ID NO: 145), and PE 252 (SEQ ID NO: 146).
  • PE 404 SEQ ID NO: 141
  • PE 395 SEQ ID NO: 142
  • PE 376 SEQ ID NO: 143
  • PE 364 SEQ ID NO: 144
  • PE 277 SEQ ID NO: 145
  • PE 252 SEQ ID NO: 146
  • a carrier of the present disclosure can comprise a receptor binding domain, and a translocation domain (e.g., a domain II), or a modified translocation domain (e.g., a modified domain II), and can further comprise a non-toxic catalytic domain (e.g., a domain III), or modified non-toxic catalytic domain (e.g., a modified domain III).
  • a translocation domain e.g., a domain II
  • a modified translocation domain e.g., a modified domain II
  • a non-toxic catalytic domain e.g., a domain III
  • modified non-toxic catalytic domain e.g., a modified domain III
  • the non-toxic catalytic domain, or modified non-toxic catalytic domain can be derived from a bacterial carrier selected from the group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin (PE), botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like toxin.
  • a bacterial carrier selected from the group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin (PE), botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like toxin.
  • translocation domain and the non-toxic catalytic domain, or modified non-toxic catalytic domain, are derived from the same bacterial toxin.
  • Cholix domain III (SEQ ID NO: 128) comprises amino acids 426-634 of SEQ ID NO: 1 and has been described as a catalytic domain responsible for cytotoxicity and includes an endoplasmic reticulum retention sequence. Domain III mediates ADP ribosylation of elongation factor 2 (“EF2”), which inactivates protein synthesis.
  • EF2 elongation factor 2
  • a carrier that“lacks endogenous ADP ribosylation activity” or a“detoxified Cholix” refers to any Cholix derived carrier described herein (including modified variants) that does not comprise the entire amino acid sequence set forth in SEQ ID NO: 128 (e.g., a portion of a domain III).
  • Such a carrier can comprise one or more modifications within SEQ ID NO: 128 in a manner which detoxifies the molecule. For example, deletion of the glutamic acid (Glu) residue at amino acid position 156 of SEQ ID NO: 128 detoxifies the molecule.
  • the portion of SEQ ID NO: 128 other than the ER retention signal can be replaced by another amino acid sequence.
  • This amino acid sequence can itself be non-immunogenic, slightly immunogenic, or highly immunogenic.
  • a highly immunogenic ER retention domain is preferable for use in eliciting a humoral immune response.
  • Cholix domain III is itself highly immunogenic and can be used in delivery constructs where a robust humoral immune response is desired.
  • PE Domain III (SEQ ID NO: 140) comprises amino acids 405- 613 of SEQ ID NO: 3) and has been described as a catalytic domain responsible for cytotoxicity and includes an endoplasmic reticulum retention sequence. Domain III mediates ADP
  • a PE derived carrier that“lacks endogenous ADP ribosylation activity” or a“detoxified PE” refers to any PE described herein (including modified variants or derivatives) that does not comprise SEQ ID NO: 140 and/or which has been modified within SEQ ID NO: 140 in a manner which detoxifies the molecule. For example, deletion of the glutamic acid (Glu) residue at amino acid position 149 of SEQ ID NO: 140 detoxifies the molecule.
  • the portion of PE domain III other than the ER retention signal can be replaced by another amino acid sequence.
  • This amino acid sequence can itself be non-immunogenic, slightly immunogenic, or highly immunogenic.
  • a highly immunogenic ER retention domain is preferable for use in eliciting a humoral immune response.
  • PE domain III is itself highly immunogenic and can be used in delivery constructs where a robust humoral immune response is desired.
  • the present disclosure contemplates carriers that can comprise a receptor binding domain polypeptide having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 137, a translocation domain having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 138, and a non-toxic catalytic domain having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 140.
  • the present disclosure contemplates carriers that can comprise a receptor binding domain polypeptide having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, a translocation domain having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 126, and a non-toxic catalytic domain having the amino acid sequence derived from the sequence set forth in SEQ ID NO: 128.
  • a carrier that is derived from a domain I of an exotoxin can further comprise the amino acid sequence set forth in SEQ ID NO: 127, or a modified sequence truncated at an amino acid residue within SEQ ID NO: 127.
  • the herein described PE domain lb (SEQ ID NO: 139) consists of amino acids 365-404 of SEQ ID NO:
  • a PE derived carrier that comprises a receptor binding domain, and a translocation domain, or a modified translocation domain, and a non-toxic catalytic domain, or modified non toxic catalytic domain, can further comprise the amino acid sequence set forth in SEQ ID NO: 139, or a modified sequence truncated at an amino acid residue within SEQ ID NO: 139.
  • a carrier of the present disclosure can comprise portion(s) of one or more of a domain II, a domain lb, or a domain III, wherein those portions (e.g., certain amino acid sequences thereof) can be part of a spacer as further described herein.
  • the methods and compositions of the present disclosure contemplate carriers that can comprise a first portion and a second portion, wherein the first portion is derived from a first exotoxin and the second portion is derived from a second exotoxin; and wherein the carrier can be coupled to a cargo (e.g., a heterologous cargo such as a biologically active cargo).
  • the first exotoxin can be Cholix
  • the second exotoxin can be PE.
  • the first portion can be derived from a domain I, a domain II, a domain lb, or a domain III of Cholix, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 133, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 148 - SEQ ID NO: 152, a functional fragment thereof, or any combination thereof.
  • the first portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11, a functional fragment thereof, or any combination thereof.
  • the second portion can be derived from a domain I, a domain II, a domain lb, or a domain III of PE, or any combination thereof.
  • the second portion can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 137 - SEQ ID NO: 145, a functional fragment thereof, or any combination thereof.
  • the first portion can be chemically coupled or recombinantly coupled to the second portion.
  • the first portion can further be directly or indirectly coupled to the second portion.
  • Such a carrier can comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence SEQ ID NO: 146 or SEQ ID NO: 147.
  • a carrier of the present disclosure can comprise a polypeptide, wherein the polypeptide can comprise at least 110 amino acid residues of a domain I of the exotoxin.
  • a carrier can comprise at least 120 amino acid residues of a domain I of the exotoxin.
  • a carrier can comprise at least 130 amino acid residues of a domain I of the exotoxin.
  • a carrier can comprise at least 140 amino acid residues of a domain I of the exotoxin.
  • a carrier can comprise at least 150 amino acid residues of a domain I of the exotoxin.
  • a carrier can comprise at least 50 contiguous amino acid residues of the domain I of the exotoxin.
  • a carrier can comprise at least 60 contiguous amino acid residues of the domain I of the exotoxin.
  • a carrier can comprise at least 75 contiguous amino acid residues of the domain I of the exotoxin.
  • a carrier can comprise at least 100 contiguous amino acid residues of the domain I of the exotoxin.
  • a carrier can comprise at least 150 contiguous amino acid residues of the domain I of the exotoxin.
  • the methods and compositions of the present disclosure contemplate carriers that can comprise on or more modifications at the N-terminal.
  • a modification can comprise at least one N-terminal methionine residue.
  • the at least one N-terminal methionine residue can be part of an N-cap as described herein.
  • a carrier comprising an N-cap can further comprise one or more amino acid variations in the first 5-10 amino acid residues compared to a reference sequences.
  • one or more of the first N-terminal amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1 can be substituted with other amino acid residues, as long as the consensus sequence that can define a functional Cholix is not altered.
  • a carrier described herein comprising an N-cap can further comprise an N- terminal methionine residue.
  • An N-cap can also only comprise an addition of an N-terminal methionine residue.
  • Exemplary carriers of the present disclosure that comprise such N-cap are set forth in any one of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO: 125.
  • functional variants of such carrier can comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO: 125, or 80% sequence identity to a functional fragment thereof.
  • a“Cholix” (also referred to herein as Cholix toxin or Cholix exotoxin) can encompass a variety of functional variants (e.g., a functional genus), wherein the functional variants can comprise one or more variations is their amino acid sequence relative to SEQ ID NO: 1 as disclosed herein.
  • the Cholix toxin having the amino acid sequence set forth in SEQ ID NO: 1 is used as the reference sequence when referred to Cholix.
  • the present disclosure is not limited to the Cholix having the amino acid sequence set forth in SEQ ID NO: 1 but instead encompasses all Cholix variants that fall within the functional genus of Cholix.
  • a first Cholix domain I polypeptide e.g., a first carrier
  • a second Cholix domain I polypeptide e.g., a second carrier
  • both the first polypeptide and the second polypeptide are capable of carrying out the same functions, e.g., transcytosis across an epithelial cell, and interact with the same receptors, such as ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan.
  • a first carrier and a second carrier can be produced in the same expression system (e.g., a bacterial expression system such as E. coli or a mammalian expression system such as a CHO cell). In other cases, and as described herein, a first carrier and a second carrier are produced in a different expression system (e.g., a bacterial or a mammalian expression system).
  • a carrier of the present disclosure can comprise properties that allow interactions with endogenous receptors and/or accessing an endogenous transport and transcytosis system.
  • a carrier of the present disclosure that is derived from Cholix domain I and comprises an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 125 or SEQ ID NO: 148 - SEQ ID NO: 152 can interact with one or more endogenous receptors.
  • endogenous receptors can include TMEM132A, GPR75, ERGIC-53, and/or perlecan, and any combination thereof.
  • Such interaction(s) can provide for (e.g., apical-to-basal) transcytosis across an epithelial cell and/or transport to the interior of an epithelial cell. These interactions allow rapid and efficient delivery. These interactions further provide transport mechanisms that may not alter the carrier of the cell that a carrier is delivered into or transported across. For example, carriers described herein do not show any chemical modifications upon release from the basal membrane of an epithelial cell, suggesting that the carriers of the present disclosure may harness one or more endogenous transport system to deliver cargo into and/or across epithelial cells.
  • a carrier as described herein can transport cargo across an epithelial cell with a transport rate of about 10 10 cm/sec to about 10 2 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of about 10 9 cm/sec to about 10 3 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of about 10 8 cm/sec to about 10 4 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of about 10 7 cm/sec to about 10 5 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of about 10 6 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 8 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 7 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 6 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 5 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 4 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 3 cm/sec.
  • a carrier can transport cargo across an epithelial cell with a transport rate of at least 10 2 cm/sec.
  • the methods and compositions of the present disclosure provide carrier molecules that rapidly and efficiently transport cargo into and/or across epithelial cells. Delivery and/or transport of cargo can be achieved by coupling the cargo to a carrier as described herein. Such a construct can be referred to herein as a“delivery construct.” As described herein, the present disclosure contemplates carriers that can comprise a small molecule, a polypeptide, an aptamer, a fragment thereof, or any combination thereof. As described herein, a carrier can be derived from an exotoxin. The exotoxin can be Cholix or PE. A carrier can be coupled directly or indirectly to the cargo. A carrier can be covalently or non-covalently coupled to the cargo.
  • a delivery construct can further comprise a spacer that links the carrier to the cargo.
  • the spacer can be any molecule that links the carrier to the cargo and can comprise oligomeric or polymeric spacers (e.g., polyethylene glycol, etc.), and amino acids.
  • a delivery construct comprising a carrier coupled to a cargo and, optionally, a spacer and/or another functional moiety can be produced synthetically or recombinantly (e.g., in E. coli or a CHO cell).
  • the terms“delivery constructs”,“delivery constructs”,“toxin- derived delivery constructs”,“chimeric constructs”,“proteins” and“fusion proteins” can be used interchangeably and can refer to constructs comprising at least one delivery or carrier domain (e.g., a Cholix or PE domain I derived carrier, a small molecule, an aptamer, or any combination thereof) and at least one heterologous cargo molecule such as a therapeutic cargo molecule.
  • the term“heterologous cargo” can be referred to as unrelated to these exotoxins.
  • toxicity e.g., intoxication of enterocytes
  • the bacterial carrier e.g., Cholix or PE
  • a carrier that is derived from a domain I e.g., a truncated version of a domain I
  • an exotoxin such as Cholix and PE is sufficient for rapid and efficient transcytosis across epithelial cell (e.g., polarized epithelial cells of a gut).
  • a delivery construct (e.g., an isolated delivery construct) comprises a carrier that provides rapid and efficient delivery and/or transport of a cargo to a certain location, wherein the location can be an organ, a tissue, a cell, or a cellular compartment.
  • the cargo molecule can be directly or indirectly coupled to the carrier.
  • the cargo that is coupled to the carrier can be a heterologous cargo (e.g., not derived from the carrier itself).
  • a delivery construct described herein can comprise a carrier coupled to a heterologous cargo.
  • the carrier can comprise certain functions that allow repaid and efficient transport of cargo to a location, e.g., a location within an epithelial cell or a location(s) within a basolateral compartment.
  • a carrier contemplated herein can be derived from an exotoxin.
  • the exotoxin can be Cholix or PE.
  • a carrier that is derived from a Cholix can comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, or at least 80% sequence identity to a functional fragment thereof.
  • a Cholix also referred to herein as Cholix toxin or Cholix exotoxin
  • can encompass a variety of functional variants e.g., a functional genus
  • the functional variants can comprise one or more variations is their amino acid sequence relative to SEQ ID NO: 1 as disclosed herein.
  • the Cholix toxin having the amino acid sequence set forth in SEQ ID NO: 1 is used as the reference sequence when referred to Cholix.
  • the present disclosure is not limited to the Cholix having the amino acid sequence set forth in SEQ ID NO: 1 but instead encompasses all Cholix variants that fall within the functional genus of Cholix.
  • a first Cholix domain I polypeptide e.g., a first carrier
  • a second Cholix domain I polypeptide e.g., a second carrier
  • both the first polypeptide and the second polypeptide are capable of carrying out the same functions, e.g., transcytosis across an epithelial cell, and interact with the same receptors, such as ribophilin 1, SEC24 (can also be referred to as COPII coat complex component), cytokeratin-8 (CK-8), transmembrane protein 132
  • TMEM132 glucose regulated protein 75 (GRP75), endoplasmatic reticulum Golgi
  • intermediate compartment 53 (ERGIC-53, the number 53 may refer to its molecular weight of approximately 53 kDa), and/or perlecan (also referred to as basement membrane- specific heparan sulfate proteoglycan core protein or HSPG).
  • a first carrier and a second carrier can be produced in the same expression system (e.g., a bacterial expression system such as E. coli or a mammalian expression system such as a CHO cell).
  • a first carrier and a second carrier can be produced in a different expression system (e.g., a bacterial or a mammalian expression system).
  • the delivery constructs contemplated herein can provide advantages over conventional delivery modalities. Such advantages can include, but are not limited to: a) aid in the production of the delivery construct; b) aid in the refolding of the chimera construct; c) aid in the formulation of the delivery construct; d) aid in reducing the sensitivity of the cargo to proteolytic destruction; e) improve the stability of the delivery construct during storage; f) in embodiments wherein the bacterial carrier elements of domain I are coupled to the heterologous (e.g., a biologically active) cargo without a spacer, or with a non-cleavable spacer, the bacterial carrier elements of domain I can function to retain the chimera to selected locations in the body following transcytosis that results in greater exposure of a biologically active (or diagnostic) cargo to specific cells to provide improved pharmacodynamics; g) in embodiments wherein the bacterial carrier elements of domain I are coupled to a heterologous (e.g., biologically active) cargo
  • the present disclosure provides methods and compositions for delivery and transport of cargo molecules across an epithelial cell (e.g., via transcytosis) and/or into the interior of an epithelial cell.
  • the methods and compositions disclosed herein can comprise a delivery construct, wherein the delivery construct comprises a carrier coupled to a heterologous cargo (e.g., via a spacer).
  • the transport and delivery processes described herein using the carriers of the present disclosure can comprise endocytosis on the apical side of an epithelial cell.
  • the transport processes can comprise the release of the delivery construct on the basal side.
  • various mechanisms can be involved in transporting cargo to those various locations.
  • delivery of cargo to an intracellular vesicle or compartment or the cytosol of an epithelial cell can comprise releasing a delivery construct comprising a carrier coupled to that cargo from a vesicle into the an intracellular vesicle or compartment or the cytosol.
  • transcytosis of a delivery construct can include vesicular transcytosis and, as such, can comprise encapsulating the delivery construct in a vesicle during transcytosis such that the delivery construct may or may not be in contact with the intracellular cytosol.
  • the methods and compositions of the present disclosure can comprise delivering a cargo to a certain location such that the cargo remains at that location for a certain amount of time.
  • a cargo molecule can be retained at an intracellular or basolateral location that has been targeted using the compositions described herein. Retention can cause the cargo molecule to elicit a certain response or biological effect (e.g., a therapeutic effect).
  • the present disclosure provides methods and compositions that allow delivery of cargo to a location within across an epithelial cell such the delivery construct (and the cargo) is retained at that location for a specific amount of time.
  • Such retention can be modulated, e.g., by allowing the cargo to be cleaved from the carrier, or by allowing the carrier to reversibly or irreversibly bind a certain protein (e.g., a receptor) that is present at that location.
  • a certain protein e.g., a receptor
  • the delivery constructs of the present disclosure can comprise a carrier, wherein the carrier can be configured to target a certain location inside or across an epithelial cell.
  • a location can be an organ, a tissue, a cell, or a cellular compartment.
  • the methods and compositions described herein can be used for various applications, e.g., those that include delivery of cargo across an intact epithelial membrane in vitro or in vivo.
  • a carrier can be coupled to a heterologous cargo in any way described herein.
  • a delivery construct comprises a carrier that is coupled to a heterologous cargo via a spacer.
  • the spacer can comprise any moiety recited herein, and can comprise any one of the amino acid sequences set forth in SEQ ID NO: 166 - SEQ ID NO: 213.
  • the spacer can be a cleavable spacer.
  • the spacer can be a non-cleavable spacer.
  • a spacer can comprise the amino acid sequence set forth in SEQ ID NO: 210, or a fragment or derivative thereof.
  • any carrier disclosed herein can be combined with any one of the cargo molecules described herein (e.g., those listed in TABLE 11 and TABLE 12), and, optionally, with any spacer described herein (e.g., those listed in TABLES 7-10 and those having an amino acid sequence set forth in SEQ ID NO: 207 - SEQ ID NO: 213) to form a delivery construct.
  • a carrier described herein can be derived from an exotoxin.
  • a carrier can be derived from a domain I of an exotoxin.
  • the exotoxin can be Cholix or PE.
  • a delivery construct contemplated herein can comprise a carrier derived from Cholix, wherein the carrier can comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 125, coupled to a heterologous cargo.
  • a carrier can comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 125.
  • a carrier can comprise an amino acid sequence having at least 99% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 1 - SEQ ID NO: 125.
  • the exotoxin that a carrier can be derived from can be PE.
  • a delivery construct comprises a carrier can comprise an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 137, or a functional fragment thereof, coupled to a heterologous cargo.
  • the methods and compositions of the present disclosure can comprise a delivery construct comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 153 - SEQ ID NO: 165, or having at least 80% sequence identity to a functional fragment thereof.
  • a delivery construct can comprise an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 153 - SEQ ID NO: 165, or having at least 90% sequence identity to a functional fragment thereof.
  • a delivery construct can comprise an amino acid sequence having at least 95% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 153— SEQ ID NO: 165, or having at least 95% sequence identity to a functional fragment thereof.
  • a delivery construct can comprise an amino acid sequence having at least 99% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 153 - SEQ ID NO: 165, or having at least 99% sequence identity to a functional fragment thereof.
  • a delivery construct can comprise an amino acid sequence having 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 153 - SEQ ID NO: 165, or having 100% sequence identity to a functional fragment thereof.
  • a delivery construct of the present disclosure can interact with one or more specific proteins, enzyme, or receptors during transport and/or delivery across an epithelial cell and/or into the interior of an epithelial cell (e.g., a polarized gut epithelial cell).
  • the one or more receptors can be endogenous receptors.
  • the delivery constructs of the present disclosure can use endogenous receptor systems that provide for rapid efficient transport and delivery of cargo across an epithelial cell or an intact epithelium (e.g., a monolayer of Caco-2 cells and/or an intact gut epithelium of a subject), and/or to the interior of an epithelial cell of an epithelium.
  • Delivery constructs comprising a carrier comprising an amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 125 can enable delivery and transport of a heterologous (e.g., a therapeutically or biologically active) cargo to the interior of an epithelial cell, e.g., to the basal side of an epithelial cell, and/or a supranuclear region (e.g., the endoplasmatic reticulum, the Golgi apparatus, and/or an endosome) of an epithelial cell.
  • the interior of an epithelial cell can be an intracellular vesicle or compartment or the cytosol of the epithelial cell.
  • a cargo (e.g., a heterologous cargo) can be delivered to the basal side of the epithelial cell (e.g., a location or compartment at the basal side).
  • a heterologous cargo can be delivered to a supranuclear region of the epithelial cell.
  • Transport of a delivery construct to the interior of an epithelial cell can comprise releasing the delivery construct from a vesicle that formed during endocytosis of the delivery construct on the apical surface of the epithelial cell.
  • Delivery and/or transport to a location in the interior of a cell can comprise retaining the delivery construct in a vesicle and/or releasing the delivery construct from that vesicle, such that the delivery construct can be in contact with the cytosol of the epithelial cell (e.g., the construct may or may not be in contact with the cytosol of the epithelial cell during transcytosis due to encapsulation in the vesicle).
  • a carrier comprising a truncated version of Cholix domain I can be released from a vesicle, e.g., those comprising an amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 107, or a functional fragment or derivative thereof.
  • Delivery constructs of the present disclosure comprising a carrier comprising an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 29, SEQ ID NO: 129 - SEQ ID NO: 133, or SEQ ID NO: 141 - SEQ ID NO: 145 can enable delivery and transport of a heterologous (e.g., a therapeutically or biologically active) cargo across an epithelial cell. Transport across an epithelial cell (e.g., a polarized gut epithelial cell) can occur via transcytosis.
  • the transcytosis mechanism utilized by the herein described delivery constructs is an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 29, SEQ ID NO: 129 - SEQ ID NO: 133, or SEQ ID NO: 141 - SEQ ID NO: 145
  • a heterologous (e.g., a therapeutically or biologically active) cargo across an epithelial cell. Transport across an epitheli
  • a carrier of a delivery construct can comprise the structural elements that allow these receptor interactions.
  • the receptors that a carrier as disclosed herein can interact with include ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and perlecan, or any combination thereof.
  • a carrier as described herein may not or may not significantly interact with clathrin or GPR78, or a combination thereof.
  • an endogenous system including those receptors can have several advantages over other transport mechanisms.
  • Using an endogenous transport system can include the following advantages: (i) an intact layer of epithelial cells such as a monolayer or an epithelium in vivo can be crossed without damaging or disrupting the cells or monolayer structure; (ii) rapid and efficient delivery and transport can be achieved; (iii) the interaction of distinct domains or regions of an exotoxin derived construct with specific receptors allows modulation of these interaction in a way that allows to specifically target certain regions or compartments within a cell or within a subject.
  • delivery and transport e.g., of a heterologous cargo
  • delivery and transport e.g., of a heterologous cargo
  • delivery and transport e.g., of a heterologous cargo
  • delivery and transport e.g., of a heterologous cargo
  • an epithelial cell can be provided by using certain truncated versions of an exotoxin domain I, such as those having an amino acid sequence set forth in any one of SEQ ID NO: 30 - SEQ ID NO: 125, or functional fragment thereof.
  • the epithelial cell can be located in the gut of a subject (e.g., a rodent or a human).
  • delivery and transport e.g., of a heterologous cargo
  • delivery and transport e.g., of a heterologous cargo
  • transcytosis e.g., by using an endogenous transcytosis system
  • certain truncated versions or derivatives of an exotoxin domain I such as those having an amino acid sequence set forth in any one SEQ ID NO: 4 - SEQ ID NO: 29, SEQ ID NO: 129 - SEQ ID NO: 133, or SEQ ID NO: 141 - SEQ ID NO: 145.
  • the ability of the herein described delivery constructs to rapidly and efficiently deliver therapeutically active and/or diagnostic cargo to those locations enables new options for treatment, prevention, and/or diagnosis of various diseases (e.g., inflammatory disease, autoimmune diseases, hormone-deficiency diseases, obesity and metabolic disorders, and cancer).
  • various diseases e.g., inflammatory disease, autoimmune diseases, hormone-deficiency diseases, obesity and metabolic disorders, and cancer.
  • Delivery constructs of the present disclosure in addition to a carrier, a cargo, and, optionally, a spacer, can further comprise one or more functional moieties.
  • a functional moiety can be a detectable agent, an affinity handle (e.g., a clickable functional groups such as an azide), a barcode (e.g., a nucleic acid barcode), cell-penetrating agents, or other functional moieties that modulate the pharmacokinetic (PK) and/or pharmacodynamic (PD) profile of the delivery construct.
  • a delivery construct can comprise a cell-penetrating agent.
  • the cell-penetrating agent can be a peptide.
  • the cell-penetrating agent can comprise polycations, polyorganic acids, endosomal releasing polymers, poly(2-propylacrylic acid), poly(2-ethylacrylic acid), Tat peptide, Arg patch, a knotted peptide, CysTAT, S19-TAT, R8 (SEQ ID NO: 73), pAntp, Pas-TAT, Pas- R8 (SEQ ID NO: 76), Pas-FHV, Pas-pAntP, F2R4 (SEQ ID NO: 79), B55, aurin, IMT-P8, BR2, OMOTAG1, OMOTAG2, pVEC, SynB3, DPV1047, C105Y, Transpotan, MTS, hLF, PFVYLI (SEQ ID NO: 93), maurocalcine, imperatoxin, hadrucalin, hemicalcin, opicalcin-l, opicalcin-2, midkin(62-l04),
  • a cell-penetrating agent can be coupled to a delivery construct as described herein via the N- or the C-terminus.
  • a cell -penetrating agent can provide access to a variety of cell types.
  • a cell-penetrating agent can provide additional functionality, e.g., for therapeutic cargo delivery, once the delivery construct has crossed and epithelial layer (e.g., an epithelium of a subject).
  • a multimer comprising multiple delivery constructs can be formed in solution.
  • a multimer can be formed by multimerization of the carrier and/or the heterologous cargo.
  • the multimer can be a heteromer or a homomer.
  • the homomer can be a homodimer.
  • the homodimer can be formed by dimerization of the heterologous cargo.
  • a delivery construct comprising the amino acid sequence set forth in SEQ ID NO: 217 can form a dimer. Dimerization of such a delivery construct can be due to dimerization of the cargo, e.g., IL-10 (e.g., SEQ ID NO: 217) in this case.
  • the methods and compositions of the present disclosure can comprise a delivery construct comprising a carrier coupled to a cargo, such as a heterologous cargo.
  • a heterologous cargo e.g., biologically active
  • the heterologous cargo can be introduced into any portion of the carrier that does not disrupt the endocytosis and/or transcytosis activity of the carrier.
  • the present disclosure provides delivery constructs that comprise a polypeptide carrier.
  • a heterologous cargo can be directly coupled to the N-terminus or C-terminus of such a polypeptide carrier (e.g., a domain I or a truncated version thereof, e.g., SEQ ID NO: 4 - SEQ ID NO: 125).
  • a heterologous cargo can be couple to the carrier via a side chain of an amino acid of the carrier receptor binding domain.
  • a heterologous cargo can be coupled to the carrier with a cleavable spacer such that cleavage at the cleavable spacer(s) separates the heterologous cargo from the remainder of the delivery construct.
  • a heterologous cargo can be also a polypeptide that comprises a short leader peptide that remains attached to the polypeptide following cleavage of the cleavable spacer.
  • the heterologous cargo can comprise a short leader peptide of greater than 1 amino acid, greater than 5 amino acids, greater than 10 amino acids, greater than 15 amino acids, greater than 20 amino acids, greater than 25 amino acids, greater than 30 amino acids, greater than 50 amino acids, or greater than 100 amino acids.
  • a biological active cargo can comprise a short leader peptide of less than 100 amino acids, less than 50 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, or less than 5 amino acids.
  • a biological active cargo can comprise a short leader peptide of between 1- 100 amino acids, between 5-10 amino acids, between 10 to 50 amino acids, or between 20 to 80 amino acids.
  • the present disclosure provides methods and compositions comprising carrier that are derived from a domain I of an exotoxin, wherein the exotoxin can be Cholix or PE.
  • the exotoxin can be Cholix or PE.
  • native Cholix e.g., SEQ ID NO: 1 or SEQ ID NO: 2
  • PE e.g., SEQ ID NO: 135
  • domain lb loop is not essential for any known activity of the toxin, including cell binding, translocation, ER retention or ADP ribosylation activity.
  • domain lb can be deleted entirely, or modified to contain a heterologous cargo, e.g., a biologically active cargo.
  • the heterologous cargo (e.g., biologically active cargo) can be inserted into Cholix or PE carrier domain lb.
  • a heterologous cargo (e.g., biologically active cargo), for example, can be inserted into a Cholix derived carrier domain lb between the cysteines at positions 395 and 402 that are not cross-linked. This can be accomplished by reducing the disulfide linkage between the cysteines, by deleting one or both of the cysteines entirely from the lb domain, by mutating one or both of the cysteines to other residues, for example, serine, or by other similar techniques.
  • the biologically active cargo can be inserted into the domain lb loop between the cysteines at positions 395 and 402. In such embodiments, the disulfide linkage between the cysteines can be used to constrain the biologically active cargo domain.
  • the methods and compositions described herein can comprise delivery constructs that are produced such that a heterologous cargo is expressed together with a carrier (and, optionally, a spacer) as a fusion protein (e.g., the delivery construct).
  • a heterologous cargo can be inserted into the delivery construct by any method known to one of skill in the art without limitation.
  • amino acids corresponding to the heterologous cargo can be directly inserted into the receptor binding domain, with or without deletion of native amino acid sequences.
  • a heterologous cargo may not be expressed together with a carrier (and, optionally, a spacer) as a fusion protein
  • the heterologous cargo can be coupled to the carrier by any suitable method known by one of skill in the art, without limitation, including peptide conjugation chemistry and/or click chemistry.
  • the methods and compositions of the present disclosure can comprise delivery constructs comprising a carrier coupled to a cargo (e.g., a heterologous cargo), wherein the carrier is capable of delivering the heterologous cargo into and/or across an epithelial cell in vitro (e.g., an epithelial cell monolayer) or in vivo (e.g., a gut epithelium of a subject).
  • a carrier can be coupled to a cargo in any way described herein.
  • the carrier can be directly or indirectly coupled the cargo.
  • the carrier can also be covalently or non-covalently coupled to the cargo.
  • a spacer can comprise any moiety recited herein.
  • a spacer can be any molecule that links the carrier to the cargo and can comprise oligomeric or polymeric spacers (e.g., polyethylene glycol, etc.), other small molecule spacer (e.g., those derived from dicarbonic acids such as succinic acid, aspartic acid, etc.) and amino acids (including short peptide sequences etc.).
  • a“spacer,” as described herein, generally refers to a chemical moiety that can be attached to or coupled to a molecule of the present disclosure.
  • a spacer can be located between a first molecule and a second molecule.
  • a spacer can connect, attach, link, or couple a first molecule (e.g., a polypeptide, small molecule, nucleic acid, etc.) to a second molecule (e.g., a polypeptide, small molecule, nucleic acid, etc.).
  • a spacer can reduce steric hindrance between the first molecule and the second molecule.
  • a spacer can be an amino acid sequence coupled to the C-terminus of a peptide or polypeptide.
  • the amino acid sequence of a spacer as disclosed herein can be between 1-100 amino acid residues long.
  • a spacer can be between 5-75 amino acid residues long.
  • a spacer can be between 5-50 amino acid residues long.
  • a spacer is between 5-25 amino acid residues long.
  • a carrier can comprise any one of the amino acid sequences set forth in SEQ ID NO: 4 - SEQ ID NO: 125 is coupled to a spacer at its C-terminus (and the spacer can be further coupled to a heterologous cargo via its C- terminus).
  • the spacer can be an amino acid spacer.
  • the spacer can comprise any of the amino acid sequences set forth in SEQ ID NO: 166 - SEQ ID NO: 213.
  • the spacer can comprise a portion of a domain II, a domain lb, or a domain III of an exotoxin, or any combination thereof.
  • a delivery construct as described herein can comprise a carrier comprising an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 125, coupled to a spacer, wherein the spacer comprises amino acid residues 1-82 of SEQ ID NO: 126.
  • a spacer can comprise an amino acid sequence.
  • a spacer can comprise at most 82 amino acid residues of any one SEQ ID NO: 126.
  • a spacer can comprise the first 82 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 126.
  • the amino acid residues of the Cholix domain II can be contiguous amino acid residues (e.g., residues 1-82 of SEQ ID NO: 126).
  • spacer that can be used in combination with the herein described methods and compositions comprise those comprising an amino acid sequences set forth in SEQ ID NO: 207
  • a spacer comprises the amino acid sequence set forth in SEQ ID NO: 210, or a fragment or derivative thereof.
  • the methods and compositions of the present disclosure can comprise spacer that can comprise a portion of a domain II, a domain lb, and/or a domain III of an exotoxin.
  • a carrier comprising an amino acid sequence set forth in any one of SEQ ID NO: 4 - SEQ ID NO: 125 can further be coupled (e.g., via the C-terminus) to a spacer, wherein the spacer comprises from about 80 to about 90 amino acid residues from any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 138 - SEQ ID NO: 140.
  • a spacer can comprise at most 85 amino acid residues of any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 137
  • a spacer can comprise at most 82 amino acid residues of any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 137 - SEQ ID NO: 139.
  • a spacer can comprise at most 80 amino acid residues of any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 137 - SEQ ID NO: 139.
  • a spacer can comprise at most 50 amino acid residues of any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 137 - SEQ ID NO: 139.
  • a spacer can comprise at most 25 amino acid residues of any one of SEQ ID NO: 126 - SEQ ID NO: 128 and/or SEQ ID NO: 137 - SEQ ID NO: 139.
  • a spacer can comprise at most 82 amino acid residues of any one SEQ ID NO: 126.
  • a spacer can comprise the first 82 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 126.
  • the amino acid residues of the Cholix domain II can be contiguous amino acid residues (e.g., residues 1-82 of SEQ ID NO: 126).
  • a spacer of the present disclosure can be a cleavable spacer.
  • a spacer of the present disclosure can be a non-cleavable spacer.
  • a heterologous cargo e.g., a biologically or therapeutically active cargo
  • a delivery construct can further comprise a spacer that can indirectly couple a carrier to a cargo (e.g., a heterologous cargo).
  • a spacer as described herein can be a cleavable spacer. The number of cleavable spacers present in a delivery construct depends, at least in part, on the location of the heterologous cargo in relation to the delivery construct and the nature of the heterologous cargo.
  • the delivery constructs can comprise a single cleavable spacer.
  • the heterologous cargo is, e.g., a dimer or other multimer
  • each subunit of the biologically active cargo can be separated from the remainder of the delivery construct and/or the other subunits of the biologically active cargo by cleavage at the cleavable spacer.
  • a cleavable spacer can be cleaved by a cleaving enzyme that is present at or near the basolateral membrane of an epithelial cell.
  • a cleaving enzyme that is present at or near the basolateral membrane of an epithelial cell.
  • the biologically active cargo can be liberated from the remainder of the delivery construct following transcytosis across the mucous membrane and release from the epithelial cell into the cellular matrix on the basolateral side of the membrane.
  • cleaving enzymes could be used that are present inside the epithelial cell, such that the cleavable spacer is cleaved prior to release of the delivery construct from the basolateral membrane, so long as the cleaving enzyme does not cleave the delivery construct before the delivery construct enters the trafficking pathway in the polarized epithelial cell that results in release of the delivery construct and biologically active cargo from the basolateral membrane of the cell.
  • a carrier of the present disclosure can be cleaved by an enzyme.
  • the enzyme that is present at a basolateral membrane of a polarized epithelial cell can be selected from, e.g., Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin All, Thrombin I, or Urokinase I. TABLE 6 presents these enzymes together with an amino acid sequence that is recognized and cleaved by the particular peptidase.
  • a cleavable spacer can exhibit a greater propensity for cleavage than the remainder of the delivery construct.
  • many peptide and polypeptide sequences can be cleaved by peptidases and proteases.
  • the cleavable spacer can be selected so that it will be preferentially cleaved relative to other amino acid sequences present in the delivery construct during administration of the delivery construct.
  • a carrier of a delivery construct can be substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about 75%) intact following delivery of the delivery construct to the bloodstream of the subject.
  • a cargo of a delivery construct can be substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about 75%) intact following delivery of the delivery construct to the bloodstream of the subject.
  • a cleavable spacer can be substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about 75%) cleaved following delivery of the delivery construct to the bloodstream of the subject.
  • a cleaving enzyme found in the plasma of the subject can be used to cleave the cleavable spacer. Any cleaving enzyme known by one of skill in the art to be present in the plasma of the subject can be used to cleave the cleavable spacer. Uses of such enzymes to cleave the cleavable spacers is less preferred than use of cleaving enzymes found near the basolateral membrane of a polarized epithelial cell because it is believed that more efficient cleavage will occur in near the basolateral membrane.
  • cleavage mediated by a plasma enzyme is sufficiently efficient to allow cleavage of a sufficient fraction of the delivery constructs to avoid adverse effects; such plasma cleaving enzymes can be used to cleave the delivery constructs.
  • the cleavable spacer can be cleaved with an enzyme that is selected from the group consisting of caspase-l, caspase-3, proprotein convertase 1, proprotein convertase 2, proprotein convertase 4, proprotein convertase 4 PACE 4, prolyl oligopeptidase, endothelin cleaving enzyme, dipeptidyl-peptidase IV, signal peptidase, neprilysin, renin, and esterase (see, e.g., U.S. Pat. No. 6,673,574, incorporated by reference in its entirety herein).
  • TABLE 7 presents these enzymes together with an amino acid sequence(s) recognized by the particular peptidase.
  • the peptidase cleaves a peptide comprising these sequences at the N-terminal side of the amino acid identified with an asterisk.
  • a cleavable spacer can be any cleavable spacer known by one of skill in the art to be cleavable by an enzyme that is present at the basolateral membrane of an epithelial cell.
  • a cleavable spacer can comprise a peptide.
  • a cleavable spacer can comprise a nucleic acid, such as RNA or DNA.
  • a cleavable spacer can comprise a carbohydrate, such as a disaccharide or a trisaccharide.
  • a cleavable spacer can be any cleavable spacer known by one of skill in the art to be cleavable by an enzyme that is present in the plasma of the subject to whom the delivery construct is administered.
  • a cleavable spacer can comprise a peptide.
  • a cleavable spacer can comprise a nucleic acid, such as RNA or DNA.
  • a cleavable spacer comprises a carbohydrate, such as a disaccharide or a trisaccharide.
  • a cleavable spacer can be selected from, or can be derived from the exemplary list presented in TABLE 8.
  • a cleavable spacer can be a spacer that comprises an amino acid sequence that is a known substrate for the tobacco etch virus (TEV) protease. Accordingly, a cleavable spacer can comprise the amino acid sequence set in forth in, e.g., GGGGSGGGENLYFQS (SEQ ID NO: 193).
  • novel delivery constructs of the present disclosure can comprise a peptide sequence (or like domain), which serves to inhibit, interfere with, or block the ability of the biologically active cargo to bind to receptors at the surface of epithelial cells. Accordingly, depending upon the biologically cargo to be delivered, the peptide sequence (or like domain) which serves to inhibit, interfere with, or block the ability of the biologically active cargo to bind to its receptor at the surface of epithelial cells will be directed specifically to the receptor to which the biologically active binds.
  • a cleavable spacer of the present disclosure can comprise a peptide sequence, which serves to inhibit, interfere with, or block the ability of the biologically active cargo to bind GM-l at the surface of epithelial cells.
  • TABLE 9 presents several examples of peptide sequences, which can be incorporated in whole, or in part, into the cleavable spacers to be used in the preparation of the delivery constructs of the present disclosure.
  • a cleavable spacer used in the preparation of the delivery constructs of the present disclosure can comprise the amino acid sequence of, e.g ., SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189 and SEQ ID NO: 190, or variants or fragments thereof, as depicted in TABLE 10 below.
  • TABLE 10 Exemplary Cleavable GM-1 Binding Peptide-Containing Spacers
  • a delivery construct of the present disclosure can comprise a carrier coupled to a heterologous cargo (e.g., a biologically active cargo), wherein the cargo can be separated from the carrier by a spacer consisting of one or more amino acids (e.g., up to 25 amino acids).
  • the spacer can be a peptide spacer or any other molecular entity that may be used to couple to link a first and a second molecule.
  • a spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them.
  • the constituent amino acids of the spacer can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
  • the spacer can be capable of forming covalent bonds to both the delivery construct and to the (e.g., biologically active) cargo.
  • Suitable spacers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon spacers, heterocyclic carbon spacers, or peptide spacers.
  • the spacer(s) can be joined to the constituent amino acids of the delivery construct and/or the biologically active cargo through their side groups (e.g., through a disulfide linkage to cysteine).
  • the spacers can be joined to the alpha carbon amino and/or carboxyl groups of the terminal amino acids of the delivery construct and/or the (e.g., biologically active) cargo.
  • a bifunctional spacer having one functional group capable of reacting with a group on the bacterial carrier and another group reactive on the biologically active cargo can be used to form the desired conjugate.
  • derivatization can involve chemical treatment of the targeting moiety.
  • Procedures for generation of, for example, free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are known (See U.S. Pat. No.
  • a cargo e.g., a biologically active cargo to be delivered to a location (e.g., a location within a subject such as a human) can be coupled to the carrier using one or more non- cleavable peptide spacers comprising, e.g., the amino acid sequence GTGGS (SEQ ID NO: 207), GGGGS (SEQ ID NO: 208), GGGGSGGGGS (SEQ ID NO: 209), GGGGSGGGGSGGGGS (SEQ ID NO: 210), or GGGGSGGG (SEQ ID NO: 211), wherein the carrier targets said cargo (e.g., biologically active cargo) to specific cells, including but not limited to, cells of the immune system such as macrophages, antigen-presenting cells and dendritic cells (e.g., upon transporting the cargo across an epithelial cell).
  • the carrier targets said cargo (e.g., biologically active cargo) to specific cells, including but not limited to, cells of the immune system such as macrophag
  • the delivery constructs of the present disclosure can be produced using a variety of methods. The selection of a production method can depend on the molecular structure of the delivery construct and/or its components (e.g., the carrier, cargo, and/or spacer). Thus, for some delivery constructs organic synthetic methods may be advantageous for producing such delivery construct.
  • a delivery construct of the present disclosure can be a polypeptide. Such polypeptides can be produced, for example, using recombinant DNA methodology. Generally, this involves creating a DNA sequence that encodes the delivery construct, placing the DNA in an expression cassette under the control of a particular promoter, expressing the molecule in a host, isolating the expressed molecule and, if required, folding of the molecule into an active conformational form.
  • DNA encoding the delivery constructs described herein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solid support method of U.S. Pat. No. 4,458,066, and the like.
  • Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences.
  • subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.
  • a DNA encoding a delivery constructs of the present disclosure can be cloned using DNA amplification methods such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the gene for the biologically active cargo is PCR amplified, using a sense primer containing the restriction site for, e.g., Ndel and an antisense primer containing the restriction site for Hindlll. This can produce a nucleic acid encoding the biologically active cargo sequence and having terminal restriction sites.
  • a delivery construct having“complementary” restriction sites can similarly be cloned and then ligated to the biologically active cargo and/or to a spacer attached to the biologically active cargo.
  • DNA encoding delivery constructs of the present disclosure is artificially synthesized by, for example, solid-phase DNA synthesis.
  • a“Cholix” also referred to herein as Cholix toxin or Cholix exotoxin
  • a“Cholix” can encompass a variety of functional variants (e.g., a functional genus), wherein the functional variants can comprise one or more variations is their amino acid sequence relative to SEQ ID NO: 1 as disclosed herein.
  • the Cholix toxin having the amino acid sequence set forth in SEQ ID NO: 1 is used as the reference sequence when referred to Cholix.
  • a variant of the Cholix exotoxin with the amino acid sequence set forth in SEQ ID NI: 1 can be a Cholix exotoxin which amino acid sequence is set forth in SEQ ID NO: 2, wherein both variants are capable of carrying out the same functions, e.g., transcytosis across an epithelial cell, and interact with the same receptors, such as ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan.
  • a polypeptide can affect, to some degree, the amino acid sequence of such polypeptide (e.g., due to post-translational modifications).
  • a first carrier and a second carrier are produced in the same expression system (e.g., a bacterial expression system such as E. coli or a mammalian expression system such as a CHO cell).
  • a first carrier and a second carrier are produced in a different expression system (e.g., a bacterial or a mammalian expression system).
  • Bacterial expression systems include E. coli
  • mammalian expression systems include CHO cells, for example.
  • a bacterially produced polypeptide can comprise an N-cap, wherein the N-cap can comprise one more modifications at the N-terminal of the polypeptide.
  • An N-cap can comprise an N-terminal methionine residue.
  • Examples of Cholix domain I derived carrier polypeptides that can be bacterially produced and that comprise such N-terminal methionine include those comprising the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO: 135.
  • the present disclosure provides methods and compositions that allow for the rapid and efficient delivery of cargo into and/or across epithelial cells (e.g., polarized epithelial cells) in vitro (e.g., a Caco-2 cell monolayer) and in vivo (e.g., a gut epithelium of a subject).
  • epithelial cells e.g., polarized epithelial cells
  • Such rapid and efficient delivery can be achieved by coupling the cargo (e.g., a biologically active cargo) to a carrier to form a delivery construct.
  • Such delivery constructs can be modified to target certain locations within an epithelial cell or to transport cargo across an epithelial cell such as an intact epithelial membrane.
  • compositions and methods of the present disclosure provide efficient transport and delivery of various cargo molecules to different locations (e.g., organs, tissues, or cells) of a subject (e.g., a rodent or a human).
  • the delivery constructs of the present disclosure can allow for delivery into epithelial cells and/or for rapid transcytosis (e.g., vesicular transcytosis) across an epithelial cell layer such as a gut epithelium of a subject.
  • the presently described delivery mechanisms allow for transport and delivery of various cargo molecules.
  • the herein described delivery constructs can be coupled to at least one, at least two, at least three, at least five or at least 10 cargo molecules.
  • the cargo can be a heterologous cargo, e.g., heterologous to the carrier.
  • a delivery construct described herein comprises a Cholix domain I derived carrier (e.g., those having the amino acid sequences set forth in SEQ ID NO: 1 - SEQ ID NO: 125) coupled to a heterologous cargo, wherein the heterologous cargo is a non-Cholix derived cargo molecule (e.g., is not derived or does not contain fragments of a Cholix toxin domain I, II, lb, and III).
  • a heterologous cargo can be a biologically active cargo.
  • a biologically active cargo can include therapeutic and/or diagnostic molecules.
  • the delivery constructs of the present disclosure can be used to deliver a biologically active cargo to a subject (e.g., a rodent or a human).
  • A“biologically active cargo” as used herein includes, but is not limited to: a
  • a biologically active cargo of the present disclosure can be a macromolecule that can perform a desirable biological activity when introduced to the bloodstream of the subject.
  • a heterologous cargo as described herein can be a biologically active cargo.
  • a biologically active cargo that is part of a delivery construct can exert its effects in biological compartments of the subject other than the subject’s blood. For example, in various
  • the biologically active cargo can exert its effects in the lymphatic system.
  • the biologically active cargo can exert its effects in an organ or tissue, such as, for example, the subject’s liver, heart, lungs, pancreas, kidney, brain, bone marrow, etc.
  • the biologically active cargo can or cannot be present in the blood, lymph, or other biological fluid at detectable concentrations, yet can still accumulate at sufficient concentrations at its site of action to exert a biological effect.
  • a biologically active cargo can be a protein that comprises more than one polypeptide subunit.
  • the protein can be a dimer, trimer, or higher order multimer.
  • a biologically active cargo to be delivered to a certain location can be selected from, e.g., cytokines and cytokine receptors such as Interleukin- 1 (IL-l), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-l l, IL-12, IL-13, IL-14, IL-15, IL-l 6, IL-17, IL-l 8, IL-l 9, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, lymphokine inhibitory factor, macrophage colony stimulating factor, platelet derived growth factor, stem cell factor, tumor growth factor-b, tumor necrosis factor, lymphotoxin, Fas, granulocyte colony stimulating factor, granulocyte macrophage colony
  • IL-l Interleukin- 1
  • erythropoietin angiogenin, hepatocyte growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, tumor growth factor-a, thrombopoietin, thyroid stimulating factor, thyroid releasing hormone, neurotrophin, epidermal growth factor, VEGF, ciliary neurotrophic factor, LDL, somatomedin, insulin growth factor, insulin-like growth factor I and II, chemokines such as ENA-78, ELC, GRO-a, GRO-b, GRO-g, HRG, LEF, IP- 10, MCP-l, MCP-2, MCP-3, MCP-4, MIP-l-a, MIR-1-b, MG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES, lymphotactin, eotaxin-l, eotaxin-2, TARC, TECK, WAP-l, WAP-2, GCP-l, GCP-2; a-
  • a biologically active cargo as described herein can be a hormone.
  • a hormone can be a growth hormone.
  • the hormone can be a human growth hormone having, for example, the amino acid sequence set forth in SEQ ID NO: 214.
  • a biologically active cargo can be a molecule affects and/or interacts with a metabolism of a subject.
  • a biologically active cargo as described herein can be a drug that can be used to prevent, treat and/or diagnose a metabolic disease or condition.
  • a biologically active cargo as described herein can be a glucagon -like peptide (GLP).
  • the GLP can be GLP-l having the amino acid sequence set forth in SEQ ID NO: 215.
  • a hormone that can be used in combination with the methods and compositions described herein can be insulin (with c- peptide element removed from mature protein), or a derivative thereof.
  • An Insulin peptide can comprise the amino acid sequence set forth in SEQ ID NO: 216.
  • a biologically active cargo can be an interleukin.
  • interleukins that can be used with the methods and compositions described herein can include IL-10 and IL-22, having the amino acid sequence set forth in SEQ ID NO: 217 and SEQ ID NO: 218, respectively.
  • the biologically active cargo disclosed herein can modulate the spatial orientation of a delivery construct.
  • a cargo molecule can induce multimerization of two or more delivery constructs.
  • Such multimers can be homomers or heteromers.
  • the multimer can be a homodimer.
  • a delivery construct comprising the amino acid sequence set forth in SEQ ID NO: 217 can form a dimer.
  • Such dimerization can be induced by IL-10 (as IL-10 can form a natural dimer and thus promote dimerization of a delivery construct comprising an IL-10 as cargo).
  • a biologically active cargo can also comprise toxin, such as endotoxins, enterotoxins or exotoxins.
  • a biologically active cargo can be an ExtB polypeptide (which will form a natural pentamer) having the amino acid sequence set forth in SEQ ID NO: 219.
  • biologically active cargo that can be delivered according to the present disclosure include, but are not limited to, antineoplastic compounds, such as
  • nitrosoureas e.g., carmustine, lomustine, semustine, strepzotocin
  • methylhydrazines e.g., procarbazine, dacarbazine
  • steroid hormones e.g, glucocorticoids, estrogens, progestins, androgens, tetrahydrodesoxycaricosterone
  • immunoactive compounds such as
  • immunosuppressives e.g, pyrimethamine, trimethopterin, penicillamine, cyclosporine, azathioprine
  • immunostimulants e.g, levamisole, diethyl dithiocarbamate, enkephalins, endorphins
  • antimicrobial compounds such as antibiotics, e.g, b-lactam, penicillin,
  • cephalosporins cephalosporins, carbapenims and monobactams, b-lactamase inhibitors, aminoglycosides, macrolides, tetracyclins, spectinomycin; antimalarials, amebicides; antiprotazoals; antifungals, e.g, amphotericin b, antivirals, e.g, acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine, gancyclovir; parasiticides; antihalmintics; radiopharmaceutics; gastrointestinal drugs;
  • norepinephrine bitartrate phenylephrine HC1, ritodrine HC1; cholinomimetic drugs, e.g, acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl; cholinesterase reactivators;
  • adrenergic blocking drugs e.g., acebutolol HC1, atenolol, esmolol HC1, labetalol HC1, metoprolol, nadolol, phentolamine mesylate, propanolol HC1; antimuscarinic drugs, e.g, anisotropine methylbromide, atropine S0 4 , clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr; neuromuscular blocking drugs; depolarizing drugs, e.g., atracurium besylate, hexafluorenium Br, metocurine iodide, succinyl choline Cl, tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g, baclofen; neurotransmitters and neurotransmitter agents, e.g, acetylcholine, adenosine,
  • hormones such as pituitary hormones, e
  • antithyroid drugs estrogenic hormones; progestins and antagonists; hormonal contraceptives; testicular hormones; gastrointestinal hormones, e.g., cholecystokinin, enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growth factor-urogastrone, gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactive intestinal peptide, or sincalide.
  • gastrointestinal hormones e.g., cholecystokinin, enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growth factor-urogastrone, gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactive intestinal peptide, or sincalide.
  • the biologically active cargo is an enzyme selected from hyaluronidase, streptokinase, tissue plasminogen activ
  • a biologically active cargo as described herein can also include a therapeutic and/or diagnostic antibody, an antibody fragment, a diabody, a minibody, or a single-chain variable fragment (e.g., scFv), or a combination thereof.
  • a biologically active cargo as described herein can be an anti-tumor necrosis factor alpha (anti-TNFa) agent.
  • An anti-TNFa agent is an anti-TNFa antibody or a functional fragment thereof.
  • An Anti-TNFa antibody can be adalimumab (Abbvie HUMIRA®, Drug Bank DB 00051) or infliximab (Centocor
  • chemotherapeutics such as chemotherapy or anti -tumor agents which are effective against various types of human cancers, including leukemia, lymphomas, carcinomas, sarcomas, myelomas etc.
  • doxorubicin such as, for example, doxorubicin, mitomycin, cisplatin, daunorubicin, bleomycin, actinomycin D, and neocarzinostatin.
  • Treg inhibitors have been extensively studied and described in the art (see, e.g., Casares et al, Journal of Immunology, 185(9): 5150-5159, 2010, and references cited therein).
  • TABLE 12 shows exemplary amino acid sequences of various heterologous cargos that can be used in combination with the herein disclosed methods and compositions.
  • any of the heterologous cargo molecules shown in TABLE 12 below can be combined with any carrier disclosed herein, e.g., those carrier listed in TABLE 2 and/or TABLE 3 above.
  • a cargo molecule of the present disclosure can comprise an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 214 - SEQ ID NO: 220, at least 80% sequence identity to a functional fragment thereof, and/or any combination of thereof.
  • a cargo molecule of the present disclosure can comprise an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 214 - SEQ ID NO: 220, at least 80% sequence identity to a functional fragment thereof, and/or any combination of thereof.
  • a cargo molecule of the present disclosure can comprise an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 214 - SEQ ID NO: 220, at least 80% sequence identity to a functional fragment thereof, and/or any combination of thereof.
  • a cargo molecule of the present disclosure can comprise an amino acid sequence having at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 214 - SEQ ID NO: 220, at least 80% sequence identity to a functional fragment thereof, and/or any
  • a cargo molecule of the present disclosure can comprise any one of the amino acid sequences set forth in SEQ ID NO: 214 - SEQ ID NO: 220, a functional fragment thereof, and/or any combination of thereof.
  • a cargo described and disclosed herein can be retained at a location that has been targeted using the compositions described herein. Retention can cause the cargo molecule to elicit a certain response or biological effect (e.g., a therapeutic effect).
  • the delivery of a molecule (e.g., a heterologous cargo) to a location can refer to the retention of the molecule at that location.
  • Retention of a molecule at a certain intracellular or extracellular region or compartment can be for a certain amount of time, e.g., at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at 30 minutes, or at least 60 minutes.
  • Retention of a molecule can depend on various factors such as the location where the molecule is retained and/or the types of molecular interactions that occur between the molecule (e.g., a carrier, a delivery construct, and/or a heterologous cargo).
  • delivery of a heterologous cargo to a basolateral compartment via transcytosis across a polarized epithelial cell can comprise retaining the heterologous cargo at the basolateral location for a time sufficient to elicit a certain effect, such as a therapeutic effect in case of a therapeutic and/or biologically active cargo.
  • a delivery construct can be configured to release a cargo at a specific location, e.g., by using pH-dependent and/or enzyme-dependent spacer.
  • the cargo molecule Upon release of a cargo from a carrier, the cargo molecule can elicit a certain effect and/or response. For example, and in the case of biologically and/or therapeutically active cargos, such cargos can elicit their therapeutic effects in vitro or in vivo upon release from the carrier.
  • a cargo may also be capable of eliciting a response when still bound to the carrier. This may depend on the cargo and/or the delivery construct.
  • a heterologous cargo can be a detectable agent such as a fluorescent molecule or a radioactive moiety.
  • a detectable agent as described herein can be used to detect the molecule that the detectable agent is coupled to in various locations, e.g., inside a subject or inside a cell.
  • a detectable agent can also have additional features and functions, such as therapeutic or other biological properties.
  • a radionuclide coupled to a carrier as described herein can allow the detection of the carrier but can also have therapeutic properties, e.g., as a therapeutic radionuclide.
  • a carrier can conjugated to, linked to, or fused with detectable agents, such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that may be used in imaging.
  • a delivery construct can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents.
  • radioisotopes that may be used as detectable agents include alpha emitters, beta emitters, positron emitters, and gamma emitters.
  • the metal or radioisotope may be selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal may be actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope may be actinium-225 or lead-2l2.
  • the near-infrared dyes that may be used in combination with the herein described chimeric binding agents may not be easily quenched by biological tissues and fluids.
  • the fluorophore may be a fluorescent agent emitting electromagnetic radiation at a wavelength between 650 nm and 4000 nm, such emissions being used to detect such agent.
  • Non-limiting examples of fluorescent dyes that may be used as a conjugating molecule in the present disclosure include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG).
  • Near infrared dyes may include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5).
  • fluorescent dyes for use as a conjugating molecule in the present disclosure may include acradine orange or yellow, Alexa Fluors (e.g., Alexa Fluor 790, 750, 700, 680, 660, and 647) and any derivative thereof, 7- actinomycin D, 8-anilinonaphthalene-l -sulfonic acid, ATTO dye and any derivative thereof, auramine-rhodamine stain and any derivative thereof, bensantrhone, bimane, 9-10- bis(phenylethynyl)anthracene, 5,12 - bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivative thereof, l-chloro-9,l0- bis(phenylethynyl)anthracene and any derivative thereof, DAP I, DiOC6, DyLight Fluors and any derivative thereof, epicocconone, e
  • TMR tetramethylrhodamine
  • coumarin and coumarin dyes e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.
  • Oregon Green Dyes e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.
  • Texas Red Texas Red-X
  • SPECTRUM RED SPECTRUM GREEN
  • cyanine dyes e.g, CY-3, Cy-5, CY-3.5, CY- 5.5, etc
  • ALEXA FLUOR dyes e.g, ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc ), BODIPY dyes
  • radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters.
  • the metal or radioisotope may be selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal may be actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope may be actinium-225 or lead-2l2. Additionally, the following radionuclides may be used for diagnosis and/or therapy: carbon (e.g., u C or 14 C), nitrogen (e.g., 13 N), fluorine (e.g., 18 F), gallium (e.g., 67 Ga or 68 Ga), copper (e.g., Cu or Cu), zirconium (e.g., Zr), lutetium (e.g., Lu).
  • carbon e.g., u C or 14 C
  • nitrogen e.g., 13 N
  • fluorine e.g., 18 F
  • gallium e.g., 67 Ga or 68 Ga
  • copper e.g., Cu or Cu
  • zirconium e.g., Zr
  • lutetium e.g
  • a delivery construct as disclosed herein can be conjugated to, coupled to, or fused to a radiosensitizer or photosensitizer.
  • radiosensitizers may include but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine).
  • photosensitizers may include but are not limited to: fluorescent molecules or beads that generate heat when
  • porphyrins and porphyrin derivatives e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines
  • metalloporphyrins
  • metallophthalocyanines angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides,
  • pyropheophorbides cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, eryth rosins, rose bengals), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5- aminolevulinic acid.
  • cyanines e.g., merocyanine 540
  • pheophytins sapphyrins,
  • this approach may allow for highly specific targeting of diseased cells (e.g., cancer cells) using both a therapeutic agent (e.g., drug) and electromagnetic energy (e.g., radiation or light) concurrently.
  • a therapeutic agent e.g., drug
  • electromagnetic energy e.g., radiation or light
  • the proteins of the present disclosure can be conjugated to, coupled to, fused with, or covalently or non-covalently coupled to the agent, e.g., directly or via a spacer.
  • a radionuclide may be attached to a carrier or delivery construct as described herein using a chelator.
  • exemplary chelator moieties may include 2,2',2"-(3-(4-(3-(l-(4-(l,2,4,5- tetrazin-3 -yl)phenyl)- 1-oco-5,8,11,14,17,20,23 -heptaoxa-2-azapentacosan-25 - yl)thioureido)benzyl)-l,4,7-triazonane-2,5,8-triyl)triacetic acid; 2,2',2"-(3-(4-(3-(l-(4-(l,2,4,5- tetrazin-3-yl)phenyl)-l-oxo-5,8,l l, l4,l7,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37- yl
  • the present disclosure provides methods and compositions for transport and/or delivery of a cargo molecule to certain location(s) within a cell (e.g., a supranuclear location) or across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a rodent or a human).
  • a cargo molecule can be directed to a set of location(s) by coupling it to a carrier molecule.
  • carrier molecule can interact with unique receptors both on the cell surface and intracellularly for the targeted delivery of the cargo.
  • carrier, cargos, and uses thereof are described herein.
  • Contemplated herein are delivery constructs that can be used to deliver a cargo to a location within a cell (e.g., epithelial cell) or across a cell (e.g., epithelial cell).
  • Such carriers can be a small molecule, a polypeptide, an aptamer, an antibody, a nucleic acid a fragment of any of the above, or a combination of any of the above.
  • the delivery constructs described herein can be used for various applications, including but not limited to, therapeutic, preventative, and/or diagnostic applications. Such therapeutic, preventative, and/or diagnostic applications can be provided if, for example, therapeutically active cargo molecules are coupled to carriers described herein that enable targeted delivery to various locations (e.g., in a subject such as a human).
  • compositions of the present disclosure relate to compositions for administration to a human subject.
  • the pharmaceutical compositions comprise the non-naturally occurring delivery constructs recited herein, alone or in combination.
  • the pharmaceutical compositions can comprise additional molecules capable of altering the characteristics of the non-naturally occurring delivery constructs, for example, stabilizing, modulating and/or activating their function.
  • the composition may, e.g., be in solid or liquid form and can be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).
  • composition of the present disclosure may, optionally and additionally, comprise a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material and any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants.
  • the pharmaceutical compositions are generally formulated appropriately for the immediate use intended for the delivery construct.
  • the delivery construct can be formulated in a composition suitable for storage.
  • a composition suitable for storage is a lyophilized preparation of the delivery construct together with a suitable stabilizer.
  • the delivery construct composition can be formulated for storage in a solution with one or more suitable stabilizers. Any such stabilizer known to one of skill in the art without limitation can be used.
  • stabilizers suitable for lyophilized preparations include, but are not limited to, sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino acids.
  • Stabilizers suitable for liquid preparations include, but are not limited to, sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino acids.
  • Specific stabilizers than can be used in the compositions include, but are not limited to, trehalose, serum albumin, phosphatidylcholine, lecithin, and arginine.
  • Other compounds, compositions, and methods for stabilizing a lyophilized or liquid preparation of the delivery constructs can be found, for example, in U.S. Pat. Nos. 6,573,237, 6,525,102, 6,391,296, 6,255,284, 6,133,229, 6,007,791, 5,997,856, and 5,917,021.
  • the pharmaceutical compositions of the present disclosure are formulated for oral delivery.
  • the pharmaceutical compositions formulated for oral administration take advantage of the bacterial toxin’s ability to mediate transcytosis across the gastrointestinal (GI) epithelium and/or delivery to the interior of a cell of the GI epithelium (e.g., gut). It is anticipated that oral administration of these pharmaceutical compositions will result in absorption of the delivery construct through polarized epithelial cells of the digestive mucosa, e.g., the intestinal mucosa, followed by release of the biologically active cargo at the basolateral side of the mucous membrane.
  • GI gastrointestinal
  • the epithelial cell is selected from the group consisting of nasal epithelial cells, oral epithelial cells, intestinal epithelial cells, rectal epithelial cells, vaginal epithelial cells, and pulmonary epithelial cells.
  • Pharmaceutical compositions of the disclosure can include the addition of a transcytosis enhancer to facilitate transfer of the fusion protein across the GI epithelium. Such enhancers are known in the art. See Xia et ah, (2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et al. (2001) Pharmaceutical Res., 18(2): 191-195, each incorporated by reference in its entirety herein.
  • the delivery constructs of the disclosure will exhibit extended half-life in serum, that is, the biologically active cargo of the delivery constructs will exhibit an extended serum half-life compared to the biologically active cargo in its non-fused state.
  • the oral formulations of the pharmaceutical compositions of the present disclosure are prepared so that they are suitable for transport to the GI epithelium and protection of the delivery construct in the stomach.
  • Such formulations can include carrier and dispersant components and can be in any suitable form, including aerosols (for oral or pulmonary delivery), syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, emulsions, cachets or pellets granulates, and dispersible powders.
  • the pharmaceutical compositions are employed in solid dosage forms, e.g., tablets, capsules, or the like, suitable for simple oral administration of precise dosages.
  • the oral formulation can comprise a delivery construct and one or more compounds that can protect the delivery construct while it is in the stomach.
  • the protective compound should be able to prevent acid and/or enzymatic hydrolysis of the delivery construct.
  • the oral formulation comprises a delivery construct and one or more compounds that can facilitate transit of the construct from the stomach to the small intestine.
  • the one or more compounds that can protect the delivery construct from degradation in the stomach can also facilitate transit of the construct from the stomach to the small intestine.
  • inclusion of sodium bicarbonate can be useful for facilitating the rapid movement of intra-gastric delivered materials from the stomach to the duodenum as described in Mrsny et al., Vaccine 17: 1425-1433, 1999.
  • compositions so that the delivery constructs can pass through the stomach and contact polarized epithelial membranes in the small intestine include, but are not limited to, enteric-coating technologies as described in DeYoung, Int J Pancreatol, 5 Suppl:3 l-6, 1989 and the methods provided in U.S. Pat. Nos. 6,613,332, 6,174,529, 6,086,918, 5,922,680, and 5,807,832, each incorporated by reference in its entirety herein.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents in order to provide a pharmaceutically elegant and palatable preparation.
  • the delivery construct is mixed with at least one
  • the tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
  • additives e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
  • DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract.
  • Compositions comprising delivery constructs can be prepared as described generally in Remington’s Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference.
  • the pharmaceutical compositions can be formulated as orally deliverable tablets containing delivery constructs in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for manufacture of tablets.
  • excipients can be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc.
  • the tablets can be uncoated or they can be coated with known techniques to delay disintegration and absorption in the gastrointestinal track and thereby provide a sustained action over a longer period of time.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax can be employed.
  • compositions can be formulated as hard gelatin capsules wherein the delivery construct is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the delivery construct is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate, or kaolin
  • an aqueous or an oil medium for example, arachis oil, peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions can contain a delivery construct in the admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose,
  • dispersing or wetting agents can be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example,
  • polyoxyethylene stearate or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecyl ethyl oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.
  • the aqueous suspensions can also contain one or more preservatives for example, ethyl or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.
  • preservatives for example, ethyl or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.
  • the pharmaceutical compositions can be in the form of oil-in-water emulsions.
  • the oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the same partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • the pharmaceutical composition can be in the form of a tablet or capsule, and the tablet or capsule can be coated or encapsulated to protect a therapeutically or biologically active cargo from enzymatic action in the stomach and to ensure that there is sufficient biologically active cargo to be absorbed by the subject to produce an effective response.
  • Such coating or encapsulation methods include, e.g., encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation in microparticles, insertion into liposomes in emulsions, and conjugation to other molecules.
  • the capsule or tablet releases the delivery construct in a pH-dependent manner.
  • Capsules or tablets used for administering a delivery construct as described herein can comprise one or more enteric coatings.
  • nanoparticles include mucoadhesive nanoparticles coated with chitosan and Carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(l):39-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutyl-vinyl alcohol) and poly(D,L- lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1): 147-160, 2000).
  • Encapsulated or coated tablets can be used that release a biologically active cargo in a layer-by-layer manner, thereby releasing biologically active cargo over a pre-determined time frame while moving along the gastrointestinal tract.
  • tablets comprising the biologically active cargo can be placed within a larger tablet, thereby protecting the inner tablet from environmental and processing conditions, such as temperature, chemical agents (e.g., solvents), pH, and moisture.
  • the outer tablet and coatings further serve to protect the
  • compositions described herein can be formulated for oral delivery using polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, Oral Delivery of Microencapsulated Proteins, in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • ETseful surface-active agents or surfactants promote absorption of polypeptides through mucosal membrane or lining.
  • ETseful surface-active agents or surfactants include fatty acids and salts thereof, bile salts, phospholipid, or an alkyl saccharide. Examples of fatty acids and salts thereof include sodium, potassium and lysine salts of caprylate (C 8 ), caprate (Ci 0 ), laurate (Ci 2 ) and myristate (Ci 4 ).
  • bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, and ursodeoxycholic acid.
  • phospholipids include single-chain phospholipids, such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine,
  • alkyl saccharides include alkyl glucosides or alkyl maltosides, such as decyl glucoside and dodecyl maltoside.
  • oral administration of the delivery constructs results in absorption of the delivery construct through polarized epithelial cells of the digestive mucosa, e.g., the intestinal mucosa, followed by cleavage of the delivery construct and release of the biologically active cargo at the basolateral side of the mucous membrane.
  • the biologically active cargo when the biologically active cargo exerts a biological activity in the liver, such as, for example, activities mediated by IL-10 binding to its cognate receptor, the biologically active cargo is believed to exert an effect in excess of what would be expected based on the plasma concentrations observed in the subject, i.e., oral administration of the delivery construct can deliver a higher effective concentration of the delivered biologically active cargo to the liver of the subject than is observed in the subject’s plasma.
  • the present disclosure relates to methods of orally administering the pharmaceutical compositions of the disclosure.
  • Such methods can include, but are not limited to, steps of orally administering the compositions by the patient or a caregiver.
  • Such administration steps can include administration on intervals such as once or twice per day depending on the delivery construct, disease or patient condition or individual patient.
  • Such methods also include the administration of various dosages of the individual delivery construct. For instance, the initial dosage of a pharmaceutical composition can be at a higher level to induce a desired effect, such as reduction in blood glucose levels. Subsequent dosages can then be decreased once a desired effect is achieved.
  • Such changes or modifications to administration protocols can be performed by the attending physician or health care worker.
  • compositions can be administered to the subject at a suitable dose.
  • the dosage regimen can be determined by the attending physician based upon specific clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient’s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgment of the ordinary clinician or physician.
  • the skilled person knows that the effective amount of a pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the biologically active cargo.
  • the length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect.
  • the particular amounts can be determined by conventional tests, which are well known to the person skilled in the art.
  • the amount of biologically active cargo is an amount effective to accomplish the purpose of the particular active agent.
  • the amount in the composition typically is a
  • the amount can be less than a pharmacologically, biologically, therapeutically, or chemically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form can contain a multiplicity of carrier/biologically or chemically active agent compositions or can contain a divided pharmacologically, biologically, therapeutically, or chemically effective amount.
  • the total effective amounts can then be administered in cumulative units containing, in total, pharmacologically, biologically, therapeutically or chemically active amounts of biologically active cargo.
  • the terms“co-administration”,“co-administered” and“in combination with”, referring to the delivery constructs of the disclosure and one or more other therapeutic agents, is intended to mean, and does refer to and include the following:
  • a combination therapy can comprise administering the isolated delivery construct composition and the second agent composition simultaneously, either in the same
  • isolated delivery construct composition and the second agent composition are administered sequentially, i.e., the isolated delivery construct composition is administered either prior to or after the administration of the second agent composition.
  • An administration of the isolated delivery construct composition and the second agent composition can be concurrent, i.e., the administration period of the isolated delivery construct composition and the second agent composition overlap with each other.
  • An administration of the isolated delivery construct composition and the second agent composition can be non-concurrent.
  • the isolated delivery construct composition and the second agent composition can be non-concurrent.
  • the second agent composition can be non-concurrent.
  • administration of the isolated delivery construct composition is terminated before the second agent composition is administered.
  • the administration second agent composition can be terminated before the isolated delivery construct composition is administered.
  • the pharmaceutical compositions formulated for oral delivery can be used to treat certain classes of diseases or medical conditions that are particularly amenable for oral formulation and delivery.
  • diseases or conditions include, e.g., viral disease or infections, cancer, a metabolic disease, obesity, autoimmune diseases, inflammatory diseases, allergy, graft-vs-host disease, systemic microbial infection, anemia, cardiovascular disease, psychosis, genetic diseases, neurodegenerative diseases, disorders of hematopoietic cells, diseases of the endocrine system or reproductive systems, gastrointestinal diseases.
  • oral formulations of the delivery constructs of the disclosure are particularly useful because they allow long-term patient care and therapy via home oral administration without reliance on injectable treatment or drug protocols.
  • a pharmaceutical composition of the present disclosure can comprise any of the delivery constructs described herein, which includes any combination of carrier, cargo, and/or spacer described herein.
  • the delivery constructs described herein allow for oral administration, which can be followed by transport of the delivery construct across or into a cell of an epithelium of a subject.
  • a delivery construct that has been transported across such an epithelial layer can subsequently reach various parts and/or organs and/or tissues within the subject.
  • a delivery construct, and in various cases the cargo that a delivery construct comprises, can elicit an effect upon reaching a submucosal compartment.
  • a biologically active cargo can be a cargo capable of eliciting an immune response, and thus a delivery construct can present such cargo to immune cell once it has reached a submucosal compartment.
  • the present disclosure relates to methods for treatment, prophylaxis and/or prevention of an inflammatory disease in a subject, comprising administering a pharmaceutical composition of the present disclosure to the subject.
  • “Inflammatory diseases” include all diseases associated with acute or chronic inflammation. Acute inflammation is the initial response of the body to harmful stimuli and results from an increased movement of plasma and leukocytes (such as e.g. granulocytes) from the blood into the injured tissues. A number of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue.
  • Prolonged inflammation is referred to as chronic inflammation, which leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
  • Inflammatory diseases can be caused by e.g. bums, chemical irritants, frostbite, toxins, infection by pathogens, physical injury, immune reactions due to hypersensitivity, ionizing radiation, or foreign bodies, such as e.g. splinters, dirt and debris. Examples of inflammatory diseases are well known in the art.
  • An inflammatory disease can be selected from the group consisting of inflammatory bowel disease, psoriasis and bacterial sepsis.
  • the term“inflammatory bowel disease”, as used herein, refers to a group of inflammatory conditions of the colon and small intestine including, for example, Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet’s syndrome and indeterminate colitis. Delivery constructs that can be used to prevent and/or treat such inflammatory disease include those comprising the amino acid sequence set forth in SEQ ID NO: 154 and/or SEQ ID NO: 155.
  • Crohn’s disease in accordance with the present disclosure, is a T-helper Type 1 (Thl) inflammatory bowel disease, which has an immune response pattern that includes an increased production of interleukin- 12, tumor necrosis factor (TNF), and interferon-g
  • Crohn’s disease Common symptoms of Crohn’s disease include diarrhea, cramping, abdominal pain, fever, and even rectal bleeding. Crohn’s disease and complications associated with it often results in the patient requiring surgery, often more than once. There is no known cure for Crohn’s disease, and long-term, effective treatment options are limited. The goals of treatment are to control inflammation, correct nutritional deficiencies, and relieve symptoms like abdominal pain, diarrhea, and rectal to bleeding. While treatment can help control the disease by lowering the number of times a person experiences a recurrence, there is no cure. Treatment can include drugs, nutrition supplements, surgery, or a combination of these options.
  • anti-inflammation drugs including sulfasalazine, cortisone or steroids, including prednisone, immune system suppressors, such as 6-mercaptopurine or azathioprine, and antibiotics.
  • Psoriasis in accordance with the present disclosure, is a disease, which affects the skin and joints. It commonly causes red scaly patches to appear on the skin. The scaly patches caused by psoriasis, called psoriatic plaques, are areas of inflammation and excessive skin production. Skin rapidly accumulates at these sites and takes a silvery-white appearance. Plaques frequently occur on the skin of the elbows and knees, but can affect any area including the scalp and genitals. Psoriasis is hypothesized to be immune-mediated and is not contagious. The disorder is a chronic recurring condition, which varies in severity from minor localized patches to complete body coverage.
  • Psoriatic nail dystrophy psoriatic nail dystrophy
  • Psoriatic arthritis Ten to fifteen percent of people with psoriasis have psoriatic arthritis.
  • bacterial sepsis refers to life-threatening conditions resulting from the circulation of bacteria in the blood stream. Sepsis results in generalized systemic production of pro-inflammatory cytokines that results in tissue damage and ultimately septic shock due to failure of the microcirculation.
  • the present disclosure relates to methods for treatment, prophylaxis and/or prevention of an autoimmune disease in a subject, comprising administering a pharmaceutical composition of the present disclosure to the subject.
  • An autoimmune disease as pertains to the present disclosure, is a disease or disorder arising from and directed against an individual’s own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,
  • thrombocytopenia purpura Grave’s disease, Sjogren’s disease, dermatomyositis, Hashimoto’s disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis, and scleroderma.
  • Exemplary delivery constructs that can be used to treat those disease can include those comprising any carrier set forth in SEQ ID NO: 4 - SEQ ID NO: 125 coupled to, for example, an anti-TNFa antibody, or a functional binding fragment thereof.
  • Rheumatoid arthritis is a chronic, systemic inflammatory disorder that can affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium (synovitis) secondary to hyperplasia of synovial cells, excess synovial fluid, and the development of pannus in the synovium. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. [0414] The present disclosure relates to methods and compositions for treatment, prophylaxis and/or prevention of a cancer in a subject, comprising administering a
  • Cancers to be treated include, but are not limited to, non-Hodgkin’s lymphomas, Hodgkin’s lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the pancreas, colon, gastric intestine, prostate, bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignant melanoma and rituximab resistant NHL and leukemia.
  • the therapeutically effective amount of a pharmaceutical composition of the present disclosure will be administered in combination with one or more other therapeutic agents.
  • therapeutic agents can be accepted in the art as a standard treatment for a particular disease state as described herein, such as inflammatory disease, autoimmune disease, or cancer.
  • exemplary therapeutic agents contemplated include, but are not limited to, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, or other active and ancillary agents.
  • the present disclosure relates to methods for treatment, prophylaxis and/or prevention of a metabolic disorder in a subject, comprising administering a pharmaceutical composition of the present disclosure to the subject.
  • the metabolic disorder is selected from the group consisting of: diabetes, obesity, diabetes as a consequence of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, and hyperlipidemia.
  • the present disclosure relates to methods for treatment, prophylaxis and/or prevention of a fatty liver disease (e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH)), a gastrointestinal disease, or a neurodegenerative disease in a subject, comprising administering a pharmaceutical composition of the present disclosure to the subject.
  • a fatty liver disease e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH)
  • NASH nonalcoholic steatohepatitis
  • the present disclosure relates to methods and compositions for treatment, prophylaxis and/or prevention of a GH deficient growth disorder in a subject, said method comprising administering a pharmaceutical composition of the present disclosure to the subject.
  • the disorder is selected from the group consisting of: growth hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome, Prader-Willi syndrome, short stature homeobox-containing gene (SHOX) deficiency, chronic renal insufficiency, and idiopathic short stature short bowel syndrome, GH deficiency due to rare pituitary tumors or their treatment, and muscle-wasting disease associated with HIV/AIDS.
  • Diagnoses as described herein can further comprise monitoring a response to a treatment (e.g., the treatment of a subject). For example, if response to a treatment correlates with a reduction of a certain marker (e.g., a biomarker), the delivery constructs of the present disclosure can be used to measure such marker at a certain location (e.g., a certain immune cell population in a submucosal compartment).
  • a certain marker e.g., a biomarker
  • the methods and compositions described herein can be used to provide biologically and/or therapeutically relevant information, e.g., upon a biopsy sample has been taken from a subject, which can be followed by
  • immunohistochemistry e.g., the detection of accumulation of a delivery constructs in a certain tissue etc.
  • Non-invasive diagnosis can comprise molecular and/or nuclear imaging.
  • a delivery construct can comprise cargo that is labeled with a fluorescent and/or radioactive compound such that the location and/or concentration of such a delivery construct can be determined in a subject after administration. Any moiety with diagnostic applicability as described herein can be used to provide diagnostic and/or theranostic (therapeutic and diagnostic) agents.
  • the expression system can comprise a polynucleotide sequence that encodes a cleavable spacer so that cleavage at the cleavable spacer separates a biologically active cargo encoded by a nucleic acid inserted into the polyspacer insertion site from the remainder of the encoded delivery construct.
  • the polynucleotide comprises one nucleotide sequence encoding a cleavable spacer between the polyspacer insertion site and the remainder of the polynucleotide.
  • the polyspacer insertion site can be flanked by nucleotide sequences that each encode a cleavable spacer.
  • Various in vitro methods that can be used to prepare a polynucleotide encoding a delivery construct useful in the delivery constructs of the disclosure include, but are not limited to, reverse transcription, the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR) and the QP replicase amplification system (QB). Any such technique known by one of skill in the art to be useful in construction of recombinant nucleic acids can be used.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • SR self-sustained sequence replication system
  • QB QP replicase amplification system
  • a polynucleotide encoding the protein or a portion thereof can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of a delivery construct or a nucleotide encoding the receptor binding domain.
  • Polynucleotides encoding a delivery construct, or a portion thereof also can be isolated by screening genomic of cDNA libraries using probes selected from the sequences of the desired polynucleotide under stringent, moderately stringent, or highly stringent hybridization conditions.
  • the polynucleotides can also encode a secretory sequence at the amino terminus of the encoded delivery construct. Such constructs are useful for producing the delivery constructs in mammalian cells as they simplify isolation of the delivery construct and/or hybrid delivery construct polypeptides.
  • polynucleotides of the disclosure also encompass derivative versions of polynucleotides encoding a delivery construct.
  • derivatives can be made by any method known by one of skill in the art without limitation.
  • derivatives can be made by site-specific mutagenesis, including substitution, insertion, or deletion of one, two, three, five, ten or more nucleotides, of polynucleotides encoding the delivery construct.
  • derivatives can be made by random mutagenesis.
  • One method for randomly mutagenizing a nucleic acid comprises amplifying the nucleic acid in a PCR reaction in the presence of 0.1 mM MnCl 2 and unbalanced nucleotide concentrations. These conditions increase the inaccuracy incorporation rate of the polymerase used in the PCR reaction and result in random mutagenesis of the amplified nucleic acid.
  • a delivery construct comprises a bacterial carrier and a biologically active cargo to be delivered to a subject; and, optionally, a non-cleavable or cleavable spacer. Cleavage at the cleavable spacer can separate the biologically active cargo from the remainder of the delivery construct.
  • the cleavable spacer can be cleaved by an enzyme that is present at a basolateral membrane of a polarized epithelial cell of the subject or in the plasma of the subject.
  • the polynucleotide can hybridize under stringent hybridization conditions to any polynucleotide of this disclosure.
  • the polynucleotide can hybridize under stringent conditions to a nucleic acid that encodes any delivery construct of the disclosure.
  • expression vectors for expressing the delivery constructs and/or hybrid delivery construct polypeptides.
  • expression vectors are recombinant polynucleotide molecules comprising expression control sequences operatively linked to a nucleotide sequence encoding a polypeptide.
  • Expression vectors can readily be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, selectable markers, etc. to result in stable transcription and translation or mRNA. Techniques for construction of expression vectors and expression of genes in cells comprising the expression vectors are well known in the art.
  • Useful promoters for use in expression vectors include, but are not limited to, a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone- inducible MMTV promoter, a SV40 promoter, a MRP pol III promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), and a constitutive CMV promoter.
  • the expression vectors should contain expression and replication signals compatible with the cell in which the delivery constructs are expressed.
  • Expression vectors useful for expressing delivery constructs include viral vectors such as retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, and the like. Viral and plasmid vectors are preferred for transfecting the expression vectors into mammalian cells.
  • the expression vector pcDNAl Invitrogen, San Diego, Calif.
  • the expression control sequence comprises the CMV promoter
  • the expression vectors can be introduced into the cell for expression of the delivery constructs by any method known to one of skill in the art without limitation. Such methods include, but are not limited to, e.g., direct uptake of the molecule by a cell from solution;
  • the expression vectors can also contain a purification moiety that simplifies isolation of the delivery construct and/or hybrid delivery construct polypeptides.
  • a polyhistidine moiety of, e.g., six histidine residues, can be incorporated at the amino terminal end of the protein.
  • the polyhistidine moiety allows convenient isolation of the protein in a single step by nickel-chelate chromatography.
  • the purification moiety can be cleaved from the remainder of the delivery construct following purification.
  • the moiety does not interfere with the function of the functional domains of the delivery construct and thus need not be cleaved.
  • the present disclosure provides a cell that can comprise an expression vector for expression of the delivery constructs and/or hybrid delivery construct polypeptides, or portions thereof.
  • the cell can be selected for its ability to express high concentrations of the delivery construct to facilitate purification of the protein.
  • the cell is a prokaryotic cell, for example, E. coli.
  • the delivery constructs are properly folded and comprise the appropriate disulfide linkages when expressed in E. coli.
  • the cell is a eukaryotic cell. ETseful eukaryotic cells include yeast and mammalian cells.
  • any mammalian cell known by one of skill in the art to be useful for expressing a recombinant polypeptide can be used to express the delivery constructs.
  • CHO Chinese hamster ovary
  • the delivery constructs and/or hybrid delivery construct polypeptides of the disclosure can be produced by recombination, as described below. However, the delivery constructs can also be produced by chemical synthesis using methods known to those of skill in the art.
  • the delivery constructs of the present disclosure can be produced using a variety of methods. The selection of a production method can depend on the molecular structure of the delivery construct and/or its components (e.g., the carrier, cargo, and/or spacer). Thus, for some delivery constructs organic synthetic methods may be advantageous for producing such delivery construct.
  • a delivery construct of the present disclosure can be a polypeptide. Such polypeptides can be produced, for example, using recombinant DNA methodology. Generally, this involves creating a DNA sequence that encodes the delivery construct, placing the DNA in an expression cassette under the control of a particular promoter, expressing the molecule in a host, isolating the expressed molecule and, if required, folding of the molecule into an active conformational form.
  • DNA encoding the delivery constructs described herein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solid support method of ET.S. Pat. No. 4,458,066, and the like.
  • Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences.
  • subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.
  • a DNA encoding a delivery constructs of the present disclosure can be cloned using DNA amplification methods such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the gene for the biologically active cargo is PCR amplified, using a sense primer containing the restriction site for, e.g., Ndel and an antisense primer containing the restriction site for Hindlll. This can produce a nucleic acid encoding the biologically active cargo sequence and having terminal restriction sites.
  • a delivery construct having“complementary” restriction sites can similarly be cloned and then ligated to the biologically active cargo and/or to a spacer attached to the biologically active cargo. Ligation of the nucleic acid sequences and insertion into a vector produces a vector encoding the biologically active cargo joined to the bacterial carrier receptor binding domain.
  • DNA encoding delivery constructs of the present disclosure is artificially synthesized by, for example, solid-phase DNA synthesis.
  • a“Cholix” also referred to herein as Cholix toxin or Cholix exotoxin
  • a“Cholix” can encompass a variety of functional variants (e.g., a functional genus), wherein the functional variants can comprise one or more variations is their amino acid sequence relative to SEQ ID NO: 1 as disclosed herein.
  • the Cholix toxin having the amino acid sequence set forth in SEQ ID NO: 1 is used as the reference sequence when referred to Cholix.
  • a variant of the Cholix exotoxin with the amino acid sequence set forth in SEQ ID NI: 1 can be a Cholix exotoxin which amino acid sequence is set forth in SEQ ID NO: 2, wherein both variants are capable of carrying out the same functions, e.g., transcytosis across an epithelial cell, and interact with the same receptors, such as ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan.
  • a polypeptide can affect, to some degree, the amino acid sequence of such polypeptide (e.g., due to post-translational modifications).
  • a first carrier and a second carrier are produced in the same expression system (e.g., a bacterial expression system such as E. coli or a mammalian expression system such as a CHO cell).
  • a first carrier and a second carrier are produced in a different expression system (e.g., a bacterial or a mammalian expression system).
  • Bacterial expression systems include E. coli
  • mammalian expression systems include CHO cells, for example.
  • a bacterially produced polypeptide can comprise an N-cap, wherein the N-cap can comprise one more modifications at the N-terminal of the polypeptide.
  • An N-cap can comprise an N-terminal methionine residue.
  • Examples of Cholix domain I derived carrier polypeptides that can be bacterially produced and that comprise such N-terminal methionine include those comprising the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO: 135.
  • transcytosis Testing The transcytosis function of the isolated delivery constructs can be tested as a function of the delivery construct’s ability to pass through an epithelial membrane. Because transcytosis first requires binding to the epithelial cell, these assays can also be used to assess the function of the delivery construct of the delivery construct.
  • transcytosis activity can be tested by any method known by one of skill in the art, without limitation.
  • transcytosis activity can be tested by assessing the ability of a delivery construct to enter a non-polarized cell to which it binds.
  • Cholix derived carrier and without intending to be bound to any particular theory or mechanism of action, it is described herein that the transcytosis function that allows a delivery construct to pass through a polarized epithelial cell and the function to enter non polarized cells resides in the same domain, i.e. the domain described herein as domain I.
  • the delivery construct’s ability to enter the cell can be assessed, for example, by detecting the physical presence of the construct in the interior of the cell.
  • the delivery construct can be labeled with, for example, a fluorescent marker, and the delivery construct exposed to the cell. Then, the cells can be washed, removing any delivery construct that has not entered the cell, and the amount of label remaining determined. Detecting the label in this traction indicates that the delivery construct has entered the cell.
  • the delivery construct’s transcytosis ability can be tested by assessing the delivery construct’s ability to pass through a polarized epithelial cell.
  • the delivery construct can be labeled with, for example, a fluorescent marker (e.g., RFP) and contacted to the apical membranes of a layer of epithelial cells. Fluorescence detected on the basolateral side of the membrane formed by the epithelial cells indicates that the transcytosis domain is functioning properly.
  • a fluorescent marker e.g., RFP
  • In vivo transcytosis can be tested using male Wistar rats.
  • Male Wistar rats can be housed 3-5 per cage with a 12/12 h light/dark cycle and can be 225-275 g (approximately 6-8 weeks old) when placed on study. Experiments can be conducted during the light phase using a non-recovery protocol that uses continuous isoflurane anesthesia. A 4-5 cm midline abdominal incision that exposes mid-jejunum regions can be conducted.
  • Stock solutions at 3.86xl0 5 M of test articles can be prepared in phosphate buffered saline (PBS), with 50 pL (per 250 g rat) being administered by intraluminal injection (ILI) using a 29-gauge needle.
  • PBS phosphate buffered saline
  • ILI intraluminal injection
  • the injection site mesentery can then be marked with a permanent marker.
  • a 3-5 mm region that captured the marked intestine segment can be isolated and processed for microscopic assessment.
  • In vivo experiments are performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986, the European Communities Council Directive of 1986 (86/609/EEC), and the ETniversity of Bath’s ethical review procedures.
  • Cleavable Spacer Cleavage Testing The function of the cleavable spacer can generally be tested in a cleavage assay. Any suitable cleavage assay known by one of skill in the art, without limitation, can be used to test the cleavable spacers. Both cell-based and cell-free assays can be used to test the ability of an enzyme to cleave the cleavable spacers.
  • An exemplary cell-free assay for testing cleavage of cleavable spacers comprises preparing extracts of polarized epithelial cells and exposing a labeled delivery construct bearing a cleavable spacer to the fraction of the extract that corresponds to membrane-associated enzymes.
  • the label can be attached to either the biologically active cargo to be delivered or to the remainder of the delivery construct.
  • these enzymes are cleavage enzymes found near the basolateral membrane of a polarized epithelial cell, as described above. Cleavage can be detected, for example, by binding the delivery construct with, for example, an antibody and washing off unbound molecules.
  • the binding agent used in the assay can be specific for the biologically active cargo, and the remainder of the construct can be labeled. In either case, cleavage can be assessed.
  • Cleavage can also be tested using cell-based assays that test cleavage by polarized epithelial cells assembled on semi-permeable membranes.
  • a labeled delivery construct, or portion of a delivery construct comprising the cleavable spacer can be contacted to either the apical or basolateral side of a monolayer of suitable epithelial cells, such as, for example, Caco-2 cells, under conditions that permit cleavage of the spacer.
  • Cleavage can be detected by detecting the presence or absence of the label using a reagent that specifically binds the delivery construct, or portion thereof.
  • an antibody specific for the delivery construct can be used to bind a delivery construct comprising a label distal to the cleavable spacer in relation to the portion of the delivery construct bound by the antibody. Cleavage can then be assessed by detecting the presence of the label on molecules bound to the antibody. If cleavage has occurred, little or no label should be observed on the molecules bound to the antibody.
  • enzymes that preferentially cleave at the basolateral membrane rather than the apical membrane can be identified, and, further, the ability of such enzymes to cleave the cleavable spacer in a delivery construct can be confirmed.
  • cleavage can also be tested using a fluorescence reporter assay as described in Ei.S. Pat. No. 6,759,207. Briefly, in such assays, the fluorescence reporter is contacted to the basolateral side of a monolayer of suitable epithelial cells under conditions that allow the cleaving enzyme to cleave the reporter. Cleavage of the reporter changes the structure of the fluorescence reporter, changing it from a non-fluorescent configuration to a fluorescent configuration. The amount of fluorescence observed indicates the activity of the cleaving enzyme present at the basolateral membrane.
  • cleavage can also be tested using an intra-molecularly quenched molecular probe, such as those described in U.S. Pat. No. 6,592,847.
  • probes generally comprise a fluorescent moiety that emits photons when excited with light of appropriate wavelength and a quencher moiety that absorbs such photons when in close proximity to the fluorescent moiety. Cleavage of the probe separates the quenching moiety from the fluorescent moiety, such that fluorescence can be detected, thereby indicating that cleavage has occurred.
  • probes can be used to identify and assess cleavage by particular cleaving enzymes by contacting the basolateral side of a monolayer of suitable epithelial cells with the probe under conditions that allow the cleaving enzyme to cleave the probe. The amount of fluorescence observed indicates the activity of the cleaving enzyme being tested.
  • Block tissue sections by pipetting 2% BSA and 2% donkey serum in 0.1% Triton x- 100 in PBS onto sections. Incubate for 2 hours at room temperature. Remove blocking solution and add primary antibodies. Dilute antibodies to required concentration in 0.05% Triton c-100 and 1% BSA in PBS. Incubate overnight at 4°C. Wash 3 x 5 minutes with PBS. Incubate with secondary antibodies. Dilute antibodies to required concentration in 0.05% Triton c-100 and 1% BSA in PBS. Incubate for 2 hours at room temperature. Wash 3 x 5 minutes with PBS. Incubate with 200 nM DAPI at room temperature for 45 minutes. Wash 3 x 5 minutes with PBS.
  • tissue sections by immersing in 70% ethanol, 100% ethanol, histoclear and 100% ethanol for 5 minutes each. Place a drop of fluorshield mounting media on each tissue section and cover with a glass coverslip. Gently apply pressure to the coverslip to remove air bubbles. Allow mounting media to dry for 4 hours. Store slides at 4°C and image using confocal fluorescent microscope.
  • Cholix Domain I Interacting Proteins In order to identify Cholix and/or PE interacting partners (e.g., receptors, enzymes, etc.) and establish the vesicular compartments where they interact with Cholix or PE exotoxins (e.g., a domain I of those exotoxins or a truncated version thereof), a series of pull-downs can be performed to identify potential interaction partners that can be followed by in silico associations using surface plasmon resonance, in vitro transcytosis studies using polarized Caco-2 human intestinal epithelial cells where genetic knockdown of specific targets can be achieved, and in vivo transcytosis studies where Cholix elements and specific receptors can be co-localized in established vesicular structures.
  • Cholix and/or PE interacting partners e.g., receptors, enzymes, etc.
  • Cholix or PE exotoxins e.g., a domain I of those exotoxins or a truncated version thereof
  • a transcytosis process can involve elements that are normally restricted within specific vesicular elements of polarized intestinal epithelial cells but can be recruited or“hijacked” by, e.g., Cholix domain I or truncated versions thereof, to leave the late endosome and avoid lysosomal degradation following apical receptor-mediated endocytosis.
  • This Example describes the exemplary preparation of delivery constructs comprising truncated Cholix carriers (truncation in domains II and/or lb) and truncated PE carriers
  • modified Cholix and/or PE carriers were prepared and used to prepare Constructs 1-12: 1) a modified Cholix carrier truncated at amino acid residue 425 of SEQ ID NO: 1 (Cholix 425 , SEQ ID NO: 129); 2) a modified Cholix carrier truncated at amino acid residue 415 of SEQ ID NO: 1 (Cholix 415 , SEQ ID NO: 130); 3) a modified Cholix carrier truncated at amino acid residue 397 of SEQ ID NO: 1 (Cholix 397 , SEQ ID NO: 131); 4) a modified Cholix carrier truncated at amino acid residue 386 of SEQ ID NO: 1 (Cholix 386 , SEQ ID NO: 132); 5) a modified Cholix carrier truncated at amino acid residue 291 of SEQ ID NO: 1 (Cholix 291 , SEQ ID NO: 133); 6) a modified Cholix carrier truncated at amino acid residue 265 of SEQ
  • the polyglycine-serine peptide spacer GTGGS (SEQ ID NO: 201) was used to conjoin red fluorescent protein (RFP, SEQ ID NO: 220) at the C-terminus of each modified toxin.
  • RFP emulated the presence of a biologically active cargo.
  • Codon-optimized genes were obtained from a commercial source and cloned into the pET26(+) expression vector that was used to transform BL2l(DE3) component E. coli cells using the manufacturer’s suggested protocol. Clones were selected using Kanamycin/Agar plates incubate overnight at 37°C. Protein expression in fermented cultures of selected clones was achieved by 1 mM IPTG induction. Pelleted bacteria were lysed to collect inclusion bodies that were extensively washed in 50 mM Tris, 20 mM EDTA, 2.5 % Triton X-100, 0.5 M NaCl, pH 8 prior to solubilization facilitated by sonication in 100 mM Tris, pH 8, 7 M Guanidine HC1.
  • proteins in the supernatant were refolded using a shuffle buffer containing 100 mM Tris, pH 8, 0.5 M L- Arginine, 1 M Urea, 2 mM EDTA, 1 mM oxidized glutathione (fresh made), and 1 mM Reduced glutathione (fresh made) that was dialyzed at 4°C against 25 mM Tris, pH 8, 0.1 M Urea, and 1 mM EDTA.
  • desired proteins were purified using ion exchange and size exclusion chromatography. Final protein samples were analyzed by SDS-polyacrylamide gel electrophoresis and stored at -80°C.
  • This Example describes an exemplary in vivo study to evaluate epithelial trafficking and delivery functions of the exotoxin derived carrier molecules described herein.
  • the mesentery adjacent to the site of injection was labeled with a marker and the intestine was returned to the abdominal cavity, with the incision being closed with clamps At specific time points, the injected intestine was retrieved, surgically isolated and flushed with a 4°C isotonic PBS solution.
  • Fluorescent secondary antibodies were diluted in 1% BSA, 0.1% Triton-XlOO in PBS and incubated for 2 hours at room temperature prior to processing for confocal microscopy. On occasion, an approximately 1 cm section of intestine at the injection site was collected for biochemical studies.
  • This Example shows an exemplary list of proteins or markers for specific cell compartments that were analyzed using immunohistochemical (IHC) staining and immuno- fluorescence confocal microscopy and when evaluating the delivery constructs of the present disclosure.
  • IHC immunohistochemical
  • This example describes trans-epithelial transport of delivery constructs comprising truncated Cholix carriers (e.g., truncation in domains II and/or lb) and truncated PE carriers (e.g., truncation in domain II and/or lb) conjoined to biologically active cargos across polarized intestinal epithelium occurred (e.g., via apical-to-basolateral transcytosis).
  • truncated Cholix carriers e.g., truncation in domains II and/or lb
  • PE carriers e.g., truncation in domain II and/or lb
  • Construct 6 (Cholix 265 -RFP), which comprises only domain I of Cholix (SEQ ID NO: 4) was evaluated.
  • Immunofluorescence assessment of Cholix 265 -RFP e.g., amino acid of SEQ ID NO: 4 or 5 coupled to SEQ ID NO: 220
  • Transport across rat small intestine in vivo produced a similar outcome as observed with PE Construct 12.
  • Evaluation using an antibody to both the Cholix and the RFP component was used to demonstrate that both elements of the chimera were being transported (see e.g., FIG. 3).
  • delivery constructs comprising the amino acid sequences set forth in SEQ ID NO: 146 (Construct 13) and SEQ ID NO: 146 (Construct 14) were prepared, and evaluated as described above in EXAMPLE 2.
  • the chimeric Cholix-PE constructs of this example comprise mixed domains I, II, lb, and III from either the Cholix exotoxin or the PE exotoxin as described herein.
  • the chimeric carrier construct 13 (SEQ ID NO: 146) comprises a Cholix domain I derived from the sequence set forth in SEQ ID NO: 1 (amino acid residues 1-265 of SEQ ID NO: 2), a PE translocation domain II derived from the sequence set forth in SEQ ID NO: 138, a PE domain lb derived from the sequence set forth in SEQ ID NO: 139, and a non-toxic PE catalytic domain III derived from the sequence set forth in SEQ ID NO: 140.
  • the chimeric carrier construct 14 (SEQ ID NO: 147) comprises a PE domain I derived from the sequence set forth in SEQ ID NO: 137, a Cholix carrier translocation domain II derived from the sequence set forth in SEQ ID NO: 126, a Cholix carrier domain lb derived from the sequence set forth in SEQ ID NO: 127, and a non-toxic Cholix carrier catalytic domain III derived from the sequence set forth in SEQ ID NO: 128.
  • the chimeric carrier constructs are capable of transport across rat jejunum and target cells in the lamina intestinal in vivo.
  • the chimeric delivery constructs comprising portions or domains from two or more different exotoxins (e.g., Cholix and PE) can efficiently deliver cargo into and across epithelial cells.
  • exotoxins e.g., Cholix and PE
  • This example demonstrates the transcytosis function of truncated Cholix derived carrier polypeptides, wherein, importantly, the truncation occurred at various locations within the domain I of the Cholix exotoxin.
  • the non-cleavable polyglycine-serine peptide spacer GGGGSGGGGSGGGGS was used to couple human growth hormone (HGH) (SEQ ID NO: 214) to the C- terminus of various modified Cholix carrier polypeptides to prepare the following delivery constructs for evaluation according to the protein production procedure described in EXAMPLE 1 above (in this bacterially produced): 1) SEQ ID NO: 160, which comprises a modified Cholix carrier truncated at amino acid residue 187 of SEQ ID NO: 5; 2) SEQ ID NO: 159, which comprises a modified Cholix carrier truncated at amino acid residue 151 of SEQ ID NO: 5; 3) SEQ ID NO: 158, which comprises a modified Cholix carrier truncated at amino acid residue 134 of SEQ ID NO: 5; 4) SEQ ID NO: 161, which comprises a modified Cholix carrier truncated at amino acid residue 206 of SEQ ID NO: 5; 5) SEQ ID NO: 160, which comprises a modified Cho
  • constructs were injected into the lumen of the small intestine of rats and the injection site was collected 5, 10 or 15 minutes after injection. The tissue was fixed and sectioned then stained with anti-Cholix and anti-HGH antibodies. Fluorescent secondary antibodies were used to visualize the protein location using confocal microscopy. As depicted in FIG. 8A-FIG. 8C, the construct comprising the amino acid sequence of SEQ ID NO: 165 was visualized in the epithelial cells and limited to an area near the membrane in the apical side of the cells at 5, 10 and 15 minutes. There was no significant movement of the protein through the cells away from this compartment and no protein appearing in the lamina limbal.
  • polypeptide carrier with the amino acid sequence of SEQ ID NO: 165 is sufficient for uptake into the epithelial cells, e.g., via its receptor binding site and/or via interactions with TMEM132 and/or LRP1, but lacks the part of the sequence for transcytosis, resulting in accumulation in the epithelial cells.
  • constructs comprising the amino acid sequences of SEQ ID NO: 161 - SEQ ID NO: 164 were shown to rapidly move across the epithelial cells following uptake and transport out of the cells into the lamina intestinal.
  • the Cholix derived construct with SEQ ID NO: 160 was also visualized in the apical compartment of the epithelial cells at 5 minutes. At 10 and 15 minutes, small amounts of the protein moved to the basal side of the cell, but did not appear in the lamina limbal.
  • This example demonstrates apical-to-basolateral transcytosis of a modified, non toxic Cholix (ntChx) across polarized intestinal epithelial cells in vitro.
  • the Cholix construct was rendered non-toxic through an amino acid variation of a specific glutamic acid residue (substituted with alanine) within the enzymatic pocket for ADP-ribosylation, resulting in the E581 A substitution and a polypeptide with the amino acid sequence set forth in SEQ ID NO: 3 (ntChx).
  • ntChx did not appear to be significantly modified (e.g., chemically modified) in its apparent size when assessed by Western blot analysis (FIG. 10B).
  • ntChx transported via an energy-requiring, highly-efficient, receptor-mediated transcytosis process.
  • apical surface receptors e.g., low density lipoprotein receptor-related protein 1 (LRP1)
  • LRP1 low density lipoprotein receptor- related protein 1
  • the apical membrane surface pH of the small intestinal epithelium can be between 5 and 7 (17). transcytosis across primary human intestinal epithelium in vitro tested for 20 pg/mL and examined after 120 min was observed to be approximately twice as efficient when the apical media was pH 7 compared to pH 5, while the basal pH of 7 or 5 did not seem to have an effect
  • ntChx as disclosed herein can be capable of efficient, consistent, and continuous transport across human intestinal epithelium through a receptor-mediated process that can not result in significant size modification to the transported protein.
  • This example demonstrates apical-to-basolateral transcytosis of ntChx (SEQ ID NO: 3) across polarized intestinal epithelial cells in vivo , examining the ability of ntChx to transport across an intestinal epithelium in vivo by direct intra-luminal injection (ILI) into rat jejunum.
  • ILI intra-luminal injection
  • ntChx identified using an anti- Cholix polyclonal antisera, entered into epithelial cells rapidly and trafficked through these epithelial cells in vesicular-like structures that involved clatherin co-localizations, with the transcytosis process being completed within minutes (FIG. 12A-FIG. 12C).
  • Time-dependent changes in the location of vesicle-like structures positive for ntChx demonstrated its distribution within compartments localized above and below the enterocyte nucleus as well as within a selected population of cells within the lamina limbal. Transcytosis of ntChx appeared to also occur through goblet cells.
  • This example demonstrates apical-to-basolateral transcytosis of ntChx (SEQ ID NO: 3) that involves accessing specific vesicular compartments (e.g., FIG. 13A-FIG. 13F).
  • Transcytosis of ntChx can involve receptor-mediated endocytosis processes and subsequent vesicular trafficking that avoids the typical fate for ligands internalized by this route of lysosomal degradation.
  • a number of obligate and facilitative intracellular pathogens are capable of subverting host cells endocytic and secretory pathways that restrict their exposure to the lysosomal through effector proteins capable or manipulating host cells processes.
  • This example examined ntChx-containing vesicles undergoing transcytosis to determine the identity of associated proteins that can define the trafficking elements and cellular compartments that can be involved in transcytosis of Chx and Chx variants as disclosed herein.
  • the trans-Golgi network can be considered to function as a secretory pathway sorting station, directing newly synthesized proteins to different cellular compartments, with TGN-38 being identified as indicative.
  • TGN-38 was not observed to co-localize with TGN-38 in enterocytes in this experiment, but did co-localize after transcytosis in non polarized cells with the lamina propria (FIG. 13C).
  • Calnexin is a lectin chaperone located in the endoplasmic reticulum (ER) that can be involved in regulating the free cytosolic Ca 2+
  • ntChx can co-localize with calnexin primarily in the apical compartment of enterocytes but with distributions that included the basal compartment (FIG. 13D).
  • Ras-related protein Rab 7 a marker late endosomes and vesicles being trafficked to lysosomes, was observed to co-localized in both the apical and basal portions of enterocytes with ntChx (FIG. 13E).
  • LAMP1 lysosome-associated membrane protein 1
  • LAMP1 lysosome-associated membrane protein 1
  • ntChx -NPs Apical application of ntChx -NPs resulted in a transcytosis process consistent with that observed for ntChx alone and without co-localization with LAMP 1 -positive structures (FIG. 13F). Once across the intestinal epithelial cell layer, ntChx -NPs was observed in LAMP1- positive structures within cells present within the lamina propria (FIG. 13C).
  • This example demonstrates the in vivo transport of Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 4) truncated carrier proteins as described in EXAMPLE 6 compared to non-toxic full-length Cholix as described in EXAMPLE 7.
  • Cholix domain I e.g., SEQ ID NO: 4 or SEQ ID NO: 4
  • ntChx SEQ ID NO: 3
  • Transcytosis of ntChx was monitored by Western blot and immunofluorescence microscopy using a polyclonal antibody raised against the full-length protein.
  • this polyclonal antibody can provide equivalent coverage across all regions of ntChx for the purpose of detecting truncated forms of the exotoxin
  • the capacity for a truncated form of Cholix to ferry a protein cargo that could be used to assess transcytosis equivalence was investigated.
  • ntChx full-length protein
  • RFP red fluorescent protein
  • This example demonstrates the in vitro apical-basolateral transcytosis and intracellular delivery functions of various truncated Cholix domain I carrier proteins conjugated to human growth hormone via a spacer as described above in EXAMPLE 6.
  • Transcytosis of SEQ ID NO: 161 and SEQ ID NO: 162 were comparable to that of SEQ ID NO: 164 at the 2 h time point of assessment in this in vitro model of human small intestine (FIG. 15B).
  • the delivery construct with the amino acid set forth in SEQ ID NO: 161 was superior in its transport capacity compared to SEQ ID NO: 164 as demonstrated by the higher relative signal of SEQ ID NO: 161 compared to SEQ ID NO: 164.
  • domain I of Cholix is sufficient for apical-to-basal transport, and that it can function as a transcytosis element to deliver various heterologous cargos across epithelial cells, wherein the heterologous cargo may replace the domains II, lb, and III of the Cholix exotoxin.
  • elements within the first 206 amino acid residues of the bacterially expressed Cholix protein e.g., SEQ ID NO: 5, or, alternatively the first 205 amino acid residues of SEQ ID NO: 4
  • the results suggest that the transcytosis efficiency of these first 206 amino acids of SEQ ID NO: 5 may even be greater than that of the entire domain I (e.g., SEQ ID NO:
  • the herein disclosed truncated Cholix domain I constructs can be used to efficiently shuttle heterologous cargo molecules such as therapeutic and/or diagnostic agents across an epithelial cell layer (e.g., the gut epithelium) enabling oral administration of therapeutic and/or diagnostic agents (e.g., larger polypeptides or proteins such as antibodies) that are otherwise administered via parenteral administration routes (e.g., intravenously of subcutaneously).
  • therapeutic and/or diagnostic agents e.g., larger polypeptides or proteins such as antibodies
  • parenteral administration routes e.g., intravenously of subcutaneously.
  • these results show that truncated versions of Cholix domain I may be used to deliver various heterologous cargo into epithelial cells using the delivery constructs as described herein.
  • This example demonstrates the in vivo transport of a Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 5) truncated protein chimeras across gut epithelial cells for delivery of heterologous cargo (in this example: human growth hormone).
  • a Cholix domain I e.g., SEQ ID NO: 4 or SEQ ID NO: 5
  • heterologous cargo in this example: human growth hormone
  • the truncation mutant M+Cholix 1 186 -(SEQ ID NO: lO)-HGH resulted in similar outcomes as observed for the construct with SEQ ID NO: 159 with one difference that the construct with SEQ ID NO: 160 appeared to access a supra-nuclear vesicular compartment that was not accessed by the construct with SEQ ID NO: 159 (FIG. 16C).
  • Cholix domain I and truncated version thereof e.g., those comprising the first 206 amino acid residues of Cholix domain I (SEQ ID NO: 5), can efficiently deliver various cargo across epithelial cells (e.g., across polarized gut epithelial cells of a subject).
  • truncated versions of Cholix domain I may be used to deliver various heterologous cargo into epithelial cells using the delivery constructs as described herein.
  • FIG. 17A depicts some functional amino acid sequences within Cholix Domain I as colored sequence fragments.
  • the peptide 134ELDQQRNIIEVPKLYSID151 was the element that differed between M+Cholix 1 150 -HGH (amino acid sequence set forth in SEQ ID NO: 159) and M+Cholix 1 133 -HGH (amino acid sequence set forth in SEQ ID NO: 158), one chimera that could undergo endocytosis and one that could not.
  • Another peptide of interest is within the first 39 amino acids (1MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLD39, SEQ ID NO: 149), which was lacking in the M+Cholix 39' 186 construct (SEQ ID NO: 165) that lacked the ability to traffic from the apical portion of the epithelial cell to the basal domain following endocytosis.
  • the peptide that provided the difference between M+Cholix 1 150 -(SEQ ID NO: 2lO)-HGH and M+Cholix 1 186 -(SEQ ID NO: 2lO)-HGH was
  • M+Cholix 1 150 -(SEQ ID NO: 2lO)-HGH showed the ability to access a supra-nuclear area of the cell that was not accessed by M+Cholix 1 150 -(SEQ ID NO: 2lO)-HGH.
  • M+Cholix 1 265 - (SEQ ID NO: 2lO)-HGH was secreted from the basal surface of intestinal epithelial cells while M+Cholix 1 186 -(SEQ ID NO: 2lO)-HGH was not; i 87 KAAQKEGSRHKRWAHWHTGLAL 2 06 (SEQ ID NO: 152) is the peptide that describes the difference in the sequences of these two chimeras (see e.g., FIG. 17A).
  • a polymer framework containing peptide sequences of amino acids from positions 1-39, 134-151, 151-178, and 178-206 of Cholix domain I with SEQ ID NO: 5 in various combinations was labeled with different of quantum dot forms.
  • Cholix sequence variants as disclosed herein retain efficient endocytosis following uptake from the lumen but lack ability to complete transcytosis, being useful to target apical or apical and basal vesicular structures. From the data presented, it appears that Cholix utilizes a receptor-mediated-type endocytosis process that involves amino acids 134-151, which provides access to an early endosomal vesicular compartment in the apical portion of enterocytes (e.g., gut epithelial cells).
  • Amino acids 151-187 of the Cholix exotoxin domain I appear to allow its movement to a supra nuclear compartment consistent with a sorting site in the cell for secretory events, and thus allow delivery of various cargos to those locations as well. Movement to the basal compartment of the cells becomes more efficient with the presence of amino acids 1-40.
  • amino acids 187- 206 provide a mechanism for secretion from the basal membrane that releases the entire and intact protein into the lamina basement where it could provide therapeutically effective concentrations of therapeutic cargo molecules (e.g., interleukins), present antigens to immune cells, and/or allow the cargo to be taken up into systemic circulation for delivery to other target organs/tissues in a subject.
  • therapeutic cargo molecules e.g., interleukins
  • Cholix domain I derived delivery constructs utilize distinct compartments for trafficking into and across (e.g., via transcytosis) epithelial cells using various marker proteins (see e.g., EXAMPLE 3) that indicate Cholix derived carrier constructs utilize specific and endogenous trafficking pathways.
  • FIG. 21 A shows that the Cholix-IL-lO delivery constructs (SEQ ID NO: 154) strongly co-localized with the EEA1 antigen in cellular locations consistent with trafficking at both the apical and basal domains of enterocytes, suggesting the presence of the Cholix derived delivery constructs in early endosome compartments.
  • Rab7 the Cholix-IL-lO delivery constructs
  • LAMP1 was identified in large, specific vesicles consistent mature lysosomes that were devoid of Cholix-IL-lO delivery constructs (SEQ ID NO: 154,
  • Cholix-IL-lO chimera also co-localizes with the LAMP1 antigen in cellular locations other than lysosome-like structures, consistent with vesicle trafficking at both the apical and basal domains of enterocytes, suggesting the presence of the Cholix derived delivery constructs in lysosomal compartments (FIG. 21C).
  • Cholix-IL-lO chimera SEQ ID NO: 1544 also strongly co-localized with clathrin-coated vesicles, particularly in areas adjacent to the nucleus and with Rab 1 predominantly in the basal compartment of enterocytes as well as in selected cells within the lamina propria (FIG. 21D).
  • Calnexin Cholix-IL-lO chimera (SEQ ID NO: 154) co-localized with the endoplasmic reticulum as demonstrated by calnexin in a pattern adjacent to the nucleus in enterocytes and in a large fraction of cells with in the lamina propria (FIG. 21E).
  • Endoplasmatic reticulum Golgi intermediate compartment Cholix-IL-lO chimera (SEQ ID NO: 154) strongly co-localizes with the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) and the LAMN1 antigen appeared to re-distribute in response to carrier endocytosis and transcytosis, as shown for 1 (FIG. 21F), 5 (FIG. 21G), 10 (FIG. 21H), and 15 minutes after injection (FIG. 211). Transcytosis of the delivery construct was demonstrated to consistently traffic in large quantities across enterocytes. Specific compartments that strongly co-localized with this transcytosis included early endosomes and late endosomes.
  • the Cholix derived carrier appeared to be associated with clathrin-coated vesicles in the vicinity of the ER-Golgi network organized adjacent to enterocyte nuclei. Co-localization of the Cholix derived carrier was observed with the ER and ERGIC, also described as LMAN1 (lectin, mannose binding 1), but limited in its association with elements of the cis-Golgi, Golgi, and trans-Golgi network. The Cholix derived carrier co-localized with recycling endosomes near the basal surface of enterocytes in a manner that might coordinate with ERGIC re-distribution.
  • ERGIC-53 can also function as an intracellular cargo receptor involved in the anterograde transport of a limited number of glycoprotein ligands in the early exocytic pathway and is used by a number of RNA viruses as part of their exocytosis strategy.
  • ELISA-based binding studies demonstrated that Cholix and Cholix-derived delivery constructs can associate with ERGIC-53 at pH 7.4, but this interaction is significantly stronger at pH 5.5. SPR studies further supported this pH-dependent interaction (FIG. 31).
  • 58K antigen The 58K antigen localized in enterocytes at a site apical to the nucleus and the Cholix-ILlO chimera shows some co-localization with this antigen in a manner that suggests a brief movement through this compartment. No 58K antigen was observed in cells within the lamina propria (FIG. 21K).
  • TGN38 antigen Cholix-IL-lO chimera (SEQ ID NO: 154) showed some level of co-localization with the TGN38 antigen (top right), which showed a cellular distribution that was restricted to the apical side of nuclei in enterocytes and adjacent to the nucleus in a few cells within the laminalitis (FIG. 21L, white light and merge images shown bottom left and bottom right, respectively).
  • Rab 1 Cholix-IL-lO chimera (SEQ ID NO: 154, top right) strongly co-localized with Rab 1 (top left) predominantly in the basal compartment of enterocytes and in selected cells within the laminalitis (FIG. 21M, white light and merge images shown bottom left and bottom right, respectively).
  • GGGGSGGGGSGGGGS spacer (SEQ ID NO: 210) were used in this study, e g., SEQ ID NO: 164.
  • SEQ ID NO: 164 a limited set of candidate proteins as carrier protein receptors have been identified through bead capture and mass spectrometry analysis studies. Then, the interactions of the Cholix carrier with these candidate proteins were assessed in vitro (e.g., using Caco-2 cell monolayers) and in vivo (e.g., in the rat jejunum).
  • nano-sized magnetic beads 25 nm or 100 nm diameter
  • these decorated beads were allowed to transport across polarized monolayers in vitro of the human colon cancer cell lines Caco2 for set periods of time before gentle cell disruption and capture of vesicles containing these magnetic beads.
  • This figure shows interaction of Cholix carrier with heparan sulfate proteoglycan (HSPG), Dickkopf-related protein 1 (DKK1), the chaperone glucose-regulated protein 75 (GRP75), and cytokeratin-8 (K8 or CK8).
  • HSPG heparan sulfate proteoglycan
  • DKK1 Dickkopf-related protein 1
  • GFP75 chaperone glucose-regulated protein 75
  • cytokeratin-8 K8 or CK8
  • FIG. 28A and FIG 28B show that the intestinal localization of GRP75 and HSPC is consistent between rat and human intestine.
  • FIG. 29 shows effects of HSPG knockout by CRISPR on transport function of the delivery construct (SEQ ID NO: 164) comprising Cholix domain I coupled to HGH and HGH alone as internal control of non-selective transport.
  • Cells were seeded at l.5xl0 5 cells/mL in transwells.
  • transepithelial/transendothelial electrical resistance (TEER) was measured and PBS containing 20 ug/mL of the carrier with SEQ ID NO: 164 was added to the apical chambers. After 3 h, basolateral samples were collected and concentrated.
  • TMEM132A is highly similar to the rat GRP78 binding protein. Without being bound by any theory, it was assumed that the similarities between GRP78 and GRP75 may provide a rational for TMEM132A interactions with GRP75 and the potential for their co-localization at the time of Cholix endocytosis.
  • Cholix derived carrier and cargo transport and delivery is an active and selective process involving distinct receptors. This may be useful for the targeted delivery and therapeutic and/or diagnostic molecules across and/or to the interior of epithelial cells (e.g., gut epithelial cells) for the treatment and diagnosis of diseases as described herein.
  • epithelial cells e.g., gut epithelial cells
  • This example demonstrates the assessment of the pH-dependence of a Cholix derived carrier protein (e.g., SEQ ID NO: 4) and one of its interacting receptors during active transcytosis, GRP75.
  • a Cholix derived carrier protein e.g., SEQ ID NO: 4
  • Binding affinities at those three pH levels were generally in the low nanomolar range, however, a significantly higher (approximately 20-fold higher) binding affinity of the Cholix carrier to GRP75 was measured at pH 6.5, indicating pH dependency of this interaction.
  • a number of truncated forms of the full-length Cholix exotoxin were also prepared to examine various aspects of these interactions: truncations at amino acid E134, D151, K187, L206, K245, or Q251, or L206 conjoined to the N-terminus of human growth hormone (HGH) through a G 4 SG 4 SG 4 S sequence with this glycine-serine spacer being identified previously for constructing genetic chimeras.
  • HGH human growth hormone
  • K187 truncation we also deleted the first 39 amino acids to produce the E40-K187 fragment of Chx domain I with SEQ ID NO: 5.
  • the construct with SEQ ID NO: 158 failed to achieve apical entry into intestinal epithelial cells
  • the construct with SEQ ID NO: 159 and SEQ ID NO: 160 underwent endocytosis to reach both apical and basal vesicular pools within enterocytes but did not to access the lamina propria
  • the construct with SEQ ID NO: 165 underwent endocytosis but failed to migrate from the apical to the basal vesicular compartment of the enterocyte
  • the construct with SEQ ID NO: 164 efficiently and rapidly completes transcytosis.
  • the first 206 amino acids of Cholix domain I were evaluated for elements that may participate in the events that resulted in apical-to-basal transcytosis.
  • TMEM132A transmembrane protein 123A
  • TMEM132A is a single-pass transmembrane protein that contains cohesin and three tandem immunoglobulin domains, thus connecting the extracellular medium with the intracellular cytoskeleton.
  • TMEM132A serine/threonine-protein phosphatase 1
  • PP1 serine/threonine-protein phosphatase 1
  • Cholix domain I (SEQ ID NO: 5, bacterially expressed) enriched the isolation of TMEM132A along with catalytic and regulatory subunits of the PP1 complex.
  • ELISA-based studies showed that Cholix domain I with SEQ ID NO: 5 interacted with the extracellular domain of TMEM132A; SPR studies showed this interaction to occur at pH 7.5 and less at pH 5.5.
  • Knock-down studies in Caco-2 cells, to generate Caco-2 TMEM132A ⁇ cells, showed that reduction of TMEM132A dramatically reduced the transport of transcytosis of Cholix domain I with SEQ ID NO: 5.
  • TMEM132A that was restricted to the apical plasma membrane of enterocytes.
  • a time course examining transcytosis of SEQ ID NO: 164 suggested that TMEM132A remained at or adjacent to the apical plasma membrane and did not redistribute significantly to other regions of these polarized epithelial cells.
  • the construct with SEQ ID NO: 158 did not significantly enter into enterocytes following apical application, the construct with SEQ ID NO: 159 did enter, suggesting that a domain of Chx critical for apical cell entry resided with the 17 amino acids that discriminated where two carrier constructs.
  • the internal pH of an early or a recycling endosome can drop from neutrality to ⁇ 6.0.
  • Cytokeratin 8 was one of several proteins in this family identified in the initial screen as a potential Chx interaction partner; CK-8 was previously identified as a cell- surface receptor for the Pet toxin secreted from enteroaggregative E. coli. ELISA-based binding studies showed CK-8 to interact with TMEM132A and also Chx. CK-8 distribution in rat enterocytes was restricted to the apical surface and at discrete domains in the apical and basal compartments.
  • ERGIC-53 can also function as an intracellular cargo receptor involved in the anterograde transport of a limited number of glycoprotein ligands in the early exocytic pathway and is used by a number of RNA viruses as part of their exocytosis strategy.
  • ELISA-based binding studies demonstrated that the construct having sequence set forth in SEQ ID NO: 164 can associate with ERGIC-53 at pH 7.4, but this interaction is significantly stronger at pH 5.5. SPR studies further supported this pH- dependent interaction.
  • Chx interactions with GPR75 may provide some function other that routing vesicles from an apical endosomal compartment to a basal endosome compartment and hypothesized that GPR75 interactions could function to minimize routing of this bacterial effector protein from vesicles to lysosomes at both locations.
  • Luminal introduction of the delivery constructs with SEQ ID NO: 164 in the rat ILI model provided data to support ERGIC-53 as an element subverted by Cholix constructs of the present disclosure to achieve efficient transcytosis.
  • ERGIC-53 Prior to and at times immediately following apical application of the construct having sequence set forth in SEQ ID NO: 164, ERGIC-53 was observed in discrete populations in enterocytes that was focused near the apical surface of the cell nucleus, a location where ERGIC is consistently located.
  • ERGIC-53 Within a few minutes of luminal application of the construct having sequence set forth in SEQ ID NO: 164, ERGIC-53 was observed to move to areas within enterocytes adjacent to the apical plasma membrane and to a basal domain.
  • Cholix carrier transcytosis and ERGIC-53 redistribution to the basal area of enterocytes was coincident.
  • Transcytosis of the construct having sequence set forth in SEQ ID NO: 164 was observed following its ILI into rat jejunum did not affect the intracellular distribution of ribophorin 1, showing it to co-localize to a limited extent in the apical vesicular compartment where ribophorin 1 was present throughout the time course during which ERGIC-53 was subverted to the basal compartment. Additionally, ERGIC-53 interacts with a constellation of proteins, including SEC24, in its role as a soluble cargo receptor. Notably, a pull-down with GRP75 as bait identified SEC24. Similarly, apical application of the construct having sequence set forth in SEQ ID NO: 164 did not induce a gross alteration of intracellular compartment organization.
  • the ERGIC is involved in sorting soluble molecules destined for secretion from the cell and ERGIC-53 undergoes a process of concentrative sorting that involves the coat protein COPII. Since both COPI and COP II are involved in vesicle trafficking at the ER-Golgi interface, the potential for these coat proteins to co-localized with the construct having sequence set forth in SEQ ID NO: 164 during the transcytosis process was investigated.

Abstract

La présente invention concerne des constructions d'administration non naturelles isolées comprenant une construction d'administration dérivée d'une toxine bactérienne couplée à un cargo thérapeutique biologiquement actif; la construction d'administration étant apte à administrer le cargo biologiquement actif par le biais d'un transport de transcytose à travers une cellule épithéliale; et la construction d'administration ne comprenant pas de domaine de translocation dérivé de toxine bactérienne ou de domaine catalytique dérivé de toxine bactérienne (cytotoxique).
PCT/US2019/021474 2018-03-08 2019-03-08 Constructions d'administration dérivées de toxines pour administration orale WO2019173787A1 (fr)

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CN201980031164.1A CN112105376A (zh) 2018-03-08 2019-03-08 用于经口递送的毒素衍生的递送构建体
EP23192492.9A EP4316586A3 (fr) 2018-03-08 2019-03-08 Constructions d'administration dérivées de toxines pour administration orale
ES19717013T ES2920428T3 (es) 2018-03-08 2019-03-08 Constructos de administración derivados de toxinas para la administración oral
EA202092126A EA202092126A1 (ru) 2018-11-07 2019-03-08 Новые полученные из токсинов конструкции доставки для пероральной доставки
SI201930291T SI3762009T1 (sl) 2018-03-08 2019-03-08 Iz toksinov izpeljani dostavni konstrukti za peroralno dostavo
CA3093386A CA3093386A1 (fr) 2018-03-08 2019-03-08 Constructions d'administration derivees de toxines pour administration orale
KR1020207028975A KR20210076881A (ko) 2018-03-08 2019-03-08 경구 전달용 독소-유래 전달 구조체
PL19717013.7T PL3762009T3 (pl) 2018-03-08 2019-03-08 Pochodzące z toksyny konstrukty dostarczające do dostarczania doustnego
EP19717013.7A EP3762009B1 (fr) 2018-03-08 2019-03-08 Constructions d'administration dérivées de toxines pour administration orale
SG11202009925PA SG11202009925PA (en) 2018-03-08 2019-03-08 Toxin-derived delivery constructs for oral delivery
DK19717013.7T DK3762009T3 (da) 2018-03-08 2019-03-08 Toxin-afledte indgivelseskonstrukter til oral indgivelse
AU2019230230A AU2019230230A1 (en) 2018-03-08 2019-03-08 Toxin-derived delivery constructs for oral delivery
EP22165874.3A EP4082558B1 (fr) 2018-03-08 2019-03-08 Constructions dérivées de toxine pour l'administration par voie orale
KR1020217017372A KR20210110800A (ko) 2018-11-07 2019-09-11 이종성 페이로드의 경구 전달용 콜릭스-유래 담체
CN201980088247.4A CN113347997A (zh) 2018-11-07 2019-09-11 用于经口递送异源有效载荷的Cholix衍生的携带体
IL282986A IL282986B2 (en) 2018-11-07 2019-09-11 Colics-derivative carriers for oral administration of heterologous cargo
CA3119179A CA3119179A1 (fr) 2018-11-07 2019-09-11 Supports derives de cholix pour administration orale de chargement heterologue
PCT/US2019/050708 WO2020096695A1 (fr) 2018-11-07 2019-09-11 Supports dérivés de cholix pour administration orale de chargement hétérologue
AU2019374703A AU2019374703A1 (en) 2018-11-07 2019-09-11 Cholix-derived carriers for oral delivery of heterologous payload
SG11202104734YA SG11202104734YA (en) 2018-11-07 2019-09-11 Cholix-derived carriers for oral delivery of heterologous payload
TW108132886A TW202031297A (zh) 2018-11-07 2019-09-11 用於經口遞送異種負載之衍生自cholix的載體
MX2021005382A MX2021005382A (es) 2018-11-07 2019-09-11 Vehiculos derivados de cholix para suministro oral de carga util heterologa.
JP2021525126A JP2022512976A (ja) 2018-11-07 2019-09-11 異種ペイロードの経口送達のためのコリックス由来担体
EP19881649.8A EP3826682A4 (fr) 2018-11-07 2019-09-11 Supports dérivés de cholix pour administration orale de chargement hétérologue
BR112021009001A BR112021009001A8 (pt) 2018-11-07 2019-09-11 Veículos derivados de cholix para administração oral de carga útil heteróloga
EP22159495.5A EP4083058A3 (fr) 2018-11-07 2019-11-07 Constructions d'administration pour la transcytose et procédés associés
BR112021009003-7A BR112021009003A2 (pt) 2018-11-07 2019-11-07 construtos de liberação para transcitose e métodos relacionados.
MX2021005346A MX2021005346A (es) 2018-11-07 2019-11-07 Constructos de suministro para transcitosis y metodos relacionados.
DK19207825.1T DK3650037T3 (da) 2018-11-07 2019-11-07 Indgivelseskonstrukter til transcytose og tilhørende fremgangsmåder
EP19207825.1A EP3650037B1 (fr) 2018-11-07 2019-11-07 Constructions d'administration pour la transcytose et procédés associés
JP2021525169A JP2022506990A (ja) 2018-11-07 2019-11-07 トランスサイトーシスのための送達構築物および関連する方法
AU2019377117A AU2019377117A1 (en) 2018-11-07 2019-11-07 Delivery constructs for transcytosis and related methods
ES19207825T ES2911075T3 (es) 2018-11-07 2019-11-07 Constructos de administración para transcitosis y métodos relacionados
PT192078251T PT3650037T (pt) 2018-11-07 2019-11-07 Construções de administração para transcitose e métodos relacionados
CA3119060A CA3119060A1 (fr) 2018-11-07 2019-11-07 Constructions d'administration pour la transcytose et methodes associees
PCT/US2019/060356 WO2020097394A1 (fr) 2018-11-07 2019-11-07 Constructions d'administration pour la transcytose et méthodes associées
TW108140533A TW202031678A (zh) 2018-11-07 2019-11-07 用於胞移作用及相關方法之遞送構築體
KR1020217017210A KR20210110294A (ko) 2018-11-07 2019-11-07 통과세포외배출용 전달 구조체 및 관련 방법
CN201980088287.9A CN113423722A (zh) 2018-11-07 2019-11-07 用于胞吞转运的递送构建体及相关方法
SG11202104721RA SG11202104721RA (en) 2018-11-07 2019-11-07 Delivery constructs for transcytosis and related methods
PL19207825T PL3650037T3 (pl) 2018-11-07 2019-11-07 Konstrukty dostarczające do transcytozy i powiązane z nimi sposoby
US16/686,671 US20200140511A1 (en) 2018-11-07 2019-11-18 Delivery constructs for transcytosis and related methods
IL277209A IL277209A (en) 2018-03-08 2020-09-08 Structures for administration of toxin derivatives for oral administration
US17/015,011 US11426466B2 (en) 2018-03-08 2020-09-08 Toxin-derived delivery constructs for pulmonary delivery
IL282987A IL282987B2 (en) 2018-11-07 2021-05-06 Delivery constructs for transcytosis and related methods
CL2021001209A CL2021001209A1 (es) 2018-11-07 2021-05-07 Constructos de suministro para transcitosis y métodos relacionados
CONC2021/0007359A CO2021007359A2 (es) 2018-11-07 2021-06-04 Vehículos derivados de cholix para suministro oral de carga útil heteróloga
CONC2021/0007402A CO2021007402A2 (es) 2018-11-07 2021-06-07 Constructos de suministro para transcitosis y métodos relacionados
US17/868,077 US20230158163A1 (en) 2018-03-08 2022-07-19 Toxin-derived delivery constructs

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10617767B2 (en) 2010-09-15 2020-04-14 Applied Molecular Transport Inc. Compositions and methods for oral delivery of therapeutic cargo
US10624956B2 (en) 2014-05-07 2020-04-21 Applied Molecular Transport Inc. Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
EP3650037A1 (fr) * 2018-11-07 2020-05-13 Applied Molecular Transport Inc. Constructions d'administration pour la transcytose et procédés associés
WO2020096695A1 (fr) * 2018-11-07 2020-05-14 Applied Molecular Transport Inc. Supports dérivés de cholix pour administration orale de chargement hétérologue
WO2021155281A1 (fr) * 2020-01-31 2021-08-05 Applied Molecular Transport Inc. Compositions et méthodes pour le traitement de la maladie cœliaque
US11160869B2 (en) 2019-08-16 2021-11-02 Applied Molecular Transport Inc. Compositions, formulations and interleukin production and purification
US11246915B2 (en) 2010-09-15 2022-02-15 Applied Molecular Transport Inc. Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo
WO2022177890A1 (fr) * 2021-02-16 2022-08-25 Applied Molecular Transport Inc. Compositions orales solides contenant du zinc
US11426466B2 (en) 2018-03-08 2022-08-30 Applied Molecular Transport Inc. Toxin-derived delivery constructs for pulmonary delivery
WO2022241167A1 (fr) * 2021-05-12 2022-11-17 Applied Molecular Transport Inc. Constructions d'administration dérivées de toxines bactériennes et utilisations associées
WO2022240407A1 (fr) * 2021-05-12 2022-11-17 Applied Molecular Transport Inc. Constructions d'administration dérivées de toxines bactériennes et utilisations associées
WO2023141527A1 (fr) * 2022-01-21 2023-07-27 Applied Molecular Transport Inc. Constructions d'administration clivables par furine
WO2023196532A1 (fr) * 2022-04-08 2023-10-12 Applied Molecular Transport Inc. Constructions d'il-10/vecteur chimériques pour le traitement de la pochite et procédés d'utilisation

Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4414148A (en) 1981-04-15 1983-11-08 Sanofi Anti-cancer drugs for the treatment of melanomas and method for preparing thereof
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4569789A (en) 1984-08-29 1986-02-11 Dana-Farber Cancer Institute, Inc. Acid-cleavable compound, use in protein conjugates and drug delivery systems
US4589071A (en) 1982-04-19 1986-05-13 Nissan Motor Co., Ltd. Method and apparatus for controlling reduction ratio of continuously variable transmission with acceleration compensation
US4659839A (en) 1984-10-10 1987-04-21 Mallinckrodt, Inc. Coupling agents for radiolabeled antibody fragments
US4671958A (en) 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
US4680338A (en) 1985-10-17 1987-07-14 Immunomedics, Inc. Bifunctional linker
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4699784A (en) 1986-02-25 1987-10-13 Center For Molecular Medicine & Immunology Tumoricidal methotrexate-antibody conjugate
US5272065A (en) 1983-10-20 1993-12-21 Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA
US5807832A (en) 1987-06-09 1998-09-15 Biotech Australia Pty Limited Oral delivery of biologically active substances bound to vitamin B12
US5917021A (en) 1995-04-07 1999-06-29 Enzon, Inc. Stabilized monomeric protein compositions
US5922680A (en) 1996-10-23 1999-07-13 Ferring, B.V. Stabilized composition for oral administration of peptides
US5997856A (en) 1988-10-05 1999-12-07 Chiron Corporation Method and compositions for solubilization and stabilization of polypeptides, especially proteins
US6007791A (en) 1994-10-10 1999-12-28 Chiron Corporation Preparation of protein microspheres, films and coatings
US6086918A (en) 1996-03-15 2000-07-11 Unigene Laboratories, Inc. Oral peptide pharmaceutical products
US6133229A (en) 1993-10-08 2000-10-17 The University Of Leeds Innovations, Ltd. Stabilization of proteins in solution
US6174529B1 (en) 1991-06-21 2001-01-16 University Of Cincinnati Oral therapy for the treatment of allergies and method of manufacture
US6255284B1 (en) 1993-02-02 2001-07-03 Xoma Corporation Pharmaceutical composition
US6391296B1 (en) 1997-08-01 2002-05-21 Toray Industries, Inc. Method of stabilizing useful protein and useful protein compositions
US6525102B1 (en) 1999-10-04 2003-02-25 Chiron Corporation Stabilized liquid polypeptide-containing pharmaceutical compositions
US6573237B2 (en) 2001-03-16 2003-06-03 Eli Lilly And Company Protein formulations
US6592847B1 (en) 1998-05-14 2003-07-15 The General Hospital Corporation Intramolecularly-quenched near infrared flourescent probes
US6613332B1 (en) 1991-06-21 2003-09-02 The University Of Cincinnati Oral administration of therapeutic proteins
US6673574B2 (en) 2000-11-30 2004-01-06 Unigene Laboratories Inc. Oral delivery of peptides using enzyme-cleavable membrane translocators
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
WO2012036746A1 (fr) * 2010-09-15 2012-03-22 Randall J Mrsny Systèmes et procédés d'administration d'agents biologiquement actifs par le biais de séquences vectrices dérivées de toxine bactérienne
US8877161B2 (en) 2011-10-19 2014-11-04 Georgia Regents Research Institute, Inc. GM1-like peptides and uses thereof
WO2015171965A2 (fr) * 2014-05-07 2015-11-12 Applied Molecular Transport Llc Molécules de fusion dérivées de la toxine cholix pour l'administration par voie orale d'une charge biologiquement active

Patent Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4414148A (en) 1981-04-15 1983-11-08 Sanofi Anti-cancer drugs for the treatment of melanomas and method for preparing thereof
US4671958A (en) 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
US4589071A (en) 1982-04-19 1986-05-13 Nissan Motor Co., Ltd. Method and apparatus for controlling reduction ratio of continuously variable transmission with acceleration compensation
US5272065A (en) 1983-10-20 1993-12-21 Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA
US4569789A (en) 1984-08-29 1986-02-11 Dana-Farber Cancer Institute, Inc. Acid-cleavable compound, use in protein conjugates and drug delivery systems
US4659839A (en) 1984-10-10 1987-04-21 Mallinckrodt, Inc. Coupling agents for radiolabeled antibody fragments
US4680338A (en) 1985-10-17 1987-07-14 Immunomedics, Inc. Bifunctional linker
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4699784A (en) 1986-02-25 1987-10-13 Center For Molecular Medicine & Immunology Tumoricidal methotrexate-antibody conjugate
US5807832A (en) 1987-06-09 1998-09-15 Biotech Australia Pty Limited Oral delivery of biologically active substances bound to vitamin B12
US5997856A (en) 1988-10-05 1999-12-07 Chiron Corporation Method and compositions for solubilization and stabilization of polypeptides, especially proteins
US6613332B1 (en) 1991-06-21 2003-09-02 The University Of Cincinnati Oral administration of therapeutic proteins
US6174529B1 (en) 1991-06-21 2001-01-16 University Of Cincinnati Oral therapy for the treatment of allergies and method of manufacture
US6255284B1 (en) 1993-02-02 2001-07-03 Xoma Corporation Pharmaceutical composition
US6133229A (en) 1993-10-08 2000-10-17 The University Of Leeds Innovations, Ltd. Stabilization of proteins in solution
US6007791A (en) 1994-10-10 1999-12-28 Chiron Corporation Preparation of protein microspheres, films and coatings
US5917021A (en) 1995-04-07 1999-06-29 Enzon, Inc. Stabilized monomeric protein compositions
US6086918A (en) 1996-03-15 2000-07-11 Unigene Laboratories, Inc. Oral peptide pharmaceutical products
US5922680A (en) 1996-10-23 1999-07-13 Ferring, B.V. Stabilized composition for oral administration of peptides
US6391296B1 (en) 1997-08-01 2002-05-21 Toray Industries, Inc. Method of stabilizing useful protein and useful protein compositions
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6592847B1 (en) 1998-05-14 2003-07-15 The General Hospital Corporation Intramolecularly-quenched near infrared flourescent probes
US6525102B1 (en) 1999-10-04 2003-02-25 Chiron Corporation Stabilized liquid polypeptide-containing pharmaceutical compositions
US6673574B2 (en) 2000-11-30 2004-01-06 Unigene Laboratories Inc. Oral delivery of peptides using enzyme-cleavable membrane translocators
US6573237B2 (en) 2001-03-16 2003-06-03 Eli Lilly And Company Protein formulations
WO2012036746A1 (fr) * 2010-09-15 2012-03-22 Randall J Mrsny Systèmes et procédés d'administration d'agents biologiquement actifs par le biais de séquences vectrices dérivées de toxine bactérienne
US8877161B2 (en) 2011-10-19 2014-11-04 Georgia Regents Research Institute, Inc. GM1-like peptides and uses thereof
WO2015171965A2 (fr) * 2014-05-07 2015-11-12 Applied Molecular Transport Llc Molécules de fusion dérivées de la toxine cholix pour l'administration par voie orale d'une charge biologiquement active

Non-Patent Citations (44)

* Cited by examiner, † Cited by third party
Title
"Current Edition, Current Protocols in Molecular Biology", GREENE PUBLISHING ASSOCIATES AND WILEY INTERSCIENCE
"Overview of principles of hybridization and the strategy of nucleic acid probe assays", ELSEVIER
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO., pages: 18042
"Remington's Pharmaceutical Sciences", 2005, MACK PUBLISHING CO
ALLURED, PROC. NATL. ACAD. SCI., vol. 83, 1986, pages 1320 - 1324
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
AUSUBEL ET AL.: "Current Edition, Current Protocols in Molecular Biology", GREENE PUBLISHING ASSOCIATES AND WILEY INTERSCIENCE
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1992, GREENE PUBLISHING ASSOCIATES
AWASTHI ET AL.: "Novel Cholix toxin variants, ADP-ribosylating toxins in Vibrio Cholerae Non-Ol/Non-0139 strains, and their pathogenicity", INFECTION AND IMMUNITY, vol. 81, no. 2, 2013, pages 531 - 541
BEAUCAGE ET AL., TETRA. LETT., vol. 22, 1981, pages 1859 - 1862
BORLINGHAUS ET AL., CANCER RES., vol. 47, 1987, pages 4071 - 4075
BROWN ET AL., METH. ENZYMOL., vol. 68, 1979, pages 109 - 151
CALIF; BARON ET AL., NUCLEIC ACIDS RES., vol. 23, 1995, pages 3605 - 06
CASARES ET AL., JOURNAL OF IMMUNOLOGY, vol. 185, no. 9, 2010, pages 5150 - 5159
DEYOUNG, INT J PANCREATOL, vol. 5, no. 31-6, 1989
DIBASE; MORREL: "Oral Delivery of Microencapsulated Proteins, in Protein Delivery: Physical Systems", 1997, PLENUM PRESS, pages: 255 - 288
ERLICH: "PCR Technology", 1989, STOCKTON PRESS
GOEDDEL ET AL.: "Methods in Enzymology", vol. 185, 1990, ACADEMIC PRESS, INC.
GOEDDEL: "Gene Expression Technology: Methods in Enzymology", vol. 185, 1990, ACADEMIC PRESS
GREEN; SAMBROOK: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
HSU C-T ET AL: "Vaccination against Gonadotropin-releasing Hormone (GnRH) Using Toxin Receptor-binding Domain-Conjugated GnRH Repeats", CANCER RESEARCH, PROCEEDINGS: AACR 107TH ANNUAL MEETING 2016; APRIL 16-20, 2016; NEW ORLEANS, LA, vol. 60, 15 July 2000 (2000-07-15), pages 3701 - 3705, XP002213279, ISSN: 0008-5472 *
JAULANG HWANG ET AL: "Structure and Function Relationship of Pseudomonas Exotoxin A", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 4, 5 February 1989 (1989-02-05), pages 2379 - 2384, XP055591345 *
JORGENSEN, R. ET AL., J BIOL CHEM, vol. 283, no. 16, 2008, pages 10671 - 10678
JUNG ET AL., EUR. J. PHARM. BIOPHARM., vol. 50, no. 1, 2000, pages 147 - 160
KARLIN; ALTSCHUL, PROC. NAT'L. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5787
KRIEGER: "Gene Transfer and Expression--A Laboratory Manual", 1990, STOCKTON PRESS
KYTE ET AL., J. MOL. BIOL., vol. 157, 1982, pages 105 - 131
MARIE-PIERRE TAUPIAC ET AL: "A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly", MOLECULAR MICROBIOLOGY., vol. 31, no. 5, 1 March 1999 (1999-03-01), GB, pages 1385 - 1393, XP055591198, ISSN: 0950-382X, DOI: 10.1046/j.1365-2958.1999.01280.x *
MIGUEL LUGO ET AL: "The Father, Son and Cholix Toxin: The Third Member of the DT Group Mono-ADP-Ribosyltransferase Toxin Family", TOXINS, vol. 7, no. 8, 24 July 2015 (2015-07-24), pages 2757 - 2772, XP055591185, DOI: 10.3390/toxins7082757 *
MRSNY ET AL., VACCINE, vol. 17, 1999, pages 1425 - 1433
MULLER B ET AL., SPRINGER SEMIN. IMMUNOPATHOL., vol. 20, 1998, pages 181 - 96
MULLIS ET AL.: "Cold Spring Harbor Symp. Quant. Biol.", vol. 51, 1987, pages: 263
NARANG ET AL., METH. ENZYMOL., vol. 68, 1979, pages 90 - 99
ROMAGNANI, INFLAMM BOWEL DIS, vol. 5, 1999, pages 285 - 94
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning--A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning--A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY, COLD SPRING HARBOR
TAKEUCHI ET AL., ADV. DRUG DELIV. REV., vol. 47, no. 1, 2001, pages 39 - 54
TIJSSEN: "Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes", 1993
XIA ET AL., J. PHARMACOL. EXPERIMENT. THERAP., vol. 295, 2000, pages 594 - 600
XIA ET AL., PHARMACEUTICAL RES., vol. 18, no. 2, 2001, pages 191 - 195
XIANG Y. HAN ET AL: "Active Site Mutations of Pseudomonas aeruginosa Exotoxin A : ANALYSIS OF THE HIS RESIDUE", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 2, 13 January 1995 (1995-01-13), US, pages 679 - 684, XP055591689, ISSN: 0021-9258, DOI: 10.1074/jbc.270.2.679 *
YOSHIHIRO JINNO ET AL: "Mutational Analysis of Domain I of Pseudomonas Exotoxin MUTATIONS IN DOMAIN I OF PSEUDOMONAS EXOTOXIN WHICH REDUCE CELL BINDING AND ANIMAL TOXICITY", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 263, no. 26, 15 September 1988 (1988-09-15), pages 13203 - 13207, XP055591707 *

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