U.S. patent application number 14/591397 was filed with the patent office on 2015-07-23 for multimeric tie 2 agonists and uses thereof in stimulating angiogenesis.
The applicant listed for this patent is Sunnybrook Health Sciences Centre. Invention is credited to Daniel Dumont, Paul Van Slyke.
Application Number | 20150202251 14/591397 |
Document ID | / |
Family ID | 39324074 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202251 |
Kind Code |
A1 |
Van Slyke; Paul ; et
al. |
July 23, 2015 |
MULTIMERIC TIE 2 AGONISTS AND USES THEREOF IN STIMULATING
ANGIOGENESIS
Abstract
The present invention provides a multimeric form of a Tie 2
binding peptide monomer, wherein the multimeric form has Tie 2
agonist activity. The multimeric form, preferably a tetramer,
stimulates angiogenesis and promotes wound healing. The present
invention also features pharmaceutical compositions comprising the
multimeric Tie 2 agonists, including those suitable for topical or
systemic administration. Methods of using the multimeric Tie 2
agonists of the invention for stimulating angiogenesis and for
promoting healing of wounds, such as diabetic ulcers or skin
grafts, are also provided.
Inventors: |
Van Slyke; Paul; (North
York, CA) ; Dumont; Daniel; (Oakville, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sunnybrook Health Sciences Centre |
Toronto |
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CA |
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|
Family ID: |
39324074 |
Appl. No.: |
14/591397 |
Filed: |
January 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12446511 |
Jan 5, 2011 |
8957022 |
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PCT/CA2007/001903 |
Oct 26, 2007 |
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14591397 |
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60854950 |
Oct 27, 2006 |
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Current U.S.
Class: |
514/7.5 |
Current CPC
Class: |
A61P 17/02 20180101;
A61P 39/00 20180101; A61K 38/10 20130101; A61K 38/16 20130101; A61K
47/60 20170801; A61K 9/0014 20130101; C12Q 1/485 20130101; A61K
38/1866 20130101; G01N 33/94 20130101; A61K 9/0019 20130101; A61K
45/06 20130101; A61K 47/62 20170801; A61L 27/60 20130101; A61K
38/08 20130101; A61K 38/1709 20130101; A61K 47/545 20170801; A61P
9/00 20180101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 47/48 20060101 A61K047/48; A61K 38/08 20060101
A61K038/08 |
Claims
1-79. (canceled)
80. A composition comprising a multimeric form of a Tie 2 binding
peptide monomer, wherein the multimeric form has Tie 2 agonist
activity.
81. The composition of claim 80, wherein the Tie 2 binding peptide
monomer binds to Tie 2 with high affinity but does not
substantially inhibit binding of an angiopoietin to Tie 2.
82. The composition of claim 81, wherein the angiopoietin is Ang
1.
83. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises an amino acid sequence that is not present in a
native Tie 2 ligand.
84. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises a T7 peptide (SEQ ID NOs: 1 or 2).
85. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises a GA3 peptide (SEQ ID NOs: 3 or 4).
86. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises a peptide selected from the group consisting of a
T4 peptide (SEQ ID NOs: 9-10), a T6 peptide (SEQ ID NOs: 7-8) and a
T8 peptide (SEQ ID NOs: 5-6).
87. The composition of claim 80, wherein the multimeric form is a
tetramer.
88. The composition of claim 80, wherein the multimeric form is a
dimer.
89. The composition of claim 80, wherein the multimeric form
comprises six, eight, ten or twelve units of the Tie 2 binding
peptide monomer.
90. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises a structure: A-B-C, wherein A comprises a Tie 2
binding peptide, B comprises a spacer and C comprises a
multimerizing group, wherein C has affinity for D, a multimer agent
comprising multiple binding sites for C.
91. The composition of claim 90, wherein the multimer agent D has
four binding sites for the multimerizing group C such that a
tetramer is formed when four Tie 2 binding peptide monomers, A-B-C,
interact with the multimer agent D.
92. The composition of claim 80, wherein the Tie 2 binding peptide
monomer comprises a structure: A-B, wherein A comprises a Tie 2
binding peptide and B comprises a spacer, wherein the multimeric
form is created by covalent linkage of multiple Tie 2 binding
peptide monomers via the spacer B.
93. The composition of claim 80, which comprises a tetramer form of
a Tie 2 binding peptide monomer, wherein the Tie 2 binding peptide
monomer comprises a structure: A-B-C, wherein: A comprises a Tie 2
binding peptide selected from a T7 peptide (SEQ ID NOs: 1 or 2) and
a GA3 peptide (SEQ ID NOs: 3 or 4); B comprises a polyethylene
glycol spacer; and C comprises a biotin group, wherein four copies
of A-B-C are associated with a tetramer agent, D, to create the
tetramer form, the tetramer agent, D, being selected from the group
consisting of avidin, streptavidin and neutravidin.
94. The composition of claim 80, which comprises a peptide dimer,
comprising: (a) a first peptide chain; (b) a second peptide chain;
and (c) a linking moiety connecting said first and second peptide
chains, wherein said peptide dimer binds to and activates the Tie 2
receptor.
95. The composition of claim 94, wherein the first peptide chain
and the second peptide chain are both T7 peptides (SEQ ID NOs: 1 or
2).
96. The composition of claim 94, wherein the linking moiety
comprises one or more water soluble polymers covalently bound to
the first peptide chain and the second peptide chain.
97. The composition of claim 96, wherein the one or more water
soluble polymers are linear polymers.
98. The composition of claim 97, wherein the water soluble polymer
is a polyethylene glycol (PEG).
99. The composition of claim 80, which comprises a peptide
tetramer, comprising: (a) a first peptide chain; (b) a second
peptide chain; (c) a third peptide chain; (d) a fourth peptide
chain; and (e) a linking moiety connecting said first, second,
third and fourth peptide chains, wherein said peptide tetramer
binds to and activates the Tie 2 receptor.
100. The composition of claim 99, wherein the first, second, third
and fourth peptide chains are T7 peptides (SEQ ID NOs: 1 or 2).
101. The composition of claim 99, wherein the linking moiety
comprises one or more soluble polymers covalently bound to the
first, second, third and fourth peptide chains.
102. The composition of claim 101, wherein the water soluble
polymer is a branched chain water soluble polymer.
103. The composition of claim 102, wherein the water soluble
polymer is a branched chain polyethylene glycol (PEG).
104. A composition comprising a Tie 2 binding peptide monomer, the
Tie 2 binding peptide monomer comprising a structure A-B-C,
wherein: A comprises a Tie 2 binding peptide; B comprises a spacer;
and C comprises a multimerizing group.
105. A method of stimulating angiogenesis at a site in a subject
comprising contacting the site with the multimeric form of a Tie 2
binding peptide monomer of claim 80 such that angiogenesis is
stimulated at the site in the subject.
106. The method of claim 105, wherein angiogenesis stimulated by
the multimeric form is characterized by at least one of the
following properties: a) recruitment of perivascular support cells;
b) non-leakiness of vessels; and c) well-defined arborization.
107. The method of claim 105, which further comprises contacting
the site in the subject with a second angiogenic agent.
108. The method of claim 105, wherein angiogenesis is stimulated in
a clinical situation selected from the group consisting of
vascularization of regenerative tissues, ischemic limb disease,
cerebral ischemia, conditions of vascular inflammation,
arteriosclerosis, avascular necrosis, stimulation of hair growth
and erectile dysfunction.
109. A method of decreasing vascular permeability at a site of
leaky vessels comprising contacting the site of leaky vessels with
the multimeric form of the Tie 2 binding peptide monomer of claim
80 such that vascular permeability is decreased.
Description
BACKGROUND OF THE INVENTION
[0001] Angiogenesis is the process by which new blood vessels are
generated. Many of the signaling pathways that drive the angiogenic
response originate at the plasma membrane and emanate from the
activation of endothelial cell receptor tyrosine kinases, including
Tie2/Tek (Jones, N. et al. (2001) Nat. Rev. Mol. Cell. Biol.
4:257-267; Olsson, A. et al. (2006) Nat. Rev. Mol. Cell. Biol.
5:359-371). Angiopoietin (Ang) members 1-4 constitute a family of
protein growth factors, all of which have been shown to activate
Tie 2 receptor activity to differing extents.
[0002] All the Ang's are characterized structurally by an
N-terminal super clustering domain (SCD) followed by a coiled-coil
domain (CCD) and a C-terminal fibrinogen-like domain (FLD) (Ward,
N. and Dumont, D. (2002) Semin. Cell. Dev. Biol. 1:19-27) and
(Tsigkos, K. et al. (2003) Expert Opin. Investig. Drugs 6:933-941).
Functional studies have highlighted a role for the SCD and CCD's in
forming high order homotypic Ang multimers (Procopio, W. et al.
(1999) J. Biol. Chem. 42:30196-30201). The specific nature of these
multimers is variable and seems to be unique to each Ang family
member. Binding specificity of the Ang's for the Tie 2 receptor has
been ascribed to the FLD. Taken together, unique structural
attributes of each Ang family member promotes binding and
differential clustering of Tie 2. The pleiotropic physiological
effects of Ang 1-4 are thought to at least in part be mediated by
appropriate and specific clustering of the receptor (Davis, S. et.
al. (2003) Nat. Struct. Biol. 1:38-44; Procopio, W. et. al. (1999)
J. Biol. Chem. 42:30196-30201; Cho, C. et. al. (2004) Proc. Natl.
Acad. Sci. USA 15:5547-5552; Ward, D. et. al. (2004) Biochem.
Biophys. Res. Commun. 3:937-946; Kim, K-T. et al. (2005) J. Biol.
Chem. 280:20126-20131). Gene ablation and transgenic approaches in
mice have highlighted an indispensable role for Ang 1 and 2 in the
development and maintenance of the blood and lymphatic vascular
systems as well as well as the hematopoietic system. Non-genetic
studies of the Ang's have been hampered by the inherent difficulty
associated with their purification, stability and solubility.
[0003] Chronic wounds represent a significant medical problem. For
example, global estimates report that 12.5 million patients
worldwide suffer from chronic wounds and a significant number of
these individuals suffer from decubitus ulcers and diabetic foot
ulcers. Wound healing involves a well choreographed series of
molecular activities that ultimately lead to wound closure. These
events are driven by three interrelated processes: inflammation,
cellular proliferation and angiogenesis. Impaired angiogenesis is
one of several primary defects reported in diabetic patients. These
patients often suffer from impaired wound healing, and as such
suffer significant morbidity associated with vascular compromise
(Dinh, T. and Veves, A. (2005) Curr. Pharm. Des. 18:145-153).
[0004] Primary defects in growth factor secretion and/or
proteolytic cleavage of growth factors in diabetic wounds has been
reported suggesting therapeutic application of these factors may be
beneficial (Wieman, T. et al. (1998) Diabetes Care 5:822-827;
Tsang, M. et. al. (2003) Diabetes Care 6:1856-1861). However, to
date, effective means for stimulating angiogenesis, such as for use
in the treatment of chronic wounds, are still lacking. Accordingly,
a need exists for agents that are effective in stimulating
angiogenesis.
SUMMARY OF THE INVENTION
[0005] This invention provides multimeric Tie 2 agonists that have
angiogenic activity and that can be used to promote wound healing.
A Tie 2 agonist of the invention is an angiopoietin mimetic that
comprises a multimeric form of a Tie 2 binding peptide monomer. The
Tie 2 agonists of the invention have been demonstrated to
specifically bind to and activate Tie 2, as evidenced by
phosphorylation of Tie 2, and to activate signaling pathways that
previously have been demonstrated to be downstream of Tie 2,
including the MAPK, AKT and eNOS pathways. The Tie 2 agonists of
the invention also have been demonstrated to stimulate
angiogenesis, resulting in well arborized vessels. Moreover, when
used to stimulate angiogenesis in combination with VEGF, the Tie 2
agonists have been shown to mitigate the tortuosity seen when
angiogenesis is stimulated by VEGF alone. Using an in vivo model of
wound healing, the Tie 2 agonists of the invention have been
demonstrated to improve wound closure time, primarily through
increased granulation tissue and neovascularization of the
wound.
[0006] Accordingly, one aspect of the invention pertains to a
composition comprising a multimeric form of a Tie 2 binding peptide
monomer, wherein the multimeric form has Tie 2 agonist activity.
Preferably, the Tie 2 binding peptide contained in the monomer
binds to Tie 2 with high affinity but does not substantially
inhibit binding of an angiopoietin (e.g., Ang 1) to Tie 2. In one
embodiment, the Tie 2 binding peptide monomer comprises an amino
acid sequence that is present in a native Tie 2 ligand. In another
embodiment, the Tie 2 binding peptide monomer comprises an amino
acid sequence that is not present in a native Tie 2 ligand.
Preferred Tie 2 binding peptides for use in the monomers of the
invention include, but are not limited to, a T7 peptide (SEQ ID
NOs: 1 or 2), a GA3 peptide (SEQ ID NOs: 3 or 4), a T6 peptide (SEQ
ID NOs: 7 or 8) and a T8 peptide (SEQ ID NOs: 5 or 6). In an
alternative embodiment, the Tie 2 binding peptide used in the
monomer competes with an angiopoietin (e.g., Ang 1) for binding to
Tie 2. A non-limiting example of such a peptide is a T 4 peptide
(SEQ ID NOs: 9 or 10).
[0007] In a preferred embodiment, the multimeric form is a
tetramer. Alternatively, the multimeric form can be, for example, a
dimer or a multimeric form that comprises six, eight, ten or twelve
units of the Tie 2 binding peptide monomer. In yet other
embodiments, the multimeric form comprises an odd number of units
of the Tie 2 binding peptide monomer, such as three, five, seven,
nine or eleven units.
[0008] In a preferred embodiment, the Tie 2 binding peptide monomer
comprises a structure: A-B-C, wherein A comprises a Tie 2 binding
peptide, B comprises a spacer and C comprises a multimerizing
group, wherein C has affinity for D, a multimer agent comprising
multiple binding sites for C. For example, the multimer agent D can
have four binding sites for the multimerizing group C such that a
tetramer is formed when four Tie 2 binding peptide monomers, A-B-C,
interact with the multimer agent D. In a preferred embodiment, C
comprises a biotin group and D comprises an agent selected from the
group consisting of avidin, streptavidin and neutravidin. In
another preferred embodiment, the spacer B comprises polyethylene
glycol (PEG).
[0009] In another embodiment, the Tie 2 binding peptide monomer
comprises a structure: A-B, wherein A comprises a Tie 2 binding
peptide and B comprises a spacer, wherein the multimeric form is
created by covalent linkage of multiple Tie 2 binding peptide
monomers via the spacer B. In a preferred embodiment, the spacer B
comprises polyethylene glycol (PEG).
[0010] In another embodiment, the multimeric form comprises a
peptide dimer, comprising: (a) a first peptide chain; (b) a second
peptide chain; and (c) a linking moiety connecting said first and
second peptide chains, wherein said peptide dimer binds to and
activates the Tie 2 receptor. Preferably, the first peptide chain
is a T7 peptide (SEQ ID NOs: 1 or 2). Preferably, the second
peptide chain is a T7 peptide (SEQ ID NOs: 1 or 2). More
preferably, both the first and second peptide chains are T7
peptides (SEQ ID NOs: 1 or 2). Preferably, the linking moiety
comprises one or more water soluble polymers covalently bound to
the first peptide chain and the second peptide chain. More
preferably, the one or more water soluble polymers are linear
polymers. In a preferred embodiment, the water soluble polymer is a
polyethylene glycol (PEG) (e.g., a linear PEG molecule).
Preferably, the PEG has a molecular weight of less than about
20,000 Daltons. More preferably, the PEG has a molecular weight of
in the range of about 3,000 Daltons to about 10,000 Daltons. In
various embodiments, the PEG has a molecular weight of about 3,000
Daltons, about 3,400 Daltons, about 5,000 Daltons or about 10,000
Daltons.
[0011] In another embodiment, the multimeric form comprises a
peptide tetramer, comprising: (a) a first peptide chain; (b) a
second peptide chain; (c) a third peptide chain; (d) a fourth
peptide chain; and (e) a linking moiety connecting said first,
second, third and fourth peptide chains, wherein said peptide
tetramer binds to and activates the Tie 2 receptor. Preferably, the
first, second, third and fourth peptide chains are T7 peptides (SEQ
ID NOs: 1 or 2). Preferably, the linking moiety comprises one or
more water soluble polymers covalently bound to the first, second,
third and fourth peptide chains. More preferably, the one or more
water soluble polymers are branched chain polymers. In a preferred
embodiment, the water soluble polymer is a polyethylene glycol
(PEG) (e.g., a branched chain PEG molecule). Preferably, the
branched PEG has a molecular weight in the range of about 3,000
Daltons to about 20,000 Daltons. In various embodiments, the
branched PEG has a molecular weight of about 3,000 Daltons, about
3,400 Daltons, about 5,000 Daltons, about 10,000 Daltons or about
20,000 Daltons.
[0012] The multimeric forms of the invention exhibit Tie 2 agonist
activity. For example, in one embodiment, the multimeric form
stimulates Tie 2 phosphorylation. In another embodiment, the
multimeric form stimulates phosphorylation of MAPK, AKT and eNOS.
Preferably, a multimeric form of the invention has at least one
effect on endothelial cells selected from the group consisting of:
stimulation of endothelial cell migration, stimulation of MMP2
release from endothelial cells and protection of endothelial cells
from serum withdrawal-induced apoptosis and even more preferably
has all three effects on endothelial cells. Preferably, the
multimeric form stimulates an angiogenic response in vivo in a
Matrigel assay. Preferably, the multimeric form stimulates wound
healing in a subject when applied topically to a wound of the
subject.
[0013] In a particularly preferred embodiment, the invention
provides a composition comprising a tetramer form of a Tie 2
binding peptide monomer, wherein the Tie 2 binding peptide monomer
comprises a structure: A-B-C, wherein:
[0014] A comprises a Tie 2 binding peptide selected from a T7
peptide (SEQ ID NOs: 1 or 2) and a GA3 peptide (SEQ ID NOs: 3 or
4);
[0015] B comprises a polyethylene glycol spacer; and
[0016] C comprises a biotin group,
wherein four copies of A-B-C are associated with a tetramer agent,
D, to create the tetramer form, the tetramer agent, D, being
selected from the group consisting of avidin, streptavidin and
neutravidin.
[0017] Another aspect of the invention pertains to pharmaceutical
compositions comprising the multimeric form of a Tie 2 binding
peptide monomer and a pharmaceutically acceptable carrier. In one
embodiment, the pharmaceutically acceptable carrier is suitable for
topical administration. In another embodiment, the pharmaceutically
acceptable carrier is suitable for systemic administration.
[0018] Another aspect of the invention pertains to methods for
making the multimeric forms of the invention. For example, the
invention provides a method of making a Tie 2 binding peptide
monomer comprising a structure: A-B-C, wherein:
[0019] A comprises a Tie 2 binding peptide;
[0020] B comprises a polyethylene glycol spacer; and
[0021] C comprises a biotin group,
the method comprising reacting a Tie 2 binding peptide comprising
an amino terminal amino acid residue comprising a first reactive
group with a reagent comprising the structure: second reactive
group-B-C, wherein the first reactive group reacts with the second
reactive group, to form A-B-C. Still further, the invention
provides a method of making a tetramer form of the Tie 2 binding
peptide monomer A-B-C, the method comprising combining the Tie 2
binding peptide monomer, A-B-C, with a tetramer agent, D, at a 4:1
ratio, the tetramer agent, D, being selected from the group
consisting of avidin, streptavidin and neutravidin.
[0022] Yet another aspect of the invention pertains to a method of
detecting a multimeric form of the invention, wherein the detection
method comprises contacting a composition of the invention with a
monoclonal antibody that specifically binds to a Tie 2 binding
peptide contained within the composition. For example, a
composition of the invention comprising a T7 peptide can be
detected using a monoclonal antibody that specifically binds to the
T7 peptide. Monoclonal antibody compositions that specifically bind
to the T7 peptide also are encompassed by the invention.
[0023] Yet another aspect of the invention pertains to a method of
activating a Tie 2 receptor comprising contacting the Tie 2
receptor with the multimeric form of a Tie 2 binding peptide
monomer of the invention such that the Tie 2 receptor is activated.
Activation of the Tie 2 receptor can be evidenced by, for example,
phosphorylation of residue tyrosine 992 (Y992) of the Tie 2
receptor or phosphorylation of MAPK, AKT or eNOS.
[0024] Yet another aspect of the invention pertains to a method of
stimulating angiogenesis at a site in a subject comprising
contacting the site with the multimeric form of a Tie 2 binding
peptide monomer of the invention such that angiogenesis is
stimulated at the site in the subject. The multimeric form can be
contacted with the site by, for example, topical administration of
the multimeric form or systemic administration of the multimeric
form. Preferably, angiogenesis stimulated by the multimeric form is
characterized by at least one of the following properties:
[0025] a) recruitment of perivascular support cells;
[0026] b) non-leakiness of vessels; and
[0027] c) well-defined arborization.
In one embodiment of the method of stimulating angiogenesis, the
method further comprises contacting the site in the subject with a
second angiogenic agent, such as VEGF, PDGF, G-CSF, recombinant
human erythropoietin, bFGF and placental growth factor (PLGF). The
method of stimulating angiogenesis can be used in clinical
situations such as vascularization of regenerative tissues,
ischemic limb disease, cerebral ischemia, conditions of vascular
inflammation, arteriosclerosis, avascular necrosis, stimulation of
hair growth and erectile dysfunction.
[0028] Another aspect of the invention pertains to a method of
decreasing vascular permeability at a site of leaky vessels. The
method comprises contacting the site of leaky vessels with a
multimeric form of a Tie 2 binding peptide monomer of the invention
such that vascular permeability is decreased. The method of
decreasing vascular permeability can be used in clinical situations
such as stroke, macular degeneration, macular edema, lymph edema,
breakdown of the blood-retinal barrier, breakdown of the
blood-brain barrier and normalization of tumor vasculature.
[0029] Another aspect of the invention pertains to a method of
protecting endothelial cells. The method comprises contacting the
endothelial cells with a multimeric form of a Tie 2 binding peptide
monomer of the invention such that the endothelial cells are
protected. The method of protecting endothelial cells can be used
in clinical situations such as kidney fibrosis, stroke, macular
degeneration and diabetic complications.
[0030] Still another aspect of the invention pertains to a method
of stimulating healing of a wound in a subject, the method
comprising contacting the wound with the multimeric form of a Tie 2
binding peptide monomer of the invention such that healing of the
wound is stimulated in the subject. The multimeric form can be
contacted with the wound by, for example, topical administration of
the multimeric form or systemic administration of the multimeric
form. In a preferred embodiment, the wound is a diabetic ulcer. In
other embodiments, the wound is, for example, a decubitus ulcer, a
pressure ulcer, a surgical incision, a traumatic tissue injury, a
burn or a skin graft.
[0031] The invention also provides various biomaterials into which
is incorporated a multimeric form of a Tie 2 binding peptide
monomer of the invention. The biomaterial can be, for example,
Matrigel, a skin substitute or a cross-linked glycosaminoglycan
hydrogel. In one embodiment of the biomaterial, a second agent is
also incorporated into the biomaterial. Such a second agent can be,
for example, VEGF, PDGF, G-CSF, recombinant human erythropoietin,
bFGF or placental growth factor (PLGF).
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1A is a schematic diagram of unclustered Vasculotide,
prepared by linkage of the T7 peptide to a PEG-spacer containing
biotin labelling reagent (Maleimide-PEO2-Biotin).
[0033] FIG. 1B is an immunoblot of an in vitro pull down assay,
showing that unclustered Vasculotide, but not irrelevant
biotinylated peptide, specifically binds Tie-2 in this assay.
[0034] FIG. 1C is an immunoblot analysis of lysates from HUVECs
stimulated with Ang 1, avidin, unclustered Vasculotide or clustered
Vasculotide at various doses, showing that treatment with clustered
Vasculotide results in activation of Tie 2 (Tek) and downstream
signalling proteins eNOS, MAPK and AKT.
[0035] FIG. 1D is a dose analysis immunoblot of lysates from
Eahy926 ECs stimulated with unclustered Vasculotide or clustered
Vasculotide, showing that clustered Vasculotide activates Tie 2
receptor phosphorylation at concentrations ranging from 200 .mu.M
to 750 pM.
[0036] FIG. 1E is an immunoblot analysis of the activation kinetics
of Tie 2 activation in HUVECs stimulated with Ang 1 or clustered
Vasculotide for various times, showing that clustered Vasculotide
activates Tie 2, AKT and MAPK, but not FAK, in HUVECs, with
activation kinetics that closely mimic that of recombinant Ang
1.
[0037] FIG. 1F is an immunoblot analysis of lysates from C166 cells
(left)) or Cos1 cells (right), which are phenotypically null for
Tie-2 expression. Cells were infected with recombinant adenovirus
encoding EGFP (Ad-EGFP lanes) or Tie 2 (Ad-Tek lane; positive
control.) Cells infected with Ad-EGFP were stimulated with
unclustered or clustered Vasculotide, neither of which were able to
stimulate phosphorylation of AKT or MAPK above basal unstimulated
levels.
[0038] FIG. 2A is a bar graph of results from a cell death ELISA
experiment, showing that clustered Vasculotide reduces serum
withdrawal-induced cell death of HUVEC cells.
[0039] FIG. 2B is a bar graph of results from a modified Boyden
chamber migration assay, showing that clustered Vaculotide promotes
chemotactic cell migration that is statistically indistinguishable
from migration induced by VEGF (100 ng/ml) or Ang 1 (500
ng/ml).
[0040] FIG. 2C is a gelatin zymographic analysis of conditioned
media from HUVEC cells stimulated with VEGF, unclustered
Vasculotide or clustered Vasculotide, indicating a role for
clustered Vasculotide in promoting release of MMP2.
[0041] FIG. 3A is a bar graph of the results of a Matrigel assay,
quantifying the number of vessels in each treatment group, showing
that clustered Vasculotide, alone or in combination with VEGF,
increases vessel number.
[0042] FIG. 3B is a bar graph of the results of a Matrigel assay,
quantifying the vessel length in each treatment group, showing that
clustered Vasculotide, alone or in combination with VEGF, increases
vessel length.
[0043] FIG. 3C is a bar graph of the results of a Matrigel assay,
quantifying the number of nodes in each treatment group, showing
that clustered Vasculotide, alone or in combination with VEGF,
increases node number.
[0044] FIG. 4A is a bar graph of the results of a wound closure
assay, quantifying total pixel counts of digital tracings of wound
margins over time, showing that treatment with clustered
Vasculotide led to decreases in wound margin as early as day 2 of
treatment.
[0045] 4B is a representative photomicrograph of hematoxylin eosin
(H&E) and Masson's trichome (MT) stained skin sections from
wounds treated with PBS, bFGF (10 .mu.g/ml) or clustered
Vasculotide (5 nM).
[0046] FIG. 4C is a bar graph of the results of histological
scoring of a wound closure assay, quantifying re-epithelialization,
granulation tissue and neovascularization and inflammation using a
0-4 scale, showing that treatment with clustered Vasculotide
resulted in delayed re-epithelialization but promoted profound
increases in regranulation and neovascularization.
[0047] FIG. 5A is a bar graph of the results from a modified Boyden
chamber migration assay, showing that a multimeric GA3
peptide-containing compound promotes chemotactic cell
migration.
[0048] FIG. 5B is a gelatin zymographic analysis of conditioned
media from HUVEC cells stimulated with a multimeric GA3
peptide-containing compound, demonstrating that the compound can
promote release of MMP2.
[0049] FIG. 6 is a schematic illustration of PEG-linked, T7
peptide-containing Tie 2 agonists, referred to herein as
PEG-Vasculotide.
[0050] FIG. 7 is an immunoblot analysis of lysates from bovine
vascular endothelial cells (bVECs) stimulated with PTE200-T7
(tetrameric PEG-linked T7, 20,000 Da MW) at 5 .mu.g/ml, showing
that treatment with PTE200-T7 results in activation of Tie 2.
[0051] FIG. 8 is an immunoblot analysis of lysates from bovine
vascular endothelial cells (bVECs) stimulated with DE100-T7
(dimeric PEG-linked T7, 10,000 Da MW) at 250 ng/ml or 500 ng/ml,
showing that treatment with DE100-T7 results in activation of Tie
2.
[0052] FIG. 9 is an immunoprecipitation/immunoblot analysis of lung
tissue lysates from CD1 mice stimulated in vivo with DE100-T7
(dimeric PEG-linked T7, 10,000 Da MW) at 5 .mu.g or 100 .mu.g,
DE200-T7 (dimeric PEG-linked T7, 20,000 Da MW) at 10 .mu.g or 50
.mu.g), PTE200-T7 (tetrameric PEG-linked T7) at 5 .mu.g or 75
.mu.g, or PBS, examining phosphorylation of Tie 2 and of downstream
pathway markers MAPK and AKT.
[0053] FIG. 10 is a dot blot analysis to assess the specificity of
mouse monoclonal antibodies engineered to detect T7 peptide using
PTE200-T7 as the antigen.
[0054] FIG. 11 is an immunoblot analysis to assess the specificity
of mouse monoclonal antibodies engineered to detect T7 peptide
using T7-BSA as the antigen.
DETAILED DESCRIPTION OF THE INVENTION
[0055] This invention pertains to multimeric forms of a Tie 2
binding peptide monomer and compositions and uses thereof. As
demonstrated herein, when a peptide known to bind Tie 2 was
multimerized, it was shown to be capable of activating Tie 2 and
its associated signalling pathways. Moreover, this activation has
been demonstrated to result in the production of angiogenic
responses, both in vitro and in vivo. Still further, it has been
demonstrated that activation of the pleiotropic Tie 2 signalling
axis by the multimeric compound of the invention can produce blood
vessels that are highly organized and well supported by myogenic
support cells. Stimulation of wound healing by the multimeric form
of the invention also has been demonstrated. Accordingly, the
multimeric forms of the invention can be applied to a wide variety
of situations in which stimulation of angiogenesis and/or wound
healing is desireable.
[0056] Prior to further describing the invention, it may be helpful
to an understanding thereof to set forth definitions of certain
terms to be used herein
[0057] As used herein, the term "Tie 2" refers to a receptor
protein tyrosine kinase that is expressed almost exclusively on
endothelial cells and that is also known in the art as TEK, p140
TEK, CD202B and VMCM. The term "Tie 2" is intended to encompass the
receptor from any species that expresses this receptor, although
human Tie 2 is preferred. The mRNA and protein sequences of human
Tie 2 are set forth at GenBank Accession Nos. NM.sub.--000459 and
NP.sub.--000450, respectively.
[0058] As used herein, the term "angiopoietin" is intended to refer
to any one of a family of protein growth factors known to be
ligands for Tie 2, including angiopoietin 1 (or Ang 1),
angiopoietin 2 (or Ang 2), angiopoietin 3 (or Ang 3) and
angiopoietin 4 (or Ang 4). The term "angiopoietin" is intended to
encompass the growth factor from any species that expresses the
growth factor, although human angiopoietin family members are
preferred. The mRNA and protein sequences of human Ang 1 are set
forth at GenBank Accession Nos. NM.sub.--001146 and
NP.sub.--001137, respectively. The mRNA and protein sequences of
human Ang 2 are set forth at GenBank Accession Nos. NM.sub.--001147
and NP.sub.--001138, respectively. The mRNA and protein sequences
of human Ang 4 are set forth at GenBank Accession Nos.
NM.sub.--015985 and NP.sub.--057069, respectively.
[0059] As used herein, the term "MAPK" is intended to refer to
mitogen activated protein kinase, also known as ERK or
extracellular signal-regulated kinase, an intracellular kinase that
is phosphorylated upon activation of Tie 2. The term "MAPK" is
intended to encompass the kinase from any species that expresses
the kinase, although human MAPK is preferred. The mRNA and protein
sequences of human MAPK are set forth at GenBank Accession Nos.
NM.sub.--002736 and NP.sub.--002745, respectively.
[0060] As used herein, the term "AKT" is intended to refer to a
protein kinase also known as v-akt murine thymoma viral oncogene
homolog, an intracellular kinase that is phosphorylated upon
activation of Tie 2. The term "AKT" is intended to encompass the
kinase from any species that expresses the kinase, although human
AKT is preferred. The mRNA and protein sequences of human AKT are
set forth at GenBank Accession Nos. NM.sub.--001014431 and
NP.sub.--001014431, respectively.
[0061] As used herein, the term "eNOS" is intended to refer to
endothelial cell nitric oxide synthetase, also known as NOS 3, NOS
III or ECNOS, an intracellular enzyme that is phosphorylated upon
activation of Tie 2. The term "eNOS" is intended to encompass the
enzyme from any species that expresses the enzyme, although human
eNOS is preferred. The mRNA and protein sequences of human eNOS are
set forth at GenBank Accession Nos. NM.sub.--000603 and
NP.sub.--000594, respectively.
[0062] As used herein, the term "MMP2" is intended to refer to
matrix metalloproteinase 2, a protein secreted by endothelial cells
that is involved in the breakdown of extracellular matrix. The term
"MMP2" is intended to encompass the protein from any species that
expresses the protein, although human MMP2 is preferred. The mRNA
and protein sequences of human MMP2 are set forth at GenBank
Accession Nos. NM.sub.--004530 and NP.sub.--004521,
respectively.
[0063] As used herein, the term "VEGF" is intended to refer to
vascular endothelial growth factor, also known as VPF or vascular
permeability factor, a growth factor involved in endothelial cell
growth and angiogenesis. The term "VEGF" is intended to encompass
the growth factor from any species that expresses the growth
factor, although human VEGF is preferred. The mRNA and protein
sequences of human VEGF (variant 1) are set forth at GenBank
Accession Nos. NM.sub.--001025366 and NP.sub.--001020537,
respectively.
[0064] As used herein, the term "PDGF" is intended to refer to
platelet derived growth factor, a mitogenic factor for cells of
mesenchymal origin. The term "PDGF" is intended to encompass the
growth factor from any species that expresses the growth factor,
although human PDGF is preferred. The protein has an alpha chain
and a beta chain and can exist as a homodimer or a heterodimer. The
mRNA and protein sequences of human PDGF alpha (isoform 1) are set
forth at GenBank Accession Nos. NM.sub.--002607 and
NP.sub.--002598, respectively. The mRNA and protein sequences of
human PDGF beta (isoform 1) are set forth at GenBank Accession Nos.
NM.sub.--002608 and NP.sub.--002599, respectively.
[0065] As used herein, the term "Tie 2 binding peptide" is intended
to encompass peptides at least two amino acids in length and
preferably no more than 100 amino acids in length that have binding
affinity for Tie 2. The term "Tie 2 binding peptide" is not
intended to encompass naturally occurring ligands for Tie 2, such
as native, full-length angiopoietin proteins. Furthermore, the term
"Tie 2 binding peptide" is intended to encompass peptides comprised
in whole or in part of L-amino acids, peptides comprised in whole
or in part of D-amino acids and peptides comprised of both L- and
D-amino acids. Still further, the term "Tie 2 binding peptide" is
intended to encompass peptides comprised in whole or in part of the
20 naturally-occurring amino acid residues, peptides comprised in
whole or in part of non-naturally-occurring amino acid residues and
peptide comprised of both naturally-occurring and
non-naturally-occurring amino acid residues.
[0066] As used herein, the term "Tie 2 binding peptide monomer" is
intended to refer to a single unit of a Tie 2 binding peptide
compound. The Tie 2 binding peptide compound, or monomer, comprises
the Tie 2 binding peptide, and may comprise other chemical moieties
(e.g., spacers, multimerizing groups and the like), but the Tie 2
binding peptide monomer comprises only one copy (or unit) of the
Tie 2 binding peptide and thus has a single valency for the Tie 2
receptor.
[0067] As used herein, the term "multimeric form" of a Tie 2
binding peptide monomer is intended to refer to forms that contain
more than one unit of the Tie 2 binding peptide monomer such that
the multimeric form (e g, dimer, tetramer and the like) comprises
more than one copy (or unit) of the Tie 2 binding peptide and thus
has multivalency for the Tie 2 receptor.
[0068] As used herein, the term "high affinity", as used with
respect to binding of a Tie 2 binding peptide to the Tie 2
receptor, is intended to mean binding of the peptide to the
receptor with K.sub.d of about 10.sup.-3 M or less, more preferably
10.sup.-4 M or less, even more preferably 10.sup.-5 M or less.
[0069] As used herein, the term "does not substantially inhibit
binding of an angiopoietin to Tie 2", as used with respect to a Tie
2 binding peptide, is intended to mean that the ability of the Tie
2 binding peptide to inhibit binding of an angiopoietin (e.g., Ang
1) to Tie 2 is essentially no greater than the ability of a
unrelated, control peptide (e.g., a peptide that does not have
measurable affinity for Tie 2) to inhibit the binding of an
angiopoietin (e.g., Ang 1) to Tie 2.
[0070] As used herein, the term "Tie 2 agonist activity" is
intended to refer to stimulating, enhancing, increasing or
upregulating Tie 2 receptor activity, as measured by any method,
technique, signal, detector or indicator that is known in the art
to be indicative of Tie 2 receptor activity. Non-limiting examples
of such indicators of Tie 2 activity include phosphorylation of
human Tie 2 at amino acid residue Y992 or phosphorylation of one or
more of MAPK, AKT and eNOS.
[0071] The invention is described in further detail in the
following subsections, which subsections are presented only for
purposes of clarity and should in no way be considered as
limitations.
I. Multimeric Forms of Tie 2 Binding Peptide Monomers
[0072] This invention provides a composition comprising a
multimeric form of a Tie 2 binding peptide monomer, wherein the
multimeric form has Tie 2 agonist activity. Thus, the composition
has two aspects to consider: the structure of the Tie 2 binding
peptide monomer itself, and the means by which the monomer is
multimerized to create the multimeric form that has Tie 2 agonist
activity, both of which aspects will be discussed further herein.
In one embodiment, the multimeric form comprises an even number of
units of the monomer. In a preferred embodiment, the multimeric
form is a tetramer. In another preferred embodiment, the multimeric
form is a dimer. In yet other embodiments, the multimeric form
comprises six, eight, ten or twelve units of the Tie 2 binding
peptide monomer. In another embodiment, the multimeric form
comprises an odd number of units of the monomer. For example, the
multimer form can be a trimer or the multimeric form can comprises
five, seven, nine or eleven units of the Tie 2 binding peptide
monomer.
[0073] The Tie 2 binding peptide monomer comprises a peptide that
has binding affinity for Tie 2. Furthermore, the monomer may
comprise other chemical moieties, which are discussed in further
detail below.
[0074] The Tie 2 binding peptide contained within the monomer is at
least two amino acids in length, more preferably is at least five
amino acids in length and even more preferably is at least seven
amino acids in length. A preferred size range for the peptide is
7-25 amino acids in length, more preferably 7-15 amino acids in
length. Other size ranges include 5-30 amino acids in length, 5-40
amino acids in length, 5-50 amino acids in length, 5-60 amino acids
in length, 5-70 amino acids in length, 5-80 amino acids in length,
5-90 amino acids in length or 5-100 amino acids in length.
Preferably, the peptide is no more than 100 amino acids in
length.
[0075] In one embodiment, the Tie 2 binding peptide within the
monomer comprises an amino acid sequence that is present in a
native Tie 2 ligand (e.g., an angiopoietin, such as Ang 1 or Ang
2). For example, a fragment of an angiopoietin that retains the
ability to bind to Tie 2 can be used as the Tie 2 binding peptide.
Alternatively, in another embodiment, the Tie 2 binding peptide
within the monomer comprises an amino acid sequence that is not
present in a native Tie 2 ligand. It has been shown that peptides
having amino acid sequences that differ from the primary sequence
of angiopoietins can be selected that have affinity for Tie 2 (see
e.g., Tournaire, R. et al. (2004) EMBO Reports 5:262-267). Such
peptides can be identified, for example, by screening of a phage
displayed peptide library (e.g., a random 7-mer library) for
peptides that bind to Tie 2 (e.g., a Tie 2-Fc fusion protein), with
confirmation of peptide binding to Tie 2 by screening of the
selected peptide for binding to Tie 2 using an ELISA assay (e.g.,
as described in Tournaire, R. et al. (2004) supra).
[0076] It is preferable that the Tie 2 binding peptide used in the
monomer binds to Tie 2 with high affinity but does not
substantially inhibit binding of an angiopoietin to Tie 2. This
embodiment is preferable so that the multimeric form does not
compete with native angiopoietins for binding to Tie 2. For
example, preferably the Tie 2 binding peptide binds to Tie 2 with
high affinity but does not substantially inhibit the binding of Ang
1 to Tie 2. Additionally or alternatively, preferably the Tie 2
binding peptide binds to Tie 2 with high affinity but does not
substantially inhibit the binding of, for example, Ang 2 or Ang 4,
to Tie 2.
[0077] In a preferred embodiment, the Tie 2 binding peptide monomer
comprises a T7 peptide, which T7 peptide comprises an amino acid
sequence: His-His-His-Arg-His-Ser-Phe (SEQ ID NO: 1). In one
embodiment, the T7 peptide has an amino terminal cysteine residue
added to it and, thus, in this embodiment, the T7 peptide comprises
an amino acid sequence: Cys-His-His-His-Arg-His-Ser-Phe (SEQ ID NO:
2).
[0078] In another preferred embodiment, the Tie 2 binding peptide
monomer comprises a GA3 peptide, which GA3 peptide comprises an
amino acid sequence:
Trp-Thr-Ile-Ile-Gln-Arg-Arg-Glu-Asp-Gly-Ser-Val-Asp-Phe-Gln-Arg-
-Thr-Trp-Lys-Glu-Tyr-Lys (SEQ ID NO: 3). In one embodiment, the GA3
peptide has an amino terminal cysteine residue added to it and,
thus, in this embodiment, the GA3 peptide comprises an amino acid
sequence:
Cys-Trp-Thr-Ile-Ile-Gln-Arg-Arg-Glu-Asp-Gly-Ser-Val-Asp-Phe-Gln-Arg-Thr-T-
rp-Lys-Glu-Tyr-Lys (SEQ ID NO: 4).
[0079] In yet another embodiment, the Tie 2 binding peptide monomer
comprises a T8 peptide, which T8 peptide comprises an amino acid
sequence: His-Pro-Trp-Leu-Thr-Arg-His (SEQ ID NO: 5). In one
embodiment, the T8 peptide has an amino terminal cysteine residue
added to it and, thus, in this embodiment, the T8 peptide comprises
an amino acid sequence: Cys-His-Pro-Trp-Leu-Thr-Arg-His (SEQ ID NO:
6).
[0080] In yet another embodiment, the Tie 2 binding peptide monomer
comprises a T6 peptide, which T6 peptide comprises an amino acid
sequence: Lys-Leu-Trp-Val-Ile-Pro-Lys (SEQ ID NO: 7). In one
embodiment, the T6 peptide has an amino terminal cysteine residue
added to it and, thus, in this embodiment, the T6 peptide comprises
an amino acid sequence: Cys-Lys-Leu-Trp-Val-Ile-Pro-Lys (SEQ ID NO:
8).
[0081] In an alternative embodiment, the Tie 2 binding peptide used
in the monomer can comprises a peptide that competes with an
angiopoietin (e.g., Ang 1) for binding to Tie 2. A non-limiting
example of such a peptide is a T4 peptide, which T4 peptide
comprises an amino acid sequence: Asn-Leu-Leu-Met-Ala-Ala-Ser (SEQ
ID NO: 9). In one embodiment, the T4 peptide has an amino terminal
cysteine residue added to it and, thus, in this embodiment, the T4
peptide comprises an amino acid sequence:
Cys-Asn-Leu-Leu-Met-Ala-Ala-Ser (SEQ ID NO: 10).
[0082] The Tie 2 binding peptides T4, T6, T7 and T8 also are
described in Tournaire, R. et al. (2004) EMBO Reports 5:262-267.
The Tie 2 binding peptide GA3 also is described in Wu, X. et al.
(2004) Biochem. Biophys. Res. Commun. 315:1004-1010.
[0083] In addition to the Tie 2 binding peptide, the Tie 2 binding
peptide monomer can comprise other chemical moieties or groups,
such as spacers and/or multimerizing groups. For example, the Tie 2
binding peptide can be linked to a spacer, which may serve one or
more functionalities. The spacer can, for example, function to
increase the distance between the monomers when they are
multimerized to facilitate interaction of the multimeric form with
the Tie 2 receptor (e.g., reduce steric hindrance). Additionally or
alternatively, the spacer can, for example, serve as a chemical
group by which the monomers can be multimerized. Moreover, the Tie
2 binding peptide monomer can comprise one or more multimerizing
groups, chemical moieties that function to facilitate
multimerization of the monomers. A preferred multimerizing group is
a biotin group, which has affinity for avidin, streptavidin and
neutravidin such that any of the three latter compounds can be used
for multimerization of monomers comprising a biotin group. Another
example of a multimerizing group is a coiled coil domain, which can
be linked to the amino terminus of the peptide through standard
recombinant DNA engineering techniques and which self-assembles
into oligomeric structures (see e.g., U.S. Patent Publications
20030220476 and 20060074230 for further description of the use of
coiled coil domains for multimerization). Non-limiting examples of
coiled coil domains suitable for use are the coiled coil domains
from the yeast transcription factor GCN4, from cartilage matrix
protein (CMP) or from cartilage oligomeric matrix protein
(COMP).
[0084] A preferred spacer is a polyethylene glycol (PEG) spacer,
which is a polymeric molecule that can contain different numbers of
units, such as 2, 4, 6, 8, 10, 11 or 12 units. PEG polymers are
also known in the art as polyethylene oxide (PEO) polymers and thus
the terms PEG and PEO as used herein are intended to be equivalent.
Numerous other suitable spacers (also known as linkers) are well
known in the art, non-limiting examples of which include other
polyalkylene glycols, polyesters and polyalkylene amines. Moreover,
a wide variety of spacers linked on one end to a reactive moiety
and on the other end to a biotin group are commercially available
(EZ-Link Biotin reagents available from Pierce Chemical Co.,
Rockford, Ill., USA) and can be used in the preparation of the Tie
2 binding peptide monomers of the invention. Non-limiting examples
of commercially available reagents of the structure: reactive
moiety-spacer-biotin include:
Sulfhydryl Reactive Reagents:
[0085] EZ-Link Biotin-BMCC
(1-Biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane)
[0086] EZ-Link Biotin-HPDP
(N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide [0087]
EZ-Link Iodoacetyl-LC-Biotin
(N-iodoacetyl-N-biotinylhexylenediamine) [0088] EZ-Link
Iodoacetyl-PEO.sub.2 Biotin
((+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine) [0089] EZ-Link
Maleimide PEO.sub.n-Biotin (n=2 or 11)
Amine Reactive Reagents:
[0089] [0090] EZ-Link NHS-PEO.sub.n-Biotin (n=4 or 12) [0091]
EZ-Link NHS--SS-Biotin (succinimidyl
2-(biotinamido)-ethyl-1,3'-dithiopropionate) [0092] EZ-Link
Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6-(biotinamido) hexanoate)
[0093] EZ-Link TFP-PEO.sub.3-Biotin (Tetrafluorophenyl Ester
PEO.sub.3-biotin)
Carboxyl Reactive Reagents:
[0093] [0094] EZ-Link 5-(Biotinamido)pentylamine [0095] EZ-Link
Amine-PEO.sub.2-Biotin Labeling Reagent
((+)-Biotinyl-3,6-dioxaoctanediamine) [0096] EZ-Link
Amine-PEO.sub.3-Biotin Labeling Reagent
((+)-Biotinyl-3,6,9-trioxaundecanediamine) [0097] EZ-Link Biotin
PEO-Amine ((+)-Biotinyl-3,6-dioxaoctanediamine) [0098] EZ-Link
Biotin-PEO-LC-Amine ((+)-Biotinyl-3,6,9-trioxaundecanediamine)
[0099] Furthermore, a branched arm spacer can be linked to multiple
copies of the Tie 2 binding peptide as a means to multimerize the
peptide. Non-limiting examples include 2 and 4 armed activated
branched PEG spacers, although spacers with more arms, such as 8 or
12 armed activated branched PEG spacers also can be used. Branched
activated PEG spacers (e.g., activated with maleimide) are
commercially available (e.g., NOF Corporation, Tokyo, Japan).
[0100] In a preferred embodiment, the Tie 2 binding peptide monomer
comprises a structure: A-B-C, wherein A comprises a Tie 2 binding
peptide, B comprises a spacer and C comprises a multimerizing
group, wherein C has affinity for D, a multimer agent comprising
multiple binding sites for C. In a particularly preferred
embodiment, the multimer agent D has four binding sites for the
multimerizing group C such that a tetramer is formed when four Tie
2 binding peptide monomers, A-B-C, interact with the multimer agent
D. A preferred multimerizing group, C, for use in creating
tetramers is a biotin group. Preferred multimer agents, D, for use
in creating tetramers are avidin, streptavidin and neutravidin. It
is well known in the art that avidin, streptavidin and neutravidin
have four binding sites for biotin and that biotin binds with high
affinity to each of avidin, streptavidin and neutravidin. A
preferred spacer, B, for use in a monomer of the structure A-B-C is
a polyethylene glycol (PEG) spacer.
[0101] In another embodiment, the Tie 2 binding peptide monomer
comprises a structure: A-B, wherein A comprises a Tie 2 binding
peptide and B comprises a spacer, wherein the multimeric form is
created by covalent linkage of multiple Tie 2 binding peptide
monomers via the spacer B. A preferred spacer, B, for use in a
monomer of the structure A-B is a polyethylene glycol (PEG)
spacer.
[0102] In a particularly preferred embodiment, the invention
provides a composition comprising a tetramer form of a Tie 2
binding peptide monomer, wherein the Tie 2 binding peptide monomer
comprises a structure: A-B-C, wherein:
[0103] A comprises a Tie 2 binding peptide selected from a T7
peptide and a GA3 peptide;
[0104] B comprises a polyethylene glycol spacer; and
[0105] C comprises a biotin group,
wherein four copies of A-B-C are associated with a tetramer agent,
D, to create the tetramer form, the tetramer agent, D, being
selected from the group consisting of avidin, streptavidin and
neutravidin. A specific example of this embodiment is the compound
Vasculotide (described in detail in the Example), in which A
comprises a T7 peptide, B comprises a polyethylene glycol spacer
and C comprises a biotin group, and wherein multimeric Vasculotide
comprises avidin as the tetramer agent D.
[0106] In another aspect, the invention provides a composition
comprising a Tie 2 binding peptide monomer, the Tie 2 binding
peptide monomer comprising a structure A-B-C, wherein:
[0107] A comprises a Tie 2 binding peptide;
[0108] B comprises a spacer; and
[0109] C comprises a multimerizing group.
[0110] Preferably, the Tie 2 binding peptide, A, comprises a T7
peptide or a GA3 peptide. Alternatively, the Tie 2 binding peptide
can comprises, for example, a T8 peptide, a T6 peptide or a T4
peptide. Preferably, the spacer, B, comprises a polyethylene glycol
spacer. Preferably, the multimerizing group, C, comprises a biotin
group.
[0111] In a preferred embodiment, the multimeric form of the
invention comprises a peptide dimer, comprising: (a) a first
peptide chain; (b) a second peptide chain; and (c) a linking moiety
connecting said first and second peptide chains, wherein said
peptide dimer binds to and activates the Tie 2 receptor.
Preferably, the first peptide chain is a T7 peptide. Preferably,
the second peptide chain is a T7 peptide. More preferably, both the
first and second peptide chains are T7 peptides. Alternatively, the
first and second peptide chains independently can be selected from
the group consisting of a T7 peptide, a GA3 peptide, a T4 peptide,
a T6 peptide and a T8 peptide, although preferably the first and
second peptide chains are both the same type of peptide chain.
Additional Tie 2 binding peptides that can be used are described in
further detail above.
[0112] Preferably, the linking moiety comprises one or more water
soluble polymers covalently bound to the first peptide chain and
the second peptide chain. More preferably, the one or more water
soluble polymers are linear polymers. In a preferred embodiment,
the water soluble polymer is a polyethylene glycol (PEG) (e.g., a
linear PEG molecule). Preferably, the PEG has a molecular weight of
less than about 20,000 Daltons. More preferably, the linear PEG has
a molecular weight in the range of about 3,000 Daltons to about
10,000 Daltons. In various embodiments, the linear PEG has a
molecular weight of about 3,000 Daltons, about 3,400 Daltons, about
5,000 Daltons or about 10,000 Daltons. It is understood that in a
given preparation of PEG, the molecular weights will typically vary
among individual molecules. Some molecules will weight more, and
some less, than the stated molecular weight. Such variation is
generally reflected by use of the word "about" to describe the
molecular weights of the PEG molecules.
[0113] As demonstrated in Example 11, T7-PEG dimers comprising PEG
having a molecular weight of 10,000 Da exhibited effective
activation of Tie 2 and downstream pathways (e.g, MAPK, AKT) in
vivo, whereas T7-PEG dimers comprising a PEG having a molecular
weight of 20,000 Da did not effectively activate Tie 2 or
downstream pathways, indicating that the longer PEG linker resulted
in a dimer in which the two peptide chains were spaced too far
apart for optimal activation of Tie 2 and, thus, the shorter
linkers may be preferably for optimal results. Accordingly, dimers
utilizing a linear PEG linker having a molecular weight less than
about 20,000 Da, more preferably having a molecular weight in the
range of about 3,000 Daltons to about 10,000 Da, are preferred.
[0114] In another embodiment, the multimeric form of the invention
comprises a peptide tetramer, comprising: (a) a first peptide
chain; (b) a second peptide chain; (c) a third peptide chain; (d) a
fourth peptide chain; and (e) a linking moiety connecting said
first, second, third and fourth peptide chains, wherein said
peptide tetramer binds to and activates the Tie 2 receptor.
Preferably, the first, second, third and fourth peptide chains are
T7 peptides. Alternatively, the first, second, third and fourth
peptide chains independently can be selected from the group
consisting of a T7 peptide, a GA3 peptide, a T4 peptide, a T6
peptide and a T8 peptide, although preferably the first, second,
third and fourth peptide chains are all the same type of peptide
chain. Additional Tie 2 binding peptides that can be used are
described in further detail above.
[0115] Preferably, the linking moiety comprises one or more water
soluble polymers covalently bound to the first, second, third and
fourth peptide chains. More preferably, the one or more water
soluble polymers are branched chain polymers. In a preferred
embodiment, the water soluble polymer is a polyethylene glycol
(PEG) (e.g., a branched chain PEG molecule). As demonstrated in
Example 11, a T7-PEG tetramer utilizing a branched chain tetrameric
PEG linker having a molecular weight of 20,000 Da was effective in
activating Tie 2 and downstream pathways (e.g., MAPK, AKT) in vivo.
Preferably, the branched PEG has a molecular weight in the range of
about 3,000 Daltons to about 20,000 Daltons. In various
embodiments, the branched PEG has a molecular weight of about 3,000
Daltons, about 3,400 Daltons, about 5,000 Daltons, about 10,000
Daltons or about 20,000 Daltons. It is understood that in a given
preparation of PEG, the molecular weights will typically vary among
individual molecules. Some molecules will weight more, and some
less, than the stated molecular weight. Such variation is generally
reflected by use of the word "about" to describe the molecular
weights of the PEG molecules.
[0116] In the PEG-containing dimers, a single, preferably linear,
PEG moiety is simultaneously attached to the termini (e.g., the
N-termini) of both peptide chains of the peptide dimer. In the PEG
containing tetramers, a single, branched chain PEG moiety is
simultaneously attached to the termini (e.g., the N-termini) of the
four peptide chains of the peptide tetramer. Such peptide dimers
and tetramers are illustrated schematically in FIG. 6. To prepare
the PEG-containing dimeric and tetrameric compounds described
above, Tie 2 binding peptides can be reacted with activated PEG
linkers (e.g., PEG dimaleimide for preparation of dimers, PEG
tetramaleimide for preparation of tetramers) as described in detail
in Example 9. Such activated PEG linkers (linear or branched chain)
are commercially available (e.g., from NOF America
Corporation).
[0117] In addition to the dimers and tetramers described above, the
invention encompasses other multimeric forms comprising two or more
Tie 2 binding peptides linked by a linking moiety, such as those
containing three, five, six, seven, eight, nine, ten, eleven or
twelve Tie 2 binding peptides covalently linked to a linking
moiety, preferably a branched linking moiety, such as a branched
chain PEG molecule. Such alternative multimeric forms can be
prepared as described for the dimers and tetramers, using linker
moieties having the appropriate number of reactive ends (e.g., six
reactive ends for a multimer containing six peptide chains) and the
appropriate ratio of peptide to linker (e.g., 6:1 for a multimer
containing six peptide chains).
[0118] While PEG linkers are preferred water soluble polymer
linkers of the invention, alternative water soluble polymer linkers
include, but are not limited to, copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers),
poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol
homopolymers, polypropylene oxide/ethylene oxide copolymers, and
polyoxyethylated polyols. For peptide dimers, preferably the
polymer linker has a molecular weight of less than 20,000 Da and
more preferably has a molecular weight of 10,000 Da. For peptide
tetramers, preferably the polymer linker has a molecular weight of
20,000 Da.
[0119] In addition to water soluble polymers, other types of
linking moieties known in the art can be used join the peptide
chains in the multimers (e.g., two peptide chains in the dimer,
four peptide chains in the tetramer). Non-limiting examples of
additional suitable linker moieties that can be used to join
multiple peptide chains to form multimers include those described
in US Publication 20070104704 and US Publication 20070027074, the
entire contents of both of which are expressly incorporated herein
by reference.
[0120] In yet another aspect, the invention provides a composition
comprising a Tie 2 binding peptide monomer, the Tie 2 binding
peptide monomer comprising a structure A-B, wherein:
[0121] A comprises a Tie 2 binding peptide; and
[0122] B comprises a spacer.
Preferably, the Tie 2 binding peptide, A, comprises a T7 peptide or
a GA3 peptide. Alternatively, the Tie 2 binding peptide can
comprises, for example, a T8 peptide, a T6 peptide or a T4 peptide.
Preferably, the spacer, B, comprises a polyethylene glycol
spacer.
[0123] The invention also provides kits comprising one or more
compositions of the invention, preferably packaged with
instructions for use of the kit. For example, in a preferred
embodiment, the invention provides a kit comprising a Tie 2 binding
peptide monomer comprising the structure A-B-C, wherein
[0124] A comprises a Tie 2 binding peptide;
[0125] B comprises a spacer; and
[0126] C comprises a multimerizing group;
packaged together with a multimer agent, D, wherein C has affinity
for D, and wherein D comprises multiple binding sites for C.
Preferred examples of A, B, C and D are as described previously
herein. Preferably, the kit is packaged with instructions for
creating a multimer form of the Tie 2 binding peptide monomer by
combining the monomer composition with the multimer agents such
that a multimeric form of the monomer is produced.
[0127] In another embodiment, the invention provides a kit
comprising a Tie 2 binding peptide monomer comprising the structure
A-B, wherein
[0128] A comprises a Tie 2 binding peptide; and
[0129] B comprises a spacer;
packaged together with instructions for creating a multimer form of
the Tie 2 binding peptide monomer by multimerization through the
spacer, B. Optionally, the kit may contain one or more reagents
that facilitate multimerization through B. Preferred examples of A
and B are as described previously herein.
II. Characteristic Properties of Multimeric Forms of Tie 2 Binding
Peptide Monomers
[0130] The multimeric forms of Tie 2 binding peptide monomers
provided by the invention exhibit Tie 2 agonist activity. This Tie
2 agonist activity can be detected using indicators of Tie 2
activation that are well established in the art and that are
described in detail in the Examples. For example, a multimeric form
of the invention can stimulate Tie 2 phosphorylation (e.g.,
phosphorylation at amino acid residue Y992 of human Tie 2).
Furthermore, a multimeric form of the invention can stimulate
phosphorylation of a molecule in a downstream signalling pathway of
Tie 2, such as phosphorylation of MAPK, AKT (e.g., phosphorylation
at amino acid residue 5473 of human AKT) and/or eNOS (e.g.,
phosphorylation at amino acid residue S1177 of eNOS). In particular
embodiments, a multimeric form of the invention does not stimulate
phosphorylation of focal adhesion kinase (FAK) (e.g.,
phosphorylation at amino acid reside Y397 of human FAK), which
differentiates the multimeric form from native Ang 1, which has
been shown to activate FAK by a Tie 2-independent manner. The
ability of a multimeric form to stimulate phosphorylation of
particular proteins can be determined used standard techniques
well-known in the art, such as immunoblot assays of cell lysates
treated with the multimeric form, as described in detail in Example
3.
[0131] In preferred embodiments, a multimeric form of the invention
has demonstrable effects on endothelial cells. For example, a
multimeric form of the invention preferably has at least one effect
on endothelial cells selected from the group consisting of:
stimulation of endothelial cell migration, stimulation of MMP2
release from endothelial cells and protection of endothelial cells
from serum withdrawal-induced apoptosis. More preferably, a
multimeric form of the invention has at least two of these effects
on endothelial cells and even more preferably has all three of
these effects on endothelial cells. The ability of a multimeric
form to have any of these effects on endothelial cells can be
determined using assays known in the art, such as a Boyden chamber
assay to assess cell migration, a zymography assay to assess MMP2
release or a cell death ELISA assay to assess serum withdrawal
induced apoptosis. Such assays are described in detail in Example
4.
[0132] In preferred embodiments, a multimeric form of the invention
has demonstrable effects on angiogenesis, as measured in an in
vitro or in vivo angiogenesis assay. A preferred assay is an in
vivo Matrigel assay (described in detail in Example 5), in which
growth factor reduced Matrigel is impregnated with the multimeric
form and injected subcutaneously into a test animal. After a period
of time (e.g., 14 days), the test animal can be treated with an
agent that facilitates vessel identification and quantitation
(e.g., FITC-lectin) and the Matrigel plug can be removed and
examined for an angiogenic response. As demonstrated in Example 5,
a multimeric form of the invention is capable of inducing a robust
angiogenic response, as evidenced by large, well branched vessels
that contain a continuum of small and large arterioles and venules
and that stain positively for the myogenic support cell marker, Sma
I. Significantly, the multimeric forms of the invention can
stimulate an angiogenic response in an in vivo assay (e.g.,
Matrigel assay) that displays more organized branching patterns and
less tortuosity that the angiogenic response stimulated by VEGF.
Still further, preferred multimeric forms of the invention, when
used in combination with VEGF, can decrease the level of vessel
tortuosity that is seen with VEGF treatment alone.
[0133] In preferred embodiments, a multimeric form of the invention
can stimulate wound healing in a subject when applied topically to
a wound of the subject. The ability of the multimeric form to
stimulate wound healing can be assessed in an animal model, such as
the B6.Cg-m(+/+)Lepr(db)/J (db/db) strain of mouse, a diabetic
strain of mouse that presents with impaired wound healing. An
excisional wound can be made on the mouse, the multimeric form,
incorporated into a topical formulation, can be applied to the
wound and wound healing can be assessed as described in Example 6.
Preferred multimeric forms of the invention can accelerate wound
closure times and/or can promote increases in collagen deposition
and neovascularization.
III. Preparation of Multimeric Forms of Tie 2 Binding Peptide
Monomers
[0134] The preparation of a multimeric form of the invention
typically involves at least two steps: first, preparation of the
Tie 2 binding peptide monomer and second, multimerization of the
monomer to create the multimer form.
[0135] The Tie 2 binding peptide contained within the monomer can
be prepared by one of many methods known in the art for peptide
synthesis, including but not limited to solid phase peptide
synthesis (SPPS) and liquid phase peptide synthesis (LPPS). The two
most common chemistries used in peptide synthesis are Fmoc and Boc;
each chemistry has its own side chain protection characteristics
that allow for selective deprotection of side chains for post
synthesis modifications. For SPPS, a summary of many techniques may
be found in J. M. Stewart and J. D. Young, Solid Phase Peptide
Synthesis, W.H. Freeman Co. (San Francisco) 1963, and J.
Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic
Press (New York), 1973. A preferred peptide synthesis method is
Fmoc-SPPS, a recent summary of which can be found in W. Chan, Fmoc
Solid Phase Peptide Synthesis: A Practical Approach, Oxford
University Press (United Kingdom) 1999. Peptides can be synthesized
using an automated peptide synthesizer, such as Applied Biosystems'
ABI433A Peptide Synthesizer (Foster City, Calif., USA).
[0136] In addition to containing the amino acid sequence that
interacts with the Tie 2 receptor, the Tie 2 binding peptide can be
modified to contain one or more additional amino acid residues that
function to facilitate further modification of the peptide. For
example, an amino terminal cysteine residue can be added during
synthesis to the Tie 2 binding peptide sequence to provide a
reactive sulfhydryl group to allow for attachment of one or more
additional chemical moieties via the sulfhydryl group. It will be
apparent to the ordinarily skilled artisan that various other
chemical modifications of the peptide can be performed to create a
reactive group that can be used to link chemical moieties to the
peptide. For example, the peptide can be modified during synthesis
to contain an amino terminal lysine residue to provide a reactive
primary amino group to allow for attachment of one or more
additional chemical moieties via the primary amine group.
Alternatively, the peptide can be modified during synthesis to
contain an amino terminal aspartic acid or glutamic acid residue to
provide a reactive carboxyl group to allow for attachment of one or
more additional chemical moieties via the carboxyl group.
Preferably, chemical modification of the peptide is carried out at
the amino terminal or carboxy terminal end of the peptide so as to
minimize any possible interference with the Tie 2 binding portion
of the peptide.
[0137] As discussed herein, the Tie 2 binding peptide monomer can
include other chemical moieties in addition to the Tie 2 binding
peptide, such as a spacer moiety and/or a multimerizing group,
which additional chemical moieties can be attached using chemical
reactions well known in the art. For example, a Tie 2 binding
peptide that contains a reactive sulfhydryl group (e.g., an amino
terminal cysteine residue) can be reacted with a maleimide group
that is linked to one or more additional chemical moieties of
interest to thereby link the additional chemical moieties to the
Tie 2 binding peptide. Nonlimiting examples of commercially
available reagents that can be used to link a spacer group and a
multimerizing group to a peptide having a reactive sulfhydryl group
include maleimide-PEO.sub.2-biotin and maleimide-PEO.sub.11-biotin
(Pierce Chemical, Rockford, Ill., USA). Nonlimiting examples of
commercially available reagents that can be used to link a spacer
group and a multimerizing group to a peptide having a reactive
primary amine group include NHS-PEO.sub.4-biotin,
NHS-PEO.sub.12-biotin and NHS-SS-biotin (Pierce Chemical, Rockford,
Ill., USA). Nonlimiting examples of commercially available reagents
that can be used to link a spacer group and a multimerizing group
to a peptide having a reactive carboxyl group include
Amine-PEO.sub.2-biotin and biotin-PEO-LC-Amine (Pierce Chemical,
Rockford, Ill., USA). Other examples of suitable reagents for
linking a spacer-biotin reagent to a Tie 2 binding peptide via
either a reactive sulfhydryl group, primary amine group or carboxyl
group are set forth in subsection I above.
[0138] In a preferred embodiment, the invention provides a method
of making a Tie 2 binding peptide monomer comprising a structure:
A-B-C, wherein:
[0139] A comprises a Tie 2 binding peptide;
[0140] B comprises a polyethylene glycol spacer; and
[0141] C comprises a biotin group,
the method comprising reacting a Tie 2 binding peptide comprising
an amino terminal amino acid residue comprising a first reactive
group with a reagent comprising the structure: second reactive
group-B-C, wherein the first reactive group reacts with the second
reactive group to form A-B-C. For example, the first reactive group
can be a sulfhydryl group (e.g., the amino terminal amino acid
residue of the peptide can be cysteine residue) and the second
reactive group can be a maleimide group. Other suitable examples of
first and second reactive groups are described above regarding
suitable commercially available spacer reagents.
[0142] In another embodiment, the invention provides a method of
making a Tie 2 binding peptide monomer comprising a structure: A-B,
wherein:
[0143] A comprises a Tie 2 binding peptide; and
[0144] B comprises a polyethylene glycol spacer;
the method comprising reacting a Tie 2 binding peptide comprising
an amino terminal amino acid residue comprising a first reactive
group with a reagent comprising the structure: second reactive
group-B to form A-B. For example, the first reactive group can be a
sulfhydryl group (e.g., the amino terminal amino acid residue of
the peptide can be cysteine residue) and the second reactive group
can be a maleimide group. Other suitable examples of first and
second reactive groups are described above regarding suitable
commercially available spacer reagents.
[0145] To create the multimeric forms of the Tie 2 binding peptide
monomers of the invention, multiple copies of the monomers are
linked together to thereby multimerize the monomers.
Multimerization can be accomplished by one of several possible
methods. For example, for monomers that comprise a multimerizing
group (e.g., monomers of the structure A-B-C), the monomers can be
combined with a multimer agent that has affinity for the
multimerizing group and that has multiple binding sites for the
multimerizing group. Depending on the number of binding sites that
the multimer agent has, the ratio of monomer to multimer agent can
be adjusted accordingly. For example, when the multimerizing group
is biotin and the multimer agent is avidin, streptavidin or
neutravidin (each of which have four binding sites for biotin), the
ratio of monomer to multimer agent can be selected as 4:1.
Accordingly, the invention provides a preferred method of making a
tetramer form of a Tie 2 binding peptide monomer of the structure
A-B-C, wherein A is a Tie 2 binding peptide, B is a spacer and C is
biotin, the method comprising combining the Tie 2 binding peptide
monomer, A-B-C, with a tetramer agent, D, at a 4:1 ratio, the
tetramer agent, D, being selected from the group consisting of
avidin, streptavidin and neutravidin.
[0146] In an alternative embodiment, differing ratios of monomer
(e.g., A-B-C) and multimer agents (e.g., D) can be combined to
create multimers having different numbers of monomer units. Thus,
when the multimer agent is a tetramer, a 4:1 ratio of monomer to
multimer agent can be used to achieve tetrameric multimers, but
lower ratios also can be used to achieve trimers or dimers. More
specifically, a 3:1 ratio of monomers to multimer agent can be used
to achieve trimers and a 2:1 ratio of monomer to multimer agent can
be used to achieve dimers. Preferably, after the monomers and
multimer agent are combined, one or more purification steps are
carried out to purify the desired multimeric form and/or to remove
unbound monomers. Nonlimiting examples of purification techniques
that can be used to isolate the desired multimeric species include
HPLC, size exclusion chromatography and avidin agarose
chromatography (to remove unbound monomers linked to a biotin
multimerizing group).
[0147] For monomers having a structure A-B, wherein A is a Tie 2
binding peptide and B is a spacer, multimerization can be
accomplished by covalent linkage or noncovalent association of
multiple copies of the monomer through the spacer B. For example,
branched activated PEG spacers (commercially available from NOF
Corporation, Tokyo, Japan) can be reacted with multiple copies of
the monomer to create the multimeric structure. Numerous suitable
reactive groups are available with which the branched arm spacer
can be activated, including but not limited to maleimide, amine,
glutaryl-NHS, carboxymethyl-NHS, carbonate and aldehyde. Following
reaction of the monomer with the branched arm spacer, preferably
one or more purification steps are carried out to purify the
desired multimeric form and/or to remove unbound monomers, as
described above.
IV. Methods of Detecting a Composition Comprising a Tie 2 Binding
Peptide
[0148] In another aspect, the invention provide a method of
detecting the compositions of the invention comprising a multimeric
form of a Tie 2 binding peptide. In the detection method, a
composition of the invention is contacted with a monoclonal
antibody that specifically binds to the Tie 2 binding peptide
contained within the multimeric form such that the Tie 2 binding
peptide is detected. Specific formats of detection assay that are
known in the art that are applicable to the detection of the Tie 2
binding peptide include, for example, enzyme linked immunosorbent
assay (ELISA) and radioimmunoassay (RIA). Preferred monoclonal
antibodies for use in the assays are those that specifically bind
to the T7 peptide contained within multimeric forms of the T7
peptide, such as those described in Example 12. Monoclonal
antibodies that bind to the Tie 2 binding peptide (e.g., T7) can be
prepared by standard techniques. Assessment of the binding of the
monoclonal antibody to the Tie 2 binding peptide also can be
performed by standard techniques, such as ELISA, RIA, dot blot
analysis, immunoblot analysis, immunoprecipitation and the like, as
described further in Example 12.
[0149] The invention also provides monoclonal antibody compositions
that specifically bind to the T7 peptide. Non-limiting examples of
such monoclonal antibodies include the antibodies secreted by the
hybridoma clones 2C11, 13D4, 3E4 and 4H6 described further in
Example 12.
V. Pharmaceutical Compositions and Administration Thereof
[0150] Another aspect of the invention pertains to pharmaceutical
compositions comprising the multimeric forms of the Tie 2 binding
peptide monomers of the invention. The pharmaceutical compositions
typically include the multimeric form of the Tie 2 binding peptide
monomer and a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers can be selected to be suitable for the desired
route of administration. For example, in one embodiment, the
pharmaceutically acceptable carrier is suitable for topical
administration. A non-limiting example of a suitable carrier for
topical administration is IntraSite Gel (commercially available
from Smith & Nephew). In another embodiment, the
pharmaceutically acceptable carrier is suitable for systemic
administration. A non-limiting example of a suitable carrier for
systemic (e.g., intravenous) administration is phosphate buffered
saline (PBS).
[0151] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for topical administration or
for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion).
Depending on the route of administration, the active compound,
i.e., multimeric form of the Tie 2 binding monomer, may be coated
in a material to protect the compound from the action of acids and
other natural conditions that may inactivate the compound.
[0152] The pharmaceutical compositions of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M. et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0153] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0154] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0155] These compositions may also contain, for example,
preservatives, wetting agents, emulsifying agents and/or dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monostearate and gelatin.
[0156] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. Sterile
injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by sterilization microfiltration. The composition can be
formulated as a solution, microemulsion, liposome, or other ordered
structure suitable to high drug concentration. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
[0157] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0158] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0159] For systemic administration of the multimeric form of the
Tie 2 binding peptide monomer, the dosage typically ranges from
about 0.0001 to 100 mg/kg, and more usually 0.001 to 5 mg/kg, of
the host body weight. For example dosages can be 1 .mu.g/kg, 5
.mu.g/kg, 50 .mu.g/kg, 0.1 mg/kg, 0.3 mg/kg body weight, 1 mg/kg
body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg
body weight or within the range of 0.01-1 mg/kg. For topical
administration, exemplary dosage ranges are from about 5 .mu.g/ml
to about 5 mg/kg, more preferably 50 .mu.g/kg to 2 mg/kg, such as 1
mg/kg.
[0160] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts. A "therapeutically effective dosage" of the
multimeric form of the Tie 2 binding peptide monomer of the
invention preferably results in increased angiogenesis, stimulation
of wound healing or both. One of ordinary skill in the art would be
able to determine such amounts based on such factors as the
subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0161] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for the multimeric forms of the invention include
non-parenteral routes, including topical, epidermal or mucosal
routes of administration, for example, intranasally, orally,
vaginally, rectally, sublingually or topically. Alternatively,
other preferred routes of administration include parental routes,
including intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, transtracheal, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0162] In certain embodiments, the multimeric forms of the Tie 2
binding peptide monomers of the invention can be administered in
combination with other therapeutic agents, such as other agents
that promote angiogenesis and/or stimulate wound healing.
Non-limiting examples of other agents with which the multimeric
forms of the invention can be combined include VEGF, PDGF, G-CSF,
recombinant human erythropoietin, bFGF and placental growth factor
(PLGF).
VI. Methods of Using Multimeric Forms of Tie 2 Binding Peptide
Monomers
[0163] Another aspect of the invention pertains to methods of using
the multimeric forms of the Tie 2 binding peptide monomers of the
invention. As discussed herein, the multimeric forms have Tie 2
agonist activity. Accordingly, the multimeric forms can be used to
activate the Tie 2 receptor, either in vitro or in vivo. Thus, in
one embodiment, the invention provides a method of activating a Tie
2 receptor comprising contacting the Tie 2 receptor with the
multimeric form of the Tie 2 binding peptide monomer such that the
Tie 2 receptor is activated. Activation of the Tie 2 receptor can
be evidenced by any of numerous possible indicators of Tie 2
activation well established in the art, including but not limited
to the various in vitro and in vivo assays described in detail in
the Examples. In one embodiment, for example, wherein activation of
the Tie 2 receptor is evidenced by phosphorylation of residue
tyrosine 992 (Y992) of the Tie 2 receptor. In another embodiment,
for example, activation of the Tie 2 receptor is evidenced by
phosphorylation of MAPK, AKT or eNOS.
[0164] Since the multimeric forms of the invention have angiogenic
activity, the invention also provides a method of stimulating
angiogenesis at a site in a subject, wherein the method comprises
contacting the site with the multimeric form of the Tie 2 binding
peptide monomer such that angiogenesis is stimulated at the site in
the subject. In one embodiment, the multimeric form is contacted
with the site by topical administration of the multimeric form. In
another embodiment, the multimeric form is contacted with the site
by systemic administration of the multimeric form. Angiogenesis can
be confirmed by the presence of one or more well established
characteristics of angiogenesis. In a preferred embodiment,
angiogenesis stimulated by the multimeric form is characterized by
at least one of the following properties:
[0165] a) recruitment of perivascular support cells;
[0166] b) non-leakiness of vessels; and
[0167] c) well-defined arborization.
Recruitment of perivascular support cells can be demonstrated by
detection of a marker of smooth muscle cells, for example by
immunostaining with an antibody against smooth muscle actin 1 (Sma
1). Non-leakiness of vessels can be assessed using vessel
permeability assays established in the art, including in vitro
and/or in vivo assays. A non-limiting example of an in vivo vessel
permeability assay is the Miles assay using either Evan's Blue or
FITC albumin. As used herein, vessels are to be considered
"non-leaky" if the degree of permeability of the vessels is less
than the degree of permeability of vessels whose growth was
stimulated by VEGF treatment. Well-defined arborization can be
demonstrated, for example, by imaging of newly formed vessels and
quantification of number of vessels and number of nodes in a
particular image field (see Example 5 for a more detailed
description). Well-defined arborization is indicated by, for
example, significant and organized branching of the vessels, such
as angiogenesis in which the ratio of the number of vessels to the
number of nodes is 1.0:0.5, more preferably 1.0:0.7 or even more
preferably 1.0:1.0. Furthermore, the flow dynamics of neovessels
can be assessed using micro Doppler ultrasound.
[0168] In the method for stimulating angiogenesis, the site can be
contacted with the multimer form alone or, alternatively, the site
can be contacted with one or more additional angiogenic agents.
Thus, in another embodiment, the angiogenesis method further
comprises contacting the site in the subject with a second
angiogenic agent. Non-limiting examples of additional angiogenic
agents that can be used in combination with the multimeric forms of
the invention include VEGF, PDGF, G-CSF, recombinant human
erythropoietin, bFGF and placental growth factor (PLGF). As
demonstrated herein, a multimeric form of the invention, when used
in combination with VEGF, has been shown to reduce the vessel
tortuosity and vascular permeability seen when VEGF is used alone
to stimulate angiogenesis. Thus, in a preferred angiogenesis method
of the invention, a multimeric form of the invention is used in
combination with VEGF.
[0169] Given the ability of the multimeric forms of the invention
to stimulate angiogenesis, the multimeric forms can be used in a
variety of clinical situations in which promotion of angiogenesis
is desirable. Non-limiting examples of such indications include
vascularization of regenerative tissues, ischemic limb disease,
cerebral ischemia, conditions of vascular inflammation including
arteriosclerosis, avascular necrosis, stimulation of hair growth
and erectile dysfunction.
[0170] Moreover, given the ability of the multimeric forms to
reduce vascular permeability induced by other agents such as VEGF,
the multimeric forms can be used clinically to counteract vascular
permeability induced by, for example, VEGF, radiation, a pathogen
or other clinical event (e.g., stroke). The ability of Ang 1 to
protect vasculature against leakage and/or counteract endothelial
permeability induced by other agents has been reported (see e.g.,
Thurston, G et al. (1999) Science 286:2511-2514; Thurston, G. et
al. (2000) Nat. Med. 6:460-463; Pizurki, L. et al. (2003) Br. J.
Pharmacol. 139:329-336; Jho, D. et al. (2005) Circ. Res.
96:1282-1290). Moreover, Ang 1 has been reported to reduce cerebral
blood vessel leakage and promote stabilization of angiogenic
vessels in experimental stroke models (Zhang, Z. G. et al. (2002)
Neurosci. 113:683-687; Zacharek, A. et al. (2006) Neurosci. Lett.
404:28-32), to inhibit breakdown of the blood-retinal barrier
(Nambu, H. et al. (2004) Gene Therap. 11:865-873) and to inhibit
retinal detachment in a model of proliferative retinopathy (Nambu,
H. et al. (2005) J. Cell. Physiol. 204:227-235). In view of the
demonstrated effects of the multimeric forms of the invention in
decreasing vascular permeability, another aspect the invention
pertains to a method of decreasing vascular permeability at a site
of leaky vessels, the method comprising contacting the site of
leaky vessels with a multimeric form of a Tie 2 binding peptide
monomer of the invention such that vascular permeability is
decreased. Such a method can be used in a variety of clinical
situations, non-limiting examples of which include stroke, macular
degeneration, macular edema, lymph edema, breakdown of the
blood-retinal barrier, breakdown of the blood-brain barrier (e.g.,
during chemotherapeutic treatment) and normalization of tumor
vasculature to facilitate drug delivery and increase radiation
sensitivity.
[0171] The multimeric forms of the invention also have been shown
to have a protective effect on endothelial cells, e.g., by
inhibiting apoptosis of endothelial cells. The ability of a Tie 2
agonist to protect endothelial cells in renal vasculature has been
reported to ameliorate renal fibrosis in an experimental model
(Kim, W. et al. (2006) J. Am. Soc. Nephrol. 17:2474-2483). In view
of the demonstrated effects of the multimeric forms of the
invention in protecting endothelial cells, another aspect the
invention pertains to a method of protecting endothelial cells, the
method comprising contacting the endothelial cells with a
multimeric form of a Tie 2 binding peptide monomer of the invention
such that the endothelial cells are protected. Such a method can be
used in a variety of clinical situations, non-limiting examples of
which include kidney fibrosis, stroke, macular degeneration and
diabetic complications (e.g., in the kidney, eye, skin and/or
limbs).
[0172] The multimeric forms of the invention also have been shown
to be effective in stimulating wound healing. Accordingly, in still
another aspect, the invention provides a method of stimulating
healing of a wound in a subject, the method comprising contacting
the wound with a multimeric form of a Tie 2 binding peptide monomer
of the invention such that healing of the wound is stimulated in
the subject. In one embodiment, the multimeric form is contacted
with the wound by topical administration of the multimeric form. In
another embodiment, the multimeric form is contacted with the wound
by systemic administration of the multimeric form. Stimulation of
wound healing can be evidenced by, for example, accelerated wound
closure time as compared to wound healing in the absence of the
multimeric form, increased granulation tissue at the wound site as
compared to no treatment with the multimeric form and/or enhanced
neovascularization of the wound as compared to no treatment with
the multimeric form.
[0173] In a preferred embodiment, the method of stimulating healing
of wound is used in the treatment of a diabetic ulcer. Currently,
there are 16 million people with diabetes in the U.S., with 798,000
new cases reported annually and a prevalence of approximately 6% of
the population. Estimates report that 10-15% of diabetics will go
on to develop foot ulcers, of which 14-20% will require amputation.
Foot ulceration is the precursor to approximately 85% of lower
extremity amputations. As demonstrated herein, a multimeric form of
the invention can stimulate wound healing in an animal model of
diabetic ulcers when the multimeric form is topically applied to
the wound (see Example 6).
[0174] In other embodiments, the method of the invention for
stimulating healing of a wound can be used in a variety of clinical
situations involving wounds, including but not limited to decubitus
ulcers, pressure ulcers, surgical incisions, traumatic tissue
injuries, burns and skin grafts.
[0175] The multimeric forms of the invention also can be
incorporated into a biomaterial that then can be implanted at a
site in a subject to thereby provide the effects of the multimeric
form at that site. Biomaterials that provide a matrix or scaffold
are suitable for use. The multimeric form can be incorporated alone
or in combination with one or more additional agents, such as VEGF,
PDGF, G-CSF, recombinant human erythropoietin, bFGF and placental
growth factor (PLGF). Non-limiting examples of suitable
biomaterials include Matrigel, skin substitutes and cross-linked
glycosaminoglycan hydrogels (e.g., as described in Riley, C. M. et
al. (2006) J. Biomaterials 27:5935-5943). Accordingly, another
aspect of the invention pertains to a biomaterial composition into
which is incorporated a multimeric form of the invention, alone or
in combination with one or more additional agents. A packaged
material that comprises the biomaterial is also encompassed by the
invention. The packaged material can be labeled for use of the
biomaterial.
[0176] Various aspects and embodiments of the present invention are
further described by way of the following Examples. The Examples
are offered by way of illustration and not by way of limitation.
The contents of all figures and all references, patents and
published patent applications cited throughout this application are
expressly incorporated herein by reference.
EXAMPLES
Example 1
Preparation of a Tetrameric Tie 2 Binding Peptide, Vasculotide
[0177] In this example, a tetrameric form of a 7mer peptide known
to bind Tie 2 was prepared, using a biotin-avidin system to cluster
the peptide as a tetramer. The peptide, referred to as T7, was
previously described in Tournaire R. et al. (2004) EMBO Reports
5:262-267 and has an amino acid sequence of
His-His-His-Arg-His-Ser-Phe (SEQ ID NO: 1).
[0178] The peptide was synthesized using Fmoc Solid Phase Peptide
Synthesis, a summary of which synthesis method can be found in W.
Chan, Fmoc Solid Phase Peptide Synthesis: A Practical Approach,
Oxford University Press (UK), 1999. The peptide was synthesized
using Applied Biosystems' ABI433A Peptide Synthesizer (Foster City,
Calif., USA) using manufacturer's instructions with a few
modifications. The modifications were: 1) coupling time was
extended to 17.5 minutes and 2) HBTU was substituted by HATU. The
resin used was Wang resin. To create a free sulfhydryl group on the
peptide for further modification, an amino terminal cysteine
residue was added to the peptide such that the final sequence of
the peptide was Cys-His-His-His-Arg-His-Ser-Phe (SEQ ID NO: 2). The
protection groups for the side chain residue were: Cys-(tBu),
His-(Trt), Arg-(Pbf), Ser-(tBu) Amino acid derivatives used here
were the L-form to mimic the natural folding conformation in
mammalian cells. The cleavage cocktail used to cleave T7 peptide
off the solid phase resin support contained: 90% Trifluoro acetic
acid (TFA), 8% TIPS, 2% EDT. The cleavage was performed under
nitrogen gas for 3 hours. The cleavage cocktail and the resin
mixture were separated by means of Kimax Fritted funnel T7 peptide
was then precipitated with cold ether and centrifuged at
2700.times.g for 5 min at 4.degree. C. The pellet was washed for a
total of four times with cold ether. Each wash was carried out by
30 seconds vortexing, and 5 min centrifugation at 2700.times.g at
4.degree. C. Finally, T7 peptide pellet was dried under nitrogen
gas.
[0179] To create the tetramer, the T7 peptide first was modified by
addition of a 29 angstrom biotinylated polyethylene glycol
(PEG)-maleimide moiety, which is illustrated schematically in FIG.
1A. Biotin was conjugated to the T7 peptide by using
EZ-link-PEO-maleimide-biotin (Pierce's catalog number 21901)
according to the manufacturer's instruction. 20 mg of T7 peptide
was dissolved in 5 mL of conjugation buffer (0.001 M EDTA, 0.2M
phosphate buffer, pH 7.4) in a 15 mL tube. 10 mg
EZ-link-PEO-maleimide-biotin was dissolved in 1 mL conjugation
buffer, then was added into the T7 peptide solution and mixed by 5
seconds vortexing. The mixture was wrapped in foil and incubated at
between 20-25.degree. C. for four hours, then was stored in
-20.degree. C. until HPLC purification.
[0180] The T7-biotin product in the above reaction mixture was
purified using reverse phase HPLC under the following
conditions:
Column: Agilent Zorbax Extended-C18 (cat#770450-902), 4.6 mm (inner
idameter).times.250 mm (length), 5 micron particle, 80A pore
size
Mobile Phase:
[0181] A: 20 mM Ammonium hydroxide (0.701 g/L) in distilled water.
B: 20 mM Ammonium hydroxide (0.701 g/L) in 90% methanol 10%
distilled water.
Detection: UV 215 and 280 nm
[0182] Column was equilibrated in 90% mobile phase A for 15 minutes
at flow rate 1 ml per minute. 200 microliter of the above
peptide-biotin conjugation reaction mixture was injected at time
zero according to the following flow scheme:
TABLE-US-00001 percentage of B Time flow rate (all steps were
isocratic) 0 minute 0.2 mL/min.sup. .sup. 10% 5 minute 0.5
mL/min.sup. .sup. 10% 10 minute 1 mL/min 33.1% 30 minute 1 mL/min
33.2% 45 minute 1 mL/min 33.3% 60 minute 1 mL/min 33.4% 70 minute 1
mL/min 0%
[0183] The eluent were collected in a tube for every one minute.
Isocratic elution of T7-biotin achieved at 33.3% of mobile phase B
at retention time of 49 minutes. This fraction was verified by mass
spectrometry (MS) to contain a parent ion with m/z=1585.69 (singly
protonated monoisotopic peak). The theoretical m/z of the expected
product is 1585.6894.
[0184] Post synthesis clustering of this biotinylated peptide-PEG,
referred to hence forth as Vasculotide, with avidin in a 4:1 ratio
(Vasculotide:avidin) gave rise to an obligate tetrameric
compound.
Example 2
Characterization of the Tie 2 Binding of Vasculotide
[0185] In this example, the ability of Vasculotide to bind the Tie
2 receptor, despite the engineered modifications, was tested using
an in vitro pull down assay. In the pull down assay, a cell lysate
of EaHy926 endothelial cells (which express high levels of Tie 2)
was prepared in Phospho Lipase C.gamma. lysis buffer (50 mM Hepes
buffer pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM
MgCl.sub.2, 1.0 mM EGTA, 10 mM NaPPi, 100 nM NaF, 2 mM
Na.sub.3VO.sub.4, lx aprotinin, lx leupeptin and 1.times.PMSF). The
whole cell lysate was mixed with either unclustered biotinylated
Vasculotide or biotinylated irrelevant peptide. The biotinylated
peptides then were isolated with avidin agarose and subsequently
tested for their ability to precipitate Tie 2 via standard
immunoblot analysis using anti-Tie 2 antibody.
[0186] The results of the immunoblot are shown in FIG. 1B. The
results demonstrated that, when purified using avidin agarose
resin, Vasculotide but not irrelevant biotinylated peptide was able
to precipitate Tie 2. Addition of a ten fold excess of Vasculotide
preclustered with soluble avidin was able to compete for available
Tie 2 and was able to abolish this interaction (FIG. 1B, final
lane).
Example 3
Characterization of the Tie 2 Activation by Vasculotide
[0187] Having established that Vasculotide was able to bind Tie 2
(see Example 2), the ability of Vasculotide, when clustered with
avidin in a 4:1 ratio (Vasculotide:avidin), to activate the Tie 2
receptor was tested by examining several well established
downstream signalling pathways in human umbilical vein endothelial
cells (HUVEC) (Cambrex, N.J.). Such signalling pathways are
described in, for example, Kim, I. et al. (2000) Circ. Res.
9:952-959; Fujikawa, K. et al. (1999) Exp. Cell. Res. 2:663-672;
Babaei, S. et al. (2003) Am. J. Pathol. 6:1927-1936.
[0188] HUVEC were grown on 6-well or 10 cm plates (Nunc) coated
with gelatin (Sigma). HUVEC were cultured in F12 growth medium
containing 10% fetal bovine serum (FBS), 0.1 mg/ml heparin
sulphate, 1.times. penicillin, 1.times. streptomycin, 1.times.
glutamine, VEGF 10 ng/ml, EGF 10 ng/ml, bFGF 5 ng/ml. All HUVEC
were used between passage 3-9.
[0189] HUVEC were stimulated with one of either Ang 1 (R&D
Systems), Vasculotide, avidin or clustered Vasculotide
(preclustered with a 4:1 molar ratio of peptide:avidin in PBS for 2
hours at 4.degree. C.) at various doses. Stimulations with
Vasculotide were performed in full serum containing growth media
for 15 minutes unless otherwise indicated. Cell lysate preparations
and immunoblot analysis were performed by standard methods.
Antibodies used for immunoblotting (in this and/or subsequent
Examples) were monoclonal anti Tie 2 (Pharminogen), polyclonal anti
pY992 Tie 2, (Cell Signaling Technology), polyclonal anti MAPK
(Cell Signaling Technology), monoclonal anti phospho MAPK (Cell
Signaling Technology), polyclonal anti AKT (Cell Signaling
Technology), polyclonal anti pS473 AKT (Cell Signaling Technology),
monoclonal anti pS1177 eNOS (BD Biosciences), polyclonal anti pY397
FAK (Biosource), polyclonal anti FAK (Santa Cruz), polyclonal anti
smooth muscle actin-Cy3 direct conjugate (Dako), polyclonal anti
PECAM (Pharminogen) and polyclonal anti smooth muscle actin
(Sigma).
[0190] The results of the immunoblot analysis are shown in FIG. 1C.
The immunoblot analysis of whole cell lysates revealed that
clustered Vasculotide activates Tie 2 (pY992 Tie 2) at
concentrations ranging from 20 nM to 750 pM, with 5-10 nM appearing
optimal. Significantly, only when preclustered with avidin was
Vasculotide able to stimulate Tie 2 phosphorylation (see avidin
alone and unclustered Vasculotide). Coincident with receptor
activation, phosphorylation of several known Tie 2-responsive
proteins including MAPK, AKT and eNOS, was observed.
[0191] Paradoxically, high concentrations of clustered Vasculotide
are not capable of activating Tie 2 receptor activity in HUVEC (see
clustered Vasculotide 50 nM). It was hypothesized that this was due
to the fact that high ratios of clustered Vasculotide:Tie 2 would
decrease receptor clustering. To test this hypothesis, Eahy926
EC's, which express much higher levels of Tie 2, were utilized. For
immunoblot analysis, Tie 2 was immunoprecipitated from the Eahy926
cells with monoclonal anti Tie 2 (Pharmingen, clone 33.1) and
protein G sepharose (Amersham) and monoclonal anti pY (Upstate
Biotechnology, clone 4G10), according to standard techniques. The
results of this immunoblot are shown in FIG. 1D. Using this cell
line, specific activation of the Tie 2 receptor at concentrations
ranging from 200 .mu.M to 750 pM was demonstrated, suggesting that
the ratio of clustered Vasculotide:Tie 2 is critical for
activation.
[0192] To examine Tie 2 activation kinetics, HUVEC were stimulated
with native Ang 1 or clustered Vasculotide for various times.
Stimulation was performed in 10% FBS. The results are shown in FIG.
1E. Low concentration clustered Vasculotide (2 nM) compared
favorably to Ang 1 (400 ng/ml) stimulation in its ability to
activate Tie 2 phosphorylation. Overall, activation of the receptor
by either Ang 1 or clustered Vasculotide followed a very similar
time course with phosphorylation returning to almost basal levels
sometime after 2 hours. Again, marked increases in pMAPK and pAKT
were observed.
[0193] Previously, Ang 1 has been shown to activate focal adhesion
kinase (FAK) in a Tie 2 independent manner through direct
engagement of .alpha.V.beta.1 integrin (Hu, B. et al. (2006)
Cancer. Res. 2:775-783; Kim, I et al. (2000) Circ. Res. 9:952-959;
Dallabrida, S. et al. (2005) Circ. Res. 4:8-24). To test whether
clustered Vasculotide was capable of activating this arm of the Ang
1 signalling cascade, the activation of FAK was examined, the
results of which are also shown in FIG. 1E (bottom panel). As
previously reported, Ang 1 time dependently activated FAK, whereas
clustered Vasculotide had no significant effect. These results are
suggestive of a more direct role for Vasculotide in eliciting Tie
2-specific signalling.
[0194] To more formally address the specificity of clustered
Vasculotide for the Tie 2 receptor, reconstitution experiments were
performed in Cos 1 fibroblast cells and C166 EC's, both of which
are phenotypically null for Tie 2. Cos 1 and C166 cells (ATCC) were
maintained on 10-cm-diameter plates (Nunc) in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% (FBS), 1.times.
penicillin, 1.times. streptomycin, and 200 mM L-glutamine (all
Gibco BRL) in a 5% CO.sub.2 incubator at 37.degree. C. Cos1 or C166
cells were infected (MOI 30) overnight with adenoviruses encoding
one of either enhanced green fluorescent protein (EGFP) or Tie 2.
Infection efficiency was confirmed by epifluorescence (EGFP), or by
immunoblot (Tie 2).
[0195] Cells infected with recombinant adenovirus encoding EGFP or
Tie 2 were either left unstimulated or stimulated with Vasculotide,
clustered Vasculotide or avidin for 16 hours, after which MTS
reagent was applied to all samples for 4 hours. Absorbance at 492
was measured using a multiwell plate reader (Power Wave X340,
Biotek Instruments Inc). To broadly examine the potential that
clustered Vasculotide was activating Tie 2-independent downstream
signalling pathways, the activation of MAPK and AKT in the EGFP
infected cells was examined. The results are shown in FIG. 1F.
Neither Vasculotide nor clustered Vasculotide significantly induced
phosphorylation of MAPK or AKT in the EGFP infected cells above
that noted in the non-stimulated samples. As expected,
overexpression of Tie 2 in either of the cell types resulted in its
constitutive activation (see pY992 Tie 2) as well as activation of
MAPK and AKT. This fact precluded us from stimulating these cells
any further with Vasculotide or clustered Vasculotide. Because MAPK
and AKT are highly activated downstream of a host of cell surface
receptors we reason that these results are highly suggestive of a
Tie 2 specific and dependent role for clustered Vasculotide in
these events.
Example 4
Effects of Vasculotide on Endothelial Cell Apoptosis and
Migration
[0196] Ang 1, and to a lesser degree Ang 2, have been shown to
protect endothelial cells (ECs) from various different
apoptosis-inducing conditions including serum withdrawal (Kwak, H
et al. (1999) FEBS Lett. 2-3:249-253; Harfouche, R. and Hussain, R.
(2006) Am. J. Physiol. Heart Circ. Physiol. 291:H1635-1645). Based
on the fact that clustered Vasculotide strongly activates the anti
apoptotic protein AKT (see Example 3), the ability of Vasculotide
to protect ECs from serum withdrawal-induced death was examined To
do so, HUVEC were maintained in F12 media plus 0.1% FBS for 16
hours in the presence of various concentrations of unclustered or
clustered Vasculotide, followed by analysis of cell apoptosis via
cell death ELISA. Analysis of apoptosis was performed using Cell
Death ELISA Plus (Roche) according to manufacturers'
specifications. The results are shown in FIG. 2A, in which values
were normalized to basal levels of cell death for HUVECs maintained
in full 10% FBS, growth factor supplemented F12 media. The results
shown represent the mean of three replicates plus or minus 1 SD.
Student's T-test was used for statistical analysis with P value
indicated. The results revealed a statistically significant
decrease in apoptosis when the ECs were treated with clustered
Vasculotide at 500 pM (p=0.002) compared to non-treated cells.
Non-clustered Vasculotide and avidin alone had no effect on
survival, further illustrating that Vasculotide is only active when
preclustered with avidin.
[0197] EC migration is an event deemed critical to
revascularization upon injury. Migration must take place in a
coordinated fashion, with the ECs responding to chemotactic signals
from the stroma and in turn secreting proteases necessary to clear
a path for their directional migration. Previous studies underline
a role for Ang 1 in promoting EC migration and release of the
matrix degrading enzyme MMP 2 (Kim, I. et al. (2000) Circ. Res.
9:952-959; Witzenbichler, B. et al. (1998) J. Biol. Chem.
29:18514-18521). To test if Vasculotide, like Ang 1, was capable of
promoting these processes, the effect of Vasculotide on EC
migration was tested using a modified Boyden chamber assay. HUVEC
were seeded at a density of 8.4.times.10.sup.4 cells in 500 .mu.l
of F12 growth media plus 0.1% FBS in the upper chamber of an 8
.mu.m pore size modified Boyden chamber (Falcon). 500 .mu.l of F12
media plus 0.1% FBS plus various growth factors (Ang 1 (R& D
Systems), unclustered or clustered Vasculotide, VEGF (R&D
Systems) or clustered Vasculotide in combination with VEGF) or
controls were placed in the bottom chamber. Cells were allowed to
migrate for 4 hours in a 37.degree. C., 5% CO.sub.2 incubator.
Nonmigrating cells were scraped off, and filters were fixed in 100%
methanol for 5 minutes, stained with Harris' Hematoxylin (BDH) for
10 minutes and washed twice with tap water for 3 minutes each.
Filters were then mounted using Aquapolymount mounting medium.
Cells that had migrated more than 50% of the way through the pore
were scored as positive for migration.
[0198] The results are shown in FIG. 2B. Student's t test was used
to test the statistical significance with a 95% confidence
interval. Stimulations were performed in triplicate and migrating
cells in 6 microscopic fields per replicate were counted. Error
bars shown in FIG. 2B represent mean+/-SD. The results showed that
clustered Vasculotide, at both doses examined (2 nM and 10 nM),
promoted significant increases in chemotactic cell migration when
compared to no treatment. Noted migration was not significantly
different than that seen in samples treated with VEGF or Ang 1. Non
clustered Vasculotide did not promote migration above that seen in
the untreated sample and the combination of VEGF and clustered
Vasculotide did not offer any additional effect beyond that
observed for either VEGF or clustered Vasculotide alone.
[0199] In vivo, ECs can not migrate until they secrete enzymes
necessary to break down components of the extra cellular matrix.
Gelatin zymography was employed to determine if clustered
Vasculotide promoted secretion of MMP2. To perform the zymography,
HUVEC were cultured in F12 media plus 0.1% FBS and stimulated for
16 h with VEGF, unclustered Vasculotide or clustered Vasculotide at
various concentrations. Conditioned media was centrifuged to remove
cellular debris and prepared for gel electrophoresis using
non-reducing conditions. Gels were washed twice for 30 minutes in
2.5% Triton X-100 to remove sodium dodecyl sulfate and were then
incubated in substrate buffer (50 mmol/L Tris-HCl, pH 8.8, 5 mmol/L
CaCl.sub.2) for 16 hours at 37.degree. C. Gels were then stained
with 0.5% Coomassie blue in 30% methanol/10% acetic acid for 2
hours at room temperature and destained in 50% methanol/10% acetic
acid. The presence of metalloproteinases was indicated by unstained
proteolytic zones in the gel. The results are shown in FIG. 2C.
Analysis of the conditioned media revealed that clustered
Vasculotide dose-dependently degraded gelatin at a molecular weight
that was consistent with pro and active forms of MMP2. Taken
together with the results of the migration analysis, this data
indicates that application of clustered Vasculotide can offer
distinct advantages in vivo to migrating endothelial cells.
Example 5
Promotion of Angiogenesis by Vasculotide in a Matrigel Assay
[0200] To further examine the in vivo angiogenic potential of
Vasculotide, a Matrigel assay was performed. More specifically,
growth factor reduced Matrigel (BD Biosciences) was impregnated
with either clustered Vasculotide or one of several different
control factors. These Matrigel samples were injected
subcutaneously into the flank region of 3 month old CD1 mice. 14
days post implantation and immediately prior to sacrifice, FITC
lectin (100 .mu.g) was injected IV and allowed to circulate for 10
minutes, to facilitate vessel identification and quantification.
Plugs were surgically resected and fixed in 4% paraformaldehyde
(PFA) for 16 h. Images of vascular topology were taken using a
Zeiss dissecting microscope at 4.times. magnification. Upon fixing,
all plugs were whole mount stained with anti-Sma1-Cy3. Plugs were
analyzed on a Zeiss Axiovert 100 M confocal microscope (Carl Zeiss)
for three dimensional image reconstruction and photos were
processed using LSM Image Browser and Adobe Photoshop 7.0.
Morphometric quantification of vessel characteristics was performed
by skeletonizing confocal image projections with Image Processing
Tool Kit 5.0 (IPTK 5.0). Student's t test was used to test the
statistical significance with a 95% confidence interval. All
experiments were performed with three replicates each, with at
least 5 random image stacks.
[0201] Upon surgical removal of the plugs, a distinct and robust
angiogenic response was seen in the membranous capsule that
surrounded the plugs impregnated with clustered Vasculotide, VEGF
or clustered Vasculotide plus VEGF. In the case of the clustered
Vasculotide samples, clear, large, well arborized vessels were
present on the surface. These vessels were well branched and
contained a continuum of small and large arterioles and venules.
Consistent with literature accounts (Connolly, D. et al. (1989) J.
Clin. Invest. 5:1470-1478), vessel growth promoted by the addition
of VEGF alone was apparent, although the nature of these vessels
was highly tortuous. Vessels located in the proximal tissue
surrounding these plugs also looked enlarged and inflamed upon
examination. Application of clustered Vasculotide in combination
with VEGF did not seem to increase the overall microvascular
density but rather contributed to decreases in the level of the
tortuosity seen with VEGF alone.
[0202] To more fully address and assign quantitative vessel
parameters to the different treatment groups, the plugs were fixed
and interrogated with laser confocal microscopy analysis combined
with post processing image analysis (Image Processing Tool Kit 5.0,
Reindeer Graphics, NC). Vessel parameters, including length, number
and branch points (nodes) were quantified on at least six
representative fields. Statistical analysis was performed by way of
unpaired Student's t test. Bar graphs of the number of vessels,
vessel length and number of nodes, for each treatment group, are
shown in FIGS. 3A, 3B and 3C, respectively. It is noted that poor
perfusion of FITC-lecithin into mice harboring 20 nM clustered
Vasculotide plus VEGF plugs prevented statistical analysis,
therefore bars are the mean of two separate fields. All other bars
shown represent mean+/-SD. The results show that no significant
vessel growth was noted in plugs containing Matrigel or Matrigel
impregnated with non clustered Vasculotide. Plugs containing VEGF,
clustered Vasculotide or clustered Vasculotide plus VEGF displayed
robust induction of angiogenesis. Statistically, VEGF, Vasculotide
or combinations thereof did not differ in total vessel number,
length or branch points. As was noted in the membranous capsule,
samples that contained Vasculotide displayed more organized
branching patterns and less tortuosity than the plugs containing
VEGF alone (however these were parameters that could not
specifically be quantified by assignment of a numerical value).
[0203] Ang 1 has been reported to potentiate the recruitment of
myogenic support cells (Suri, C. et al. (1996) Cell 87:1171-1180)
and inhibit VEGF induced vascular permeability through mechanisms
that are not well understood (Thurston, G. et al. (2000) Nat. Med.
6:460-463). To determine if Vasculotide, like Ang 1, was capable of
initiating pericyte recruitment, whole mount immunofluorescence was
performed against smooth muscle actin 1 (Sma1). Matrigel plug
analysis of VEGF driven angiogenesis revealed a relative absence of
Sma1 positive staining cells. Samples containing clustered
Vasculotide alone or in combination with VEGF displayed a
significant increase in overall Sma 1 staining. Sma 1 staining was
tightly associated with the exterior of the vessels and was
enriched on larger vessels. Taken together these results
demonstrate potent in vivo angiogenic effects of clustered
Vasculotide and indicate that, when combined with VEGF, clustered
Vasculotide is capable of counteracting VEGF induced permeability
and aberrant vessel formation.
Example 6
Wound Healing by Topically Administered Vasculotide
[0204] Chronic wounds, such as neuropathic or neuroischemic foot
ulcers, are prevalent in diabetic patients. These wounds may arise
from the patient's inability to sense injury (neuropathic) or due
to microvascular defects (neuroischemic). Animal models of type II
diabetes exist and closely recapitulate wound healing defects seen
in human diabetics. Here, B6.Cg-m(+/+)Lepr(db)/J (db/db) mice, a
strain of diabetic mice that presents with impaired wound healing,
were used to test if clustered Vasculotide could improve wound
closure times.
[0205] Pathogen free, nine week old B6.Cg-m(+/+)Lepr(db)/J (db/db)
mice were purchased from Jackson Laboratories. Circular, full
excisional, 6 mm diameter wounds were made, according to animal
committee approval guidelines, equal distant apart on the dorsal
side of the mice. Various factors were suspended in sterile
Intrasite Topical Gel (Smith and Nephew) at noted concentrations.
Treatments were as follows: Vehicle n=8, bFGF (10 ng/ml) n=8,
clustered Vasculotide (20 nM) n=4, clustered Vasculotide (5 nM)
n=4. Preparations were applied at day 0 (time of wounding), day 2,
4 and 6. Standardized images were taken on days 0, 2, 4, 6 and 7
using a Canon EOS digital Rebel camera. Image J (NIH) was used to
manually outline wound perimeter from which total pixel counts
(wound area) were determined Mice were sacrificed on day 7 and full
wound beds including margin were excised and fixed in 4% PFA.
Wounds were cut in half at the widest point and each half was
prepared for either paraffin or frozen sections. Wax sections were
processed according to standard protocols for H&E and Masson's
Trichrome stain. Images were captured using a Leica DMLS compound
light microscope and Pixel Link camera at 5.times. and 20.times..
Student's t test was used to test the statistical significance.
[0206] The wound closure data is summarized in the bar graph of
FIG. 4A, in which the data is presented as pixel counts of digital
tracings of the wound margins over time. All bars shown represent
mean+/-S.D. Significant decreases in wound margin in the clustered
Vasculotide samples (20 nM, p=0.05, n=4) were noted as early as day
2. Coincident with improved wound closure was a change in the
appearance of the wounds treated with clustered Vasculotide. These
wounds presented with a lighter more mucoid looking scab. Margin
closure in the clustered Vasculotide treated wounds continued at an
accelerated pace for the duration of the assay. As was the case in
vitro, clustered Vasculotide applied at lower concentration seemed
to offer more benefit. Although this may seem unusual, similar
results were also noted with topical application of Regranex, a
topical preparation of modified PDGF (Mustoe, T. et al. (1994)
Arch. Surg. 129:213-219). Moreover, these results further highlight
the need to optimize the Vasculotide:Tie 2 ratio to facilitate
appropriate clustering of the receptor. Although Vasculotide
provided the fastest healing at early time points, by day 7 of the
treatment wound closure promoted by bFGF at 10 .mu.g/ml was
statistically indistinguishable from clustered Vasculotide at 20 nM
or 5 nM.
[0207] Paraffin embedded cross sections of all wounds stained with
hematoxylin and eosin (H & E) and Masson's trichrome were
examined by a pathologist for independent blind analysis. A
representative photomicrograph of the hematoxylin eosin (H&E)
and Masson's trichrome (MT) stained skin sections from wounds
treated with PBS, bFGF (10 .mu.g/ml) or clustered Vasculotide (5
nM) is shown in FIG. 4B. The H&E and MT staining showed
dramatic increases in granulation tissue production in all
Vasculotide treated wounds. Additionally, the MT staining revealed
marked collagen deposition in the Vasculatide treated samples.
[0208] For histological scoring, sections were fixed in 4%
paraformaldehyde, embedded in paraffin and sectioned and stained
with H & E and Masson's trichrome. Sections were evaluated by a
pathologist for re-epithelialization, granulation tissue formation,
and inflammation, with criteria used for scoring skin as
follows:
Re-Epithelialization:
[0209] 0--none 1--minimal (0-20% regrowth from wound margins)
2--mild (25% to 50% regrowth) 3--moderate (>50% regrowth, up to
100% but not >2 cells thick along length) 4--complete regrowth
(epithelium>1 cell thick along length, keratinized)
Granulation Tissue (GT) and Neovascularization:
[0210] 0--none 1--minimal (1-3 small, isolated islands of GT at
margins of defect) 2--mild (multifocal, patchy islands of GT
underlying tissue defect, <10 new blood vessels) 3--moderate
(locally extensive bands of GT underlying length of defect)
4--marked (dense bands of GT at margins and underlying length of
defect with collagen fibrils and numerous blood vessels)
Inflammation (within Defect): 0--none 1--minimal (a few scattered
neutrophils at margins of defect) 2--mild (multifocal aggregates of
up to 5 neutrophils underlying defect) 3--moderate (multifocal
aggregates of up to 10 neutrophils and occasional macrophages,
minimal to mild edema) 4--marked (locally extensive, dense
neutrophilic infiltrates with lesser numbers of macrophages,
mild-moderate edema)
[0211] The results of the histological scoring are illustrated in
FIG. 4C. Differences between vehicle and bFGF, although apparent in
wound closure times, existed only at the level of inflammation
(p=0.0486). Application of clustered Vaculotide resulted in delayed
re-epithelialization (20 nM, p=0.0004 and 5 nM, p=0.029) but
promoted profound increases in regranulation and neovascularization
(20 nM and 5 nM p=0.0006). Inflammation noted in the clustered
Vasculotide samples was only slightly increased at 20 nM (p=0.034)
and was not significantly different from vehicle at the 5 nM dose
(p=0.0979).
[0212] Immunohistochemical analysis of the wounds with antibodies
directed against PECAM1, ICAM 1, Pan EC, podoplannin, and smooth
muscle actin demonstrated clear differences between clustered
Vasculotide-treated and untreated samples. First, it is noted that
histopathological examination of the stained tissues from 5 nM and
20 nM clustered Vasculotide treatments revealed no statistically
significant differences in the results for the doses of Vasculotide
used. At both Vasculotide doses, increases in total endothelial
cells (PECAM staining), activation of endothelial cells (ICAM1
staining) and fibroblast activation (Sma 1 staining) were observed
in all Vasculotide treated wounds. Furthermore, wounds treated with
clustered Vasculotide displayed dramatic increases in the number of
PECAM1- and Pan EC-positive vessels within the granulation tissue.
Importantly many of the new vessels in the granulation tissue were
accompanied by SMA-positive support cells. Furthermore, these
vessels were also positive for ICAM 1, a marker of activated
endothelial cells, demonstrating an active wound healing process
was occurring. The increase in vessel density was surprisingly not
accompanied by an increase in lymphangiogenesis, as detailed by
similar numbers of podoplannin-positive vessels. Overall collagen
deposition, as assessed by Masson's Trichrome staining, was
dramatically increased at both of the clustered Vasculotide
concentrations compared to the vehicle and bFGF samples. Collagen
deposition, primarily mediated by activated fibroblasts, plays a
necessary role in contracting the wound margins, offers tensile
strength, provides a scaffold for neovascularisation and
facilitates cellular signalling to migrating cells via integrin
engagement. Based on pathological and immunohistochemical analysis
we conclude that the improved wound closure times noted in the
clustered Vasculotide samples are a likely consequence of strong
contraction effects brought about by increases in collagen
deposition and neovascularisation.
Example 7
Systemic Administration of Vasculotide
[0213] In this example, the effect of systemically administered
Vasculotide on circulating endothelial cells was examined Three
month old CD1 mice were injected intravenously with either PBS
(vehicle) or 50 ng of clustered Vasculotide. Twenty four hours post
injection, peripheral blood was collected on heparin via cardiac
puncture and nonhematopoietic circulating endothelial cells (CECs)
were sorted using a four channel FACS approach. CECs were defined
as CD13.sup.+/VEGFR-2.sup.+/CD45.sup.-, Viable (propidium iodide).
Cell numbers reported are number of viable cells/.mu.l of
peripheral blood. Total white blood cell count was also determined
using a hemocytometer.
[0214] The results demonstrated that systemic delivery of
Vasculotide was well tolerated and resulted in a decrease in the
number of circulating endothelial cells. This has also been
described for Ang-1 delivered by adenoviral infection, suggesting
that Vasculotide has similar properties in the stem cell niche as
Ang-1.
Example 8
Multimeric Tie 2 Agonist Comprising a GA3 Peptide
[0215] In this example, an alternative form of the Vasculotide
molecule was prepared in which a GA3 peptide was used instead of a
T7 peptide. The amino acid sequence of the GA3 peptide was as
follows:
Trp-Thr-Ile-Ile-Gln-Arg-Arg-Glu-Asp-Gly-Ser-Val-Asp-Phe-Gln-Arg-Thr-Trp-L-
ys-Glu-Tyr-Lys (SEQ ID NO: 3). To facilitate further modification
of the peptide, it was synthesized, as described in Example 1, with
an additional amino terminal cysteine residue such that the amino
acid sequence of the peptide used in the multimeric form was as
follows:
Cys-Trp-Thr-Ile-Ile-Gln-Arg-Arg-Glu-Asp-Gly-Ser-Val-Asp-Phe-Gln-Arg-Thr-T-
rp-Lys-Glu-Tyr-Lys (SEQ ID NO: 4). The cysteine-containing peptide
was conjugated to biotin using EZ-link-PEO-maleimide-biotin
(Pierce's catalog number 21901) as described in Example 1. Post
synthesis clustering of this biotinylated peptide-PEG with avidin
in a 4:1 ratio gave rise to an obligate tetrameric compound.
[0216] The tetrameric GA3-containing compound was tested in the
endothelial cell migration assay as described in detail in Example
4. The results are shown in Figure SA, which demonstrate that the
tetrameric GA3-containing compound was effective in promoting
endothelial cell migration.
[0217] The tetrameric GA3-containing compound also was tested in
the zymography assay as described in detail in Example 4. The
results are shown in FIG. 5B, which demonstrate that the tetrameric
GA3-containing compound was effective in promoting MMP2
release.
[0218] These results demonstrate the agonist activity of a second
multimeric Tie 2 binding peptide containing compound, wherein the
compound contains a GA3 peptide rather than a T7 peptide.
Example 9
Preparation of PEG-Linked Multimeric Tie 2 Agonists
[0219] In this example, multimeric Tie 2 agonists were prepared
using polyethylene glycol (PEG) linkers to covalently join the
peptide components of the agonists. Three different PEG-linked Tie
2 agonists were prepared: (i) an agonist in which two T7 peptides
were joined using a 10,000 Dalton MW PEG linker; (ii) an agonist in
which two T7 peptides were joined using a 20,000 Dalton MW PEG
linker; and (iii) an agonist in which four T7 peptides were joined
using a 20,000 Dalton MW tetrameric PEG linker. To prepare these
molecules, activated branched arm PEGs were purchased from NOF
America. PEGs used in this example were Sunbright DE-100MA (PEG
dimaleimide MW 10,000 Da, terminal activity 79%), Sunbright
DE-200MA (PEG dimaleimide MW 20,000 Da, terminal activity 85.1%)
and Sunbright PTE-200MA (PEG tetramaleimide MW 20,000 Da, terminal
activity 94%). T7 peptide was reacted with activated PEGs as
follows: In the case of the two dimaleimide PEGs exactly two molar
equivalents of T7 peptide and 1 molar equivalent of dimaleimide PEG
were dissolved in phosphate buffered saline (PBS), pH 7.2. In the
case of tetramaleimide PEG exactly four molar equivalents of T7
peptide and 1 molar equivalent of tetramaleimide PEG were dissolved
in PBS, pH 7.2. Reactions were allowed to proceed at 21.degree. C.
for 16 hours The structures of the resultant multimeric Tie 2
agonists are illustrated schematically in FIG. 6. These PEG-linked,
T7 peptide-containing multimeric Tie 2 agonists are referred to
herein as PEG-T7 Vasculotide.
Example 10
Characterization of the Tie 2 Activation by PEG-Vasculotide
[0220] In this example, the ability of the PEG-T7 Vasculotide
compounds, prepared as described in Example 9, to activate the Tie
2 receptor was examined in an in vitro Tie 2 activation assay.
PEG-T7 Vasculotide compounds were used in endothelial cell
stimulations as follows. Purified bovine vascular endothelial cells
(bVEC) were grown on 6-well plates (Nunc) in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% (FBS), 1.times.
penicillin, 1.times. streptomycin, and 200 mM L-glutamine (all
Gibco BRL) in a 5% CO.sub.2 incubator at 37.degree. C. The bVECs
were stimulated with either PBS, tetrameric PEG-T7 Vasculotide MW
20,000 Daltons (PTE200-T7; 5 .mu.g/ml) or dimeric PEG-T7
Vasculotide MW 10,000 Daltons (DE100-T7; 250 ng/ml or 500 ng/ml)
for 10 minutes. Cell lysates were prepared in RIPA lysis buffer (50
mM Tris pH 7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulphate, 1.times. aprotinin, 1.times.
leupeptin and 1.times.PMSF). Equal quantities of cellular protein
were electrophoretically resolved and transferred to PVDF membrane
for immunoblot analysis. Immunoblot analysis was performed
according to standard practices, using monoclonal anti-Tie 2
antibody (Pharminogen) and polyclonal anti-pY992 Tie 2 antibody
(Cell Signaling Technology), and employing horse radish peroxidise
detection methods.
[0221] The results for stimulation with 5 .mu.g/ml of PTE200-T7 are
shown in FIG. 7 and the results for stimulation with either 250
ng/ml or 500 ng/ml DE100-T7 are shown in FIG. 8. The results show
that increased levels of Tie2 activation, as detected by
phosphorylation at Y992, occurred in the samples treated with
PTE200-T7 or DE100-T7 but not in the samples treated with PBS,
demonstrating the both PEG-T7 Vasculotide compounds are capable of
activating the Tie 2 receptor in vitro.
Example 11
In Vivo Tie 2 Activation by PEG-Vasculotide
[0222] In this example, the ability of the PEG-T7 Vasculotide
compounds, prepared as described in Example 9, to activate the Tie
2 receptor in vivo was examined by administering the compounds to
mice, followed by examination of lung cell tissue for activation of
Tie 2 and downstream pathways, since lung tissue contains a very
high vascular density and thus serves as an ideal organ to examine
activation of Tie 2.
[0223] Twelve week old wild type CD1 mice were injected
intravenously in the tail vein with PTE200-T7 Vaculotide (5 .mu.g
or 75 .mu.g), DE200-T7 Vasculotide (10 .mu.g or 50 .mu.g) or
DE100-T7 Vasculotide (5 .mu.g or 100 .mu.g) suspended in sterile
PBS or PBS alone. After 20 minutes the mice were sacrificed by way
of cervical dislocation and the lungs were removed and frozen at
-80.degree. C. for analysis. Defrosted lung tissue was lysed in
RIPA lysis buffer (described further in Example 10). Protein
concentrations were determined for each sample and equal quantities
of protein were immunoprecipitated for Tie 2 (Pharminogen, anti-Tie
2 clone 33.1 and protein G sepharose, Amersham). Immunoprecipitates
were electrophoretically resolved and transferred to PVDF membrane
for immunoblot analysis. Total phosphorylation of Tie 2 was
determined using an anti pY antibody (Upstate Biotechnology, clone
4G10). Additionally equal quantities of whole cell lysate were
separated electrophoretically and transferred to PVDF membrane for
immunoblot analysis with anti pan Mapk, anti phospho Mapk, anti pan
Aid, anti pS473 Akt, as described previously in Example 3.
[0224] The results are shown in FIG. 9. Increases in total Tie 2
activation were noted in mice that received dimeric PEG-T7, MW
10,000 Da (DE100-T7) and tetrameric PEG-T7, MW 20,000 Da
(PTE200-T7). As well, pathways downstream of Tie 2 including Mapk
and Akt were activated upon treatment with dimeric PEG-T7, MW
10,000 Da and tetrameric PEG-T7, MW 20,000 Da, but not dimeric
PEG-T7, MW 20,000 (DE200-T7), suggesting that appropriate T7
peptide spacing is critical for optimal Tie 2/Mapk/Akt activation
(see pMapk and pS473Akt compared to PBS control). Furthermore,
consistent with data generated using the avidin-biotin tetrameric
Vasculotide, it was observed that lower doses of PEG-T7 Vasculotide
promoted a more roboust activation of Tie 2 and downstream
signaling events.
Example 12
Preparation and Characterization of Monoclonal Antibodies Specific
for the T7 Peptide
[0225] In this example, monoclonal antibodies specific for the T7
peptide were prepared and characterized. T7 peptide was covalently
conjugated to BSA or KLH using Pierce Chemical Compnay's "Imject
Maleimide Activated Immunogen Conjugation Kit with mcKLH and BSA"
according to kit instructions. Injection of the immunogen into 4
week old female BALB/C mice, collection of serum, preparation of
hybridomas and screening of antibodies were all performed according
to kit instructions and under the animal care guidelines of
Sunnybrook Research Institute (Toronto, Ontario, Canada).
[0226] Hybridoma supernatants initially were screened by standard
ELISA using T7-BSA as the antigen, the results of which are shown
below in Table 1:
TABLE-US-00002 TABLE 1 ELISA Screening Results for Hybridoma
Supernatants Hybridoma Single Cell ELISA for T7 Clone result
Background F11.4F2 0.258 0.037 F11.4H6 yes 0.641 0.039 F11.2C11 yes
0.307 0.043 F11.9G8 0.379 0.033 F11.15F3 0.374 0.033 F11.3E4 yes
0.249 0.047 F11.7G4 0.182 0.4 F11.7E7 0.339 0.035 F11.7B12 0.186
0.035 F11.7D6 0.168 0.036 F11.13G12 0.117 0.034 F11.12B7 0.278
0.037 F11.13D4 yes 0.955 0.04 F11.14A4 195 0.036 F11.9B11 0.105
0.034 F11.2G6 0.208 0.051
All clones that tested positive during initial screening are
presented in Table 1. All single cell clones were further tested to
assess specificity.
[0227] Dot blot analysis was performed to assess the specificity of
mouse monoclonal hybridomas engineered to detect the T7 peptide.
Tetrameric PEG-T7 (PTE200-T7) was spotted onto a PVDF membrane at
17 ng, 83 ng and 333 ng (these quantities represent the overall T7
content applied to the membrane and do not account for the weight
of the PEG). The membrane was allowed to dry at which point it was
block in 5% non-fat skim milk and probed with the 2C11, 13D4, 7E7,
3E4 and 4H6 hybridoma supernatants at 1:500 dilution. Detection was
performed according to standard practices using a goatn anti-mouse
horse radish peroxidase secondary antibody at 1:10,000 dilution.
The results are shown in FIG. 10. All clones except for 7E7 were
capable of detecting tetrameric PEG-T7 at the tested
concentrations. Tissue whole cell lysate (WCL, 70 .mu.g) was
included as a specificity control and the results indicate that the
secondary antibody used in the detection non-specifically interacts
with some component of the whole cell lysate.
[0228] To further assess the binding of the 2C11, 13D4, 3E4 and 4H6
anti-T7 mouse monoclonal antibodies, an immunoblot analysis was
performed with T7-BSA as the antigen. To prepare T7-BSA antigen, T7
peptide was covalently conjugated to bovine serum albumin using the
"Imject Maleimide Activated Immunogen Conjugation Kit with mcKLH
and BSA" supplied by Pierce according to manufacturers
instructions. The resultant T7-BSA, alone or spiked with 100 ng of
mouse lung whole cell lysate, was electrophoretically resolved (in
non-reducing conditions) and transferred to PVDF membrane for
immunoblot analysis. The PVDF membranes were probed with the 2C11,
13D4, 3E4 and 4H6 hybridoma supernatant according to standard
immunoblot practice. The results are shown in FIG. 11. All clones
tested were capable of detecting T7-BSA (total amount of T7 was
equal to 75 ng) to varying degrees. The immunoblots showed distinct
bands at approximately 70 kDa, 140 kDa, and 210 kDa which are
thought to represent non-reduced aggregates of BSA-T7. Tested
clones also detected a single protein in the lung whole cell lysate
of approximately 200 kDa.
Sequence CWU 1
1
1017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1His His His Arg His Ser Phe 1 5 28PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Cys
His His His Arg His Ser Phe 1 5 322PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Trp
Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg 1 5 10
15 Thr Trp Lys Glu Tyr Lys 20 423PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 4Cys Trp Thr Ile Ile Gln
Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 1 5 10 15 Arg Thr Trp Lys
Glu Tyr Lys 20 57PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5His Pro Trp Leu Thr Arg His 1 5
68PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Cys His Pro Trp Leu Thr Arg His 1 5
77PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Lys Leu Trp Val Ile Pro Lys 1 5 88PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Cys
Lys Leu Trp Val Ile Pro Lys 1 5 97PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 9Asn Leu Leu Met Ala Ala
Ser 1 5 108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Cys Asn Leu Leu Met Ala Ala Ser 1 5
* * * * *