Methods and compositions for needleless delivery of macromolecules
Methods and compositions for needleless delivery of macromolecules to the bloodstream of a subject are provided herein. In one aspect, the invention provides a delivery construct, comprising a receptor binding domain, a transcytosis domain, a macromolecule to be delivered to a subject, and a cleavable linker. Generally, the cleavable linker is cleavable by an enzyme present in higher concentration at or near the basal-lateral membrane of a polarized epithelial cell or in the plasma than elsewhere in the body, for example, at the apical side of the polarized epithelial cell. In other aspects, the invention provides nucleic acids encoding delivery constructs of the invention, kits comprising delivery constructs of the invention, cells expressing delivery constructs of the invention, and methods of using delivery constructs of the invention.
1 . A delivery construct, comprising:
a)- a receptor binding domain,
b)- a transcytosis domain,
c)- a macromolecule to be delivered to a subject, and
d)- a cleavable linker,
wherein cleavage at said cleavable linker separates said macromolecule from the remainder of said construct, and wherein said cleavable linker is cleavable by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a polarized epithelial cell of said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma of said subject than at an apical membrane of the polarized epithelial cell of the subject.
2 . The delivery construct of claim 1 , further comprising a second cleavable linker.
3 . The delivery construct of claim 1 , wherein said cleavable linker comprises an amino acid sequence that is selected from the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.:10).
4 . The delivery construct of claim 1 , wherein said enzyme that is present at a basal-lateral membrane of a polarized epithelial cell is selected from the group consisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I.
5 . The delivery construct of claim 1 , wherein said receptor binding domain is selected from the group consisting of receptor binding domains from Pseudomonas exotoxin A, cholera toxin, botulinum toxin, diptheria toxin, shiga toxin, or shiga-like toxin; monoclonal antibodies; polyclonal antibodies; single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8.
6 . The delivery construct of claim 1 , wherein said receptor binding domain binds to a cell-surface receptor that is selected from the group consisting of α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.
7 . The delivery construct of claim 5 , wherein said receptor binding domain of Pseudomonas exotoxin A is Domain Ia of Pseudomonas exotoxin A.
8 . The delivery construct of claim 7 , wherein said receptor binding domain of Pseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:1.
9 . The delivery construct of claim 1 , wherein said transcytosis domain is selected from the group consisting of transcytosis domains from Pseudomonas exotoxin A, botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin.
10 . The delivery construct of claim 9 , wherein said transcytosis domain is Pseudomonas exotoxin A transcytosis domain.
11 . The delivery construct of claim 10 , wherein said Pseudomonas exotoxin A transcytosis domain has an amino acid sequence that is SEQ ID NO.:2.
12 . The delivery construct of claim 1 , wherein said macromolecule is selected from the group of a nucleic acid, a peptide, a polypeptide, a protein, and a lipid.
13 . The delivery construct of claim 12 , wherein said polypeptide is selected from the group consisting of polypeptide hormones, cytokines, chemokines, growth factors, and clotting factors.
14 . The delivery construct of claim 13 , wherein said polypeptide is selected from the group consisting of IGF-I, IGF-II, IGF-III, EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin, calcitonin, parathyroid hormone, luteinizing hormone-releasing factor, tissue plasminogen activators, proinsulin, insulin, glucocorticoid, amylin, adrenocorticototropin, enkephalin, and glucagon-like peptide 1.
15 . The delivery construct of claim 14 , wherein said polypeptide is human growth hormone.
16 . The delivery construct of claim 12 , wherein said protein is human insulin.
17 . The delivery construct of claim 12 , wherein said protein is human IFN-α.
18 . The delivery construct of claim 12 , wherein said protein is human IFN-α2b.
19 . The delivery construct of claim 12 , wherein said protein is human proinsulin.
20 . The delivery construct of claim 12 , further comprising a second macromolecule that is selected from the group of a nucleic acid, a peptide, a polypeptide, a protein, a lipid, or a small organic molecule and a second cleavable linker, wherein cleavage at said second cleavable linker separates said second macromolecule from the remainder of said construct.
21 . The delivery construct of claim 17 , wherein said macromolecule is a first polypeptide and said second macromolecule is a second polypeptide.
22 . The delivery construct of claim 21 , wherein said first polypeptide and said second polypeptide associate to form a multimer.
23 . The delivery construct of claim 22 , wherein said multimer is a dimer, tetramer, or octamer.
24 . The delivery construct of claim 23 , wherein said dimer is an antibody.
25 . A polynucleotide that encodes a delivery construct, said delivery construct comprising:
a)- a receptor binding domain,
b)- a transcytosis domain,
c)- a macromolecule to be delivered to a subject, and
d)- a cleavable linker,
wherein cleavage at said cleavable linker separates said macromolecule from the remainder of said construct, and wherein said cleavable linker is cleavable by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a polarized epithelial cell of said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma of said subject than at an apical membrane of the polarized epithelial cell of the subject.
26 . A polynucleotide that hybridizes under stringent hybridization conditions to the polynucleotide of claim 25 .
27 . The polynucleotide of claim 25 , wherein said delivery construct further comprises a second cleavable linker.
28 . The polynucleotide of claim 25 , wherein said cleavable linker comprises an amino acid sequence that is selected from the group consisting of Ala-Ala-Pro-Phe (SEQ ED NO.:4), Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.:10).
29 . The polynucleotide of claim 25 , wherein said enzyme that is present at a basal-lateral membrane of a polarized epithelial cell is selected from the group consisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I.
30 . The polynucleotide of claim 25 , wherein said receptor binding domain is selected from the group consisting of receptor binding domains from Pseudomonas exotoxin A, cholera toxin, botulinum toxin, diptheria toxin, shiga toxin, or shiga-like toxin; monoclonal antibodies; polyclonal antibodies; single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8.
31 . The polynucleotide of claim 25 , wherein said receptor binding domain binds to a cell-surface receptor that is selected from the group consisting of α2-macroglobulin receptor, EGFR, IGFR, transferrin receptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.
32 . The polynucleotide of claim 30 , wherein said receptor binding domain of Pseudomonas exotoxin A is Domain Ia of Pseudomonas exotoxin A.
33 . The polynucleotide of claim 31 , wherein said receptor binding domain of Pseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:1.
34 . The polynucleotide of claim 25 , wherein said transcytosis domain is selected from the group consisting of transcytosis domains from Pseudomonas exotoxin A, botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin.
35 . The polynucleotide of claim 34 , wherein said transcytosis domain is Pseudomonas exotoxin A transcytosis domain.
36 . The polynucleotide of claim 35 , wherein said Pseudomonas exotoxin A transcytosis domain has an amino acid sequence that is SEQ ID NO.: 2.
37 . The polynucleotide of claim 25 , wherein said macromolecule is selected from the group of a peptide, a polypeptide, and a protein.
38 . The polynucleotide of claim 37 , wherein said polypeptide is selected from the group consisting of polypeptide hormones, cytokines, chemokines, growth factors, and clotting factors.
39 . The polynucleotide of claim 38 , wherein said polypeptide is selected from the group consisting of IGF-I, IGF-II, IGF-1H, EGF, IFN-α, IFN-α, 2b, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin, calcitonin, parathyroid hormone, luteinizing hormone-releasing factor, tissue plasminogen activators, proinsulin, insulin, glucocorticoid, amylin, adrenocorticototropin, enkephalin, and glucagon-like peptide 1.
40 . The polynucleotide of claim 39 , wherein said polypeptide is human growth hormone.
41 . The polynucleotide of claim 39 , wherein said protein is human insulin.
42 . A polynucleotide that encodes a delivery construct, said polynucleotide comprising:
a)- a nucleic acid sequence encoding a receptor binding domain,
b)- a nucleic acid sequence encoding a transcytosis domain,
c)- a nucleic acid sequence encoding a cleavable linker, and
d)- a nucleic acid sequence comprising a polylinker insertion site,
wherein cleavage at said cleavable linker separates said macromolecule from the remainder of said construct, and wherein said cleavable linker is cleavable by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a polarized epithelial cell of said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma of said subject than at an apical membrane of the polarized epithelial cell of the subject.
43 . An expression vector comprising the polynucleotide of claim 25 .
44 . A cell comprising the expression vector of claim 43 .
45 . A composition comprising a delivery construct, said delivery construct comprising:
a)- a receptor binding domain,
b)- a transcytosis domain,
c)- a macromolecule to be delivered to a subject, and
d)- a cleavable linker,
wherein cleavage at said cleavable linker separates said macromolecule from the remainder of said construct, and wherein said cleavable linker is cleavable by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a polarized epithelial cell of said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma of said subject than at an apical membrane of the polarized epithelial cell of the subject.
46 . The composition of claim 45 , wherein said composition further comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier.
47 . The composition of claim 45 , wherein said composition is formulated for nasal or oral administration.
48 . A method for delivering a macromolecule to a subject, comprising contacting an apical surface of a polarized epithelial cell of the subject with a delivery construct, wherein said delivery construct comprises a receptor binding domain, a transcytosis domain, a cleavable linker, and the macromolecule, wherein the transcytosis domain transcytosis the macromolecule to and through the basal-lateral membrane of said epithelial cell, wherein cleavage at said cleavable linker separates said macromolecule from the remainder of said construct, and wherein said cleavable linker is cleavable by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a polarized epithelial cell of said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma of said subject than at an apical membrane of the polarized epithelial cell of the subject, and wherein cleavage at the cleavable linker separates the macromolecule from the remainder of the delivery construct, thereby delivering the macromolecule to the subject.
49 . The method of claim 48 , wherein said receptor binding domain is selected from the group consisting of receptor binding domains from Pseudomonas exotoxin A, cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin; monoclonal antibodies; polyclonal antibodies; single-chain antibodies; TGF α; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8.
50 . The method of claim 48 , wherein said receptor binding domain binds to a cell surface receptor selected from the group consisting of α2-macroglobulin receptor, EGFR, IGFR, transferrin receptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.
51 . The method of claim 48 , wherein said transcytosis domain is selected from the group consisting of transcytosis domains from Pseudomonas exotoxin A, botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin.
52 . The method of claim 48 , wherein said macromolecule is selected from the group consisting of a peptide, a polypeptide, a protein, a nucleic acid, and a lipid.
53 . The method of claim 48 , wherein said enzyme that is present at a basal-lateral membrane of a polarized epithelial cell is selected from the group consisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I.
54 . The method of claim 48 , wherein said cleavable linker comprises an amino acid sequence that is selected from the group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:4), Gly-Gly-Phe (SEQ ID NO.:5), Ala-Ala-Pro-Val (SEQ ID NO.:6), Gly-Gly-Leu (SEQ ID NO.:7), Ala-Ala-Leu (SEQ ID NO.:8), Phe-Val-Arg (SEQ ID NO.:9), Val-Gly-Arg (SEQ ID NO.: 10).
55 . The method of claim 48 , wherein 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.
56 . The method of claim 48 , wherein said mammal is a human.
57 . The method of claim 48 , wherein said delivery construct contacts the apical membrane of the epithelial cell.
58 . A method for delivering a macromolecule to the bloodstream of a subject, comprising contacting the delivery construct of claim 1 to an apical surface of a polarized epithelial cell of the subject, such that the macromolecule is delivered to the bloodstream of the subject, wherein a lower titer of antibodies specific for the macromolecule is induced in the serum of the subject than is induced by subcutaneously administering the macromolecule to a subject separately from the remainder of the delivery construct.
59 . The method of claim 58 , wherein the macromolecule is selected from the group consisting of a peptide, a polypeptide, a protein, a nucleic acid, and a lipid.
60 . The method of claim 58 , wherein the macromolecule is selected from the group consisting of polypeptide hormones, cytokines, chemokines, growth factors, and clotting factors.
61 . The method of claim 58 , wherein the macromolecule is selected from the group consisting of IGF-I, IGF-II, IGF-III, EGF, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12, EPO, growth hormone, factor VII, vasopressin, calcitonin, parathyroid hormone, luteinizing hormone-releasing factor, tissue plasminogen activators, proinsulin, insulin, glucocorticoid, amylin, adrenocorticototropin, enkephalin, and glucagon-like peptide 1.
62 . The method of claim 58 , wherein the macromolecule is human growth hormone.
63 . The method of claim 58 , wherein the macromolecule is human insulin.
64 . The method of claim 58 , wherein the subject is a mouse, dog, goat, or human.
65 . The method of claim 58 , wherein the titer of antibodies specific for the macromolecule induced in the serum of the subject by the macromolecule delivered by with the delivery construct is less than about 75% of the titer of antibodies induced by subcutaneously administering the macromolecule to a subject separately from the remainder of the delivery construct.
66 . The method of claim 58 , wherein the titer of antibodies specific for the macromolecule induced in the serum of the subject by the macromolecule delivered by the delivery construct is less than about 50% of the titer of antibodies induced by subcutaneously administering the macromolecule to a subject separately from the remainder of the delivery construct.
67 . The method of claim 58 , wherein the titer of antibodies specific for the macromolecule induced in the serum of the subject by the macromolecule delivered by the delivery construct is less than about 25% of the titer of antibodies induced by subcutaneously administering the macromolecule to a subject separately from the remainder of the delivery construct.
68 . The method of claim 58 , wherein the titer of antibodies specific for the macromolecule induced in the serum of the subject by the macromolecule delivered by the delivery construct is less than about 10% of the titer of antibodies induced by subcutaneously administering the macromolecule to a subject separately from the remainder of the delivery construct.