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. In certain aspects, the cleavable linker can be 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. An isolated delivery construct, comprising:
a)—a receptor binding domain,
b)—a transcytosis domain,
c)—a peptide, polypeptide, or protein to be delivered to a subject, and
d)—a cleavable linker,
wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject, and said peptide, polypeptide, or protein is separated from the receptor binding domain and the transcytosis domain, by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell in said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
2. The delivery construct of claim 1 , further comprising a second cleavable linker, wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said second cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject, and said peptide, polypeptide, or protein is separated from the receptor binding domain and the transcytosis domain, by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell in said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
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 normal, polarized epithelial cell is selected from the group consisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin Al, Subtilisin All, 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; 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 peptide, polypeptide, or protein is selected from the group consisting of a peptide and a polypeptide.
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 1 , further comprising a second peptide, polypeptide, or protein and a second cleavable linker, wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said second cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject, and said second peptide, polypeptide, or protein is separated from the receptor binding domain and the transcytosis domain, by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell in said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
21. The delivery construct of claim 20 , wherein the peptide polypeptide, or protein and said second peptide, polypeptide, or protein associate to form a multimer.
22. The delivery construct of claim 21 , wherein said multimer is a dimer, tetramer, or octamer.
23. The delivery construct of claim 22 , wherein said dimer is an antibody.
24. A polynucleotide that encodes an isolated delivery construct, said delivery construct comprising:
a)—a receptor binding domain,
b)—a transcytosis domain,
c)—a peptide, polypeptide, or protein to be delivered to a subject, and
d)—a cleavable linker,
wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject, and said peptide, polypeptide, or protein is separated from the receptor binding domain and the transcytosis domain, by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell in said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
25. 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 said polylinker insertion site is oriented relative to said nucleic acid sequence encoding a cleavable linker to allow cleavage of the cleavable linker to separate a peptide, polypeptide, or protein that is encoded by a nucleic acid inserted into said polylinker insertion site from the receptor binding domain and the transcytosis domain, and wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell of a subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
26. An expression vector comprising the polynucleotide of claim 24 or 25 .
27. A cell comprising the expression vector of claim 26 .
28. A composition comprising a delivery construct, said delivery construct comprising:
a)—a receptor binding domain,
b)—a transcytosis domain,
c)—a peptide, polypeptide, or protein to be delivered to a subject, and
d)—a cleavable linker,
wherein upon transcytosis of said delivery construct across a normal, polarized epithelial cell in said subject, said cleavable linker is cleaved at a basal-lateral membrane of the polarized epithelial cell or in the plasma in said subject, and said peptide, polypeptide, or protein is separated from the receptor binding domain and the transcytosis domain, by an enzyme that i) exhibits greater activity at a basal-lateral membrane of a normal, polarized epithelial cell in said subject than at an apical membrane of the polarized epithelial cell, or ii) exhibits greater activity in the plasma in said subject than at an apical membrane of the polarized epithelial cell in the subject.
29. The composition of claim 28 , wherein said composition further comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier.
30. The composition of claim 28 , wherein said composition is formulated for nasal or oral administration.
31. The delivery construct of claim 5 , wherein said receptor binding domain of antibodies is a receptor binding domain of a monoclonal antibody or a single chain antibody.