U.S. patent application number 11/664787 was filed with the patent office on 2009-11-19 for methods and compositions for needleless delivery of macromolecules.
This patent application is currently assigned to Trinity Biosystems, Inc.. Invention is credited to Randall J. Mrsny.
Application Number | 20090285771 11/664787 |
Document ID | / |
Family ID | 36203399 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285771 |
Kind Code |
A1 |
Mrsny; Randall J. |
November 19, 2009 |
Methods and compositions for needleless delivery of
macromolecules
Abstract
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.
Inventors: |
Mrsny; Randall J.; (Los
Altos Hills, CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Trinity Biosystems, Inc.
Menlo Park
CA
|
Family ID: |
36203399 |
Appl. No.: |
11/664787 |
Filed: |
October 4, 2005 |
PCT Filed: |
October 4, 2005 |
PCT NO: |
PCT/US05/35803 |
371 Date: |
December 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60615970 |
Oct 4, 2004 |
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60684484 |
May 24, 2005 |
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60718907 |
Sep 19, 2005 |
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Current U.S.
Class: |
424/85.2 ;
424/178.1; 424/85.5; 424/85.6; 424/85.7; 435/252.33; 435/254.2;
435/320.1; 435/325; 514/1.1; 514/44R; 536/23.1 |
Current CPC
Class: |
Y02A 50/473 20180101;
A61K 47/6415 20170801; A61P 5/06 20180101; C07K 2319/55 20130101;
A61K 47/65 20170801; A61K 38/28 20130101; C07K 2319/74 20130101;
Y02A 50/471 20180101; C07K 2319/50 20130101; A61K 38/27 20130101;
Y02A 50/30 20180101; A61K 38/212 20130101 |
Class at
Publication: |
424/85.2 ; 514/2;
424/178.1; 514/12; 514/44.R; 514/3; 424/85.5; 424/85.6; 424/85.7;
536/23.1; 435/320.1; 435/252.33; 435/254.2; 435/325 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 39/395 20060101 A61K039/395; A61K 38/18 20060101
A61K038/18; A61K 38/20 20060101 A61K038/20; A61K 31/7088 20060101
A61K031/7088; A61K 38/21 20060101 A61K038/21; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 5/10 20060101
C12N005/10 |
Claims
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 .alpha.; 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 .alpha.2-macroglobulin receptor, epidermal
growth factor receptor, transferrin receptor, chemokine receptor,
CD25, CD11B, CD11C, CD80, CD86, TNF.alpha. 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-.alpha., IFN-.beta., IFN-.gamma., 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-.alpha..
18. The delivery construct of claim 12, wherein said protein is
human IFN-.alpha.2b.
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 .alpha.; 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 .alpha.2-macroglobulin receptor, EGFR, IGFR,
transferrin receptor, chemokine receptor, CD25, CD11B, CD11C, CD80,
CD86, TNF.alpha. 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-.alpha., IFN-.alpha., 2b, IFN-.beta., IFN-.gamma., 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 .alpha.; 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 .alpha.2-macroglobulin receptor, EGFR, IGFR, transferrin
receptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86,
TNF.alpha. 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-.alpha., IFN-.beta., IFN-.gamma., 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.
Description
[0001] This application is entitled to and claims benefit of U.S.
Provisional Application No. 60/615,970, filed Oct. 4, 2004, of U.S.
Provisional Application No. 60/684,484, filed May 24, 2005, and of
U.S. Provisional Application No. 60/718,907, filed Sep. 19, 2005,
each of which is hereby incorporated by reference in its
entirety.
1. FIELD OF THE INVENTION
[0002] The present invention relates, in part, to methods and
compositions for needleless delivery of macromolecules to a
subject. In one aspect, the methods and compositions involve
administering to the subject a delivery construct comprising the
macromolecule to be delivered, wherein the macromolecule is linked
to the remainder of the construct with a linker that is cleavable
at a basal-lateral membrane of a polarized epithelial cell.
2. BACKGROUND
[0003] Advances in biochemistry and molecular biology have resulted
identification and characterization of many therapeutic
macromolecules, including, for example, growth hormone,
erythropoietin, insulin, IGF, and the like. Administration of these
molecules can result in drastic improvements in quality of life for
subjects afflict with a wide range of ailments.
[0004] However, administration of these therapeutic macromolecules
remains problematic. Currently, therapeutic macromolecules are
typically administered by injection. Such injections require
penetration of the subject's skin and tissues and are associated
with pain. Further, penetration of the skin breaches one effective
nonspecific mechanism of protection against infection, and thus can
lead to potentially serious infection.
[0005] Previous attempts have been made for needleless delivery of
macromolecules to subjects. See, e.g., International Patent
Publication No. WO 01/30,392. In these efforts, delivery vehicles
are used to deliver macromolecules to a subject through polarized
epithelial cells. However, these derivatives lack a cleavable
linker that is cleavable by an enzyme at the basal-lateral membrane
of a polarized epithelial cell. Without cleavage, the probability
of induction of an immune response against the delivery vehicle
and/or the macromolecule to be delivered is increased. Also, steric
hindrance between the delivery vehicle and the macromolecule can
reduce the activity of the macromolecule, reducing efficacy the
treatment.
[0006] Accordingly, there is an unmet need for new methods and
compositions that can be used to administer macromolecules to
subjects without breaching the skin of the subject. This and other
needs are met by the methods and compositions of the present
invention.
3. SUMMARY OF THE INVENTION
[0007] The delivery constructs of the invention comprise a
macromolecule for delivery to a subject that is linked to the
remainder of the construct with a cleavable linker. The linker is
cleavable by an enzyme or an environmental cue that is present at
the basal-lateral membrane of an epithelial cell.
[0008] Accordingly, in certain aspects, 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. Cleavage at the cleavable linker can
separate the macromolecule from the remainder of the delivery
construct. In certain embodiments, the cleavable linker can be
cleavable by an enzyme that is present at a basal-lateral membrane
of a polarized epithelial cell of the subject. In other
embodiments, the cleavable linker can be cleavable by an enzyme
that is present in the plasma of said subject.
[0009] In another aspect, the invention provides a polynucleotide
that encodes a delivery construct comprising a receptor binding
domain, a transcytosis domain, a macromolecule to be delivered to a
subject, and a cleavable linker. Cleavage at the cleavable linker
can separate the macromolecule from the remainder of the delivery
construct. In certain embodiments, the cleavable linker can be
cleavable by an enzyme that is present at a basal-lateral membrane
of a polarized epithelial cell of the subject. In other
embodiments, the cleavable linker can be cleavable by an enzyme
that is present in the plasma of said subject.
[0010] In other embodiments, the polynucleotide that encodes a
delivery construct comprises a nucleic acid sequence encoding a
receptor binding domain, a nucleic acid sequence encoding a
transcytosis domain, a nucleic acid sequence encoding a cleavable
linker, and a nucleic acid sequence comprising a polylinker
insertion site. The polylinker insertion site can be oriented
relative to the nucleic acid sequence encoding a cleavable linker
to allow to cleavage of the cleavable linker to separate a
macromolecule that is encoded by a nucleic acid inserted into the
polylinker insertion site from the remainder of the encoded
delivery construct. In certain embodiments, the cleavable linker
can be cleavable by an enzyme that is present at a basal-lateral
membrane of a polarized epithelial cell of the subject. In other
embodiments, the cleavable linker can be cleavable by an enzyme
that is present in the plasma of said subject.
[0011] In another aspect, the invention provides an expression
vector comprising a polynucleotide of the invention.
[0012] In still another aspect, the invention provides a cell
comprising an expression vector of the invention.
[0013] In yet another aspect, the invention provides a composition
comprising a delivery construct of the invention. In certain
embodiments, the composition is a pharmaceutical composition.
[0014] In still another aspect, the invention provides a method for
delivering a macromolecule to a subject. The method comprises
contacting an apical surface of a polarized epithelial cell of the
subject with a delivery construct of the invention. The delivery
construct can comprise a receptor binding domain, a transcytosis
domain, a cleavable linker, and the macromolecule to be delivered.
The transcytosis domain can transcytose the macromolecule to and
through the basal-lateral membrane of the epithelial cell. In
certain embodiments, the cleavable linker can be cleaved by an
enzyme that is present at a basal-lateral membrane of a polarized
epithelial cell of the subject. In other embodiments, the cleavable
linker can be cleavable by an enzyme that is present in the plasma
of the subject. Cleavage at the cleavable linker can separate the
macromolecule from the remainder of the delivery construct, and can
deliver the macromolecule to the subject free from the remainder of
the construct.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1D present micrographs showing adhesion to and
transport across mouse tracheal epithelium of an exemplary delivery
construct comprising green fluorescent protein (GFP) (Panels A-C),
while GFP alone does not adhere to the epithelial cells (Panel
D).
[0016] FIG. 2 presents a time course of serum nt-PE-GFP
concentrations following intranasal administration of 100 .mu.g
nt-PE-GFP to anesthetized mice.
[0017] FIG. 3 presents the amino acid sequence of an exemplary
PE.
[0018] FIG. 4 presents a western blot snowing transport and
cleavage of an exemplary delivery construct comprising rat growth
hormone (rGH) by human intestinal epithelial cell monolayers. The
delivery construct was incubated in contact with the apical side of
the epithelial cell monolayer for 4 hours. Media isolated from the
basolateral side of the membrane post-incubation (lane 1) contained
rGH of native apparent molecular weight, while media from the
apical side of the membrane (lane 2) contained intact delivery
construct. Lanes 3 and 4 show intact Delivery Construct 2 and
recombinant rGH, respectively.
[0019] FIG. 5 presents serum rGH concentrations in BALB/c mice
which were dosed by subcutaneous (SC) injection of 30 .mu.g of
non-glycosylated recombinant rat GH. Individual mice sera were
tested at a dilution of 1:10, and the group average of rGH
concentration were reported (n=4 mice per time point). Standard
error of the mean (SEM) was indicated by the error bars.
[0020] FIG. 6 presents serum rGH concentrations in BALB/c mice
which were dosed orally with 100 .mu.g of Delivery Construct 2.
Individual mice sera were tested at a dilution of 1:10, and the
group average of rGH concentration were reported (n=4 mice per time
point). Standard error of the mean (SEM) was indicated by the error
bars.
[0021] FIG. 7 presents a graphical representation comparing
pharmacokinetics of rGH delivered subcutaneously and with Delivery
Construct 2.
[0022] FIG. 8 shows expression levels of IGF-1-BP3 mRNA in the
liver of female BALB/c mice treated with 30 .mu.g recombinant rGH
by subcutaneous injection or with 100 .mu.g of Delivery Construct 2
by oral gavage. Total RNA extracted from the liver was subjected to
quantitative RT-PCR using primers specific for IGF-1-BP3, as
described above. Values were normalized to glyceraldehyde-3
phosphate dehydrogenase (GAPDH) and expressed as % of control.
[0023] FIG. 9 shows expression levels of growth hormone (GH)
receptor mRNA in the liver of female BALB/c mice treated with rGH
by subcutaneous injection (30 .mu.g) or Delivery Construct 2 by
oral gavage (100 .mu.g). Total RNA extracted from the liver was
subjected to quantitative RT-PCR using primers specific for GH
receptor, shown above. Values were normalized to glyceraldehyde-3
phosphate dehydrogenase (GAPDH) and expressed as % of control.
[0024] FIG. 10 snows expression levels or insulin-like growth
factor I (TGF-I); mRNA in the liver of female BALB/c mice treated
with rGH by subcutaneous injection (30 .mu.g) or Delivery Construct
2 by oral gavage (100 .mu.g). Total RNA extracted from the liver
was subjected to quantitative RT-PCR using primers specific for
IGF-I, shown above. Values were normalized to glyceraldehyde-3
phosphate dehydrogenase (GAPDH) and expressed as % of control.
[0025] FIGS. 11A and B show serum anti-rGH IgG antibodies (diluted
1:25 in FIG. 11A and 1:200 in FIG. 11B) of mice orally administered
3,10, or 30 .mu.g Delivery Construct 2 (F2) or subcutaneously
administered 3 or 10 .mu.g rGH in graphs with error bars, while
FIGS. 11C and D presents the same data from individual animals.
[0026] FIG. 12 shows a nucleotide sequence that encodes Delivery
Construct 6 (SEQ ID NO:34), an exemplary Delivery Construct for
delivering human growth hormone (hGH).
[0027] FIGS. 13A and B show the amino acid sequence of Delivery
Construct 6 (SEQ ID NO:35), an exemplary Delivery Construct for
delivering hGH.
[0028] FIG. 14 shows a nucleotide sequence that encodes Delivery
Construct 7 (SEQ ID NO:36), an exemplary Delivery Construct for
delivering interferon-.alpha. (IFN-.alpha.).
[0029] FIGS. 15A and B show the amino acid sequence of Delivery
Construct 7 (SEQ ID NO:37), an exemplary Delivery Construct for
delivering IFN-.alpha..
[0030] FIG. 16 shows 6 presents serum IFN-.alpha. concentrations in
BALB/c mice which were dosed orally with 100 .mu.g of Delivery
Construct 8. Individual mice sera were tested at a dilution of
1:10, and the group average of IFN-.alpha. concentration were
reported (n=3 mice per time point).
[0031] FIGS. 17A and B show the amino acid sequence of Delivery
Construct 8 (SEQ ID NO:38), an exemplary Delivery Construct for
delivering proinsulin.
[0032] FIGS. 18 A and B shows the amino acid sequence of the two
amino acid chains of Delivery Construct 9 (SEQ ID NOs:39 and 40,
respectively), an exemplary Delivery Construct for delivering
insulin. Disulfide bonds are formed between the cysteine at
position 7 of SEQ ID NO:40 (shown in FIG. 18B) and the cysteine at
position 381 of SEQ ID NO:39 (shown in FIG. 18A) and between the
cysteine at position 20 of SEQ ID NO:40 and the cysteine at
position 393 of SEQ ID NO:39.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Definitions
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. As used herein,
the following terms have the meanings ascribed to them unless
specified otherwise.
[0034] A "ligand" is a compound that specifically binds to a target
molecule. Exemplary ligands include, but are not limited to, an
antibody, a cytokine, a substrate, a signaling molecule, and the
like.
[0035] A "receptor" is compound that specifically binds to a
ligand.
[0036] A ligand or a receptor (e.g., an antibody) "specifically
binds to" or "is specifically immunoreactive with" another molecule
when the ligand or receptor functions in a binding reaction that
indicates the presence of the molecule in a sample of heterogeneous
compounds. Thus, under designated assay (e.g., immunoassay)
conditions, the ligand or receptor binds preferentially to a
particular compound and does not bind in a significant amount to
other compounds present in the sample. For example, a
polynucleotide specifically binds under hybridization conditions to
another polynucleotide comprising a complementary sequence and an
antibody specifically binds under immunoassay conditions to an
antigen bearing an epitope used to induce the antibody.
[0037] "Immunoassay" refers to a method of detecting an analyte in
a sample involving contacting the sample with an antibody that
specifically binds to the analyte and detecting binding between the
antibody and the analyte. A variety of immunoassay formats may be
used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity. In one
example, an antibody that binds a particular antigen with an
affinity (K.sub.m) of about 10 .mu.M specifically binds the
antigen.
[0038] "Linker" 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 linker" refers to a
linker that can be degraded or otherwise severed to separate the
two components connected by the cleavable linker. Cleavable linkers
are generally cleaved by enzymes, typically peptidases, proteases,
nucleases, lipases, and the like. Cleavable linkers may also be
cleaved by environmental cues, such as, for example, changes in
temperature, pH, salt concentration, etc. when there is such a
change in environment following transcytosis of the delivery
construct across a polarized epithelial membrane.
[0039] "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 pharmacological result.
"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, 21st Ed. 2005, Mack Publishing
Co., Easton. A "pharmaceutically acceptable salt" is a salt that
can be formulated into a compound for pharmaceutical use including,
e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and
salts of ammonia or organic amines.
[0040] Preferred pharmaceutical carriers depend upon the intended
mode of administration of the active agent. Typical modes of
administration include enteral (e.g., oral, intranasal, rectal, Or
vaginal) or parenteral (e.g., subcutaneous, intramuscular,
intravenous or intraperitoneal injection; or topical (e.g.,
transdermal, or transmucosal administration).
[0041] "Small organic molecule" refers to organic molecules of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes organic biopolymers (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, up to about 2000 Da, or up to
about 1000 Da.
[0042] A "subject" of diagnosis, treatment, or administration is a
human or non-human animal, including a mammal or a primate, and
preferably a human.
[0043] "Treatment" refers to prophylactic treatment or therapeutic
treatment. A "prophylactic" treatment is a treatment administered
to a subject who does not exhibit signs of a disease or exhibits
only early signs for the purpose of decreasing the risk of
developing pathology. A "therapeutic" treatment is a treatment
administered to a subject who exhibits signs of pathology for the
purpose of diminishing or eliminating those signs.
[0044] "Pseudomonas exotoxin A" or "PE" is secreted by Pseudomonas
aeruginosa as a 67 kD protein composed of three prominent globular
domains (Ia, II, and III) and one small subdomain (Ib) that
connects domains II and III. See A. S. Allured et al., 1986, Proc.
Natl. Acad. Set 83:1320-1324. Without intending to be bound to any
particular theory or mechanism of action, domain Ia of PE is
believed to mediate cell binding because domain Ia specifically
binds to the low density lipoprotein receptor-related protein
("LRP"), also known as the .alpha.2-macroglobulin receptor
(".alpha.2-MR") and CD-91. See M. Z. Kounnas et al., 1992, J. Biol.
Chem. 267:12420-23. Domain Ia spans amino acids 1-252. Domain II of
PE is believed to mediate transcytosis to the interior of a cell
following binding of domain Ia to the .alpha.2-MR. Domain II spans
amino acids 253-364. Certain portions of this domain may be
required for secretion of PE from Pseudomonas aeruginosa after its
synthesis. See, e.g., Vouloux et al., 2000, J. Bacterol.
182:4051-8. Domain Ib has no known function and spans amino acids
365-399. Domain III mediates cytotoxicity of PE and includes an
endoplasmic reticulum retention sequence. PE cytotoxicity is
believed to result from ADP ribosylation of elongation factor 2,
which inactivates protein synthesis. Domain III spans amino acids
400-613 of PE. Deleting amino acid E553 (".DELTA.E553") from domain
III eliminates EF2 ADP ribosylation activity and detoxifies PE. PE
having the mutation .DELTA.E553 is referred to herein as
"PE.DELTA.E553." Genetically modified forms of PE are described in,
e.g., U.S. Pat. Nos. 5,602,095; 5,512,658 and 5,458,878 Pseudomonas
exotoxin, as used herein, also includes genetically modified,
allelic, and chemically inactivated forms of PE within this
definition. See, e.g., Vasil et al., 1986, Infect. Immunol.
52:538-48. Further, reference to the various domains of PE is made
herein to the reference PE sequence presented as FIG. 3. However,
one or more domain from modified PE, e.g., genetically or
chemically modified PE, or a portion of such domains, can also be
used in the chimeric immunogens of the invention so long as the
domains retain functional activity. One of skill in the art can
readily identify such domains of such modified PE based on, for
example, homology to the PE sequence exemplified in FIG. 3 and test
for functional activity using, for example, the assays described
below.
[0045] "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. Thus, 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. Such polynucleotides
can be synthesized, for example, using an automated DNA
synthesizer. The term "nucleic acid" typically refers to large
polynucleotides. The term "oligonucleotide" typically refers to
short polynucleotides, generally no greater than about 50
nucleotides. It will be understood that when a 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."
[0046] Conventional notation is used herein to describe
polynucleotide sequences: the left-hand end of a single-stranded
polynucleotide sequence is the 5'-end; the left-hand direction of a
double-stranded polynucleotide sequence is referred to as the
5'-direction.
[0047] The direction of 5' to 3' addition of nucleotides to nascent
RNA transcripts is referred to as the transcription direction. 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."
[0048] "Complementary" refers to the topological compatibility or
matching together of interacting surfaces of two polynucleotides.
Thus, 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.
Thus, the polynucleotide whose sequence 5'-TATAC-3' is
complementary to a polynucleotide whose sequence is
5'-GTATA-3'.
[0049] The term "% 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. For example, as used herein, 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. Exemplary levels of sequence
identity include, but are not limited to, 60, 70, 80, 85, 90, 95,
98% or more sequence identity to a given sequence.
[0050] The term "% 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. For example, as used herein, 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.
Exemplary levels of sequence homology include, but are not limited
to, 60, 70, 80, 85, 90, 95, 98% or more sequence homology to a
given sequence.
[0051] 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, and TBLASTX, BLASTP
and TBLASTN, publicly available on the Internet at the NCBI
website. See also Altschul et al., 1990, J. Mol. Biol. 215:403-10
(with special reference to the published default setting, i.e.,
parameters w=4, t=17) and Altschul et al., 1997, Nucleic Acids
Res., 25:3389-3402. 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.
[0052] A preferred alignment of selected sequences in order to
determine "% identity" between two or more sequences, is performed
using for example, the CLUSTAL-W program in MacVector version 6.5,
operated with default parameters, including an open gap penalty of
10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity
matrix.
[0053] "Polar Amino Acid" refers to a hydrophilic amino acid having
a side chain that is uncharged at physiological pH, but which has
at least one bond in which the pair of electrons shared in common
by two atoms is held more closely by one of the atoms. Genetically
encoded polar amino acids include Asn (N), Gln (Q) Ser (S) and Thr
(T).
[0054] "Nonpolar Amino Acid" refers to a hydrophobic amino acid
having a side chain that is uncharged at physiological pH and which
has bonds in which the pair of electrons shared in common by two
atoms is generally held equally by each of the two atoms (i.e., the
side chain is not polar). Genetically encoded nonpolar amino acids
include Ala (A), Gly (G), He (I), Leu (L), Met (M) and Val (V).
[0055] "Hydrophilic Amino Acid" refers to an amino acid exhibiting
a hydrophobicity of less than zero according to the normalized
consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol.
Biol. 179:125-142. Genetically encoded hydrophilic amino acids
include Arg (R), Asn (N), Asp (D), Glu (E), Gln (Q), His (H), Lys
(K), Ser (S) and Thr (T).
[0056] "Hydrophobic Amino Acid" refers to an amino acid exhibiting
a hydrophobicity of greater than zero according to the normalized
consensus hydrophobicity scale of Eisenberg et al., 1984, J Mol.
Biol. 179:125-142. Genetically encoded hydrophobic amino acids
include Ala (A), Gly (G), He (I), Leu (L), Met (M), Phe (F), Pro
(P), Trp (W), Tyr (Y) and Val (V).
[0057] "Acidic Amino Acid" refers to a hydrophilic amino acid
having a side chain pK value of less than 7. Acidic amino acids
typically have negatively charged side chains at physiological pH
due to loss of a hydrogen ion. Genetically encoded acidic amino
acids include Asp (D) and Glu (E).
[0058] "Basic Amino Acid" refers to a hydrophilic amino acid having
a side chain pK value of greater than 7. Basic amino acids
typically have positively charged side chains at physiological pH
due to association with a hydrogen ion. Genetically encoded basic
amino acids include Arg (R), His (H) and Lys (K).
[0059] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA produced by that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and
non-coding strand, used as the template for transcription, of a
gene or cDNA can be referred to as encoding the protein or other
product of that gene or cDNA. Unless otherwise specified, a
"nucleotide sequence encoding an amino acid sequence" includes all
nucleotide sequences that are degenerate versions of each other and
that encode the same amino acid sequence. Nucleotide sequences that
encode proteins and RNA may include introns.
[0060] "Amplification" refers to any means by which a
polynucleotide sequence is copied and thus expanded into a larger
number of polynucleotide molecules, e.g., by reverse transcription,
polymerase chain reaction, ligase chain reaction, and the like.
[0061] "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 complementary
polynucleotide template, and an agent for polymerization such as
DNA polymerase. A primer is typically single-stranded, but may 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.
[0062] "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 complementary polynucleotide, a probe can also
be a primer.
[0063] "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.
[0064] The term "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. An extensive guide to the hybridization of nucleic
acids can be found in Tijssen, 1993, Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, part I, chapter 2, "Overview of principles of hybridization
and the strategy of nucleic acid probe assays", Elsevier, N.Y.;
Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, 3.sup.rd ed., NY; and Ausubel et al.,
eds., Current Edition, Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience, NY.
[0065] Generally, highly stringent hybridization and wash
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH. The 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.
[0066] One 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.degree.
C., with the hybridization being carried out overnight. An example
of highly stringent wash conditions is 0.15 M NaCl at 72.degree. C.
for about 15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. 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.times.SSC at 45.degree. C.
for 15 minutes. An exemplary low stringency wash for a duplex of,
e.g., more than about 100 nucleotides, is 4-6.times.SSC at
40.degree. C. for 15 minutes. In general, a signal to noise ratio
of 2.times. (or higher) than that observed for an unrelated probe
in the particular hybridization assay indicates detection of a
specific hybridization.
[0067] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof linked via
peptide bonds, related naturally occurring structural variants, and
synthetic non-naturally occurring analogs thereof. Synthetic
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer. Conventional notation is used herein to
portray polypeptide sequences; the beginning of a polypeptide
sequence is the amino-terminus, while the end of a polypeptide
sequence is the carboxyl-terminus.
[0068] The term "protein" typically refers to large polypeptides,
for example, polypeptides comprising more than about 50 amino
acids. The term "protein" can also refer to dimers, trimers, and
multimers that comprise more than one polypeptide.
[0069] "Conservative 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:
[0070] Alanine (A), Serine (S), and Threonine (T)
[0071] Aspartic acid (D) and Glutamic acid (E)
[0072] Asparagine (N) and Glutamine (Q)
[0073] Arginine (R) and Lysine (K)
[0074] Isoleucine (I), Leucine (L), Methionine (M), and Valine
(V)
[0075] Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).
[0076] The term "about," as used herein, unless otherwise
indicated, refers to a value that is no more than 10% above or
below the value being modified by the term. For example, the term
"about 5 .mu.g/kg" means a range of from 4.5 .mu.g/kg to 5.5
.mu.g/kg. As another example, "about 1 hour" means a range of from
48 minutes to 72 minutes.
5.2. Delivery Constructs
[0077] Generally, the delivery constructs of the present invention
are polypeptides that have structural domains corresponding to
domains Ia and II of PE. These structural domains perform certain
functions, including, but not limited to, cell recognition and
transcytosis, that correspond to the functions of the domains of
PE.
[0078] In addition to the portions of the molecule that correspond
to PE functional domains, the delivery constructs of this invention
can further comprise a macromolecule for delivery to a biological
compartment of a subject. The macromolecule can be introduced into
any portion of the delivery construct that does not disrupt a
cell-binding or transcytosis activity. The macromolecule is
connected with the remainder of the delivery construct with a
cleavable linker.
[0079] Accordingly, the delivery constructs of the invention
generally comprise the following structural elements, each element
imparting particular functions to the delivery construct: (1) a
"receptor binding domain" that functions as a ligand for a cell
surface receptor and that mediates binding of the construct to a
cell; (2) a "transcytosis domain" that mediates transcytosis from a
lumen bordering the apical surface of a mucous membrane to the
basal-lateral side of a mucous membrane; (3) the macromolecule; and
(4) a cleavable linker that connects the macromolecule to the
remainder of the delivery construct.
[0080] The delivery constructs of the invention offer several
advantages over conventional techniques for local or systemic
delivery of macromolecules to a subject. Foremost among such
advantages is the ability to deliver the macromolecule without
using a needle to puncture the skin of the subject. Many subjects
require repeated, regular doses of macromolecules. For example,
diabetics must inject insulin several times per day to control
blood sugar concentrations. Such subjects' quality of life would be
greatly improved if the delivery of a macromolecule could be
accomplished without injection, by avoiding pain or potential
complications associated therewith.
[0081] Furthermore, many embodiments of the delivery constructs can
be constructed and expressed in recombinant systems. Recombinant
technology allows one to make a delivery construct having an
insertion site designed for introduction of any suitable
macromolecule. Such insertion sites allow the skilled artisan to
quickly and easily produce delivery constructs for delivery of new
macromolecules, should the need to do so arise.
[0082] In addition, connection of the macromolecule to the
remainder of the delivery construct with a linker that is cleaved
by an enzyme present at a basal-lateral membrane of an epithelial
cell allows the macromolecule to be liberated from the delivery
construct and released from the remainder of the delivery construct
soon after transcytosis across the epithelial membrane. Such
liberation reduces the probability of induction of an immune
response against the macromolecule. It also allows the
macromolecule to interact with its target free from the remainder
of the delivery construct.
[0083] Other advantages of the delivery constructs of the invention
will be apparent to those of skill in the art.
[0084] In certain embodiments, the invention provides a delivery
construct that comprises a receptor binding domain, a transcytosis
domain, a macromolecule to be delivered to a subject, and a
cleavable linker. Cleavage at the cleavable linker separates the
macromolecule from the remainder of the construct. The cleavable
linker is cleavable by an enzyme that is present at a basal-lateral
membrane of a polarized epithelial cell of the subject or in the
plasma of the subject. In certain embodiments, the enzyme that is
at a basal-lateral membrane of a polarized epithelial cell exhibits
higher activity on the basal-lateral side of a polarized epithelial
cell than it does on the apical side of the polarized epithelial
cell. In certain embodiments, the enzyme that is in the plasma of
the subject exhibits higher activity in the plasma than it does on
the apical side of a polarized epithelial cell.
[0085] In certain embodiments, the delivery construct further
comprises a second cleavable linker. In certain embodiments, the
first and/or the second 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). In certain embodiments, the first and/or the
second 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) and is cleavable by an
enzyme that exhibits higher activity on the basal-lateral side of a
polarized epithelial cell than it does on the apical side of the
polarized epithelial cell. In certain embodiments, the first and/or
the second 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) and is cleavable by an
enzyme that exhibits higher activity in the plasma than it does on
the apical side of a polarized epithelial cell.
[0086] In certain embodiments, the 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.
[0087] In certain embodiments, the 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 .alpha.; EGF;
IGF-I; IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b;
MCAF; and IL-8. In certain embodiments, the receptor binding domain
binds to a cell-surface receptor that is selected from the group
consisting of .alpha.2-macroglobulin receptor, epidermal growth
factor receptor, transferrin receptor, chemokine receptor, CD25,
CD11B, CD11C, CD80, CD86, TNF.alpha. receptor, TOLL receptor, M-CSF
receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.
In further embodiments, the receptor binding domain of Pseudomonas
exotoxin A is Domain Ia of Pseudomonas exotoxin A. In yet further
embodiments, the receptor binding domain of Pseudomonas exotoxin A
has an amino acid sequence that is SEQ ID NO.:1.
[0088] In certain embodiments, the 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. In further embodiments, the transcytosis domain
is Pseudomonas exotoxin A transcytosis domain. In still further
embodiments, the Pseudomonas exotoxin A transcytosis domain has an
amino acid sequence that is SEQ ID NO.:2.
[0089] In certain embodiments, the macromolecule is selected from
the group of a nucleic acid, a peptide, a polypeptide, a protein,
and a lipid, hi further embodiments, the polypeptide is selected
from the group consisting of polypeptide hormones, cytokines,
chemokines, growth factors, and clotting factors. In yet further
embodiments, the polypeptide is selected from the group consisting
of IGF-I, IGF-II, IGF-III, EGF, IFN-.alpha., IFN-.beta.,
IFN-.gamma., 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, adrenocorticototropin, enkephalin, and
glucagon-like peptide 1. In still further embodiments, the
polypeptide is human growth hormone. In other embodiments, the
protein is human insulin.
[0090] In certain embodiments, the delivery constructs further
comprise a second macromolecule that is selected from the group
consisting of a nucleic acid, a peptide, a polypeptide, a protein,
a lipid, and 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. In
certain embodiments, the first macromolecule is a first polypeptide
and said second macromolecule is a second polypeptide. In certain
embodiments, the first polypeptide and the second polypeptide
associate to form a multimer. In certain embodiments, the multimer
is a dimer, tetramer, or octamer. In further embodiments, the dimer
is an antibody.
[0091] 5.2.1. Receptor Binding Domain
[0092] The delivery constructs of the invention generally comprise
a receptor binding domain. The receptor binding domain can be any
receptor binding domain known to one of skill in the art without
limitation to bind to a cell surface receptor that is present on
the apical membrane of an epithelial cell. Preferably, the receptor
binding domain binds specifically to the cell surface receptor. The
receptor binding domain should bind to the cell surface receptor
with sufficient affinity to allow endocytosis of the delivery
construct.
[0093] In certain embodiments, the receptor binding domain can
comprise a peptide, a polypeptide, a protein, a lipid, a
carbohydrate, or a small organic molecule, or a combination
thereof. Examples of each of these molecules that bind to cell
surface receptors present on the apical membrane of epithelial
cells are well known to those of skill in the art. Suitable
peptides or polypeptides include, but are not limited to, bacterial
toxin receptor binding domains, such as the receptor binding
domains from PE, cholera toxin, botulinum toxin, diptheria toxin,
shiga toxin, shiga-like toxin, etc.; antibodies, including
monoclonal, polyclonal, and single-chain antibodies, or derivatives
thereof, growth factors, such as EGF, IGF-I, IGF-II, IGF-III etc.;
cytokines, such as IL-1, IL-2, IL-3, IL-6, etc; chemokines, such as
MIP-1a, MIP-1b, MCAF, IL-8, etc.; and other ligands, such as CD4,
cell adhesion molecules from the immunoglobulin superfamily,
integrins, ligands specific for the IgA receptor, etc. See, e.g.,
Pastane et al., 1992, Annu. Rev. Biochem. 61:331-54; and U.S. Pat.
Nos. 5,668,255, 5,696,237, 5,863,745, 5,965,406, 6,022,950,
6,051,405, 6,251,392, 6,440,419, and 6,488,926. The skilled artisan
can select the appropriate receptor binding domain based upon the
expression pattern of the receptor to which the receptor binding
domain binds.
[0094] Lipids suitable for receptor binding domains include, but
are not limited to, lipids that themselves bind cell surface
receptors, such as sphingosine-1-phosphate, lysophosphatidic acid,
sphingosylphosphorylcholine, retinoic acid, etc.; lipoproteins such
as apolipoprotein E, apolipoprotein A, etc., and glycolipids such
as lipopolysaccharide, etc.; glycosphingolipids such as
globotriaosylceramide and galabiosylceramide; and the like.
Carbohydrates suitable for receptor binding domains include, but
are not limited to, monosaccharides, disaccharides, and
polysaccharides that comprise simple sugars such as glucose,
fructose, galactose, etc.; and glycoproteins such as mucins,
selectins, and the like. Suitable small organic molecules for
receptor binding domains include, but are not limited to, vitamins,
such as vitamin A, B.sub.1, B.sub.2, B.sub.3, B.sub.6, B.sub.9,
B.sub.12, C, D, E, and K, amino acids, and other small molecules
that are recognized and/or taken up by receptors present on the
apical surface of epithelial cells. U.S. Pat. No. 5,807,832
provides an example of such small organic molecule receptor binding
domains, vitamin B.sub.12.
[0095] In certain embodiments, the receptor binding domain can bind
to a receptor found on an epithelial cell. In further embodiments,
the receptor binding domain can bind to a receptor found on the
apical membrane of an epithelial cell. The receptor binding domain
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. For example, the receptor binding domain can bind to
.alpha.2-MR, EGFR, or IGFR. An example of a receptor binding domain
that can bind to .alpha.2-MR is domain Ia of PE. Accordingly, in
certain embodiments, the receptor binding domain is domain Ia of
PE. In other embodiments, the receptor binding domain is a portion
of domain Ia of PE that can bind to .alpha.2-MR. Exemplary receptor
binding domains that can bind to EGFR include, but are not limited
to, EGF and TGF.alpha.. Examples of receptor binding domains that
can bind to IGFR include, but are not limited to, IGF-I, IGF-II, or
IGF-III. Thus, in certain embodiments, the receptor binding domain
is EGF, IGF-I, IGF-II, or IGF-III. In other embodiments, the
receptor binding domain is a portion of EGF, IGF-I, IGF-II, or
IGF-III that can bind to the EGF or IGF receptor.
[0096] In certain embodiments, the receptor binding domain binds to
a receptor that is highly expressed on the apical membrane of a
polarized epithelial cell but is not expressed or expressed at low
levels on antigen presenting cells, such as, for example, dendritic
cells. Exemplary receptor binding domains that have this kind of
expression pattern include, but are not limited to, TGF.alpha.,
EGF, IGF-I, IGF-II, and IGF-III.
[0097] In certain embodiments, the delivery constructs of the
invention comprise more than one domain that can function as a
receptor binding domain. For example, the delivery construct can
comprise PE domain Ia in addition to another receptor binding
domain.
[0098] The receptor binding domain can be attached to the remainder
of the delivery construct by any method or means known by one of
skill in the art to be useful for attaching such molecules, without
limitation. In certain embodiments, the receptor binding domain is
expressed together with the remainder of the delivery construct as
a fusion protein. Such embodiments are particularly useful when the
receptor binding domain and the remainder of the construct are
formed from peptides or polypeptides.
[0099] In other embodiments, the receptor binding domain is
connected with the remainder of the delivery construct with a
linker. In yet other embodiments, the receptor binding domain is
connected with the remainder of the delivery construct without a
linker. Either of these embodiments are useful when the receptor
binding domain comprises a peptide, polypeptide, protein, lipid,
carbohydrate, nucleic acid, or small organic molecule.
[0100] In certain embodiments, the linker can form a covalent bond
between the receptor binding domain and the remainder of the
delivery construct. In certain embodiments, the covalent bond can
be a peptide bond. In other embodiments, the linker can link the
receptor binding domain to the remainder of the delivery construct
with one or more non-covalent interactions of sufficient affinity.
One of skill in the art can readily recognize linkers that interact
with each other with sufficient affinity to be useful in the
delivery constructs of the invention. For example, biotin can be
attached to the receptor binding domain, and streptavidin can be
attached to the remainder of the molecule. In certain embodiments,
the linker can directly link the receptor binding domain to the
remainder of the molecule. In other embodiments, the linker itself
comprises two or more molecules that associate in order to link the
receptor binding domain to the remainder of the molecule. Exemplary
linkers include, but are not limited to, straight or branched-chain
carbon linkers, heterocyclic carbon linkers, substituted carbon
linkers, unsaturated carbon linkers, aromatic carbon linkers,
peptide linkers, etc.
[0101] In embodiments where a linker is used to connect the
receptor binding domain to the remainder of the delivery construct,
the linkers can be attached to the receptor binding domain and/or
the remainder of the delivery construct by any means or method
known by one of skill in the art without limitation. For example,
the linker can be attached to the receptor binding domain and/or
the remainder of the delivery construct with an ether, ester,
thioether, thioester, amide, imide, disulfide, peptide, or other
suitable moiety. The skilled artisan can select the appropriate
linker and method for attaching the linker based on the physical
and chemical properties of the chosen receptor binding domain and
the linker. The linker can be attached to any suitable functional
group on the receptor binding domain or the remainder of the
molecule. For example, the linker can be attached to sulfhydryl
(--S), carboxylic acid (COOH) or free amine (--NH.sub.2) groups,
which are available for reaction with a suitable functional group
on a linker. These groups can also be used to connect the receptor
binding domain directly connected with the remainder of the
molecule in the absence of a linker.
[0102] Further, the receptor binding domain and/or the remainder of
the delivery construct can be derivatized in order to facilitate
attachment of a linker to these moieties. For example, such
derivatization can be accomplished by attaching suitable derivative
such as those available from Pierce Chemical Company, Rockford,
Ill. Alternatively, derivatization may involve chemical treatment
of the receptor binding domain and/or the remainder of the
molecule. For example, glycol cleavage of the sugar moiety of a
carbohydrate or glycoprotein receptor binding domain with periodate
generates free aldehyde groups. These free aldehyde groups may be
reacted with free amine or hydrazine groups on the remainder of the
molecule in order to connect these portions of the molecule. See,
e.g., U.S. Pat. No. 4,671,958. Further, the skilled artisan can
generate free sulfhydryl groups on proteins to provide a reactive
moiety for making a disulfide, thioether, thioester, etc. linkage.
See, e.g., U.S. Pat. No. 4,659,839.
[0103] Any of these methods for attaching a linker to a receptor
binding domain and/or the remainder of a delivery construct can
also be used to connect a receptor binding domain with the
remainder of the delivery construct in the absence of a linker. In
such embodiments, the receptor binding domain is coupled with the
remainder of the construct using a method suitable for the
particular receptor binding domain. Thus, any method suitable for
connecting a protein, peptide, polypeptide, nucleic acid,
carbohydrate, lipid, or small organic molecule to the remainder of
the delivery construct known to one of skill in the art, without
limitation, can be used to connect the receptor binding domain to
the remainder of the construct. In addition to the methods for
attaching a linker to a receptor binding domain or the remainder of
a delivery construct, as described above, the receptor binding
domain can be connected with the remainder of the construct as
described, for example, in U.S. Pat. Nos. 6,673,905; 6,585,973;
6,596,475; 5,856,090; 5,663,312; 5,391,723; 6,171,614; 5,366,958;
and 5,614,503.
[0104] In certain embodiments, the receptor binding domain can be a
monoclonal antibody. In some of these embodiments, the chimeric
immunogen is expressed as a fusion protein that comprises an
immunoglobulin heavy chain from an immunoglobulin specific for a
receptor on a cell to which the chimeric immunogen is intended to
bind. The light chain of the immunoglobulin then can be
co-expressed with the chimeric immunogen, thereby forming a light
chain-heavy chain dimer. In other embodiments, the antibody can be
expressed and assembled separately from the remainder of the
chimeric immunogen and chemically linked thereto.
[0105] 5.2.2. Transcytosis Domain
[0106] The delivery constructs of the invention also comprise a
transcytosis domain. The transcytosis domain can be any
transcytosis domain known by one of skill in the art to effect
transcytosis of chimeric proteins that have bound to a cell surface
receptor present on the apical membrane of an epithelial cell. In
certain embodiments, the transcytosis domain is a transcytosis
domain from PE, diptheria toxin, pertussis toxin, cholera toxin,
heat-labile E. coli enterotoxin, shiga toxin, or shiga-like toxin.
See, for example, U.S. Pat. Nos. 5,965,406, and 6,022,950. In
preferred embodiments, the transcytosis domain is domain II of
PE.
[0107] The transcytosis domain need not, though it may, comprise
the entire ammo acid sequence of domain II of native PE, which
spans residues 253-364 of PE. For example, the transcytosis domain
can comprise a portion of PE that spans residues 280-344 of domain
II of PE. The amino acids at positions 339 and 343 appear to be
necessary for transcytosis. See Siegall et al., 1991, Biochemistry
30:7154-59. Further, conservative or nonconservative substitutions
can be made to the amino acid sequence of the transcytosis domain,
as long as transcytosis activity is not substantially eliminated. 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 below.
[0108] Without intending to be limited to any particular theory or
mechanism of action, the transcytosis domain is believed to permit
the trafficking of the delivery construct through a polarized
epithelial cell after the construct binds to a receptor present on
the apical surface of the polarized epithelial cell. Such
trafficking through a polarized epithelial cell is referred to
herein as "transcytosis." This trafficking permits the release of
the delivery construct from the basal-lateral membrane of the
polarized epithelial cell.
[0109] 5.2.3. Macromolecules for Delivery
[0110] The delivery constructs of the invention can also comprise a
macromolecule. The macromolecule can be attached to the remainder
of the delivery construct by any method known by one of skill in
the art, without limitation. In certain embodiments, the
macromolecule is expressed together with the remainder of the
delivery construct as a fusion protein. In such embodiments, the
macromolecule can be inserted into or attached to any portion of
the delivery construct, so long as the receptor binding domain, the
transcytosis domain, and macromolecule retain their activities. The
macromolecule is connected with the remainder of the construct with
a cleavable linker, or a combination of cleavable linkers, as
described below.
[0111] In native PE, the Ib loop (domain Ib) spans amino acids 365
to 399, and is structurally characterized by a disulfide bond
between two cysteines at positions 372 and 379. This portion of PE
is not essential for any known activity of PE, including cell
binding, transcytosis, ER retention or ADP ribosylation activity.
Accordingly, domain Ib can be deleted entirely, or modified to
contain a macromolecule.
[0112] Thus, in certain embodiments, the macromolecule can be
inserted into domain Ib. If desirable, the macromolecule can be
inserted into domain Ib wherein the cysteines at positions 372 and
379 are not cross-linked, this can be accomplished by reducing the
disulfide linkage between the cysteines, by deleting the cysteines
entirely from the Ib domain, by mutating the cysteines to other
residues, such as, for example, serine, or by other similar
techniques. Alternatively, the macromolecule can be inserted into
the Ib loop between the cysteines at positions 372 and 379. In such
embodiments, the disulfide linkage between the cysteines can be
used to constrain the macromolecule if desirable. In any event, in
embodiments where the macromolecule is inserted into domain Ib of
PE, or into any other portion of the delivery construct, the
macromolecule should be flanked by cleavable linkers such that
cleavage at the cleavable linkers liberates the macromolecule from
the remainder of the construct.
[0113] In other embodiments, the macromolecule can be connected
with the N-terminal or C-terminal end of a polypeptide portion of
the delivery construct. In such embodiments, the method of
connection should be designed to avoid interference with other
functions of the delivery construct, such as receptor binding or
transcytosis. In yet other embodiments, the macromolecule can be
connected with a side chain of an amino acid of the delivery
construct. The macromolecule is connected with the remainder of the
delivery construct with a cleavable linker, as described below. In
such embodiments, the macromolecule to be delivered can be
connected with the remainder of the delivery construct with one or
more cleavable linkers such that cleavage at the cleavable
linker(s) separates the macromolecule from the remainder of the
delivery construct. It should be noted that, in certain
embodiments, the macromolecule of interest can also comprise a
short (1-20 amino acids, preferably 1-10 amino acids, and more
preferably 1-5 amino acids) leader peptide in addition to the
macromolecule of interest that remains attached to the
macromolecule following cleavage of the cleavable linker.
Preferably, this leader peptide does not affect the activity or
immunogenicity of the macromolecule.
[0114] In embodiments where the macromolecule is expressed together
with another portion of the delivery construct as a fusion protein,
the macromolecule can be can be inserted into the delivery
construct by any method known to one of skill in the art without
limitation. For example, amino acids corresponding to the
macromolecule can be inserted directly into the delivery construct,
with or without deletion of native amino acid sequences. In certain
embodiments, all or part of the Ib domain of PE can be deleted and
replaced with the macromolecule. In certain embodiments, the
cysteine residues of the Ib loop are deleted so that the
macromolecule remains unconstrained. In other embodiments, the
cysteine residues of the Ib loop are linked with a disulfide bond
and constrain the macromolecule.
[0115] The macromolecule can be any macromolecule that is desired
to be introduced into a subject. Thus, the macromolecule can be a
peptide, a polypeptide, a protein, a nucleic acid, a carbohydrate,
a lipid, a glycoprotein, synthetic organic and inorganic compounds,
or any combination thereof. The macromolecule can also be a
detectable compound such as a radiopaque compound, including air
and barium and magnetic compounds. In certain embodiments, the
macromolecule can be either soluble or insoluble in water. In
certain embodiments, the macromolecule can be a macromolecule that
can perform a desirable biological activity when introduced to the
bloodstream of the subject. For example, the macromolecule can have
receptor binding activity, enzymatic activity, messenger activity
(i.e., act as a hormone, cytokine, neurotransmitter, or other
signaling molecule), luminescent or other detectable activity, or
regulatory activity, or any combination thereof. In, for example,
diagnostic embodiments, the macromolecule can be conjugated to or
can itself be a pharmaceutically acceptable gamma-emitting moiety,
including but not limited to, indium and technetium, magnetic
particles, radiopaque materials such as air or barium and
fluorescent compounds.
[0116] In other embodiments, the macromolecule that is delivered
can exert its effects in biological compartments of the subject
other than the subject's blood. For example, in certain
embodiments, the macromolecule can exert its effects in the
lymphatic system. In other embodiments, the macromolecule 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. In such embodiments, the macromolecule may or may not
be present in the blood, lymph, or other biological fluid at
detectable concentrations, yet may still accumulate at sufficient
concentrations at its site of action to exert a biological
effect.
[0117] Further, the macromolecule can be a protein that comprises
more than one polypeptide subunit. For example, the protein can be
a dimer, trimer, or higher order multimer. In certain embodiments,
two or more subunits of the protein can be connected with a
covalent bond, such as, for example, a disulfide bond. In other
embodiments, the subunits of the protein can be held together with
non-covalent interactions. One of skill in the art can routinely
identify such proteins and determine whether the subunits are
properly associated using, for example, an immunoassay. Exemplary
proteins that comprise more than one polypeptide chain that can be
delivered with a delivery construct of the invention include, but
are not limited to, antibodies, insulin, IGF I, and the like.
[0118] Accordingly, in certain embodiments, the macromolecule is a
peptide, polypeptide, or protein. In certain embodiments, the
macromolecule comprises a peptide or polypeptide that comprises
about 5, about 8, about 10, about 12, about 15, about 17, about 20,
about 25, about 30, about 40, about 50, or about 60, about 70,
about 80, about 90, about 100, about 200, about 400, about 600,
about 800, or about 1000 amino acids. In certain embodiments, the
macromolecule is a protein that comprises 1, 2, 3, 4, 5, 6, 7, 8,
or more polypeptides. In certain embodiments, the peptide,
polypeptide, or protein is a molecule that is commonly administered
to subjects by injection. Exemplary peptides or polypeptides
include, but are not limited to, IGF-I, IGF-II, IGF-III, EGF,
IFN-.alpha., IFN-.beta., IFN-.gamma., G-CSF, GM-CSF, IL-1, IL-2,
IL-3, IL-6, IL-8, IL-12, EPO, growth hormone, clotting factors such
as factor VII, vasopressin, calcitonin parathyroid hormone,
luteinizing hormone-releasing factor, tissue plasminogen
activators, adrenocorticototropin, enkephalin, glucagon-like
peptide 1, asparaginase, and the like. In a preferred embodiment,
the macromolecule is insulin. In certain preferred embodiments, the
polypeptide is growth hormone. In even more preferred embodiments,
the polypeptide is human growth hormone. In an equally preferred
embodiment, the polypeptide is IFN-.alpha., more preferably
IFN.alpha.-2b. In an equally preferred embodiment, the polypeptide
is insulin or proinsulin. In other embodiments, the polypeptide is
green fluorescent protein. The sequences of all of these
macromolecules are well known to those in the art, and attachment
of these macromolecules to the delivery constructs is well within
the skill of those in the art using standard techniques, as
discussed below.
[0119] In certain embodiments, the macromolecule can be selected to
not be cleavable by an enzyme present at the basal-lateral membrane
of an epithelial cell. For example, the assays described in the
examples can be used to routinely test whether such a cleaving
enzyme can cleave the macromolecule to be delivered. If so, the
macromolecule can be routinely altered to eliminate the offending
amino acid sequence recognized by the cleaving enzyme. The altered
macromolecule can then be tested to ensure that it retains activity
using methods routine in the art.
[0120] Other examples of macromolecules that can be delivered
according to the present invention 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.,
pyrimemamine, trimethopterin, pemcillamine, cyclosporine,
azathioprine; and immunostimulants, e.g., levamisole, diethyl
dithiocarbamate, enkephalins, endorphins; antimicrobial compounds
such as antibiotics, e.g., .beta.-lactam, penicillin,
cephalosporins, carbapenims and monobactams, .beta.-lactamase
inhibitors, aminoglycosides, macrolides, tetracyclins,
spectinomycin; antimalarials, amebicides; antiprotazoals;
antifungals, e.g., amphotericin .beta., antivirals, e.g.,
acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine,
gancyclovir; parasiticides; antihalmintics; radiopharmaceutics;
gastrointestinal drugs; hematologic compounds; immunoglobulins;
blood clotting proteins, e.g., antihemophilic factor, factor IX
complex; anticoagulants, e.g., dicumarol, heparinNa; fibrolysin
inhibitors, e.g., tranexamic acid; cardiovascular drugs; peripheral
anti-adrenergic drugs; centrally acting antihypertensive drugs,
e.g., methyldopa, methyldopa HCl; antihypertensive direct
vasodilators, e.g., diazoxide, hydralazine HCl; drugs affecting
renin-angiotensin system; peripheral vasodilators, e.g.,
phentolamine; anti-anginal drugs; cardiac glycosides; inodilators,
e.g., amrinone, milrinone, enoximone, fenoximone, imazodan,
sulmazole; antidysrhythmics; calcium entry blockers; drugs
affecting blood lipids, e.g., ranitidine, bosentan, rezulin;
respiratory drugs; sypamomimetic drugs, e.g., albuterol, bitolterol
mesylate, dobutamine HCl, dopamine HCl, ephedrine So, epinephrine,
fenfluramine HCl, isoproterenol HCl, methoxamine HCl,
norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl;
cholmomimetic drugs, e.g., acetylcholine Cl; anticholinesterases,
e.g., edrophonium Cl; cholinesterase reactivators; adrenergic
blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl,
labetalol HCl, metoprolol, nadolol, phentolamine mesylate,
propanolol HCl; antimuscarinic drugs, e.g., anisotropine
methylbromide, atropine SO.sub.4, clinidium Br, glycopyrrolate,
ipratropium Br, scopolamine HBr; neuromuscular blocking drugs;
depolarizing drugs, e.g., atracurium besylate, hexafluorenium Br,
metocurine iodide, succinylcholine Cl, tubocurarine Cl, vecuronium
Br; centrally acting muscle relaxants, e.g., baclofen;
neurotransmitters and neurotransmitter agents, e.g., acetylcholine,
adenosine, adenosine triphosphate; amino acid neurotransmitters,
e.g., excitatory amino acids, GABA, glycine; biogenic amine
neurotransmitters, e.g., dopamine, epinephrine, histamine,
norepinephrine, octopamine, serotonin, tyramine; neuropeptides,
nitric oxide, K.sup.+ channel toxins; antiparkinson drugs, e.g.,
amaltidine HCl, benztropine mesylate, carbidopa; diuretic drugs,
e.g., dichlorphenamide, methazolamide, bendroflumethiazide,
polythiazide; antimigraine drugs, e.g, carboprost tromethamine
mesylate, methysergide maleate.
[0121] Still other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
hormones such as pituitary hormones, e.g., chorionic gonadotropin,
cosyntropin, menotropins, somatotropin, iorticotropin, protirelin,
thyrotropin, vasopressin, lypressin; adrenal hormones, e.g.,
beclomethasone dipropionate, betamethasone, dexarnethasone,
triamcinolone; pancreatic hormones, e.g., glucagon, insulin;
parathyroid hormone, e.g., dihydrochysterol; thyroid hormones,
e.g., calcitonin etidronate disodium, levothyroxine Na,
liothyronine Na, liotrix, thyroglobulin, teriparatide acetate;
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, sincalide.
[0122] Still other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
enzymes such as hyaluronidase, streptokinase, tissue plasminogen
activator, urokinase, PGE-adenosine deaminase; intravenous
anesthetics such as droperidol, etomidate, fetanyl
citrate/droperidol, hexobarbital, ketamine HCl, methohexital Na,
thiamylal Na, thiopental Na; antiepileptics, e.g., carbamazepine,
clonazepam, divalproex Na, ethosuximide, mephenyloin,
paramethadione, phenyloin, primidone.
[0123] Still other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
peptides and proteins such as ankyrins, arrestins, bacterial
membrane proteins, clathrin, connexins, dystrophin, endothelin
receptor, spectrin, selectin, cytokines; chemokines; growth
factors, insulin, erythropoietin (EPO), tumor necrosis factor
(TNF), neuropeptides, neuropeptide Y, neurotensin, transforming
growth factor .alpha., transforming growth factor .beta.,
interferon (IFN); hormones, growth inhibitors, e.g., genistein,
steroids etc; glycoproteins, e.g., ABC transporters, platelet
glycoproteins, GPIb-IX complex, GPIIb-IIIa complex, vitronectin,
thrombomodulin, CD4, CD55, CD58, CD59, CD44, lymphocye
function-associated antigen, intercellular adhesion molecule,
vascular cell adhesion molecule, Thy-1, antiporters, CA-15-3
antigen, fibronectins, laminin, myelin-associated glycoprotein,
GAP, GAP-43.
[0124] Yet other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
cytokines and cytokine receptors such as Interleukin-1 (IL-1),
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 receptor,
IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6
receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-10
receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14
receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18
receptor, lymphokine inhibitory factor, macrophage colony
stimulating factor, platelet derived growth factor, stem cell
factor, tumor growth factor .beta., tumor necrosis factor,
lymphotoxin, Fas, granulocyte colony stimulating factor,
granulocyte macrophage colony stimulating factor, interferon
.alpha., interferon .beta., and interferon .gamma..
[0125] Still other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
growth factors and protein hormones such as erythropoietin,
angiogenin, hepatocyte growth factor, fibroblast growth factor,
keratinocyte growth factor, nerve growth factor, tumor growth
factor .alpha., 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-.alpha., GRO-.beta., GRO-.gamma., HRG, LEF, IP-10,
MCP-1, MCP-2, MCP-3, MCP-4, MIP-1.alpha., MIP-1.beta., MG, MDC,
NT-3, NT-4, SCF, LIF, leptin, RANTES, lymphotactin, eotaxin-1,
eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1, GCP-2;
.alpha.-chemokine receptors, e.g., CXCR1, CXCR2, CXCR3, CXCR4,
CXCR5, CXCR6, CXCR7; and .beta.-chemokine receptors, e.g., CCR1,
CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.
[0126] Yet other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
chemotherapeutics, such as chemotherapy or anti-tumor agents which
are effective against various types of human cancers, including
leukemia, lymphomas, carcinomas, sarcomas, myelomas etc., such as,
for example, doxorubicin, mitomycin, cisplatin, daunorubicin,
bleomycin, actinomycin D, and neocarzinostatin.
[0127] Still other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
antibodies such as anti-cluster of differentiation antigen CD-1
through CD-166 and the ligands or counter receptors for these
molecules; anti-cytokine antibodies, e.g., anti-IL-1 through
anti-IL-18 and the receptors for these molecules; anti-immune
receptor antibodies; antibodies against T cell receptors, major
histocompatibility complexes I and II, B cell receptors, selectin
killer inhibitory receptors, killer activating receptors, OX-40,
MadCAM-1, Gly-CAM2, integrins, cadherens, sialoadherens, Fas,
CTLA-4, Fc .gamma.-receptors, Fc .alpha.-receptors, Fc
.epsilon.-receptors, Fc .mu.-receptors, and their ligands;
anti-metalloproteinase antibodies, e.g., antibodies specific for
collagenase, MMP-1 through MMP-8, TIMP-1, TIMP-2; anti-cell
lysis/proinflammatory molecules, e.g., perforin, complement
components, prostanoids, nitron oxide, thromboxanes; and
anti-adhesion molecules, e.g., carcioembryonic antigens, lamins,
fibronectins.
[0128] Yet other examples of macromolecules that can be delivered
according to the present invention include, but are not limited to,
antiviral agents such as reverse transcriptase inhibitors and
nucleoside analogs, e.g., ddI, ddC, 3TC, ddA, AZT; protease
inhibitors, e.g., Invirase, ABT-538; and inhibitors of in RNA
processing, e.g., ribavirin.
[0129] Further, specific examples of macromolecules that can be
delivered with the delivery constructs of the present invention
Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,
Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid,
Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS,
Estrace, Glucophage (Bristol-Myers Squibb); Ceclor, Lorabid,
Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog, Axid, Gemzar,
Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor,
Prinivil/Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec,
Primaxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, Trusopt,
Proscar, Fosamax, Sinemet, Crixivan, Propecia, Vioxx, Singulair,
Maxalt, Ivermectin (Merck & Co.); Diflucan, Unasyn, Sulperazon,
Zithromax, Trovan, Procardia XL, Cardura, Norvasc, Dofetilide,
Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec, Eletriptan, Viagra,
Droloxifene, Aricept, Lipitor (Pfizer); Vantin, Rescriptor,
Vistide, Genotropin, Micronase/Glyn./Glyb., Fragmin, Total Medrol,
Xanax/alprazolam, Sermion, Halcion/triazolam, Freedox, Dostinex,
Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar, Remisar,
Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan,
Rogaine (Pharmacia & Upjohn); Lopid, Accrupil, Dilantin,
Cognex, Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin,
Lipitor, Omnicef, FemHRT, Suramin, and Clinafloxacin (Warner
Lambert).
[0130] Yet further examples of macromolecules which may be
delivered by the delivery constructs of the present invention may
be found in: Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 9th ed. McGraw-Hill 1996, incorporated herein by
reference in its entirety.
[0131] In certain embodiments, the macromolecule can be inactive or
in a less active form when administered, then be activated in the
subject. For example, the macromolecule can be a peptide or
polypeptide with a masked active site. The peptide or polypeptide
can be activated by removing the masking moiety. Such removal can
be accomplished by peptidases or proteases in the cases of peptide
or polypeptide masking agents. Alternatively, the masking agent can
be a chemical moiety that is removed by an enzyme present in the
subject. This strategy can be used when it is desirable for the
macromolecule to be active in limited circumstances. For example,
it may be useful for a macromolecule to be active only in the liver
of the subject. In such cases, the macromolecule can be selected to
have a masking moiety that can be removed by an enzyme that is
present in the liver, but not in other organs or tissues. Exemplary
methods and compositions for making and using such masked
macromolecules can be found in U.S. Pat. Nos. 6,080,575, 6,265,540,
and 6,670,147.
[0132] In another example of such embodiments, the macromolecule
can be a pro-macromolecule that is activated by a biological
activity, for example by processing, present in the subject. For
example, the exemplary macromolecule proinsulin can be delivered
with a delivery construct of the present invention. Following
delivery of the pro-macromolecule, it can be activated in the
subject by appropriate processing enzymes. While it is believed
that proinsulin is processed by enzymes (the endoproteases PC2 and
PC3) present in highest concentration in secretory granules of
pancreatic beta-cells, it is also believed that such enzyme are
present in sufficient concentration in other compartments to permit
activation of the pro-macromolecule into its fully active form.
Further, it should be noted that many pro-macromolecules,
including, for example, proinsulin, also exhibit activity similar
to that of the fully active molecule. See, for example, Desbuquois
et al., 2003, Endocrinology 12:5308-5321. Thus, even if not all of
the pro-macromolecule is converted to the fully active form, the
pro-molecule can in many cases still exert a desirable biological
activity in the subject.
[0133] 5.2.4. Cleavable Linkers
[0134] In the delivery constructs of the invention, the
macromolecule to be delivered to the subject is connected with the
remainder of the delivery construct with one or more cleavable
linkers. The number of cleavable linkers present in the construct
depends, at least in part, on the location of the macromolecule in
relation to the remainder of the delivery construct and the nature
of the macromolecule. When the macromolecule is inserted into the
delivery construct, the macromolecule can be flanked by cleavable
linkers, such that cleavage at both linkers separates the
macromolecule. The flanking cleavable linkers can be the same or
different from each other. When the macromolecule can be separated
from the remainder of the delivery construct with cleavage at a
single linker, the delivery constructs can comprise a single
cleavable linker. Further, where the macromolecule is, e.g., a
dimer or other multimer, each subunit of the macromolecule can be
separated from the remainder of the delivery construct and/or the
other subunits of the macromolecule by cleavage at the cleavable
linker.
[0135] The cleavable linkers are generally cleavable by a cleaving
enzyme that is present at or near the basal-lateral membrane of an
epithelial cell. By selecting the cleavable linker to be cleaved by
such enzymes, the macromolecule can be liberated from the remainder
of the construct following transcytosis across the mucous membrane
and release from the epithelial cell into the cellular matrix on
the basal-lateral side of the membrane. Further, cleaving enzymes
could be used that are present inside the epithelial cell, such
that the cleavable linker is cleaved prior to release of the
delivery construct from the basal-lateral 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 macromolecule from the basal-lateral membrane of the cell.
[0136] In certain embodiments, the cleaving enzyme is a peptidase.
In other embodiments, the cleaving enzyme is an RNAse. In yet other
embodiments, the cleaving enzyme can cleave carbohydrates.
Preferred peptidases include, but are not limited to, Cathepsin GI,
Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin
I, and Urokinase I. Table 1 presents these enzymes together with an
amino acid sequence that is recognized and cleaved by the
particular peptidase.
TABLE-US-00001 TABLE 1 Peptidases Present Near Basal-Lateral Mucous
Membranes Amino Acid Sequence Peptidase Recognized and Cleaved
Cathepsin GI Ala-Ala-Pro-Phe (SEQ ID NO.: 4) Chymotrypsin I
Gly-Gly-Phe (SEQ ID NO.: 5) Elastase I Ala-Ala-Pro-Val (SEQ ID NO.:
6) Subtilisin AI Gly-Gly-Leu (SEQ ID NO.: 7) Subtilisin AII
Ala-Ala-Leu (SEQ ID NO.: 8) Thrombin I Phe-Val-Arg (SEQ ID NO.: 9)
Urokinase I Val-Gly-Arg (SEQ ID NO.: 10)
[0137] In certain embodiments, the delivery construct can comprise
more than one cleavable linker, wherein cleavage at either
cleavable linker can separate the macromolecule to be delivered
from the delivery construct. In certain embodiments, the cleavable
linker can be selected based on the sequence, in the case of
peptide, polypeptide, or protein macromolecules for delivery, to
avoid the use of cleavable linkers that comprise sequences present
in the macromolecule to be delivered. For example, if the
macromolecule comprises AAL, the cleavable linker can be selected
to be cleaved by an enzyme that does not recognize this
sequence.
[0138] Further, the cleavable linker preferably exhibits a greater
propensity for cleavage than the remainder of the delivery
construct. As one skilled in the art is aware, many peptide and
polypeptide sequences can be cleaved by peptidases and proteases.
In certain embodiments, the cleavable linker is selected to be
preferentially cleaved relative to other amino acid sequences
present in the delivery construct during administration of the
delivery construct. In certain embodiments, the receptor binding
domain is 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. In certain
embodiments, the translocation domain is 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. In certain embodiments, the
macromolecule is 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. In
certain embodiments, the cleavable linker is 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.
[0139] In other embodiments, the cleavable linker is cleaved by a
cleaving enzyme found in the plasma of the subject. 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 linker. Use of
such enzymes to cleave the cleavable linkers is less preferred than
use of cleaving enzymes found near the basal-lateral membrane of a
polarized epithelial cell because it is believed that more
efficient cleavage will occur in near the basal-lateral membrane.
However, if the skilled artisan determines that 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. Accordingly, in certain embodiments, the
cleavable linker can be cleaved with an enzyme that is selected
from the group consisting of caspase-1, 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. Table 2 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.
TABLE-US-00002 TABLE 2 Plasma Peptidases Amino Acid Sequence
Peptidase Recognized and Cleaved Caspase-1 Tyr-Val-Ala-Asp-Xaa*
(SEQ ID NO.: 11) Caspase-3 Asp-Xaa-Xaa-Asp-Xaa* (SEQ ID NO.: 12)
Proprotein convertase Arg-(Xaa).sub.n-Arg-Xaa*; 1 n = 0, 2, 4 or 6
(SEQ ID NO.: 13) Proprotein convertase Lys-(Xaa).sub.n-Arg-Xaa*; 2
n = 0, 2, 4, or 6 (SEQ ID NO.: 14) Proprotein convertase
Glp-Arg-Thr-Lys-Arg-Xaa* 4 (SEQ ID NO.: 15) Proprotein convertase
Arg-Val-Arg-Arg-Xaa* 4 PACE 4 (SEQ ID NO.: 16)
Decanoyl-Arg-Val-Arg-Arg-Xaa* (SEQ ID NO.: 17) Prolyloligopeptidase
Pro-Xaa*-Trp-Val-Pro-Xaa Endothelin cleaving (SEQ ID NO.: 18)
enzyme in combination with dipeptidyl- peptidase IV Signal
peptidase Trp-Val*-Ala-Xaa (SEQ ID NO.: 19) Neprilysin in com-
Xaa-Phe*-Xaa-Xaa bination with (SEQ ID NO.: 20)
dipeptidyl-peptidase Xaa-Tyr*-Xaa-Xaa IV (SEQ ID NO.: 21)
Xaa-Trp*-Xaa-Xaa (SEQ ID NO.: 22) Renin in combination
Asp-Arg-Tyr-Ile-Pro-Phe-His- with dipeptidyl- Leu*-Leu-(Val, Ala or
Pro)- peptidase IV Tyr-(Ser, Pro, or Ala) (SEQ ID NO.: 23)
[0140] Thus, in certain more preferred embodiments, the cleavable
linker can be any cleavable linker known by one of skill in the art
to be cleavable by an enzyme that is present at the basal-lateral
membrane of an epithelial cell. In certain embodiments, the
cleavable linker comprises a peptide. In other embodiments, the
cleavable linker comprises a nucleic acid, such as RNA or DNA. In
still other embodiments, the cleavable linker comprises a
carbohydrate, such as a disaccharide or a trisaccharide. In certain
embodiments, the cleavable linker is a peptide that 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).
[0141] Alternatively, in less preferred embodiments, the cleavable
linker can be any cleavable linker 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. In certain
embodiments, the cleavable linker comprises a peptide. In other
embodiments, the cleavable linker comprises a nucleic acid, such as
RNA or DNA. In still other embodiments, the cleavable linker
comprises a carbohydrate, such as a disaccharide or a
trisaccharide. In certain embodiments, the cleavable linker is a
peptide that comprises an amino acid sequence that is selected from
the group consisting of amino acid sequences presented in Table
2.
[0142] In certain embodiments, the delivery construct comprises
more than one cleavable linker. In certain embodiments, cleavage at
any of the cleavable linkers will separate the macromolecule to be
delivered from the remainder of the delivery construct. In certain
embodiments, the delivery construct comprises a cleavable linker
cleavable by an enzyme present at the basal-lateral side of a
polarized epithelial membrane and a cleavable linkers cleavable by
an enzyme that is present in the plasma of the subject to whom the
delivery construct is administered.
[0143] Further, Tables 4 and 5, below, present results of
experiments testing the ability of peptidases to cleave substrates
when applied to the basal-lateral or apical surface of a polarized
epithelial membrane. The sequences recognized by these enzymes are
well-known in the art. Thus, in certain embodiments, the delivery
construct comprises a cleavable linker that is cleavable by an
enzyme listed in Tables 4 and 5. Preferred peptidases exhibit
higher activity on the basolateral side of the membrane.
Particularly preferred peptidases exhibit much higher (e.g., 100%,
200%, or more increase in activity relative to the apical side) on
the basolateral side. Thus, in certain embodiments, the cleavable
linker is cleavable by an enzyme that exhibits 50% higher activity
on the basal-lateral side of the membrane than on the apical side
of the membrane. In certain embodiments, the cleavable linker is
cleavable by an enzyme that exhibits 100% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 200% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 500% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 1,000% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 2,000% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 3,000% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 5,000% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane. In certain embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 10,000% higher activity on the
basal-lateral side of the membrane than on the apical side of the
membrane.
[0144] Still further, the results in Tables 4 and 5 indicate that
certain enzymes are present in higher concentration or exhibit
greater activity in certain epithelial lineages as compared to
other epithelial lineages. Thus, the experiments described below
can be used to test whether the particular epithelial cell lineage
through which a macromolecule will be delivered exhibits the
desired cleavage activity. In certain embodiments, the cleavage
activity is present in tracheal epithelial cells, but not
intestinal epithelial cells. In other embodiments, the cleavage
activity is present in intestinal epithelial cells out not tracheal
epithelial cells. In certain embodiments, the cleavage activity is
present in intestinal epithelial cells and tracheal epithelial
cells.
[0145] In certain embodiments, the cleavable linker may be
cleavable by any enzyme that preferentially cleaves at the
basolateral side of an epithelial membrane as compared to the
apical side of the membrane. Example 6.4, below, describes an assay
that can be used to assess the activity of such enzymes, while
Table 7, appended to the end of this document, provides short names
and accession numbers for every known human protease or peptidase.
Any cleavage sequence recognized by such proteases or peptidases
that preferentially cleaves a test substrate on the basolateral
side of an epithelial membrane, or in the plasma, as compared to
the apical side of such a membrane can also be used in the methods
and compositions of the present invention. In such embodiments, one
of skill in the art can readily determine the amino acid sequence
recognized by such peptidases or proteases according to standard
procedures known in the art or according to the known sequences,
recognized by the proteases and peptidases.
[0146] The examples below provide methods for identifying cleaving
enzymes that are present at or near the basal-lateral membrane of a
polarized epithelial cell. The skilled artisan can routinely use
such methods to identify additional cleaving enzymes and the
chemical structure(s) identified and cleaved by such cleaving
enzymes. Delivery constructs comprising such cleavable linkers and
methods of delivering macromolecules using delivery constructs
comprising such cleavable linkers are also within the scope of the
present invention, whether or not such cleaving enzymes are
presented in Table 7.
[0147] In other embodiments, the cleavable linker can be a
cleavable linker that is cleaved following a change in the
environment of the delivery construct. For example, the cleavable
linker can be a cleavable linker that is pH sensitive and is
cleaved by a change in pH that is experienced when the delivery
construct is released from the basal-lateral membrane of a
polarized epithelial cell. For instance, the intestinal lumen is
strongly alkaline, while plasma is essentially neutral. Thus, a
cleavable linker can be a moiety that is cleaved upon a shift from
alkaline to neutral pH. The change in the environment of the
delivery construct that cleaves the cleavable linker can be any
environmental change that that is experienced when the delivery
construct is released from the basal-lateral membrane of a
polarized epithelial cell known by one of skill in the art, without
limitation.
5.3. Methods for Delivering a Macromolecule
[0148] In another aspect, the invention provides methods for local
or systemic delivery of a macromolecule to a subject. These methods
generally comprise administering a delivery construct of the
invention to a mucous membrane of the subject to whom the
macromolecule is delivered. The delivery construct is typically
administered in the form of a pharmaceutical composition, as
described below.
[0149] Thus, in certain aspects, the invention provides a method
for delivering a macromolecule to a subject. The method comprises
contacting an apical surface of a polarized epithelial cell of the
subject with a delivery construct. In certain embodiments, the
delivery construct comprises a receptor binding domain, a
transcytosis domain, a cleavable linker, and the macromolecule to
be delivered. The transcytosis domain can transcytose the
macromolecule to and through the basal-lateral membrane of said
epithelial cell. The cleavable linker can be cleaved by ah enzyme
that is present at a basal-lateral membrane of a polarized
epithelial cell of the subject or in the plasma of the subject.
Cleavage at the cleavable linker separates the macromolecule from
the remainder of the delivery construct, thereby delivering the
macromolecule to the subject.
[0150] In certain embodiments, the enzyme that is present at or
near 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. In certain embodiments, the 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).
[0151] In certain embodiments, the 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 .alpha.; EGF; IGF-I;
IGF-II; IGF-III; IL-1; IL-2; IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and
IL-8. In certain embodiments, the receptor binding domain binds to
a cell surface receptor selected from the group consisting of
.alpha.2-macroglobulin receptor, EGFR, IGFR, transferrin receptor,
chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNF.alpha.
receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger
receptor, and VEGF receptor.
[0152] In certain embodiments, the transcytosis domain is selected
from the group consisting of transcytosis domains from Pseudomonas
exotoxin A, diptheria toxin, pertussis toxin, cholera toxin,
heat-labile E. coli enterotoxin, shiga toxin, and shiga-like
toxin.
[0153] In certain embodiments, the macromolecule is selected from
the group consisting of a peptide, a polypeptide, a protein, a
nucleic acid, and a lipid. In a preferred embodiment, the
macromolecule is growth hormone. Even more preferably, the
macromolecule is human growth hormone.
[0154] In certain embodiments, the invention provides a method for
delivering a macromolecule to the bloodstream of a subject that
results in at least about 30% bioavailability of the macromolecule,
comprising administering a delivery construct comprising the
macromolecule to the subject, thereby delivering at least about 30%
of the total macromolecule administered to the blood of the subject
in a bioavailable form of the macromolecule. In certain
embodiments, at least about 10% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 15% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 20% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 25% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 35% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 40% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 45% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 50% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 55% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 60% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 65% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 70% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 75% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 80% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 85% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, at least about 90% of the total macromolecule
administered is bioavailable to the subject, in certain
embodiments, at least about 95% of the total macromolecule
administered is bioavailable to the subject. In certain
embodiments, the percentage of bioavailability of the macromolecule
is determined by comparing the amount of macromolecule present in a
subject's blood following administration of a delivery construct
comprising the macromolecule to the amount of macromolecule present
in a subject's blood following administration of the macromolecule
through another route of administration. In certain embodiments,
the other route of administration is injection, e.g., subcutaneous
injection, intravenous injection, intra-arterial injection, etc. In
other embodiments, the percentage of bioavailability of the
macromolecule is determined by comparing the amount of
macromolecule present in a subject's blood following administration
of a delivery construct comprising the macromolecule to the total
amount of macromolecule administered as part of the delivery
construct.
[0155] In certain embodiments, peak plasma concentrations of the
delivered macromolecule in the subject are achieved about 10
minutes after administration. In certain embodiments, peak plasma
concentrations of the delivered macromolecule in the subject are
achieved about 15 minutes after administration. In certain
embodiments, peak plasma concentrations of the delivered
macromolecule in the subject are achieved about 5 minutes after
administration. In certain embodiments, peak plasma concentrations
of the delivered macromolecule in the subject are achieved about 20
minutes after administration. In certain embodiments, peak plasma
concentrations of the delivered macromolecule in the subject are
achieved about 25 minutes after administration. In certain
embodiments, peak plasma concentrations of the delivered
macromolecule in the subject are achieved about 30 minutes after
administration. In certain embodiments, peak plasma concentrations
of the delivered macromolecule in the subject are achieved about 35
minutes after administration. In certain embodiments, peak plasma
concentrations of the delivered macromolecule in the subject are
achieved about 40 minutes after administration. In certain
embodiments, peak plasma concentrations of the delivered
macromolecule in the subject are achieved about 45 minutes after
administration. In certain embodiments, peak plasma concentrations
of the delivered macromolecule in the subject are achieved about 50
minutes after administration. In certain embodiments, peak plasma
concentrations of the delivered macromolecule in the subject are
achieved about 55 minutes after administration. In certain
embodiments, peak plasma concentrations of the delivered
macromolecule in the subject are achieved about 60 minutes after
administration. In certain embodiments, peak plasma concentrations
of the delivered macromolecule in the subject are achieved about 90
minutes alter administration. In certain embodiments, peak plasma
concentrations of the delivered macromolecule in the subject are
achieved about 120 minutes after administration.
[0156] In certain embodiments, the peak plasma concentration of the
delivered macromolecule is between about 0.01 ng/ml plasma and
about 10 .mu.g/ml plasma. In certain embodiments, the peak plasma
concentration of the delivered macromolecule is between about 0.01
ng/ml plasma and about 1 .mu.g/ml plasma. In certain embodiments,
the peak plasma concentration of the delivered macromolecule is
between about 0.01 ng/ml plasma and about 0.1 .mu.g/ml plasma. In
certain embodiments, the peak plasma concentration of the delivered
macromolecule is between about 0.01 ng/ml plasma and about 10 ng/ml
plasma. In certain embodiments, the peak plasma concentration of
the delivered macromolecule is between about 1 ng/ml plasma and
about 10 .mu.g/ml plasma. In certain embodiments, the peak plasma
concentration of the delivered macromolecule is between about 1
ng/ml plasma and about 1 .mu.g/ml plasma. In certain embodiments,
the peak plasma concentration of the delivered macromolecule is
between about 1 ng/ml plasma and about 0.5 .mu.g/ml plasma. In
certain embodiments, the peak plasma concentration of the delivered
macromolecule is between about 1 ng/ml plasma and about 0.1
.mu.g/ml plasma. In certain embodiments, the peak plasma
concentration of the delivered macromolecule is between about 10
ng/ml plasma and about 1 .mu.g/ml plasma. In certain embodiments,
the peak plasma concentration of the delivered macromolecule is
between about 10 ng/ml plasma and about 0.5 .mu.g/ml plasma.
[0157] In certain embodiments, the peak plasma concentration of the
delivered macromolecule is at least about 10 ng/ml plasma. In
certain embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 5 .mu.g/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 1 .mu.g/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 500 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 250 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 100 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 50 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 10 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 5 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 1 ng/ml plasma. In certain
embodiments, the peak plasma concentration of the delivered
macromolecule is at least about 0.1 ng/ml plasma.
[0158] Moreover, without intending to be bound to any particular
theory or mechanism of action, it is believed that oral
administration of a delivery construct can deliver a higher
effective concentration of the delivered macromolecule to the liver
of the subject than is observed in the subject's plasma. "Effective
concentration," in this context, refers to the concentration
experienced by targets of the macromolecule and can be determined
by monitoring and/or quantifying downstream effects of
macromolecule-target interactions. While still not bound to any
particular theory, it is believed that oral administration of the
delivery construct 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 construct and
release of the macromolecule at the basolateral side of the mucous
membrane. As one of skill in the art will recognize, the blood at
the basolateral membrane of such digestive mucosa is carried from
this location to the liver via the portal venous system. Thus, when
the macromolecule exerts a biological activity in the liver, such
as, for example, activities mediated by growth hormone, insulin,
IGF-I, etc. binding to their cognate receptors, the macromolecule
is believed to exert an effect in excess of what would be expected
based on the plasma concentrations observed in the subject.
Accordingly, in certain embodiments, the invention provides a
method of administering a macromolecule to a subject that comprises
orally administering a delivery construct comprising the
macromolecule to the subject, wherein the macromolecule is
delivered to the subject's liver at a higher effective
concentration than observed in the subject's plasma.
[0159] In certain embodiments, 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.
[0160] In certain embodiments, the subject is a mammal. In further
embodiments, the subject is a rodent, a lagomorph, or a primate. In
yet further embodiments, the rodent is a mouse or rat. In other
embodiments, the lagomorph is a rabbit. In still other embodiments,
the primate is a human, monkey, or ape. In a preferred embodiment,
the subject is a human.
[0161] In another aspect, the invention provides a method for
delivering a macromolecule to the bloodstream of a subject that
induces a lower titer of antibodies against the macromolecule than
other routes of administration. Without intending to be bound by
any particular theory or mechanism of action, it is believed that
entry of the macromolecule through a mucous membrane, e.g., through
the intestinal mucosa, causes the immune system to tolerate the
macromolecule better than if the macromolecule were, for example,
injected. Thus, a lower titer of antibodies against the
macromolecule can be produced in the subject by delivering the
macromolecule with a delivery construct of the invention through
the mucosa rather than injecting the macromolecule, for example,
subcutaneously, intravenously, intra-arterially, intraperitoneally,
or otherwise. Generally, the time at which the lower titer of
antibodies detected for the alternate routes of administration is
detected should be roughly comparable; for example, the titer of
antibodies can be determined at about 1 week, at about 2 weeks, at
about 3 weeks, at about 4 weeks, at about 2 months, or at about 6
months following administration of the macromolecule with the
delivery construct or by injection.
[0162] Accordingly, in certain embodiments, the invention provides
a method for delivering a macromolecule to the bloodstream a
subject that comprises contacting a delivery construct of the
invention that comprises the macromolecule to be delivered to an
apical surface of a polarized epithelial cell of the subject, such
that the macromolecule is administered 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
separately from the remainder of the delivery construct to a
subject.
[0163] In certain embodiments, the macromolecule is selected from
the group consisting of a peptide, a polypeptide, a protein, a
nucleic acid, and a lipid. In certain embodiments, the
macromolecule is selected from the group consisting of polypeptide
hormones, cytokines, chemokines, growth factors, and clotting
factors. In certain embodiments, the macromolecule is selected from
the group consisting of IGF-I, IGF-II, IGF-III, EGF, IFN-.alpha.,
IFN-.beta., IFN-.gamma., 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, adrenocorticototropin,
enkephalin, and glucagon-like peptide 1. In certain embodiments,
the macromolecule is human growth hormone. In certain embodiments,
the macromolecule is human insulin. In certain embodiments, the
subject is a mouse, rat, dog, goat, or human.
[0164] In certain embodiments, 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 95% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 90% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct. In certain embodiments,
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 85% of the titer of
antibodies induced by subcutaneously administering the
macromolecule separately from the remainder of the delivery
construct. In certain embodiments, 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 80% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 75% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct.
[0165] In certain embodiments, 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 70% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 65% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct. In certain embodiments,
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 60% of the titer of
antibodies induced by subcutaneously administering the
macromolecule separately from the remainder of the delivery
construct. In certain embodiments, 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 55% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 55% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct.
[0166] In certain embodiments, 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 separately from the remainder of
the delivery construct. In certain embodiments, 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 45% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct. In certain embodiments,
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 40% of the titer of
antibodies induced by subcutaneously administering the
macromolecule separately from the remainder of the delivery
construct. In certain embodiments, 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 35% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 30% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct.
[0167] In certain embodiments, 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 separately from the remainder of
the delivery construct. In certain embodiments, 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 20% of the titer of antibodies induced by
subcutaneously administering the macromolecule separately from the
remainder of the delivery construct. In certain embodiments, 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 15% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct. In certain embodiments,
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 separately from the remainder of the delivery
construct. In certain embodiments, 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 5% of the titer of antibodies induced by subcutaneously
administering the macromolecule separately from the remainder of
the delivery construct. In certain embodiments, 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 1% of the titer of antibodies induced
by subcutaneously administering the macromolecule separately from
the remainder of the delivery construct.
[0168] 5.3.1. Methods of Administration
[0169] The delivery constructs of the invention can be administered
to a subject by any method known to one of skill in the art. In
certain embodiments, the delivery constructs are contacted to a
mucosal membrane of the subject. For example, the mucosal membrane
can be present in the eye, nose, mouth, trachea, lungs, esophagus,
stomach, small intestine, large intestine, rectum, anus, sweat
glands, vulva, vagina, or penis of the subject. Preferably, the
mucosal membrane is a mucosal membrane present in the digestive
tract of the subject, such as a mucosal membrane in the mouth,
esophagus, stomach, small intestine, large intestine, or rectum of
the subject.
[0170] In such embodiments, the delivery constructs are preferably
administered to the subject orally. Thus, the delivery construct
can be formulated to protect the delivery construct from
degradation in the acid environment of the stomach, if necessary.
For example, many embodiments of the delivery constructs of the
invention comprise polypeptide domains with defined activities.
Unless such delivery constructs are protected from acid and/or
enzymatic hydrolysis in the stomach, the constructs will generally
be digested before delivery of substantial amounts of the
macromolecule to be delivered. Accordingly, composition
formulations that protect the delivery construct from degradation
can be used in administration of these delivery constructs.
[0171] 5.3.2. Dosage
[0172] Generally, a pharmaceutically effective amount of the
delivery construct of the invention is administered to a subject.
The skilled artisan can readily determine if the dosage of the
delivery construct is sufficient to deliver an effective amount of
the macromolecule, as described below. In certain embodiments,
between about 1 .mu.g and about 1 g of delivery construct is
administered. In other embodiments, between about 10 .mu.g and
about 500 mg of delivery construct is administered. In still other
embodiments, between about 10 .mu.g and about 100 mg of delivery
construct is administered. In yet other embodiments, between about
10 .mu.g and about 1000 .mu.g of delivery construct is
administered. In still other embodiments, between about 10 .mu.g
and about 250 .mu.g of delivery construct is administered. In yet
other embodiments, between about 10 .mu.g and about 100 .mu.g of
delivery construct is administered. Preferably, between about 10
.mu.g and about 50 .mu.g of delivery construct is administered.
[0173] The volume of a composition comprising the delivery
construct that is administered will generally depend on the
concentration of delivery construct and the formulation of the
composition. In certain embodiments, a unit dose of the delivery
construct composition is between about 0.05 ml and about 1 ml,
preferably about 0.5 ml. The delivery construct compositions can be
prepared in dosage forms containing between 1 and 50 doses (e.g.,
0.5 ml to 25 ml), more usually between 1 and 10 doses (e.g., 0.5 ml
to 5 ml)
[0174] The delivery construct compositions of the invention can be
administered in one dose or in multiple doses. A dose can be
followed by one or more doses spaced by about 1 to about 6 hours,
by about 6 to about 12 hours, by about 12 to about 24 hours, by
about 1 day to about 3 days, by about 1 day to about 1 week, by
about 1 week to about 2 weeks, by about 2 weeks to about 1 month,
by about 4 to about 8 weeks, by about 1 to about 3 months, or by
about 1 to about 6 months.
[0175] The macromolecules to be delivered are generally
macromolecules for which a large amount of knowledge regarding
dosage, frequency of administration, and methods for assessing
effective concentrations in subjects has accumulated. Such
knowledge can be used to assess efficiency of delivery, effective
concentration of the macromolecule in the subject, and frequency of
administration. Thus, the knowledge of those skilled in the art can
be used to determine whether, for example, the amount of
macromolecule delivered to the subject is an effective amount, the
dosage should be increased or decreased, the subject should be
administered the delivery construct more or less frequently, and
the like.
[0176] 5.3.3. Determining Amounts of Macromolecule Delivered
[0177] The methods of the invention can be used to deliver, either
locally or systemically, a pharmaceutically effective amount of a
macromolecule to a subject. The skilled artisan can determine
whether the methods result in delivery of such a pharmaceutically
effective amount of the macromolecule. The exact methods will
depend on the macromolecule that is delivered, but generally will
rely on either determining the concentration of the macromolecule
in the blood of the subject or in the biological compartment of the
subject where the macromolecule exerts its effects. Alternatively
or additionally, the effects of the macromolecule on the subject
can be monitored.
[0178] For example, in certain embodiments of the present
invention, the macromolecule that is delivered is insulin, e.g.,
human insulin. In such embodiments, the skilled artisan can
determine whether a pharmaceutically effective amount of human
insulin had been delivered to the subject by, for example, taking a
plasma sample from the subject and determining the concentration of
human insulin therein. One exemplary method for determining the
concentration of human insulin is by performing an ELISA assay, but
any other suitable assay known to the skilled artisan can be
used.
[0179] Alternatively, one of skill in the art can determine if an
effective amount of human insulin had been delivered to the subject
by monitoring the blood sugar concentrations of the subject. As is
well-known in the art, human insulin, among other activities, acts
on hepatocytes to promote glycogen formation, thereby reducing
plasma glucose concentrations. Accordingly, the subject's plasma
glucose concentration can be monitored to determine whether an
effective amount of insulin had been delivered.
[0180] Any effect of a macromolecule that is administered that is
known by one of skill in the art, without limitation, can be
assessed in determining whether an effective amount of the
macromolecule has been administered. Exemplary effects include, but
are not limited to, receptor binding, receptor activation,
downstream effects of receptor binding, downstream effects of
receptor activation, coordination of compounds, effective blood
clotting, bone growth, wound healing, cellular proliferation, etc.
The exact effect that is assessed will depend on the macromolecule
that is delivered.
5.4. Polynucleotides Encoding Delivery Constructs
[0181] In another aspect, the invention provides polynucleotides
comprising a nucleotide sequence encoding the delivery constructs.
These polynucleotides are useful, for example, for making the
delivery constructs. In yet another aspect, the invention provides
an expression system that comprises a recombinant polynucleotide
sequence encoding a receptor binding domain, a transcytosis domain,
and a polylinker insertion site for a polynucleotide sequence
encoding a macromolecule. The polylinker insertion site can be
anywhere in the polynucleotide sequence so long as the polylinker
insertion does not disrupt the receptor binding domain or the
transcytosis domain. The polylinker insertion site should be
oriented near a polynucleotide sequence that encodes a cleavable
linker so that cleavage at the cleavable linker separates a
macromolecule encoded by a nucleic acid inserted into the
polylinker insertion site from the remainder of the encoded
delivery construct. Thus, in embodiments where the polylinker
insertion site is at an end of the encoded construct, the
polynucleotide comprises one nucleotide sequence encoding a
cleavable linker between the polylinker insertion site and the
remainder of the polynucleotide. In embodiments where the
polylinker insertion site is not at the end of the encoded
construct, the polylinker insertion site can be flanked by
nucleotide sequences that each encode a cleavable linker.
[0182] In certain embodiments, the recombinant polynucleotides are
based on polynucleotides encoding PE, or portions or derivatives
thereof. In other embodiments, the recombinant polynucleotides are
based on polynucleotides that hybridize to a polynucleotide that
encodes PE under stringent hybridization conditions. A nucleotide
sequence encoding PE is presented as SEQ ID NO.:3. This sequence
can be used to prepare PCR primers for isolating a nucleic acid
that encodes any portion of this sequence that is desired. For
example, PCR can be used to isolate a nucleic acid that encodes one
or more of the functional domains of PE. A nucleic acid so isolated
can then be joined to nucleic acids encoding other functional
domains of the delivery constructs using standard recombinant
techniques.
[0183] Other in vitro methods that can be used to prepare a
polynucleotide encoding PE, PE domains, or any other functional
domain useful in the delivery constructs of the invention 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 (3SR) 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. For example, 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 PE or a
nucleotide encoding a receptor binding domain.
[0184] Guidance for using these cloning and in vitro amplification
methodologies are described in, for example, U.S. Pat. No.
4,683,195; Mullis et al., 1987, Cold Spring Harbor Symp. Quant.
Biol. 51:263; and Erlich, ed., 1989, PCR Technology, Stockton
Press, NY. Polynucleotides encoding a delivery construct or a
portion thereof also can be isolated by screening genomic or cDNA
libraries with probes selected from the sequences of the desired
polynucleotide under stringent, moderately stringent, or highly
stringent hybridization conditions.
[0185] Construction of nucleic acids encoding the delivery
constructs of the invention can be facilitated by introducing an
insertion site for a nucleic acid encoding the macromolecule into
the construct. In certain embodiments, an insertion site for the
macromolecule can be introduced between the nucleotides encoding
the cysteine residues of domain Ib. In other embodiments, the
insertion site can be introduced anywhere in the nucleic acid
encoding the construct so long as the insertion does not disrupt
the functional domains encoded thereby. In certain embodiments, the
insertion site can be in the ER retention domain.
[0186] In more specific embodiments, a nucleotide sequence encoding
a portion of the Ib domain between the cysteine-encoding residues
can be removed and replaced with a nucleotide sequence that
includes a cloning site cleaved by a restriction enzyme. For
example, the cloning site can be recognized and cleaved by PstI. In
such examples, a polynucleotide encoding macromolecule that is
flanked by PstI sequences can be inserted into the vector.
[0187] Further, 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 immunogen.
[0188] Furthermore, the polynucleotides of the invention also
encompass derivative versions of polynucleotides encoding a
delivery construct. Such derivatives can be made by any method
known by one of skill in the art without limitation. For example,
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. Alternatively, 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.sub.2 and unbalanced nucleotide
concentrations. These conditions increase the misincorporation rate
of the polymerase used in the PCR reaction and result in random
mutagenesis of the amplified nucleic acid.
[0189] Several site-specific mutations and deletions in chimeric
molecules derived from PE have been made and characterized. For
example, deletion of nucleotides encoding amino acids 1-252 of PE
yields a construct referred to as "PE40." Deleting nucleotides
encoding amino acids 1-279 of PE yields a construct referred to as
"PE37." See U.S. Pat. No. 5,602,095. In both of these constructs,
the receptor binding domain of PE, i.e., domain Ia, has been
deleted. Nucleic acids encoding a receptor binding domain can be
ligated to these constructs to produce delivery constructs that are
targeted to the cell surface receptor recognized by the receptor
binding domain. Of course, these recombinant polynucleotides are
particularly useful for expressing delivery constructs that have a
receptor binding domain that is not domain Ia of PE. The
recombinant polynucleotides can optionally encode an amino-terminal
methionine to assist in expression of the construct. In certain
embodiments, the receptor binding domain can be ligated to the 5'
end of the polynucleotide encoding the transcytosis domain.
[0190] Other nucleic acids encoding mutant forms of PE that can be
used as a source of nucleic acids for constructing the delivery
constructs of the invention include, but are not limited to,
PE.DELTA.553 and those described in U.S. Pat. Nos. 5,602,095;
5,512,658 and 5,458,878, and in Vasil et al., 1986, Infect. Immunol
52:538-48.
[0191] Accordingly, in certain embodiments, the invention provides
a polynucleotide that encodes a delivery construct. The delivery
construct comprises a receptor binding domain, a transcytosis
domain, a macromolecule to be delivered to a subject, and a
cleavable linker. Cleavage at the cleavable linker can separate the
macromolecule from the remainder of the construct. The cleavable
linker can be cleaved by an enzyme that is present at a
basal-lateral membrane of a polarized epithelial cell of the
subject or in the plasma of the subject.
[0192] In certain embodiments, the polynucleotide hybridizes under
stringent hybridization conditions to any polynucleotide of this
invention. In further embodiments, the polynucleotide hybridizes
under stringent conditions to a nucleic acid that encodes any
delivery construct of the invention.
[0193] In certain embodiments, the polynucleotide encodes a
delivery construct that further comprises a second cleavable
linker. In certain embodiments, the first and/or second 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). In certain embodiments, the first
and/or second cleavable linker encoded by the polynucleotide is
cleavable by an enzyme that is selected from the group consisting
of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI,
Subtilisin AII, Thrombin I, and Urokinase I.
[0194] In certain embodiments, the receptor binding domain encoded
by the polynucleotide 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 .alpha.; EGF; IGF-I; IGF-II; IGF-III; IL-1; IL-2;
IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In certain embodiments,
the receptor binding domain encoded by the polynucleotide binds to
a cell-surface receptor that is selected from the group consisting
of .alpha.2-macroglobulin receptor, EGFR, IGFR, transferrin
receptor, chemokine receptor, CD25, CD11B, CD11C, CD80, CD86,
TNF.alpha. receptor, TOLL receptor, M-CSF receptor, GM-CSF
receptor, scavenger receptor, and VEGF receptor. In further
embodiments, the receptor binding domain encoded by the
polynucleotide is Domain Ia of Pseudomonas exotoxin A. In yet
further embodiments, the receptor binding domain encoded by the
polynucleotide has an amino acid sequence that is SEQ ID NO.:
1.
[0195] In certain embodiments, the transcytosis domain encoded by
the polynucleotide is selected from the group consisting of
transcytosis domains from Pseudomonas exotoxin A, diptheria toxin,
pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin,
shiga toxin, and shiga-like toxin. In further embodiments, the
transcytosis domain is Pseudomonas exotoxin A transcytosis domain.
In still further embodiments, the Pseudomonas exotoxin A
transcytosis domain has an amino acid sequence that is SEQ ID
NO.:2.
[0196] In certain embodiments, the macromolecule encoded by the
polynucleotide is selected from the group of a peptide, a
polypeptide, and a protein. In certain embodiments, the polypeptide
is selected from the group consisting of polypeptide hormones,
cytokines, chemokines, growth factors, and clotting factors. In
further embodiments, the polypeptide is selected from the group
consisting of IGF-I, IGF-II, IGF-III, EGF, IFN-.alpha., IFN-.beta.,
IFN-.gamma., 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, adrenocorticototropin, enkephalin, and
glucagon-like peptide 1. In still further embodiments, the
polypeptide is human growth hormone. In other embodiments, the
protein is human insulin.
[0197] In other embodiments, the invention provides a
polynucleotide that encodes a delivery construct that comprises a
nucleic acid sequence encoding a receptor binding domain, a nucleic
acid sequence encoding a transcytosis domain, a nucleic acid
sequence encoding a cleavable linker, and a nucleic acid sequence
comprising a polylinker insertion site. The polylinker insertion
site can be oriented relative to the nucleic acid sequence encoding
a cleavable linker to allow to cleavage of the cleavable linker to
separate a macromolecule that is encoded by a nucleic acid inserted
into the polylinker insertion site from the remainder of said
delivery construct. The cleavable linker can be cleavable by an
enzyme that is present at a basal-lateral membrane of a polarized
epithelial cell of said subject or in the plasma of said
subject.
5.5. Expression Vectors
[0198] In still another aspect, the invention provides expression
vectors for expressing the delivery constructs. Generally,
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. See, e.g., Sambrook et al.,
2001, Molecular Cloning--A Laboratory Manual, 3.sup.rd edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and
Ausubel et al., eds., Current Edition, Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley
Interscience, NY.
[0199] 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.
[0200] 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 adenoassociated viruses, plasmid vectors, cosmids,
and the like. Viral and plasmid vectors are preferred for
transfecting the expression vectors into mammalian cells. For
example, the expression vector pcDNA1 (Invitrogen, San Diego,
Calif.), in which the expression control sequence comprises the CMV
promoter, provides good rates of transfection and expression into
such cells.
[0201] 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; facilitated uptake through lipofection using, e.g.,
liposomes or immunoliposomes; particle-mediated transfection; etc.
See, e.g., U.S. Pat. No. 5,272,065; Goeddel et al., eds, 1990,
Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger,
1990, Gene Transfer and Expression--A Laboratory Manual, Stockton
Press, NY; Sambrook et al., 1989, Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al.,
eds., Current Edition, Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience, NY.
[0202] The expression vectors can also contain a purification
moiety that simplifies isolation of the delivery construct. For
example, 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. In certain
embodiments, the purification moiety can be cleaved from the
remainder of the delivery construct following purification. In
other embodiments, the moiety does not interfere with the function
of the functional domains of the delivery construct and thus need
not be cleaved.
5.6. Cell for Expressing a Delivery Construct
[0203] In yet another aspect, the invention provides a cell
comprising an expression vector for expression of the delivery
constructs, or portions thereof. The cell is preferably selected
for its ability to express high concentrations of the delivery
construct to facilitate purification of the protein. In certain
embodiments, the cell is a prokaryotic cell, for example, E. coli.
As described in the examples, the delivery constructs are properly
folded and comprise the appropriate disulfide linkages when
expressed in E. coli.
[0204] In other embodiments, the cell is a eukaryotic cell. Useful
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, without limitation, can be used to express
the delivery constructs. For example, Chinese hamster ovary (CHO)
cells can be used to express the delivery constructs.
5.7. Compositions Comprising Delivery Constructs
[0205] The delivery constructs of the invention can be formulated
as compositions. The compositions are generally formulated
appropriately for the immediate use intended for the delivery
construct. For example, if the delivery construct is not to be
administered immediately, the delivery construct can be formulated
in a composition suitable for storage. One such composition is a
lyophilized preparation of the delivery construct together with a
suitable stabilizer. Alternatively, 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. For example,
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 may 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.
[0206] Further, the delivery construct compositions of the
invention can be formulated for administration to a subject. Such
vaccine compositions generally comprise one or more delivery
constructs of the invention and a pharmaceutically acceptable
excipient, diluent, carrier, or vehicle. Any such pharmaceutically
acceptable excipient, diluent, carrier, or vehicle known to one of
skill in the art without limitation can be used. Examples of a
suitable excipient, diluent, carrier, or vehicle can be found in
Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing
Co., Easton.
[0207] In certain embodiments, the delivery construct compositions
are formulated for oral administration. In such embodiments, the
compositions are formulated to protect the delivery construct from
acid and/or enzymatic degradation in the stomach. Upon passage to
the neutral to alkaline environment of the duodenum, the delivery
construct then contacts a mucous membrane and is transported across
the polarized epithelial membrane. The delivery constructs may be
formulated in such compositions by any method known by one of skill
in the art, without limitation.
[0208] In certain embodiments, the oral formulation comprises a
delivery construct and one or more compounds that can protect the
delivery construct while it is in the stomach. For example, the
protective compound should be able to prevent acid and/or enzymatic
hydrolysis of the delivery construct. In certain embodiments, 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. In certain embodiments, 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. Preferably, the
oral formulation comprises one or more compounds that can protect
the delivery construct from degradation in the stomach and
facilitate transit of the construct from the stomach to the small
intestine. For example, inclusion of sodium bicarbonate can be
useful in facilitating the rapid movement of intra-gastric
delivered materials from the stomach to the duodenum as described
in Mrsny et al., 1999, Vaccine 17:1425-1433.
[0209] Other methods for formulating 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, 1989, Int J Pancreatol. 5 Suppl:31-6, 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.
[0210] 5.7.1. Kits Comprising Compositions
[0211] In yet another aspect, the invention provides a kit that
comprises a composition of the invention. In certain embodiments,
the kit further comprises instructions that direct administration
of the composition to a mucous membrane of the subject to whom the
composition is administered. In certain embodiments, the kit
further comprises instructions that direct oral administration of
the composition to the subject to whom the composition is
administered.
[0212] In certain embodiments, the kit comprises a composition of
the invention in more or more containers. In certain embodiments,
the composition can be in a unit dosage form, e.g., a tablet,
lozenge, capsule, etc. In certain embodiments, the composition can
be provided in or with a device for administering the composition,
such as, for example, a device configured to administer a
single-unit dose of the composition, e.g., an inhaler.
5.8. Making and Testing Delivery Constructs
[0213] The delivery constructs of the invention are preferably
produced recombinantly, as described below. However, the delivery
constructs may also be produced by chemical synthesis using methods
known to those of skill in the art.
[0214] 5.8.1. Manufacture of Delivery Constructs
[0215] Methods for expressing and purifying the delivery constructs
of the invention are described extensively in the examples below.
Generally, the methods rely on introduction of an expression vector
encoding the delivery construct to a cell that can express the
delivery construct from the vector. The delivery construct can then
be purified for administration to a subject.
[0216] 5.8.2. Testing Delivery Constructs
[0217] Having selected the domains of the delivery construct, the
function of these domains, and of the delivery constructs as a
whole, can be routinely tested to ensure that the constructs can
deliver a macromolecule across mucous membranes of a subject free
from the remainder of the construct, For example, the delivery
constructs can be tested for cell recognition, transcytosis and
cleavage using routine assays. The entire chimeric protein can be
tested, or, the function of various domains can be tested by
substituting them for native domains of the wild-type toxin.
[0218] 5.8.2.1. Receptor binding/Cell recognition
[0219] Receptor binding domain function can be tested by monitoring
the delivery construct's ability to bind to the target receptor.
Such testing can be accomplished using cell-based assays, with the
target receptor present on a cell surface, or in cell-free assays.
For example, delivery construct binding to a target can be assessed
with affinity chromatography. The construct can be attached to a
matrix in an affinity column, and binding of the receptor to the
matrix detected, or vice versa. Alternatively, if antibodies have
been identified that bind to either the receptor binding domain or
its cognate receptor, the antibodies can be used, for example, to
detect the receptor binding domain in the delivery construct by
immunoassay, or in a competition assay for the cognate receptor. An
exemplary cell-based assay that detects delivery construct binding
to receptors on cells comprises labeling the construct and
detecting its binding to cells by, e.g., fluorescent cell sorting,
autoradiography, etc.
[0220] 5.8.2.2. Transcytosis
[0221] The function of the transcytosis domain 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 cell, these assays can also be used to assess the function of
the cell recognition domain.
[0222] The delivery construct's transcytosis activity can be tested
by any method known by one of skill in the art, without limitation.
In certain embodiments, transcytosis activity can be tested by
assessing the ability of a delivery construct to enter a
non-polarized cell to which it binds. Without intending to be bound
to any particular theory or mechanism of action, it is believed
that the same property that allows a transcytosis domain to pass
through a polarized epithelial cell also allows molecules bearing
the transcytosis domain to enter non-polarized cells. Thus, 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. For example, 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.
[0223] In other embodiments, the delivery construct's transcytosis
ability can be tested by assessing the delivery construct's ability
to pass through a polarized epithelial cell. For example, the
delivery construct can be labeled with, for example, a fluorescent
marker and contacted to the apical membranes of a layer of
epithelial cells. Fluorescence detected on the basal-lateral side
of the membrane formed by the epithelial cells indicates that the
transcytosis domain is functioning properly.
[0224] 5.8.2.3. Cleavable Linker Cleavage
[0225] The function of the cleavable linker 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 linkers. Both cell-based and cell-free assays can be used
to test the ability of an enzyme to cleave the cleavable
linkers.
[0226] An exemplary cell-free assay for testing cleavage of
cleavable linkers comprises preparing extracts of polarized
epithelial cells and exposing a labeled delivery construct bearing
a cleavable linker to the fraction of the extract that corresponds
to membrane-associated enzymes. In such assays, the label can be
attached to either the macromolecule to be delivered or to the
remainder of the delivery construct. Among these enzymes are
cleavage enzymes found near the basal-lateral 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. If label is
attached to the macromolecule to be delivered, then little or no
label should be observed on the molecule bound to the antibodies.
Alternatively, the binding agent used in the assay can be specific
for the macromolecule, and the remainder of the construct can be
labeled. In either case, cleavage can be assessed.
[0227] Cleavage can also be tested using cell-based assays that
test cleavage by polarized epithelial cells assembled into
membranes. For example, a labeled delivery construct, or portion of
a delivery construct comprising the cleavable linker, can be
contacted to either the apical or basolateral side of a monolayer
of suitable epithelial cells, such as, for example, Coco-2 cells,
under conditions that permit cleavage of the linker. 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. For example, an antibody specific for the delivery
construct can be used to bind a delivery construct comprising a
label distal to the cleavable linker 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. By
performing such experiments, 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 linker in a delivery construct can be confirmed.
[0228] Further, cleavage can also be tested using a fluorescence
reporter assay as described in U.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.
[0229] Further, cleavage can also be tested using an
intra-molecularly quenched molecular probe, such as those described
in U.S. Pat. No. 6,592,847. Such 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. Thus, such 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.
6. EXAMPLES
[0230] The following examples merely illustrate the invention, and
are not intended to limit the invention in any way.
[0231] 6.1. Construction of a Delivery Construct
[0232] Five exemplary delivery construct expression vectors for
delivering rat growth hormone (rGH) were constructed according to
the following protocol. First, the rGH gene was amplified by PCR,
incorporating restriction enzymes pairs of NdeI and EcoRI, PstI and
PstI, AgeI and EcoRI, or PstI and EcoRI sites at two ends of the
PCR products. After restriction enzyme digestion, the PCR products
were cloned into pPE64-PstI-.DELTA.553, which was digested with the
corresponding restriction enzyme pairs. The resulting constructs
were named as pPE-RGH(NdeI-EcoRI), pntPE-RGH(PstI),
pntPE-RGH(AgeI-EcoRI), and pPE-RGH(PstI-EcoRI). These constructs
thus comprise sequences encoding Domains I and II of ntPE (amino
acids 26-372 as shown in FIG. 3) and rGH (Accession No. P01244; see
Seeburg et al., 1977, Nature 270:486-494 and Page et al., 1981,
Nucleic Acids Res. 9:2087-2104), and are also tagged with a 6-His
motif at the N-terminus of the polypeptide to facilitate
purification. The final plasmids were verified by restriction
enzyme digestions and DNA sequencing.
[0233] Expression vectors comprising cleavable linkers were
constructed by introducing sequences encoding the appropriate amino
acid sequence. To do so, oligonucleotides that encode sequences
complementary to appropriate restriction sites and one of the
following amino acid sequences were synthesized, then ligated into
an expression vector prepared as described above between the ntPE
sequences and the rGH sequences. For Delivery Construct 1, the
cleavable linker sequence was RQPRGGL. For Delivery Construct 2,
the cleavable linker sequence was GGLRQPR. For Delivery Construct
3, the cleavable linker sequence was RQPREGR. For Delivery
Construct 4, the cleavable linker sequence was RQPRVGR. For
Delivery Construct 5, the cleavable linker sequence was
RQPRARR.
[0234] To separate rGH from PE protein in the event that the fusion
protein is taken up by antigen presenting cells, a protease furin
site was also inserted between the cleavable linker and rGH. To do
so, constructs containing a sequence encoding the furin site with
the five different cleavable linkers were made. Oligonucleotide
sequences for the five cleavable linkers and a furin clip site are
shown in Table 3 below. Each of the oligo duplexes was inserted
into PstI site of pPE-RGH(PstI-EcoRI). The final constructs, named
as pPE-RGH-F1, pPE-RGH-F2, pPE-RGH-F3, pPE-RGH-F4, pPE-RGH-F5 were
confirmed by restriction enzyme digestion and DNA sequencing.
TABLE-US-00003 TABLE 3 Oligonucleotide pairs for introducing a
furin cleavage site and protease cleavage site pPE-RGH-1 1 F:
AACTGCAGCGCCAGCCTCGAGGAGGATTACTGCAGAA (SEQ ID NO: 24) 1 R:
TTCTGCAGTAATCCTCCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 25) pPE-RGH-2 2
F: AACTGCAGGGAGGCTTACGCCAGCCTCGACTGCAGAA (SEQ ID NO: 26) 2 R:
TTCTGCAGTCGAGGCTGGCGTAAGCCTCCCTGCAGTT (SEQ ID NO: 27) pPE-RGH-3 3
F: AACTGCAGCGCCAGCCTCGAGAGGGCCGTCTGCAGAA (SEQ ID NO: 28) 3 R:
TTCTGCAGACGGCCCTCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 29) pPE-RGH-4 4
F: AACTGCAGCGCCAGCCTCGAGTCGGCCGTCTGCAGAA (SEQ ID NO: 30) 4 R:
TTCTGCAGACGGCCGACTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 31) pPE-RGH-5 5
F: AACTGCAGCGCCAGCCTCGAGCACGTCGTCTGCAGAA (SEQ ID NO: 32) 5 R:
TTCTGCAGACGACGTGCTCGAGGCTGGCGCTGCAGTT (SEQ ID NO: 33)
[0235] 6.2. Expression of Delivery Constructs
[0236] E. coli BL21(DE3) pLysS competent cells (Novagen, Madison,
Wis.) were transformed using a standard heat-shock method in the
presence of the appropriate plasmid to generate ntPE-rat Growth
Hormone (rGH) expression cells, selected on ampicillin-containing
media, and isolated and grown in Luria-Bertani broth (Difco; Becton
Dickinson, Franklin Lakes, N.J.) with antibiotic, then induced for
protein expression by the addition of 1 mM
isopropyl-D-thiogalactopyranoside (IPTG) at OD 0.6. Two hours
following IPTG induction, cells were harvested by centrifugation at
5,000 rpm for 10 min. Inclusion bodies were isolated following cell
lysis and proteins were solubilized in the buffer containing 100 mM
Tris_HCl (pH 8.0), 2 mM EDTA, 6 M guanidine HCl, and 65 mM
dithiothreitol. Solubilized His ntPE-rGH is refolded in the
presence of 0.1 M Tris, pH=7.4, 500 mM L-arginine, 0.9 mM GSSG, 2
mM EDTA. The refolded proteins were purified by Q sepharose Ion
Exchange and Superdex 200 Gel Filtration chromatography (Amersham
Biosciences, Inc., Sweden). The purity of proteins was assessed by
SDS-PAGE and analytic HPLC (Agilent, Inc. Palo Alto, Calif.).
[0237] 6.3. Characterization of a Delivery Construct
[0238] The following procedures can be used to assess proper
refolding of a delivery construct. The protein refolding process is
monitored by measuring, e.g., Delivery Construct 1 binding activity
with ntPE binding receptor, CD 91 receptors, and rGH binding
proteins on a Biacore SPR instrument (Biacore, Sweden) according to
the manufacturer's instructions. Proper refolding of other
macromolecules in exemplary constructs can be tested in similar
binding assays with appropriate binding agents. By testing such
binding affinities, the skilled artisan can assess the proper
folding of each portion of the delivery construct.
[0239] 6.4. Delivery Construct Cleavage Assays
[0240] This example describes experiments performed to identify and
verify enzymes that can be used to cleave the cleavable linkers of
the delivery constructs described herein. First, Caco-2 (ATCC
Accession No. HTB-37) cells in passage 21 were obtained from
American Type Culture Collection (Manassas, Va.). Human tracheal
epithelial (HTE) cells were obtained from J. Whiddecombe of the
Department of Physiology at the University of California, Davis
Medical School. Caco-2 cells were routinely grown on 75 cm.sup.2
plastic culture flasks (Becton Dickinson, Franklin Lakes, N.J.) in
DMEM containing 10% fetal bovine serum and 1%
penicillin-streptomycin at 37.degree. C. in a 5% CO.sub.2/95% air
atmosphere. HTE cells were grown as described in Yamaya et al.,
1992, Am J Physiol 262(6 Pt 1):L713-24.
[0241] To identify suitable cleavable linkers, HTE or Caco-2 cells
were seeded at a density of 5.times.10.sup.4 cells/cm.sup.2 onto
24-well collagen-coated polycarbonate transwell filters (Corning,
Acton, Mass.) for 12-14 days. Confluent monolayers achieved a
transepithelial resistance (TER) of >500 ohmcm.sup.2, as
measured using an EVOM epithelial voltohmmeter and STX2 electrode
(World Precision Instruments, Sarasota, Fla.). To determine
specific enzyme activity, substrates specific for the tested
peptidase (500 .mu.M or 1 mM substrate in 250 .mu.l DMEM without
FBS or antibiotics) were added to either the apical (AP) or
basolateral (BL) side of the monolayers. Peptidase substrates were
obtained from Calbiochem, Inc. (Division of EMD Biosciences, Inc.,
San Diego, Calif.). Cells were incubated for 2 hrs at 37.degree. C.
in a 5% CO.sub.2/95% air atmosphere. Both the apical and
basolateral media was then measured for its specific enzyme
activity according to the manufacturer's instruction. Cleavage was
assessed by detecting fluorescence of the substrates, which
reflects cleavage because it separates of the quenching agent from
the fluorescent agent present on the substrate, which separation
allows fluorescence to be detected.
[0242] Table 4 presents a summary of the results of these assays
using HTE cells, while Table 5 presents a summary of the results of
these assays using Caco-2 cells. For all results, baseline control
values were subtracted from substrate values before percentages
were determined and tests were performed at least in duplicate. The
percentages presented in the tables represent the percent increase
observed in assay in the apical or basolateral media, which depends
on which side of the membrane exhibits higher peptidase activity.
It should be noted that, even when substrate was added to the media
on the apical side of the membrane, peptidase activity can be
observed on the basolateral side of the membrane because of
diffusion of the substrate across the membrane.
TABLE-US-00004 TABLE 4 AP BL Peptidase tested in % % A405 A405 HTE
cells AP > BL BL > AP nm nm AP-500 uM Cathepsin B I 86% 0.37
0.20 BL-500 uM Cathepsin B I 64% 0.35 0.21 AP-500 uM Cathepsin G I
882% 0.04 0.004 BL-500 uM Cathepsin G I 6% 0.02 0.02 AP-500 uM
Cathepsin G II 371% 0.02 0.01 BL-500 uM Cathepsin G II 0% 0% 0.02
0.02 AP-500 uM Cathepsin G III 11% 0.02 0.02 BL-500 uM Cathepsin G
III 400% 0.01 0.03 AP-500 uM Chymotrypsin I 74% 0.04 0.02 BL-500 uM
Chymotrypsin I 36% 0.03 0.04 AP-500 uM Elastase I 49% 0.05 0.03
BL-500 uM Elastase I 23% 0.02 0.03 AP-500 uM Elastase II 43% 0.29
0.20 BL-500 uM Elastase II 31% 0.18 0.13 AP-500 uM Elastase III 89%
0.04 0.02 BL-500 uM Elastase III 967% 0.03 0.003 AP-500 uM Elastase
IV 84% 0.35 0.19 BL-500 uM Elastase IV 65% 0.23 0.14 AP-500 uM
Elastase VIII 529% 0.16 0.03 BL-500 uM Elastase VIII 181% 0.09 0.03
AP-500 uM Papain 57% 0.02 0.02 BL-500 uM Papain 5% 0.03 0.03 AP-500
uM Subtilisin A I 9% 0.02 0.02 BL-500 uM Subtilisin A I 3000% 0.001
0.03 AP-500 uM Subtilisin A II 21% 0.02 0.02 BL-500 uM Subtilisin A
II 55% 0.01 0.02 AP-500 uM Thrombin I 42% 0.15 0.11 BL-500 uM
Thrombin I 15% 0.09 0.10 AP-500 uM Thrombin II 445% 0.40 0.07
BL-500 uM Thrombin II 741% 0.41 0.05 AP-500 uM Urokinase I 8% 0.11
0.10 BL-500 uM Urokinase I 4% 0.13 0.13 AP-1 mM Cathepsin B I 17%
0.18 0.15 BL-1 mM Cathepsin B I 42% 0.24 0.17 AP-1 mM Cathepsin G I
114% 0.05 0.02 BL-1 mM Cathepsin G I 47% 0.02 0.03 AP-1 mM
Cathepsin G II 138% 0.05 0.02 BL-1 mM Cathepsin G II 19% 0.03 0.03
AP-1 mM Cathepsin G III 225% 0.07 0.02 BL-1 mM Cathepsin G III 54%
0.02 0.03 AP-1 mM Chymotrypsin I 35% 0.06 0.04 BL-1 mM Chymotrypsin
I 90% 0.04 0.07 AP-1 mM Elastase I 108% 0.02 0.03 BL-1 mM Elastase
I 864% 0.01 0.09 AP-1 mM Elastase II 62% 0.28 0.17 BL-1 mM Elastase
II 42% 0.33 0.23 AP-1 mM Elastase III 318% 0.02 0.01 BL-1 mM
Elastase III 131% 0.04 0.02 AP-1 mM Elastase IV 94% 0.41 0.21 BL-1
mM Elastase IV 61% 0.30 0.19 AP-1 mM Elastase VIII 233% 0.14 0.04
BL-1 mM Elastase VIII 41% 0.06 0.05 AP-1 mM Papain 424% 0.02 0.004
BL-1 mM Papain 141% 0.02 0.01 AP-1 mM Subtilisin A I 18% 0.03 0.03
BL-1 mM Subtilisin A I 290% 0.01 0.04 AP-1 mM Subtilisin A II 17%
0.03 0.02 BL-1 mM Subtilisin A II 318% 0.01 0.04 AP-1 mM Thrombin I
27% 0.17 0.14 BL-1 mM Thrombin I 28% 0.17 0.13 AP-1 mM Thrombin II
20% 0.12 0.10 BL-1 mM Thrombin II 13% 0.05 0.06 AP-1 mM Urokinase I
14% 0.19 0.21 BL-1 mM Urokinase I 4% 0.21 0.20
TABLE-US-00005 TABLE 5 AP BL Peptidase tested in % % A405 A405
Caco-2 cells AP > BL BL > AP nm nm AP-500 uM Cathepsin B I
150% 0.14 0.06 BL-500 uM Cathepsin B I 34% 0.17 0.12 AP-10 mM
Cathepsin G I 195% 0.31 0.10 BL-10 mM Cathepsin G I 145% 0.42 1.03
AP-500 uM Cathepsin G III 35% 0.014 0.01 BL-500 uM Cathepsin G III
185% 0.03 0.01 AP-500 uM Cathepsin G I 232% 0.05 0.01 BL-500 uM
Cathepsin G I 1709% 0.01 0.15 AP-500 uM Cathepsin G II 3900% 0.01
0.0003 BL-500 uM Cathepsin G II 1330% 0.003 0.04 AP-500 uM
Chymotrypsin I 342% 0.01 0.04 BL-500 uM Chymotrypsin I 403% 0.03
0.16 AP-500 uM Elastase I 73% 0.01 0.01 BL-500 uM Elastase I 295%
0.04 0.01 AP-500 uM Elastase II 59% 0.12 0.07 BL-500 uM Elastase II
89% 0.01 0.02 AP-500 uM Elastase III 85% 0.01 0.07 BL-500 uM
Elastase III 320% 0.003 0.01 AP-500 uM Elastase IV 32% 0.11 0.08
BL-500 uM Elastase IV 12% 0.02 0.02 AP-500 uM Elastase VIII 16%
0.02 0.02 BL-500 uM Elastase VIII 115% 0.01 0.02 AP-500 uM Papain
19% 0.018 0.02 BL-500 uM Papain 339% 0.07 0.02 AP-500 uM Subtilisin
A I ***23% -- 0.05 BL-500 uM Subtilisin A I ***94% -- 0.20 AP-500
uM Subtilisin A II N/A -- -- BL-500 uM Subtilisin A II ***11% --
0.02 AP-500 uM Thrombin I 81% 0.04 0.02 BL-500 uM Thrombin I 254%
0.01 0.04 AP-Thrombin II 500 uM 42% 0.08 0.06 BL-Thrombin II 500 uM
62% 0.09 0.06 AP-500 uM Urokinase I 111% 0.12 0.06 BL-500 uM
Urokinase I 1044% 0.005 0.05 AP-1 mM Cathepsin B I 109% 0.27 0.13
BL-1 mM Cathepsin B I 58% 0.12 0.2 AP-20 mM Cathepsin G I 129% 0.10
0.23 BL-20 mM Cathepsin G I 540% 0.11 0.70 AP-1 mM Cathepsin G III
37% 0.01 0.01 BL-1 mM Cathepsin G III 103% 0.07 0.03 AP-1 mM
Cathepsin G I 107% 0.08 0.04 BL-1 mM Cathepsin G I 144% 0.12 0.05
AP-1 mM Cathepsin G II 11% 0.05 0.06 BL-1 mM Cathepsin G II 7850%
0.00 0.04 AP-1 mM Chymotrypsin I 107% 0.08 0.04 BL-1 mM
Chymotrypsin I 288% 0.02 0.07 AP-1 mM Elastase I 217% 0.03 0.001
BL-1 mM Elastase I 880% 0.003 0.02 AP-1 mM Elastase II 27% 0.17
0.14 BL-1 mM Elastase II 34% 0.02 0.03 AP-1 mM Elastase III 192%
0.02 0.01 BL-1 mM Elastase III 77% 0.02 0.01 AP-1 mM Elastase IV
42% 0.16 0.11 BL-1 mM Elastase IV 10% 0.04 0.05 AP-1 mM Elastase
VIII 70% 0.05 0.03 BL-1 mM Elastase VIII 332% 0.11 0.03 AP-1 mM
Papain 61% 0.02 0.01 BL-1 mM Papain 0% 0.005 0.005 AP-1 mM
Subtilisin A I ***61% -- 0.13 BL-1 mM Subtilisin A I ***44% -- 0.09
AP-1 mM Subtilisin A II N/A -- -- BL-1 mM Subtilisin A II N/A -- --
AP-1 mM Thrombin I 420% 0.11 0.02 BL-1 mM Thrombin I 3400% 0.005
0.16 AP-Thrombin II 1 mM 163% 0.14 0.05 BL-Thrombin II 1 mM 29%
0.11 0.09 AP-1 mM Urokinase I 57% 0.17 0.11 BL-1 mM Urokinase I
230% 0.05 0.15 ***denotes % over baseline control only "--" denotes
values below baseline control
[0243] 6.5. Delivery of an Exemplary Macromolecule--Green
Fluorescent Protein
[0244] The following example describes experiments performed to
assess the transcytosis of an exemplary delivery construct for
delivering green fluorescent protein ("GFP") across a mouse
epithelial membrane. It is noted that this exemplary delivery
construct does not comprise a cleavable linker; however, the
presence or absence of the cleavable linker should not affect
transcytosis of the delivery construct.
[0245] Briefly, a nt-PE-GFP construct was applied to the trachea of
anesthetized female balb/c mice which were approximately 8 weeks of
age. The mice were anesthetized with inhaled isoflurane and the
trachea was exposed. A small hole was made on the trachea to allow
application of our GFP material. In our experiments, 100 .mu.g of
GFP alone or ntPE-GFP was used, respectively. The GFP material was
slowly dripped directly onto the exposed trachea in a 100 .mu.l
volume. After 15 minutes, the mice were euthanized by CO.sub.2
asphyxiation. The trachea was removed and frozen in OCT
(cat#25608-930-Tissue Tek) using biopsy cryomolds (cat#4565-Tissue
Tek). The samples were sectioned onto slides and visualized by
fluorescence microscopy (Nikon model Eclipse E400).
[0246] Micrographs of the epithelial sections are presented as
FIGS. 1A-1C. FIG. 1A shows the nt-PE-GFP construct adhering
strongly to the apical surface of the trachea epithelium. FIG. 1B
shows transcytosis of the nt-PE-GFP construct across the trachea
epithelium. FIG. 1C shows release of the nt-PE-GFP construct from
the basolateral side of the trachea epithelium. FIG. 1D presents a
micrograph of a negative control, a tracheal epithelial section
from a mouse contacted with GFP alone. The tissues exposed for 15
minutes to obtain these micrographs.
[0247] The micrographs demonstrate that nt-PE-GFP interacts
strongly with receptors on the apical surface of mouse tracheal
epithelium, transcytoses across such epithelial tissue, and
releases from the basolateral surface of the mouse tracheal
epithelium.
[0248] In addition, plasma concentrations of the nt-PE-GFP
construct were determined following administration of the delivery
construct using an ELISA assay as described in Example 6.6.1,
below. Serum samples were taken from anesthetized mice that had
received intranasal administration of 100 .mu.g of the nt-PE-GFP
delivery construct every 30 minutes following administration. FIG.
2 presents the results of this experiment, demonstrating that peak
plasma levels of the delivery construct reached between 500-900
ng/ml, indicating that the delivery construct displayed
approximately 22% bioavailability following intranasal
administration.
[0249] 6.6. Detection of Growth Hormone Protein in Tissue by
Histological Examination
[0250] This example describes histological detection in tissues of
a representative macromolecule for delivery, growth hormone.
Following administration of a delivery construct, animals are
euthanized by CO.sub.2 asphyxiation and exanguinated by cardiac
puncture. Specific tissues (lymph nodes, trachea, brain, spleen
liver, GI tract) are removed, briefly rinsed in PBS to remove any
residual blood and frozen in OCT. Sections (5 microns thick) are
placed onto slides. Slides are fixed in acetone for 10 min and
rinsed with PBS. Slides are incubated with 3% peroxidase for 5 min.
Slides are then blocked with protein for an additional 5 min.
Primary growth hormone antibody is incubated onto slides for 30 min
at a 1:100 dilution followed by PBS washes. Biotin-labeled
secondary antibody is then incubated for approximately 15 minutes
followed by PBS washes. Streptavidin HRP label is incubated onto
slides for 15 min followed by PBS washes. HRP Chromagen is applied
for 5 min followed by several rinses in distilled H.sub.20.
Finally, the slides are counterstained with hematoxylin for 1 min,
coverslipped, and examined for the presence of GH.
[0251] 6.7. Transport and Cleavage of an Exemplary Delivery
Construct in an in Vitro System
[0252] This example describes transport and cleavage of an
exemplary delivery construct, Delivery Construct 2, comprising rat
growth hormone (rGH) in an in vitro system using human tracheal
epithelial cells.
[0253] 6.7.1. Growth of Human Tracheal Epithelial Cells
[0254] Human tracheal epithelial (HTE) cells were isolated from
tracheas as previously described and cultured on semi-permeable
filter systems (0.45 um pore size; Corning, Acton, Mass.) coated
with human placental collagen. See Yamaya et al., 1992, Am J
Physiol, 262:L713-24 and Sachs et al., 2003, In Vitro Cell Dev Biol
Anim, 39:56-62. Cell sheets were used at >10 days following
plating, at which time they had a transepithelial resistance (TER)
of >100 .OMEGA.ohmscm.sup.2 as measured with a "chopstick"
voltohmeter (Millicell ERS, Manassas, Va.).
[0255] Caco-2 cells in passage 21 were obtained from American Type
Culture Collection (Manassas, Va.). Cells were routinely grown on
75 cm.sup.2 plastic culture flasks (Becton Dickinson, Franklin
Lakes, N.J.) in DMEM containing 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin at 37.degree. C. in a 5% CO.sub.2/95% air
atmosphere. For the transport and cleavage studies, Caco-2 cells
were seeded at a density of 5.times.10.sup.4 cells/cm.sup.2 onto
24-well collagen-coated polycarbonate transwell filters (Corning,
Acton Mass.) for 12-14 days. Confluent monolayers achieved a
transepithelial resistance (TER) of >500 ohm/cm.sup.2, as
measured using the EVOM and STX2 electrode (World Precision
Instruments).
[0256] 6.7.2. Transport and Cleavage Assays
[0257] To determine transport and cleavage activity, Delivery
Constructs 1 and 2 proteins (10 .mu.g in 100 .mu.l DMEM without
phenol red, FBS or antibiotics) were added to the apical side of
the epithelial monolayer. Cells were incubated for 4 hrs at
37.degree. C. in a 5% CO.sub.2/95% air atmosphere. Both the apical
and basolateral media was then assayed for its transport and
cleavage activity by testing for the presence of rGH cleaved from
the delivery construct by western blot analysis, as described
below. As a control, 10 .mu.g of dextran fluorescein was also added
to the apical wells in order to check for leakage.
[0258] Following the 4 hour incubation described above, apical and
basolateral media samples were precipitated by the addition of
trichloroacetic acid (TCA). The amount of TCA added to each sample
was twenty percent relative to the sample volume. Each sample was
vortexed and placed on ice for 30 minutes. After samples this
incubation on ice, samples were centrifuged at 14,000 RPM for 10
minutes. Next, the supernatants from each sample were aspirated and
tubes containing the remaining pellet were left open to air dry. 4
.mu.l 0.2M NaOH was added to each pellet. Five minutes after the
addition of NaOH, pellets were resuspended in 36 .mu.l 8M Urea.
[0259] Next, 10 .mu.l Sample Buffer containing DTT (Invitrogen
NP0007, NP0009) was added to each sample. Samples were then placed
on a 100.degree. C. heating block for 5 minutes. Half (19 .mu.l) of
each sample was then loaded on to a 4%-12% Tris Bis Gel (Invitrogen
NP3022BOX). For controls, recombinant rat GH (RDI R0125) and
Delivery Construct 1 or Delivery Construct 2 proteins were also
loaded directly into the gel. Electrophoresis was at 150V for 30
minutes. From the gel, samples were transferred onto nitrocellulose
at 30V for 1 hour.
[0260] The Blocking Solution, Antibody Diluents, and Antibody Wash
Solution from Invitrogen's WesternBreeze were used in subsequent
steps. The nitrocellulose membrane was placed in blocking buffer
and incubated at 4.degree. C., overnight. The membrane was washed 3
times for 3 minutes each time. The membrane was then incubated at
RT with 10 ml of 1:2000 rabbit anti growth hormone (RDI
RDIRtGHabr). After one hour, the membrane was again rinsed 3 times
for 3 minutes each. Membrane was incubated for 1 hour at room
temperature with 10 ml of goat anti rabbit IgG AP (Pierce 31340) at
1:5000. The membrane was rinsed 3 times for 3 minutes per wash. 5
.mu.l of substrate (Pierce 34042) was added to the membrane. After
color development reached desired intensity, reaction was halted by
the removal of substrate and the addition of purified water.
Finally, the membrane was washed in purified water for 30 minutes
and air dried.
[0261] Results of the Western Blot analysis are presented in FIG.
4. As seen in FIG. 4, media from the basolateral side of the
epithelial cell layer contained protein consistent with cleaved rGH
separated from the remainder of Delivery Construct 2. In contrast,
media from the apical side of the epithelial cell layer contained
largely intact delivery construct. Thus, application of Delivery
Construct 2 to the apical side of the human epithelial cell
membrane resulted in both transport to the basolateral side of the
membrane and proper cleavage of the construct as shown by release
of rGH detectable with anti-r(1H antibody and of proper apparent
molecular weight. Similar results were also observed for Delivery
Construct 1 (data not shown).
[0262] 6.8. Delivery of an Exemplary Macromolecule in an In Vivo
System
[0263] This example describes use of exemplary Delivery Construct 2
in a mouse model, showing effective transport and cleavage of the
delivery construct in vivo and the bioactivity of the macromolecule
delivered by Delivery Construct 2, rGH.
[0264] 6.8.1. Administration of a Delivery Construct Comprising Rat
Growth Hormone
[0265] Using an animal feeding needle, 100 .mu.g of Delivery
Construct 2 (in 250 .mu.l total volume) was orally delivered to
female BALB/c mice, 5-6 weeks of age (Charles River Laboratories,
Wilmington, Mass.). Delivery Construct 2 was diluted in 1 mg/ml
bovine serum albumin (BSA) and phosphate buffered saline (PBS). As
a positive control, control mice were subcutaneously (SC) injected
on their dorsal side with 30 .mu.g of recombinant rat growth
hormone (rGH) diluted in PBS (100 .mu.l total volume). At specific
times after oral gavage and SC administration, mice were euthanized
by CO.sub.2 asphyxiation and exsanguinated. Whole blood and liver
were collected and analyzed as described below. Because in the
difference in molecular weights between Delivery Construct 2 and
rGH, essentially the same number of rGH molecules were administered
in both routes.
[0266] 6.8.2. Pharmacokinetics of an Exemplary Macromolecule
Administered with a Delivery Construct in an In Vivo System
[0267] To assess the pharmacokinetics of an exemplary macromolecule
delivered with a delivery construct, ELISA assays were used to
measure serum concentrations of rGH at defined timepoints following
administration. The serum concentration data thus obtained was used
to compare the pharmacokinetics of rGH administered with Delivery
Construct 2 to those observed with conventional subcutaneous
administration. The ELISA assays were performed as follows.
[0268] Costar 9018 E.I.A./R.I.A. 96-well plates were coated
overnight with 200 ng/well of goat anti-rGH (Diagnostics Systems
Laboratories, Cat. No. R01235) in 0.2M
NaHCO.sub.3--Na.sub.2CO.sub.3, pH 9.4. Each 96-well plate was
washed four times with PBS containing 0.05% Tween 20-0.01%
thimerosal (wash buffer); blocked for 1 h with 200 .mu.l/well of
PBS/Tween 20 containing 0.5% BSA-0.01% thimerosal (assay buffer).
The standard curve was prepared using recombinant rat GH
(Diagnostics Systems Laboratories, Cat. No. R01205) diluted in
assay buffer (PBST-0.5% BSA). The first point of the standard curve
was prepared by adding 50 .mu.l recombinant rat GH to 10 ml assay
buffer (1:200), vortexed, and moved 200 .mu.l to 800 .mu.l assay
buffer (1:5). For each plate, 0.5 ml was moved to 0.5 ml assay
buffer by doing 1:2 serial dilutions for all the subsequent points.
The 10 points of the standard curve are: 100, 50, 25, 12.5, 6.25,
3.125, 1.56, 0.78, 0.39 and 0.195 ng/well. Samples were diluted at
1:10 with assay buffer, loaded 100 .mu.l/well in triplicates onto a
96-well plate, and incubated overnight. Each 96-well plate was then
washed four times with wash buffer, loaded 100 .mu.l/well of
2.sup.nd Ab (rabbit anti-rGH, Cell Sciences, Cat. No. PAAC1) at
1:300 in assay buffer (PBST-0.5% BSA) and incubate at RT for four
hours. Each 96-well plate was then washed four times with wash
buffer, loaded 100 .mu.l/well of 3rd Ab (goat anti-rabbit
IgG-horseradish peroxidase (HRP), Pierce, Cat. No. 31460) at 1:2000
in assay buffer (PBST-0.5% BSA) and incubated at room temperature
for two hours. All incubation and coating steps were performed at
room temperature on a shaker at 6 RPM. The HRP substrate, TMB
(3,3',5,5''tetramethylbenzidine), used to quantify bound antibody,
was measured at 450 nm.
[0269] ELISA results are reported as the averages of the triplicate
OD (450 nm) value of each sample. Rat GH concentrations were
determined by the exceeding mean value plus three times the
standard error of the mean (SEM) of the appropriate control
value.
[0270] The results of the ELISA assays are presented in FIGS. 5-7.
FIG. 5 presents serum rGH concentrations in BALB/c mice which were
dosed by subcutaneous (SC) injection of 30 .mu.g of
non-glycosylated recombinant rat GH. Individual mice sera were
tested at a dilution of 1:10, and the group average of rGH
concentration were reported (n=4 mice per time point). Standard
error of the mean (SEM) was indicated by the error bars. As shown
in FIG. 5, peak serum concentration of about 240 ng/ml rGH was
observed 30 minutes after administration of subcutaneous rGH.
[0271] FIG. 6 presents serum rGH concentrations in BALB/c mice
which were dosed orally with 100 .mu.g of Delivery Construct 2.
Individual mice sera were tested at a dilution of 1:10, and the
group average of rGH concentration were reported (n=4 mice per time
point). Standard error of the mean (SEM) was indicated by the error
bars. As shown in FIG. 5, peak serum concentration of about 280
ng/ml rGH was observed 20 minutes after administration of Delivery
Construct 2.
[0272] FIG. 7 presents a graphical representation comparing
pharmacokinetics of rGH delivered subcutaneously and with Delivery
Construct 2. The curve fitting comparison between rGH (SC) and
Delivery Construct 2(Oral) was performed by evaluating ELISA data
as described above using PK Solutions 2.0, Pharmacokinetics Data
Analysis (Summit Research Services, Montrose, Colo.). As shown in
FIG. 7, Delivery Construct 2 yields substantially higher peak serum
rGH concentrations than subcutaneous administrations of rGH.
Further, the peak serum concentration is achieved faster with
Delivery Construct 2 relative to subcutaneous rGH. The
bioavailability of rGH delivered with Delivery Construct 2 observed
was about 60% relative to rGH administered by subcutaneous
injection.
[0273] 6.8.3. Assays Demonstrating Activity of a Macromolecule
Following Delivery with a Delivery Construct
[0274] This example describes analysis of the biological effects of
an exemplary macromolecule, rGH, delivered with Delivery Construct
2 in an in vivo system. In brief, insulin-like growth factor
I-binding protein 3 (IGF-1-BP3), growth hormone (GH) receptor and
insulin-like growth factor I (IGF-I) expression levels were
assessed in liver tissue from mice administered either Delivery
Construct 2 or subcutaneous rGH as described above. These
transcripts were analyzed because of the well-characterized effects
of GH binding to its receptor on IGF-1-BP3 and GH receptor levels.
In particular, functional activation of the GH receptor following
binding by GH is known to result in upregulation of IGF-1-BP3 and
downregulation of GH receptor expression. Of these, upregulation of
IGF-1-BP3 mRNA expression is believed to be the most reliable
indicator of GH receptor activation. See, e.g., Sondergaard et al.,
2003, Am J Physiol Endocrinol Metab 285:E427-32.
[0275] Thus, Quantitative Real Time PCR was used to detect and
quantify the amount of IGF-1-BP3, GH receptor, IGF-I, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in
approximately 30 mg of mouse liver tissue prepared as described
above. Collected liver tissue was stored at -70.degree. C. until
further processing. Real-time detection of PCR was performed using
the Applied Biosystems 7300 Real Time PCR system (Applied
Biosystems, Foster City, Calif.). Total RNA from mouse liver was
isolated according to the RNeasy Protect Mini Kit (Qiagen). Total
RNA was used to generate cDNA for oligo dT oligodeoxynucleotide
primer (T12-18) following the protocol for Omniscript Reverse
Transcriptase (Qiagen). The primers used to amplify the cDNA were
designed using Primer Express software (Applied Biosystems),
synthesized by Operon (Alameda, Calif.), and are shown in Table
6:
TABLE-US-00006 TABLE 6 RT-PCR Primers IGF-I-BP3 (forward):
CGCAGAGAAATGGAGGACACA; IGF-I-BP3 (reverse): GGACGCCTCTGGGACTCA; GH
receptor GTTGACGAAATAGTGCAACCTGAT; (forward): GH receptor
CACGAATCCCGGTCAAACTAA; (reverse): IGF-I (forward):
GCTATGGCTCCAGCATTCG; IGF-I (reverse): GCTCCGGAAGCAACACTCA GAPDH
(forward): GCAACAGGGTGGTGGACCT GAPDH (reverse):
GGATAGGGCCTCTCTTGCTCA
[0276] Equal amounts of cDNA were used in duplicate and amplified
with the SYBR Green I Master Mix (Applied Biosystems). The thermal
cycling parameters were as follows: thermal activation for 10 min
at 95.degree. C., and 40 cycles of PCR (melting for 15 s at
95.degree. C. and annealing/extension for 1 min at 60.degree. C.).
A standard curve was constructed with a dilution curve (1:10,
1:100, 1:500, 1:1,000, 1:2,000) of total RNA from a control mouse
liver sample. A "no template control" was included with each PCR.
Amplification efficiencies were validated and normalized against
GAPDH. Correct PCR product size was confirmed by electrophoresis
through a 1% agarose gel stained with ethidium bromide. Purity of
the amplified PCR products was determined by a heat-dissociation
protocol.
[0277] The results of this analysis are shown in FIGS. 8-10. FIG. 8
shows expression levels of IGF-1-BP3 mRNA in the liver of female
BALB/c mice treated with 30 .mu.g recombinant rGH by subcutaneous
injection or with 100 .mu.g of Delivery Construct 2 by oral gavage.
Total RNA extracted from the liver was subjected to quantitative
RT-PCR using primers specific for IGF-1-BP3, as described above.
Values were normalized to glyceraldehyde-3 phosphate dehydrogenase
(GAPDH) and expressed as % of control. As shown in FIG. 8,
administration of subcutaneous rGH resulted in an about 250%
increase in IGF-1-BP3 mRNA expression levels in the liver at 60
minutes following administration. In contrast, Delivery Construct 2
caused an almost 400% increase in IGF-1-BP3 mRNA expression levels
in the liver 30 minutes following administration. Thus, oral
administration of Delivery Construct 2 effectively delivered rGH to
the bloodstream of the test mice, thereby demonstrating that a
delivery construct can effectively deliver an active macromolecule
across a mucous membrane in an in vivo system. Moreover, Delivery
Construct 2 delivered more active rGH to the liver than
subcutaneous administration, and the effects of administration of
rGH were observed substantially faster than possible with
subcutaneous administration of rGH.
[0278] FIG. 9 shows expression levels of growth hormone (GH)
receptor mRNA in the liver of female B ALB/c mice treated with
recombinant rat growth hormone (rGH) by subcutaneous injection (30
.mu.g) or Delivery Construct 2 by oral gavage (100 .mu.g). Total
RNA extracted from the liver was subjected to quantitative RT-PCR
using primers specific for GH receptor, shown above. Values were
normalized to glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and
expressed as % of control. As shown in FIG. 9, administration of
subcutaneous rGH resulted in a reduction in GH receptor mRNA
expression levels in the liver at 60 minutes following
administration to about 65% of those observed prior to
administration. In contrast, Delivery Construct 2 caused such mRNA
levels to decrease to about 15% of those observed prior to
administration. Thus, these results confirm that oral
administration of Delivery Construct 2 is effective to deliver rGH
to the bloodstream of a subject, and further, that Delivery
Construct 2 delivers significantly more active rGH to mouse liver
than conventional subcutaneous administration of rGH as shown by
the enhanced downregulation of GH receptor mRNA expression.
[0279] FIG. 10 shows expression levels of insulin-like growth
factor I (IGF-I) mRNA in the liver of female BALB/c mice treated
with recombinant rat growth hormone (rGH) by subcutaneous injection
(30 .mu.g) or Delivery Construct 2 by oral gavage (100 .mu.g).
Total RNA extracted from the liver was subjected to quantitative
RT-PCR using primers specific for IGF-I, shown above. Values were
normalized to glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and
expressed as % of control. As shown in FIG. 10, administration of
either subcutaneous rGH or Delivery Construct 2 resulted in a
reduction in IGF-1 mRNA expression levels in the liver at 30
minutes following administration to about 20% of those observed
prior to administration. Thus, both subcutaneous rGH and
orally-administered Delivery Construct 2 yielded the same effects,
demonstrating that Delivery Construct 2 can deliver active rGH to
the bloodstream of a
[0280] 6.9. Reduced Immunogenicity of Macromolecules Administered
with a Delivery Construct
[0281] This example shows that an exemplary macromolecule, rGH,
administered orally with Delivery Construct 2, is less immunogenic
than rGH administered subcutaneously.
[0282] To assess the relative immunogenicity in mice for rGH
administered a delivery construct relative to subcutaneous
administration, the serum titer of anti-rGH IgG antibodies from
oral administration of 3, 10, or 30 .mu.g Delivery Construct 2 or 3
or 10 .mu.g subcutaneous rGH was determined in an ELISA assay. To
do so, 100 .mu.l 1 ng/.mu.l recombinant rGH diluted in coating
buffer (0.2 M NaHCO.sub.3--Na.sub.2CO.sub.3, pH 9.4) was added to
Costar EIA/RIA plates, then incubated at room temperature for 16-24
hours. Next, the plates were washed 4 times with 300 .mu.l wash
buffer (phosphate-buffered saline). The plates were then blocked
with 200 .mu.l blocking buffer (0.5% BSA in phosphate-buffered
saline) and incubated at room temperature for 1 hour. Next, the
plates were again washed four times with 300 .mu.l wash buffer.
[0283] After preparation of the plates, 100 .mu.l diluted samples,
standard positive control, or assay buffer as negative control was
added to the appropriate well and incubated for one hour. Mouse
serum samples and positive control (anti-rGH IgG) were diluted 1:20
in assay buffer (0.5% BSA in phosphate-buffered saline). The plates
were then washed four times with 300 .mu.l wash buffer. Next, 100
.mu.l secondary antibody (goat anti-mouse IgG conjugated to
horseradish peroxidase, 0.4 mg/ml, Pierce #31430, diluted at 1:6000
in assay buffer and incubated at room temperature for one hour.
Next, the plates were again washed four times with 300 .mu.l wash
buffer. Next, 100 .mu.l 3,3',5,5'-Tetramethylbenzidine substrate
(Sigma) was added to each well and incubated for 2-10 minutes,
depending on color development. 100 .mu.l/well 1M H.sub.2SO.sub.4
was then added to stop the reaction and absorbance read at 450 nm.
All assays were performed in triplicate and the results
averaged.
[0284] Representative results of the ELISAs are shown in FIGS.
11A-D. The graphs presented in these figures demonstrate that 3,
10, and 30 .mu.g Delivery Construct 2 administered orally elicited
a lower titer of anti-rGH IgG antibodies than either 3 or 10 .mu.g
subcutaneous rGH. In particular, subcutaneous administration of 10
.mu.g rGH caused a substantial anti-rGH IgG response in all eight
mice, while the eight mice administered 3, 10, or 30 .mu.g Delivery
Construct 2 by oral gavage had minimal anti-rGH IgG responses.
Further, these observations were consistent whether the sera were
diluted 1:25 (FIGS. 11A and 11C) or 1:200 (FIGS. 11B and 11D).
Finally, it should be noted that each mouse administered 3, 10, or
30 .mu.g Delivery Construct 2 experienced a minimal immune response
against rGH, as shown by the tight clustering of the data points in
FIGS. 11C and D. Thus, these results demonstrate that oral
administration of Delivery Construct 2 not only delivers more
active rGH to the liver than possible with subcutaneous injection,
but further, the active rGH is less immunogenic when administered
orally with Delivery Construct 2 compared to subcutaneous
administration.
[0285] 6.10. Exemplary Delivery Construct for Delivery of Human
Growth Hormone
[0286] This example describes construction of an exemplary delivery
construct for delivering human growth hormone, termed Delivery
Construct 6. Techniques similar to those described in Example 6.1,
above, were used to construct a plasmid used to express Delivery
Construct 6. The nucleotide sequence of the portion of this plasmid
that encodes the exemplary delivery construct is presented as FIG.
12, while the amino acid sequence of the delivery construct is
presented as FIG. 13.
[0287] 6.11. Exemplary Delivery Construct for Delivering Inferferon
Alpha
[0288] This example describes construction of an exemplary delivery
construct for delivering IFN.alpha. (in this case, IFN.alpha.-2b),
termed Delivery Construct 7. Techniques similar to those described
in Example 6.1, above, were used to construct a plasmid used to
express Delivery Construct 7. The nucleotide sequence of the
portion of this plasmid that encodes the exemplary Delivery
Construct 7 is presented as FIG. 14, while the amino acid sequence
of Delivery Construct 7 is presented as FIG. 15.
[0289] 6.12. Delivery of Interferon Alpha in an In Vivo System
[0290] This example demonstrates the use of Delivery Construct 7 to
deliver IFN.alpha.-2b to the bloodstream of a subject in a mouse
model system.
[0291] Using an animal feeding needle, 100 .mu.g of Deli very
Construct 7 (in 250 .mu.l total volume) was orally administered to
female BALB/c mice, 5-6 weeks of age (Charles River Laboratories,
Wilmington, Mass.). Delivery Construct 7 was diluted in 1 mg/ml
bovine serum albumin (BSA) and phosphate buffered saline (PBS). At
specific times after oral gavage and SC administration, mice were
euthanized by CO.sub.2 asphyxiation and exsanguinated. Whole blood
was collected and analyzed as described below.
[0292] ELISA assays were used to measure serum concentrations of
IFN.alpha.-2b in one mouse immediately following administration and
in three mice at 15, 30, and 75 minutes following administration.
The ELISA assays were performed using R&D Systems Serum ELISA
Kit No. 41110-1 according to the manufacturer's instructions.
[0293] ELISA results are reported as the averages of the triplicate
OD (450 nm) value of each sample. The results of the ELISA assays
are presented in FIG. 16. As shown in FIG. 16, IFN.alpha.-2b was
detected at low (about 3 ng/ml) concentration 15 minutes after
administration. 30 minutes following administration, serum
concentration of IFN.alpha.-2b was about 43 ng/ml. 45 minutes
following administration, serum concentration of IFN.alpha.-2b had
fallen to about 13 ng/ml.
[0294] 6.13. Exemplary Delivery Construct for Delivering
Proinsulin
[0295] This example describes construction of an exemplary delivery
construct for delivering proinsulin, termed Delivery Construct 8.
Techniques similar to those described in Example 6.1, above, are
used to construct a plasmid used to express Delivery Construct 8.
The amino acid sequence of Delivery Construct 8 is presented as
FIG. 17.
[0296] 6.14. Exemplary Delivery Construct for Delivering
Insulin
[0297] This example describes construction of an exemplary delivery
construct for delivering insulin, termed Delivery Construct 9.
Techniques similar to those described in Example 6.1, above, are
used to construct a plasmid used to express Delivery Construct 9,
with certain modifications.
[0298] In particular, the scheme used to express Delivery Construct
9 is modified because insulin comprises two separate amino acid
chains. In this example, the B-chain of insulin is expressed
together with the remainder of the Delivery Construct, constructed
according to the general scheme presented in Example 6.1. Amino
acids corresponding to the A-chain are made either synthetically
(e.g., chemically synthesizing the A-chain peptide from amino
acids) or recombinantly (e.g., expressed in a suitable recombinant
system such as, for example, E. coli, yeast, etc.) The two
polypeptides are then combined under conditions that permit
association of the A-chain and B-chain. Then, disulfide bonds are
made between the two chains of insulin as found in native insulin
by application of mildly oxidizing conditions. The amino acid
sequence of the two amino acid chains of Delivery Construct 9 is
presented as FIG. 17.
[0299] The present invention provides, inter alia, delivery
constructs and methods of inducing an immune response in a subject.
While many specific examples have been provided, the above
description is intended to illustrate rather than limit the
invention. Many variations of the invention will become apparent to
those skilled in the art upon review of this specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0300] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. Citation of these
documents is not an admission that any particular reference is
"prior art" to this invention.
TABLE-US-00007 TABLE 7 Human Peptidases by Class Aspartic-type
peptidases BAE1_HUMAN (P56817) BAE2_HUMAN (Q9Y5Z0) CATD_HUMAN
(P07339) CATE_HUMAN (P14091) NAP1_HUMAN (O96009) PEPA_HUMAN (P00790
PEPC_HUMAN (P20142) RENI_HUMAN (P00797) VPRT_HUMAN (P10265) Other
Peptidases FAC2_HUMAN (Q9Y256) Cysteine-type peptidases BLMH_HUMAN
(Q13867) CATB_HUMAN (P07858) CATC_HUMAN (P53634) CATF_HUMAN
(Q9UBX1) CATH_HUMAN (P09668) CATK_HUMAN (P43235) CATL_HUMAN
(P07711) CATO_HUMAN (P43234) CATS_HUMAN (P25774) CATW_HUMAN
(P56202) CATZ_HUMAN (Q9UBR2) CSL2_HUMAN (O60911) TNAG_HUMAN
(Q9UJW2) CAN1_HUMAN (P07384) CAN2_HUMAN (P17655) CAN3_HUMAN
(P20807) CAN5_HUMAN (O15484) CAN6_HUMAN (Q9Y6Q1) CAN7_HUMAN
(Q9Y6W3) CAN9_HUMAN (O14815) CANA_HUMAN (Q9HC96) CANB_HUMAN
(Q9UMQ6) UBL1_HUMAN (P09936) UBL3_HUMAN (P15374) UBL5_HUMAN
(Q9Y5K5) GPI8_HUMAN (Q92643) LGMN_HUMAN (Q99538) CFLA_HUMAN
(O15519) I1BC_HUMAN (P29466) ICE2_HUMAN (P42575) ICE3_HUMAN
(P42574) ICE4_HUMAN (P49662) ICE5_HUMAN (P51878) ICE6_HUMAN
(P55212) ICE7_HUMAN (P55210) ICE8_HUMAN (Q14790) ICE9_HUMAN
(P55211) ICEA_HUMAN (Q92851) ICEE_HUMAN (P31944) MLT1_HUMAN
(Q9UDY8) PGPI_HUMAN (Q9NXJ5) FAFX_HUMAN (Q93008) FAFY_HUMAN
(O00507) UB10_HUMAN (Q14694) UB11_HUMAN (P51784) UB12_HUMAN
(O75317) UB13_HUMAN (Q92995) UB14_HUMAN (P54578) UB15_HUMAN
(Q9Y4E8) UB16_HUMAN (Q9Y5T5) UB18_HUMAN (Q9UMW8) UB19_HUMAN
(O94966) UB20_HUMAN (Q9Y2K6) UB21_HUMAN (Q9UK80) UB22_HUMAN
(Q9UPT9) UB24_HUMAN (Q9UPU5) UB25_HUMAN (Q9UHP3) UB26_HUMAN
(Q9BXU7) UB28_HUMAN (Q96RU2) UB29_HUMAN (Q9HBJ7) UB32_HUMAN
(Q8NFA0) UB33_HUMAN (Q8TEY7) UB35_HUMAN (Q9P2H5) UB36_HUMAN
(Q9P275) UB37_HUMAN (Q86T82) UB38_HUMAN (Q8NB14) UB40_HUMAN
(Q9NVE5) UB42_HUMAN (Q9H9J4) UB44_HUMAN (Q9H0E7) UB46_HUMAN
(P62068) UBP1_HUMAN (O94782) UBP2_HUMAN (O75604) UBP3_HUMAN
(Q9Y6I4) UBP4_HUMAN (Q13107) UBP5_HUMAN (P45974) UBP6_HUMAN
(P35125) UBP7_HUMAN (Q93009) UBP8_HUMAN (P40818) GGH_HUMAN (Q92820)
SEN1_HUMAN (Q9P0U3) SEN3_HUMAN (Q9H4L4) SEN5_HUMAN (Q96HI0)
SEN6_HUMAN (Q9GZR1) SEN7_HUMAN (Q9BQF6) SEN8_HUMAN (Q96LD8)
SNP2_HUMAN (Q9HC62) ESP1_HUMAN (Q14674) Metallopeptidases
AMPB_HUMAN (Q9H4A4) AMPE_HUMAN (Q07075) AMPN_HUMAN (P15144)
ART1_HUMAN (Q9NZ08) LCAP_HUMAN (Q9UIQ6) LKHA_HUMAN (P09960)
PSA_HUMAN (P55786) RNPL_HUMAN (Q9HAU8) THDE_HUMAN (Q9UKU6)
ACET_HUMAN (P22966) ACE_HUMAN (P12821) MEPD_HUMAN (P52888)
NEUL_HUMAN (Q9BYT8) PMIP_HUMAN (Q99797) MM01_HUMAN (P03956)
MM02_HUMAN (P08253) MM03_HUMAN (P08254) MM07_HUMAN (P09237)
MM08_HUMAN (P22894) MM09_HUMAN (P14780) MM10_HUMAN (P09238)
MM11_HUMAN (P24347) MM12_HUMAN (P39900) MM13_HUMAN (P45452)
MM14_HUMAN (P50281) MM15_HUMAN (P51511) MM16_HUMAN (P51512)
MM17_HUMAN (Q9ULZ9) MM19_HUMAN (Q99542) MM20_HUMAN (O60882)
MM21_HUMAN (Q8N119) MM24_HUMAN (Q9Y5R2) MM25_HUMAN (Q9NPA2)
MM26_HUMAN (Q9NRE1) MM28_HUMAN (Q9H239) BMP1_HUMAN (P13497)
MEPA_HUMAN (Q16819) MEPB_HUMAN (Q16820) AD02_HUMAN (Q99965)
AD07_HUMAN (Q9H2U9) AD08_HUMAN (P78325) AD09_HUMAN (Q13443)
AD10_HUMAN (O14672) AD11_HUMAN (O75078) AD12_HUMAN (O43184)
AD15_HUMAN (Q13444) AD17_HUMAN (P78536) AD18_HUMAN (Q9Y3Q7)
AD19_HUMAN (Q9H013) AD20_HUMAN (O43506) AD21_HUMAN (Q9UKJ8)
AD22_HUMAN (Q9P0K1) AD28_HUMAN (Q9UKQ2) AD29_HUMAN (Q9UKF5)
AD30_HUMAN (Q9UKF2) AD33_HUMAN (Q9BZ11) AT10_HUMAN (Q9H324)
AT12_HUMAN (P58397) AT14_HUMAN (Q8WXS8) AT15_HUMAN (Q8TE58)
AT16_HUMAN (Q8TE57) AT17_HUMAN (Q8TE56) AT18_HUMAN (Q8TE60)
AT19_HUMAN (Q8TE59) AT20_HUMAN (P59510) ATS1_HUMAN (Q9UHI8)
ATS2_HUMAN (O95450) ATS3_HUMAN (O15072) ATS4_HUMAN (O75173)
ATS5_HUMAN (Q9UNA0) ATS6_HUMAN (Q9UKP5) ATS7_HUMAN (Q9UKP4)
ATS8_HUMAN (Q9UP79) ATS9_HUMAN (Q9P2N4) ECE1_HUMAN (P42892)
ECE2_HUMAN (O60344) ECEL_HUMAN (O95672) KELL_HUMAN (P23276)
NEP_HUMAN (P08473) PEX_HUMAN (P78562) CBP1_HUMAN (P15085)
CBP2_HUMAN (P48052) CBP4_HUMAN (Q9UI42) CBP5_HUMAN (Q8WXQ8)
CBP6_HUMAN (Q8N4T0) CBPB_HUMAN (P15086) CBPC_HUMAN (P15088)
CBPD_HUMAN (O75976) CBPE_HUMAN (P16870) CBPM_HUMAN (P14384)
CBPN_HUMAN (P15169) CPX2_HUMAN (Q8N436) CPXM_HUMAN (Q96SM3)
IDE_HUMAN (P14735) MPPA_HUMAN (Q10713) MPPB_HUMAN (O75439)
NRDC_HUMAN (O43847) UCR1_HUMAN (P31930) UCR2_HUMAN (P22695)
AMPL_HUMAN (P28838) PEL1_HUMAN (Q8NDH3) DNPE_HUMAN (Q9ULA0)
MDP1_HUMAN (P16444) CGL1_HUMAN (Q96KP4) CGL2_HUMAN (Q96KN2)
ACY1_HUMAN (Q03154) GCP_HUMAN (Q9NPF4) AMP1_HUMAN (P53582)
PEPD_HUMAN (P12955) XPP2_HUMAN (O43895) AMP2_HUMAN (P50579)
P2G4_HUMAN (Q9UQ80) FOH1_HUMAN (Q04609) NLD2_HUMAN (Q9Y3Q0)
NLDL_HUMAN (Q9UQQ1) TFR1_HUMAN (P02786) TFR2_HUMAN (Q9UP52)
AF31_HUMAN (O43931) AF32_HUMAN (Q9Y4W6) SPG7_HUMAN (Q9UQ90)
YME1_HUMAN (Q96TA2) PAPA_HUMAN (Q13219) FAC1_HUMAN (O75844)
DPP3_HUMAN (Q9NY33) MS2P_HUMAN (O43462) Serine-type peptidases
ACRL_HUMAN (P58840) ACRO_HUMAN (P10323) APOA_HUMAN (P08519)
BSS4_HUMAN (Q9GZN4) C1R_HUMAN (P00736) C1S_HUMAN (P09871)
CAP7_HUMAN (P20160) CATG_HUMAN (P08311) CFAB_HUMAN (P00751)
CFAD_HUMAN (P00746) CFAI_HUMAN (P05156) CLCR_HUMAN (Q99895)
CO2_HUMAN (P06681) CORI_HUMAN (Q9Y5Q5) CRAR_HUMAN (P48740)
CTRB_HUMAN (P17538) CTRL_HUMAN (P40313) DES1_HUMAN (Q9UL52)
EL1_HUMAN (Q9UNI1) EL2A_HUMAN (P08217) EL2B_HUMAN (P08218)
EL3A_HUMAN (P09093) EL3B_HUMAN (P08861) ELNE_HUMAN (P08246)
ENTK_HUMAN (P98073) FA10_HUMAN (P00742) FA11_HUMAN (P03951)
FA12_HUMAN (P00748) FA7_HUMAN (P08709) FA9_HUMAN (P00740)
GRAA_HUMAN (P12544) GRAB_HUMAN (P10144) GRAH_HUMAN (P20718)
GRAK_HUMAN (P49863) GRAM_HUMAN (P51124) HATT_HUMAN (O60235)
HEPS_HUMAN (P05981) HGFA_HUMAN (Q04756) HGFL_HUMAN (P26927)
HGF_HUMAN (P14210) HPTR_HUMAN (P00739) HPT_HUMAN (P00738) KAL_HUMAN
(P03952) KLK1_HUMAN (P06870) KLK2_HUMAN (P20151) KLK3_HUMAN
(P07288) KLK4_HUMAN (Q9Y5K2) KLK5_HUMAN (Q9Y337) KLK6_HUMAN
(Q92876) KLK7_HUMAN (P49862) KLK8_HUMAN (O60259) KLK9_HUMAN
(Q9UKQ9) KLKA_HUMAN (O43240) KLKB_HUMAN (Q9UBX7) KLKC_HUMAN
(Q9UKR0) KLKD_HUMAN (Q9UKR3) KLKE_HUMAN (Q9P0G3) KLKF_HUMAN
(Q9H2R5) LCLP_HUMAN (P34168) MAS2_HUMAN (O00187) MCT1_HUMAN
(P23946) NETR_HUMAN (P56730) PLMN_HUMAN (P00747) PR27_HUMAN
(Q9BQR3) PRN3_HUMAN (P24158) PRTC_HUMAN (P04070) PRTZ_HUMAN
(P22891) PS23_HUMAN (O95084) PSS8_HUMAN (Q16651) ST14_HUMAN
(Q9Y5Y6) TEST_HUMAN (Q9Y6M0) THRB_HUMAN (P00734) TMS2_HUMAN
(O15393) TMS3_HUMAN (P57727) TMS4_HUMAN (Q9NRS4) TMS5_HUMAN
(Q9H3S3) TMS6_HUMAN (Q8IU80) TPA_HUMAN (P00750) TRB1_HUMAN (Q15661)
TRB2_HUMAN (P20231) TRY1_HUMAN (P07477) TRY2_HUMAN (P07478)
TRY3_HUMAN (P35030) TRYA_HUMAN (P15157) TRYD_HUMAN (Q9BZJ3)
TRYG_HUMAN (Q9NRR2) TS50_HUMAN (Q9UI38) UROK_HUMAN (P00749)
HRA1_HUMAN (Q92743) HRA2_HUMAN (O43464) HRA3_HUMAN (P83110)
HRA4_HUMAN (P83105) FURI_HUMAN (P09958) MS1P_HUMAN (Q14703)
NEC1_HUMAN (P29120) NEC2_HUMAN (P16519) PCK5_HUMAN (Q92824)
PCK6_HUMAN (P29122) PCK7_HUMAN (Q16549) PCK9_HUMAN (Q8NBP7)
TPP2_HUMAN (P29144) PPCE_HUMAN (P48147) DPP4_HUMAN (P27487)
DPP6_HUMAN (P42658) SEPR_HUMAN (Q12884) ACPH_HUMAN (P13798)
CPVL_HUMAN (Q9H3G5) PRTP_HUMAN (P10619) RISC_HUMAN (Q9HB40)
CLPP_HUMAN (Q16740) LONM_HUMAN (P36776) SPC3_HUMAN (Q9BY50)
SPC4_HUMAN (P21378) DPP2_HUMAN (Q9UHL4) PCP_HUMAN (P42785)
TSSP_HUMAN (Q9NQE7) HYEP_HUMAN (P07099) TPP1_HUMAN (O14773)
RHB1_HUMAN (O75783) RHB2_HUMAN (Q9NX52) RHB4_HUMAN (P58872)
Threonine-type peptidases PS7L_HUMAN (Q8TAA3) PSA1_HUMAN (P25786)
PSA2_HUMAN (P25787) PSA3_HUMAN (P25788) PSA4_HUMAN (P25789)
PSA5_HUMAN (P28066) PSA6_HUMAN (P60900) PSA7_HUMAN (O14818)
PSB1_HUMAN (P20618) PSB2_HUMAN (P49721) PSB3_HUMAN (P49720)
PSB4_HUMAN (P28070) PSB5_HUMAN (P28074) PSB6_HUMAN (P28072)
PSB7_HUMAN (Q99436) PSB8_HUMAN (P28062) PSB9_HUMAN (P28065)
PSBA_HUMAN (P40306)
Sequence CWU 1
1
541266PRTP. aeruginosareceptor binding domain of Pseudomonas
exotoxin A 1Met His Leu Ile Pro His Trp Ile Pro Leu Val Ala Ser Leu
Gly Leu1 5 10 15Leu Ala Gly Gly Ser Ser Ala Ser Ala Ala Glu Glu Ala
Phe Asp Leu 20 25 30Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu
Lys Asp Gly Val 35 40 45Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile
Ala Asp Thr Asn Gly 50 55 60Gln Gly Val Leu His Tyr Ser Met Val Leu
Glu Gly Gly Asn Asp Ala65 70 75 80Leu Lys Leu Ala Ile Asp Asn Ala
Leu Ser Ile Thr Ser Asp Gly Leu 85 90 95Thr Ile Arg Leu Glu Gly Gly
Val Glu Pro Asn Lys Pro Val Arg Tyr 100 105 110Ser Tyr Thr Arg Gln
Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val 115 120 125Pro Ile Gly
His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu 130 135 140Leu
Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile145 150
155 160Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr
Phe 165 170 175Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr
Leu Ala Ile 180 185 190Ser His Ala Gly Val Ser Val Val Met Ala Gln
Thr Gln Pro Arg Arg 195 200 205Glu Lys Arg Trp Ser Glu Trp Ala Ser
Gly Lys Val Leu Cys Leu Leu 210 215 220Asp Pro Leu Asp Gly Val Tyr
Asn Tyr Leu Ala Gln Gln Arg Cys Asn225 230 235 240Leu Asp Asp Thr
Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn 245 250 255Pro Ala
Lys His Asp Leu Asp Ile Lys Pro 260 2652153PRTP.
aeruginosatranscytosis domain of Pseudomonas exotoxin A 2Thr Val
Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala1 5 10 15Ala
Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr 20 25
30Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr
35 40 45Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser
Trp 50 55 60Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro
Gly Ser65 70 75 80Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro
Glu Gln Ala Arg 85 90 95Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu
Arg Phe Val Arg Gln 100 105 110Gly Thr Gly Asn Asp Glu Ala Gly Ala
Ala Asn Ala Asp Val Val Ser 115 120 125Leu Thr Cys Pro Val Ala Ala
Gly Glu Cys Ala Gly Pro Ala Asp Ser 130 135 140Gly Asp Ala Leu Leu
Glu Arg Asn Tyr145 15031839DNAP. aeruginosaPolynucleotide
3gccgaagaag ctttcgacct ctggaacgaa tgcgccaaag cctgcgtgct cgacctcaag
60gacggcgtgc gttccagccg catgagcgtc gacccggcca tcgccgacac caacggccag
120ggcgtgctgc actactccat ggtcctggag ggcggcaacg acgcgctcaa
gctggccatc 180gacaacgccc tcagcatcac cagcgacggc ctgaccatcc
gcctcgaagg cggcgtcgag 240ccgaacaagc cggtgcgcta cagctacacg
cgccaggcgc gcggcagttg gtcgctgaac 300tggctggtac cgatcggcca
cgagaagccc tcgaacatca aggtgttcat ccacgaactg 360aacgccggca
accagctcag ccacatgtcg ccgatctaca ccatcgagat gggcgacgag
420ttgctggcga agctggcgcg cgatgccacc ttcttcgtca gggcgcacga
gagcaacgag 480atgcagccga cgctcgccat cagccatgcc ggggtcagcg
tggtcatggc ccagacccag 540ccgcgccggg aaaagcgctg gagcgaatgg
gccagcggca aggtgttgtg cctgctcgac 600ccgctggacg gggtctacaa
ctacctcgcc cagcaacgct gcaacctcga cgatacctgg 660gaaggcaaga
tctaccgggt gctcgccggc aacccggcga agcatgacct ggacatcaaa
720cccacggtca tcagtcatcg cctgcacttt cccgagggcg gcagcctggc
cgcgctgacc 780gcgcaccagg cttgccacct gccgctggag actttcaccc
gtcatcgcca gccgcgcggc 840tgggaacaac tggagcagtg cggctatccg
gtgcagcggc tggtcgccct ctacctggcg 900gcgcggctgt cgtggaacca
ggtcgaccag gtgatccgca acgccctggc cagccccggc 960agcggcggcg
acctgggcga agcgatccgc gagcagccgg agcaggcccg tctggccctg
1020accctggccg ccgccgagag cgagcgcttc gtccggcagg gcaccggcaa
cgacgaggcc 1080ggcgcggcca acgccgacgt ggtgagcctg acctgcccgg
tcgccgccgg tgaatgcgcg 1140ggcccggcgg acagcggcga cgccctgctg
gagcgcaact atcccactgg cgcggagttc 1200ctcggcgacg gcggcgacgt
cagcttcagc acccgcggca cgcagaactg gacggtggag 1260cggctgctcc
aggcgcaccg ccaactggag gagcgcggct atgtgttcgt cggctaccac
1320ggcaccttcc tcgaagcggc gcaaagcatc gtcttcggcg gggtgcgcgc
gcgcagccag 1380gacctcgacg cgatctggcg cggtttctat atcgccggcg
atccggcgct ggcctacggc 1440tacgcccagg accaggaacc cgacgcacgc
ggccggatcc gcaacggtgc cctgctgcgg 1500gtctatgtgc cgcgctcgag
cctgccgggc ttctaccgca ccagcctgac cctggccgcg 1560ccggaggcgg
cgggcgaggt cgaacggctg atcggccatc cgctgccgct gcgcctggac
1620gccatcaccg gccccgagga ggaaggcggg cgcctggaga ccattctcgg
ctggccgctg 1680gccgagcgca ccgtggtgat tccctcggcg atccccaccg
acccgcgcaa cgtcggcggc 1740gacctcgacc cgtccagcat ccccgacaag
gaacaggcga tcagcgccct gccggactac 1800gccagccagc ccggcaaacc
gccgcgcgag gacctgaag 183944PRTArtificial Sequenceone of the amino
acid sequences from which the first and/or second cleavable linker
of the delivery construct is selected 4Ala Ala Pro
Phe153PRTArtificial Sequenceone of the amino acid sequences from
which the first and/or second cleavable linker of the delivery
construct is selected 5Gly Gly Phe164PRTArtificial Sequenceone of
the amino acid sequences from which the first and/or second
cleavable linker of the delivery construct is selected 6Ala Ala Pro
Val173PRTArtificial Sequenceone of the amino acid sequences from
which the first and/or second cleavable linker of the delivery
construct is selected 7Gly Gly Leu183PRTArtificial Sequenceone of
the amino acid sequences from which the first and/or second
cleavable linker of the delivery construct is selected 8Ala Ala
Leu193PRTArtificial Sequenceone of the amino acid sequences from
which the first and/or second cleavable linker of the delivery
construct is selected 9Phe Val Arg1103PRTArtificial Sequenceone of
the amino acid sequences from which the first and/or second
cleavable linker of the delivery construct is selected 10Val Gly
Arg1115PRTUnknownAmino acid sequence recognized and cleaved by
caspase-1 11Tyr Val Ala Asp Xaa1 5125PRTUnknownAmino acid sequence
recognized and cleaved by caspase-3 12Asp Xaa Xaa Asp Xaa1
5139PRTUnknownAmino acid sequence recognized and cleaved by
proprotein convertase 1 13Arg Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa1
5149PRTUnknownAmino acid sequence recognized and cleaved by
proprotein convertase 2 14Lys Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa1
5156PRTUnknownAmino acid sequence recognized and cleaved by
proprotein convertase 4 15Gly Arg Thr Lys Arg Xaa1
5165PRTUnknownAmino acid sequence recognized and cleaved by
proprotein convertase 4 PACE 4 16Arg Val Arg Arg Xaa1
5175PRTUnknownAmino acid sequence recognized and cleaved by
proprotein convertase 4 PACE 4 17Xaa Val Arg Arg Xaa1
5186PRTUnknownAmino acid sequence recognized and cleaved by
prolyloligopeptidase endothelin cleaving enzyme in combination with
dipeptidyl-peptidase IV 18Pro Xaa Trp Val Pro Xaa1
5194PRTUnknownAmino acid sequence recognized and cleaved by signal
peptidase 19Trp Val Ala Xaa1204PRTUnknownAmino acid sequence
recognized and cleaved by neprilysin in combination with
dipeptidyl-peptidase IV 20Xaa Phe Xaa Xaa1214PRTUnknownAmino acid
sequence recognized and cleaved by neprilysin in combination with
dipeptidyl-peptidase IV 21Xaa Tyr Xaa Xaa1224PRTUnknownAmino acid
sequence recognized and cleaved by neprilysin in combination with
dipeptidyl-peptidase IV 22Xaa Trp Xaa Xaa12312PRTUnknownAmino acid
sequence recognized and cleaved by renin in combination with
dipeptidyl-peptidase IV 23Asp Arg Tyr Ile Pro Phe His Leu Leu Xaa
Tyr Xaa1 5 102437DNAArtificial SequenceOligonucleotide pPE-RGH-1
(1F) for introducing a furin cleavage site and protease cleavage
site 24aactgcagcg ccagcctcga ggaggattac tgcagaa 372537DNAArtificial
SequenceOligonucleotide pPE-RGH-1 (1R) for introducing a furin
cleavage site and protease cleavage site 25ttctgcagta atcctcctcg
aggctggcgc tgcagtt 372637DNAArtificial SequenceOligonucleotide
pPE-RGH-2 (2F) for introducing a furin cleavage site and protease
cleavage site 26aactgcaggg aggcttacgc cagcctcgac tgcagaa
372737DNAArtificial SequenceOligonucleotide pPE-RGH-2 (2R) for
introducing a furin cleavage site and protease cleavage site
27ttctgcagtc gaggctggcg taagcctccc tgcagtt 372837DNAArtificial
SequenceOligonucleotide pPE-RGH-3 (3F) for introducing a furin
cleavage site and protease cleavage site 28aactgcagcg ccagcctcga
gagggccgtc tgcagaa 372937DNAArtificial SequenceOligonucleotide
pPE-RGH-3 (3R) for introducing a furin cleavage site and protease
cleavage site 29ttctgcagac ggccctctcg aggctggcgc tgcagtt
373037DNAArtificial SequenceOligonucleotide pPE-RGH-4 (4F) for
introducing a furin cleavage site and protease cleavage site
30aactgcagcg ccagcctcga gtcggccgtc tgcagaa 373137DNAArtificial
SequenceOligonucleotide pPE-RGH-4 (4R) for introducing a furin
cleavage site and protease cleavage site 31ttctgcagac ggccgactcg
aggctggcgc tgcagtt 373237DNAArtificial SequenceOligonucleotide
pPE-RGH-5 (5F) for introducing a furin cleavage site and protease
cleavage site 32aactgcagcg ccagcctcga gcacgtcgtc tgcagaa
373337DNAArtificial SequenceOligonucleotide pPE-RGH-5 (5R) for
introducing a furin cleavage site and protease cleavage site
33ttctgcagac gacgtgctcg aggctggcgc tgcagtt 37341698DNAArtificial
Sequencenucleotide sequence encodes Delivery Construct 6 - an
exemplary Delivery Construct for delivering hGH 34atggccgaag
aagctttcga cctctggaac gaatgcgcca aagcctgcgt gctcgacctc 60aaggacggcg
tgcgttccag ccgcatgagc gtcgacccgg ccatcgccga caccaacggc
120cagggcgtgc tgcactactc catggtcctg gagggcggca acgacgcgct
caagctggcc 180atcgacaacg ccctcagcat caccagcgac ggcctgacca
tccgcctcga aggcggcgtc 240gagccgaaca agccggtgcg ctacagctac
acgcgccagg cgcgcggcag ttggtcgctg 300aactggctgg taccgatcgg
ccacgagaag ccctcgaaca tcaaggtgtt catccacgaa 360ctgaacgccg
gcaaccagct cagccacatg tcgccgatct acaccatcga gatgggcgac
420gagttgctgg cgaagctggc gcgcgatgcc accttcttcg tcagggcgca
cgagagcaac 480gagatgcagc cgacgctcgc catcagccat gccggggtca
gcgtggtcat ggcccagacc 540cagccgcgcc gggaaaagcg ctggagcgaa
tgggccagcg gcaaggtgtt gtgcctgctc 600gacccgctgg acggggtcta
caactacctc gcccagcaac gctgcaacct cgacgatacc 660tgggaaggca
agatctaccg ggtgctcgcc ggcaacccgg cgaagcatga cctggacatc
720aaacccacgg tcatcagtca tcgcctgcac tttcccgagg gcggcagcct
ggccgcgctg 780accgcgcacc aggcttgcca cctgccgctg gagactttca
cccgtcatcg ccagccgcgc 840ggctgggaac aactcgagca gtgcggctat
ccggtgcagc ggctggtcgc cctctacctg 900gcggcgcggc tgtcgtggaa
ccaggtcgac caggtgatcc gcaacgccct ggccagcccc 960ggcagcggcg
gcgacctggg cgaagcgatc cgcgagcagc cggagcaggc ccgtctggcc
1020ctgaccctgg ccgccgccga gagcgagcgc ttcgtccggc agggcaccgg
caacgacgag 1080gccggcgcgg caaacctgca gggaggatta cgccagcctc
gattcccgac catcccgctg 1140tcccgtctgt tcgacaacgc tatgctgcgt
gctcaccgtc tgcaccagct ggctttcgac 1200acctaccagg agttcgaaga
agcatacatc ccgaaagaac agaaatactc cttcctgcaa 1260aacccgcaga
cctccctgtg cttctccgaa tcgatcccga ccccgtccaa ccgtgaagaa
1320acccagcaga aatccaacct ggagctcctg cgtatctccc tgctgctgat
ccagtcctgg 1380ctcgagccgg ttcagttcct gcgttccgtt ttcgctaact
ccctggttta cggtgctagc 1440gactccaacg tttacgacct gctgaaagac
ctggaagaag gtatccagac cctgatgggt 1500cgtctggaag acggttcccc
gcgtaccggt cagatcttca aacagaccta ctccaaattc 1560gacaccaact
cccacaacga cgacgctctg ctgaaaaact acggtctgct gtactgcttc
1620cgtaaagaca tggacaaagt tgaaaccttc ctgcgtatcg ttcagtgccg
ttccgttgaa 1680ggttcctgcg gtttctaa 169835565PRTArtificial
Sequenceamono acid sequence of Delivery Construct 6- an exemplary
Delivery Construct for delivering hGH 35Met Ala Glu Glu Ala Phe Asp
Leu Trp Asn Glu Cys Ala Lys Ala Cys1 5 10 15Val Leu Asp Leu Lys Asp
Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30Pro Ala Ile Ala Asp
Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met 35 40 45Val Leu Glu Gly
Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala 50 55 60Leu Ser Ile
Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val65 70 75 80Glu
Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly 85 90
95Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser
100 105 110Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln
Leu Ser 115 120 125His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp
Glu Leu Leu Ala 130 135 140Lys Leu Ala Arg Asp Ala Thr Phe Phe Val
Arg Ala His Glu Ser Asn145 150 155 160Glu Met Gln Pro Thr Leu Ala
Ile Ser His Ala Gly Val Ser Val Val 165 170 175Met Ala Gln Thr Gln
Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala 180 185 190Ser Gly Lys
Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn 195 200 205Tyr
Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys 210 215
220Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp
Ile225 230 235 240Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro
Glu Gly Gly Ser 245 250 255Leu Ala Ala Leu Thr Ala His Gln Ala Cys
His Leu Pro Leu Glu Thr 260 265 270Phe Thr Arg His Arg Gln Pro Arg
Gly Trp Glu Gln Leu Glu Gln Cys 275 280 285Gly Tyr Pro Val Gln Arg
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu 290 295 300Ser Trp Asn Gln
Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro305 310 315 320Gly
Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln 325 330
335Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val
340 345 350Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Leu
Gln Gly 355 360 365Gly Leu Arg Gln Pro Arg Phe Pro Thr Ile Pro Leu
Ser Arg Leu Phe 370 375 380Asp Asn Ala Met Leu Arg Ala His Arg Leu
His Gln Leu Ala Phe Asp385 390 395 400Thr Tyr Gln Glu Phe Glu Glu
Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 405 410 415Ser Phe Leu Gln Asn
Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile 420 425 430Pro Thr Pro
Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 435 440 445Leu
Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 450 455
460Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala
Ser465 470 475 480Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu
Glu Gly Ile Gln 485 490 495Thr Leu Met Gly Arg Leu Glu Asp Gly Ser
Pro Arg Thr Gly Gln Ile 500 505 510Phe Lys Gln Thr Tyr Ser Lys Phe
Asp Thr Asn Ser His Asn Asp Asp 515 520 525Ala Leu Leu Lys Asn Tyr
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 530 535 540Asp Lys Val Glu
Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu545 550 555 560Gly
Ser Cys Gly Phe 565361620DNAArtificial Sequencenucleotide sequence
encodes Delivery Construct 7 - an exemplary Delivery Construct for
delivering IFN-alpha 36atggccgaag aagctttcga cctctggaac gaatgcgcca
aagcctgcgt gctcgacctc 60aaggacggcg tgcgttccag ccgcatgagc gtcgacccgg
ccatcgccga caccaacggc 120cagggcgtgc tgcactactc catggtcctg
gagggcggca acgacgcgct caagctggcc 180atcgacaacg ccctcagcat
caccagcgac ggcctgacca tccgcctcga aggcggcgtc 240gagccgaaca
agccggtgcg ctacagctac acgcgccagg cgcgcggcag ttggtcgctg
300aactggctgg taccgatcgg ccacgagaag ccctcgaaca tcaaggtgtt
catccacgaa 360ctgaacgccg gcaaccagct cagccacatg tcgccgatct
acaccatcga gatgggcgac 420gagttgctgg cgaagctggc gcgcgatgcc
accttcttcg tcagggcgca cgagagcaac 480gagatgcagc cgacgctcgc
catcagccat gccggggtca gcgtggtcat ggcccagacc 540cagccgcgcc
gggaaaagcg ctggagcgaa tgggccagcg gcaaggtgtt gtgcctgctc
600gacccgctgg acggggtcta caactacctc gcccagcaac gctgcaacct
cgacgatacc 660tgggaaggca agatctaccg ggtgctcgcc ggcaacccgg
cgaagcatga cctggacatc 720aaacccacgg tcatcagtca tcgcctgcac
tttcccgagg gcggcagcct ggccgcgctg 780accgcgcacc aggcttgcca
cctgccgctg gagactttca cccgtcatcg ccagccgcgc 840ggctgggaac
aactcgagca gtgcggctat ccggtgcagc ggctggtcgc cctctacctg
900gcggcgcggc tgtcgtggaa ccaggtcgac caggtgatcc gcaacgccct
ggccagcccc 960ggcagcggcg gcgacctggg cgaagcgatc cgcgagcagc
cggagcaggc ccgtctggcc 1020ctgaccctgg ccgccgccga gagcgagcgc
ttcgtccggc agggcaccgg caacgacgag 1080gccggcgcgg caaacctgca
gggaggctta cgccagcctc gatgcgatct gcctcagacc 1140cacagcctgg
gcagcaggag gaccctgatg ctgctggctc agatgaggag aatcagcctg
1200tttagctgcc tgaaggatag gcacgatttt ggctttcctc aagaggagtt
tggcaaccag 1260tttcagaagg ctgagaccat ccctgtgctg cacgagatga
tccagcagat ctttaacctg 1320tttagcacca aggatagcag cgctgcttgg
gatgagaccc tgctggataa gttttacacc 1380gagctgtacc agcagctgaa
cgatctggag gcttgcgtga tccagggcgt gggcgtgacc 1440gagacccctc
tgatgaagga ggatagcatc ctggctgtga ggaagtactt tcagaggatc
1500accctgtacc tgaaggagaa gaagtacagc ccctgcgctt gggaagtcgt
gagggctgag 1560atcatgagga gctttagcct gagcaccaac ctgcaagaga
gcttgaggtc taaggagtaa 162037539PRTArtificial Sequenceamino acid
sequence of Delivery Construct 7 - an exemplary Delivery Construct
for delivering IFN-alpha 37Met Ala Glu Glu Ala Phe Asp Leu Trp Asn
Glu Cys Ala Lys Ala Cys1 5 10 15Val Leu Asp Leu Lys Asp Gly Val Arg
Ser Ser Arg Met Ser Val Asp 20 25 30Pro Ala Ile Ala Asp Thr Asn Gly
Gln Gly Val Leu His Tyr Ser Met 35 40 45Val Leu Glu Gly Gly Asn Asp
Ala Leu Lys Leu Ala Ile Asp Asn Ala 50 55 60Leu Ser Ile Thr Ser Asp
Gly Leu Thr Ile Arg Leu Glu Gly Gly Val65 70 75 80Glu Pro Asn Lys
Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly 85 90 95Ser Trp Ser
Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser 100 105 110Asn
Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser 115 120
125His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala
130 135 140Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu
Ser Asn145 150 155 160Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala
Gly Val Ser Val Val 165 170 175Met Ala Gln Thr Gln Pro Arg Arg Glu
Lys Arg Trp Ser Glu Trp Ala 180 185 190Ser Gly Lys Val Leu Cys Leu
Leu Asp Pro Leu Asp Gly Val Tyr Asn 195 200 205Tyr Leu Ala Gln Gln
Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys 210 215 220Ile Tyr Arg
Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile225 230 235
240Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser
245 250 255Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu
Glu Thr 260 265 270Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu Glu Gln Cys 275 280 285Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
Tyr Leu Ala Ala Arg Leu 290 295 300Ser Trp Asn Gln Val Asp Gln Val
Ile Arg Asn Ala Leu Ala Ser Pro305 310 315 320Gly Ser Gly Gly Asp
Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln 325 330 335Ala Arg Leu
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val 340 345 350Arg
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Leu Gln Gly 355 360
365Gly Leu Arg Gln Pro Arg Cys Asp Leu Pro Gln Thr His Ser Leu Gly
370 375 380Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu385 390 395 400Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
Phe Pro Gln Glu Glu 405 410 415Phe Gly Asn Gln Phe Gln Lys Ala Glu
Thr Ile Pro Val Leu His Glu 420 425 430Met Ile Gln Gln Ile Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala 435 440 445Ala Trp Asp Glu Thr
Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln 450 455 460Gln Leu Asn
Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr465 470 475
480Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
485 490 495Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys 500 505 510Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser
Phe Ser Leu Ser 515 520 525Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys
Glu 530 53538460PRTArtificial Sequenceamino acid sequence of
Delivery Construct 8 - an exemplary Delivery Construct for
delivering proinsulin 38Met Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu
Cys Ala Lys Ala Cys1 5 10 15Val Leu Asp Leu Lys Asp Gly Val Arg Ser
Ser Arg Met Ser Val Asp 20 25 30Pro Ala Ile Ala Asp Thr Asn Gly Gln
Gly Val Leu His Tyr Ser Met 35 40 45Val Leu Glu Gly Gly Asn Asp Ala
Leu Lys Leu Ala Ile Asp Asn Ala 50 55 60Leu Ser Ile Thr Ser Asp Gly
Leu Thr Ile Arg Leu Glu Gly Gly Val65 70 75 80Glu Pro Asn Lys Pro
Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly 85 90 95Ser Trp Ser Leu
Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser 100 105 110Asn Ile
Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser 115 120
125His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala
130 135 140Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu
Ser Asn145 150 155 160Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala
Gly Val Ser Val Val 165 170 175Met Ala Gln Thr Gln Pro Arg Arg Glu
Lys Arg Trp Ser Glu Trp Ala 180 185 190Ser Gly Lys Val Leu Cys Leu
Leu Asp Pro Leu Asp Gly Val Tyr Asn 195 200 205Tyr Leu Ala Gln Gln
Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys 210 215 220Ile Tyr Arg
Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile225 230 235
240Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser
245 250 255Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu
Glu Thr 260 265 270Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu Glu Gln Cys 275 280 285Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
Tyr Leu Ala Ala Arg Leu 290 295 300Ser Trp Asn Gln Val Asp Gln Val
Ile Arg Asn Ala Leu Ala Ser Pro305 310 315 320Gly Ser Gly Gly Asp
Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln 325 330 335Ala Arg Leu
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val 340 345 350Arg
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Leu Gln Gly 355 360
365Gly Leu Arg Gln Pro Arg Phe Val Asn Gln His Leu Cys Gly Ser His
370 375 380Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe
Phe Tyr385 390 395 400Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu
Gln Val Gly Gln Val 405 410 415Glu Leu Gly Gly Gly Pro Gly Ala Gly
Ser Leu Gln Pro Leu Ala Leu 420 425 430Glu Gly Ser Leu Gln Lys Arg
Gly Ile Val Glu Gln Cys Cys Thr Ser 435 440 445Ile Cys Ser Leu Tyr
Gln Leu Glu Asn Tyr Cys Asn 450 455 46039404PRTArtificial
Sequenceamino acid sequence of Delivery Construct 9 - an exemplary
Delivery Construct for delivering insulin 39Met Ala Glu Glu Ala Phe
Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys1 5 10 15Val Leu Asp Leu Lys
Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 20 25 30Pro Ala Ile Ala
Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met 35 40 45Val Leu Glu
Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala 50 55 60Leu Ser
Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val65 70 75
80Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly
85 90 95Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro
Ser 100 105 110Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn
Gln Leu Ser 115 120 125His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly
Asp Glu Leu Leu Ala 130 135 140Lys Leu Ala Arg Asp Ala Thr Phe Phe
Val Arg Ala His Glu Ser Asn145 150 155 160Glu Met Gln Pro Thr Leu
Ala Ile Ser His Ala Gly Val Ser Val Val 165 170 175Met Ala Gln Thr
Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala 180 185 190Ser Gly
Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn 195 200
205Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys
210 215 220Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu
Asp Ile225 230 235 240Lys Pro Thr Val Ile Ser His Arg Leu His Phe
Pro Glu Gly Gly Ser 245 250 255Leu Ala Ala Leu Thr Ala His Gln Ala
Cys His Leu Pro Leu Glu Thr 260 265 270Phe Thr Arg His Arg Gln Pro
Arg Gly Trp Glu Gln Leu Glu Gln Cys 275 280 285Gly Tyr Pro Val Gln
Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu 290 295 300Ser Trp Asn
Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro305 310 315
320Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln
325 330 335Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg
Phe Val 340 345 350Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala
Asn Leu Gln Gly 355 360 365Gly Leu Arg Gln Pro Arg Phe Val Asn Gln
His Leu Cys Gly Ser His 370 375 380Leu Val Glu Ala Leu Tyr Leu Val
Cys Gly Glu Arg Gly Phe Phe Tyr385 390 395 400Thr Pro Lys
Thr4021PRTArtificial Sequenceamino acid sequence of Delivery
Construct 9 - an exemplary Delivery Construct for delivering
insulin 40Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr
Gln Leu1 5 10 15Glu Asn Tyr Cys Asn 20417PRTArtificial
Sequencecleavable linker sequence of Delivery Construct 1 41Arg Gln
Pro Arg Gly Gly Leu1 5427PRTArtificial Sequencecleavable linker
sequence of Delivery Construct 2 42Gly Gly Leu Arg Gln Pro Arg1
5437PRTArtificial Sequencecleavable linker sequence of Delivery
Construct 3 43Arg Gln Pro Arg Glu Gly Arg1 5447PRTArtificial
Sequencecleavable linker sequence of Delivery Construct 4 44Arg Gln
Pro Arg Val Gly Arg1 5457PRTArtificial Sequencecleavable linker
sequence of Delivery Construct 5 45Arg Gln Pro Arg Ala Arg Arg1
54621DNAArtificial SequenceRT-PCR primer IGF-I-BP3 (forward)
46cgcagagaaa tggaggacac a 214718DNAArtificial SequenceRT-PCR primer
IGF-I-BP3 (reverse) 47ggacgcctct gggactca 184824DNAArtificial
SequenceRT-PCR primer GH receptor (forward) 48gttgacgaaa tagtgcaacc
tgat 244921DNAArtificial SequenceRT-PCR primer GH receptor
(reverse) 49cacgaatccc ggtcaaacta a 215019DNAArtificial
SequenceRT-PCR primer IGF-I (forward) 50gctatggctc cagcattcg
195119DNAArtificial SequenceRT-PCR primer IGF-I (reverse)
51gctccggaag caacactca 195219DNAArtificial SequenceRT-PCR primer
GAPDH (forward) 52gcaacagggt ggtggacct 195321DNAArtificial
SequenceRT-PCR primer GAPDH (reverse) 53ggatagggcc tctcttgctc a
2154637PRTP. aeruginosaPseudomonas aeruginosa exotoxin A 54Met His
Leu Ile Pro His Trp Ile Pro Leu Val Ala Ser Leu Gly Leu1 5 10 15Leu
Ala Gly Gly Ser Ser Ala Ser Ala Ala Glu Glu Ala Phe Asp Leu 20 25
30Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val
35 40 45Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn
Gly 50 55 60Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn
Asp Ala65 70 75 80Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr
Ser Asp Gly Leu 85 90 95Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn
Lys Pro Val Arg Tyr 100 105 110Ser Tyr Thr Arg Gln Ala Arg Gly Ser
Trp Ser Leu Asn Trp Leu Val 115 120 125Pro Ile Gly His Glu Lys Pro
Ser Asn Ile Lys Val Phe Ile His Glu 130 135 140Leu Asn Ala Gly Asn
Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile145 150 155 160Glu Met
Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe 165 170
175Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile
180 185 190Ser His Ala Gly Val Ser Val Val Met Ala Gln Thr Gln Pro
Arg Arg 195 200 205Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val
Leu Cys Leu Leu 210 215 220Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu
Ala Gln Gln Arg Cys Asn225 230 235 240Leu Asp Asp Thr Trp Glu Gly
Lys Ile Tyr Arg Val Leu Ala Gly Asn 245 250 255Pro Ala Lys His Asp
Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg 260 265 270Leu His Phe
Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln 275 280 285Ala
Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gly Pro Arg 290 295
300Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu
Val305 310 315 320Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln
Val Asp Gln Val 325 330 335Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser
Gly Gly Asp Leu Gly Glu 340 345 350Ala Ile Arg Glu Gln Pro Glu Gln
Ala Arg Leu Ala Leu Thr Leu Ala 355 360 365Ala Ala Glu Ser Glu Arg
Phe Val Arg Gln Gly Thr Gly Asn Asp Glu 370 375 380Ala Gly Ala Ala
Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala385 390 395 400Ala
Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu 405 410
415Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
420 425 430Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg
Leu Leu 435 440 445Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val
Phe Val Gly Tyr 450 455 460His Gly Thr Phe Leu Glu Ala Ala Gln Ser
Ile Val Phe Gly Gly Val465 470 475 480Arg Ala Arg Ser Gln Asp Leu
Asp Ala Ile Trp Arg Gly Phe Tyr Ile 485 490 495Ala Gly Asp Pro Ala
Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro 500 505 510Asp Ala Arg
Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val 515 520 525Pro
Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala 530 535
540Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro
Leu545 550 555 560Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu
Glu
Glu Gly Gly Arg 565 570 575Leu Glu Thr Ile Gly Trp Pro Leu Ala Glu
Arg Thr Val Val Ile Pro 580 585 590Ser Ala Ile Pro Thr Asp Pro Arg
Asn Val Gly Gly Asp Leu Asp Pro 595 600 605Ser Ser Ile Pro Asp Lys
Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr 610 615 620Ala Ser Gln Pro
Gly Lys Pro Pro Arg Glu Asp Leu Lys625 630 635
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