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COMPOSITIONS AND METHODS FOR PROMOTING ATTACHMENT OF CELLS OF
ENDOTHELIAL CELL LINEAGE TO MEDICAL DEVICES
FIELD OF THE INVENTION
The present invention relates to compositions and methods for promoting the
attachment of cells of endothelial cell lineage to an intravascular device.
BACKGROUND OF THE INVENTION
Atherosclerosis causes stenosis and occlusion of arteries. Stenting and bypass
surgery are often used to treat severe disease in small caliber arteries
(defined as less
than 6 mm in diameter). Arterial bypass procedures are limited by the
availability of a
vascular conduit, such as internal mammary artery or saphenous vein.
Unfortunately,
synthetic conduits made from polytetrafluoroethylene (PTFE) or polyethylene
terephthalate
(PET) suffer from unacceptably high rates of thrombosis in small caliber
grafts due to their
lack of an adherent, quiescent endothelium. Hence, developing a non-
thrombogenic, small
caliber arterial replacement has emerged as one of the most important-goals of
cardiovascular intervention in the elderly population.
Intravascular devices are placed within body vasculature; typically, at a site
of
occlusion in a vessel or the heart, or to replace or support a vessel or
portion of the heart.
Intravascular devices are normally manufactured from biologically inert
materials intended
to reduce the complications of insertion of a foreign object into the
vasculature, such as
stainless steel, titanium, polymers, or a combination thereof. However
numerous problems
have been reported to be associated with these devices, including thrombosis,
neointima
formation, and restenosis. Attempts have been made to reduce or eliminate the
complications of intravascular devices. For example, to address the problem of
thrombosis,
an individual with an intravascular device may receive an anticoagulant and
antiplatelet
drugs, such as ticlopidin or aspirin.
One approach to overcome complications associated with intravascular devices
is a
strategy to promote rapid endothelialization of the surface of the device in
contact with
vasculature and/or blood. In that regard, U.S. Patent No. 7,037,332 describes
a medical
device having a matrix coating made by cross-linking to the matrix an antibody
having
binding specificity for an endothelial cell antigen, for promoting attachment
of endothelial
cells to the medical device. U.S. Patent No. 6,897,218 discloses metal
complexes of a
piperazine derivative, which are described as promoting re-endothelialization,
but which do
not appear to directly bind to a device, and appear to rely on large volumes
of a blood-
circulating composition to be effective. U.S. Patent No. 6,140,127 describes a
method of
coating a stent by applying a polymer layer, applying pyridine and tresyl
chloride, and
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applying a five amino acid peptide (glycine-arginine-glutamic acid-aspartic
acid-valine;
SEQ ID NO:50) for adhering cells to the stent. U.S. Patent No. 5,929,060
discloses
derivatives of the steroid DHEA, which are described as useful for re-
endothelialization.
U.S. Patent No. 5,643,712 discloses coating of vessels of an organ or tissue
to be grafted
with a partially polymerized extracellular matrix preparation derived from
endothelial cells,
which may serve as a surface promoting re-endothelialization. Device design
may be
modified to promote the occurrence of re-endothelialization. U.S. Patent No.
6,436,132
discloses an intraluminal prosthesis for treating a stenotic region in a blood
vessel. The
openings in the stent are said to allow for re-endothelialization of the blood
vessel.
Cells of the endothelial cell lineage include endothelial cells and
endothelial
progenitor cells. Endothelial cells line all parts of the vasculature, where
they regulate
coagulation, inflammation, vascular permeability, and nutrient exchange
between the blood
and the interstitium. In areas where the endothelium is focally denuded,
coagulation
rapidly ensues. Focal coagulation of a blood vessel can lead to thrombosis and
vascular
occlusion, or other thromboembolic events. Endothelial progenitor cells have
been shown
to contribute to angiogenesis and vasculogenesis in a variety of model
systems, and also
to contribute to endothelialization of endovascular grafts in animal models.
However,.
spontaneous endothelialization of endovascular grafts is rare in human
patients, perhaps
because the graft materials are engineered to resist molecular adhesion and
coagulation,
and endothelial progenitor cells have no ability to adhere, survive, and
proliferate on such
materials. Thus, there still remains a need for methods to promote
endothelialization of
intravascular devices such as by treating the devices so as to promote
colonization and/or
growth of nascent endothelium on the treated devices.
At least two types of endothelial progenitor cells can be isolated from
peripheral
blood: "early" endothelial progenitor cells, which live for 2 to 4 weeks in
vitro and secrete
potent angiogenic factors; and "9ate" endothelial progenitor cells, which grow
out at 3
weeks and can replicate for up to 100 population doublings. Early endothelial
progenitor
cells are derived from bone marrow angioblasts under the influence of vascular
endothelial
growth factor (VEGF). Early endothelial progenitor cells have the phenotype
CD133+1-,
CD34+, VEGFR-2+, CD31+, vWF-, VE-cadherin -, E-selectin-, eNOS-, and
telomerase +.
Late *endothelial progenitor cells have the phenotype CD133+/-, CD34+, VEGFR-
2+,
CD31+, vWF+, VE-cadherin +, E-selectin+, eNOS+, and telomerase +.
Differentiated
endothelial progenitor cells are similar to late endothelial progenitor cells,
except that the
former are CD133(-) and telomerase(-). Other endothelial progenitor cell
subpopulations,
and their phenotypic markers, are being described in the art.
Desired Is an approach that can do one or more of attach, recruit, support,
and
differentiate a nascent layer of cells of endothelial cell lineage on an
intravascular device
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surface. For example, it is desired to have an intravascular device with a
coating capable
of capturing circulating cells of an endothelial cell lineage so that they are
seeded on the
surface of an intravascular device, with the intended benefit of reducing the
occurrence of
complications associated with that type of intravascular device, such as one
or more of
thrombosis, neointima formation, and restenosis.
SUMMARY OF THE INVENTION
The present invention provides biofunctional coating compositions comprising
at
least one binding domain that specifically binds to a metallic surface of a
medical device
(for ease of reference, this binding domain is referred to herein as: "surface-
binding
domain") which is coupled to at least one binding domain that specifically
binds to cells of
endothelial cell lineage (for ease of reference, this binding domain is
referred to herein as:
"endothelial-binding domain"); wherein the surface-binding domain and the
endothelial-
binding domain consist essentially of the amino acid sequences illustrated
herein in Tables
1 and-3, respectively. The surface-binding domain and the endothelial cell-
binding domain
may be coupled together directly (e.g., during synthesis, or by chemical
means) or may be
coupled via a linker, to form a single molecule of the biofunctional coating
composition of
the present invention.
The present invention also provides surface-binding domains comprised of
peptides
consisting essentially of SEQ ID NOs:1-8; and polynucleotides encoding such
surface-
binding domains.
The present invention also provides endothelial-binding domains comprised of
peptides consisting essentially of SEQ ID NOs:9-46; and polynucleotides
encoding such
endothelial-binding domains.
Using the compositions according to the present invention, the present
invention
also provides: methods for coating a metallic surface of a medical device so
as to render
the coated surface capable of adhering to cells of endothelial cell lineage
(e.g., one or
more of endothelial cells, and endothelial progenitor cells) when the coated
surface is
contacted by cells of endothelial cell lineage; methods for promoting
adherence of cells of
endothelial cell lineage to at least one metallic surface of a medical device;
and methods
for promoting endothelialization of at least one metallic surface of a medical
device by
coating the at least one surface to promote attachment of cells of the
endothelial cell
lineage. These methods comprise contacting the at least one metallic surface
of the
medical device to be coated with a biofunctional coating composition (also
known as an
"interfacial biomaterial") comprising at least one surface-binding domain of
the present
invention which is coupled to at least one endothelial-binding domain of the
present
invention. The biofunctional coating composition is contacted with and applied
to at least
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one metallic surface of a medical device in forming a coating on the medical
device, and
wherein the at least one endothelial-binding domain is in an amount effective
in the coating
for adhering cells of endothelial cell lineage to, and preferably for
promoting
endothelialization of, the at least one coated surface of the medical device.
The methods
may further comprise the step of contacting the coated device with cells of
endothelial cell
lineage in promoting one or more of attachment, adherence, support for growth,
and
support for differentiation. This latter step may occur in vitro (e.g.,
attaching the endothelial
cells prior to implantation of the device); or may occur.in vivo (e.g., once
implanted, the
individual's endothelial cells migrate from adjacent arterial areas of intact
endothelium to,
or as circulating cells, come in contact with, and adhere to, the surface of
the device coated
by the biofunctional coating composition).
With respect to the methods and compositions according to the present
invention,
at least one endothelial-binding domain may comprise a single type (e.g., that
binds
specifically to a subset of cells of endothelial cell lineage; for example, to
endothelial cells
only; or with broad specificity (e.g., in general, for both endothelial cells
and endothelial
progenitor cells)), or may comprise muitiple types (e.g., one type that binds
specifically to
endothelial cells; and another type that binds specifically to endothelial
progenitor cells).
Using the compositions according to the present invention, the invention also
relates to a method of promoting the adherence of cells of endothelial cell
lineage to a
medical device, and more preferably an intravascular device. Also provided is
a method
for manufacturing a medical device. These methods comprise contacting at least
one
metallic surface of a medical device with a biofunctional coating composition
(which binds
specifically to cells of endothelial cell lineage) in forming at least one
coated metallic
surface; and contacting the at least one coated surface with cells of
endothelial cell lineage
(e.g., in promoting adherence of cells of endothelial cell lineage to the at
least one coated
surface); wherein the biofunctional coating composition comprises at least one
surface-
binding domain and at least one endothelial-cell binding domain; and wherein
the at least
one surface-binding domain and the at least one endothelial-cell binding
domain are
coupled together. Contacting of cells of endothelial cell lineage with the
biofunctional
coating composition on the medical device can be by any method known in the
art for
promoting binding interactions between an affinity molecule and its ligand,
such as, for
example, incubating, dipping, spraying, or brushing a solution containing
cells of
endothelial cell lineage on the medical device comprising the biofunctional
coating
composition. Also provided is a medical device comprising a coating formed by
applying
an effective amount of the biofunctional coating composition to a metallic
surface of the
medical device, in rendering the medical device compatible for attachment of
endothelial
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cells, and more preferably for the attachment of endothelial cells with
subsequent
endothelialization of the coated surface.
Altematively, provided is a method for promoting endothelialization of a
vascular
device so that to a selected metallic surface of the device, once that surface
is coated and
the device implanted, promoted is attachment of cells of endothelial cell
lineage. The
method comprises the steps of: (a) contacting a biofunctional coating
composition
described herein to at least one metallic surface of a vascular device to be
endothelialized,
so that the biofunctional coating composition binds to the at least one
metallic surface, in
forming a coated metallic surface on the vascular device; wherein the
biofunctional coating
composition comprises at least one surface-binding domain having an amino acid
sequence consisting essentially of SEQ ID NOs:1 -8, coupled to at least one
endothelial-cell
binding domain having an amino acid sequence consisting essentially of SEQ ID
NOs:9-
46; and (b) implanting the device into an individual (human or non-human) in
need of the
device; wherein cells of endothelial cell lineage (produced by the individual)
contact, attach
and adhere to the coated surface of the device (primarily mediated by the
cells binding to
the at least one endothelial binding domain of the biofunctional coating
composition), in
promoting spread of cells of endothelial cell lineage over the coated metallic
surface of the
device, and in promoting endothelialization of the vascular device. Promoting
endothelialization on the implanted device may further promote one or more of
healing of
tissue or vasculature adjacent to the implanted device, promote incorporation
(integration)
of the implanted device into the adjacent tissue, and reduce occurrence of
thrombosis as
related to the implanted device.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions for an improved coating for
medical
devices, methods of coating medical devices using those compositions, and a
metallic
surface of a medical device which is coated with a biofunctional coating
composition of the
present invention.
Definition Section While the following terms are believed to be well
understood by one of
ordinary skill in the art, the following definitions are set forth to
facilitate explanation of the
invention.
The term "effective amount" is used herein, in referring to the biofunctional
coating
composition according to the present invention and for purposes of the
specification and
claims, to mean an amount sufficient of the biofunctional coating composition
is applied to
the at least one metallic surface to be coated (via contact of the at least
one surface to the
biofunctional coating) so as to (a) mediate binding of the biofunctional
coating composition
to the at least one metallic surface of the medical device in forming a
coating; and (b)
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promote adherence of endothelial cells to, and more preferably,
endothelialization of, the
coated surface.
The term "cells of endothelial cell lineage" is used herein for purposes of
the
specification and claims, to mean endothelial cells at any development stage
(e.g., ranging
from early stages of development, such as an endothelial stem cell or
progenitor cell, to a
mature stage of development such as a fully differentiated, tissue specific
endothelial cell);
and including stem cells capable of differentiating into endothelial
progenitor cells and/or
endothelial cells, such stem cells sharing at least one surface molecule or
receptor in
common with endothelial cells (e.g., bone marrow angioblast; a cardiac Sca-1 +
stem cell
(which can be differentiated into endothelial cells in the presence of
leukemia inhibitory
factor (LIF)), an adipose-derived stem cell); or a combination thereof. Thus,
cells of
endothelial cell lineage include endothelial cells, endothelial progenitor
cells, and stem cells
capable of differentiating into endothelial cells and/or endothelial
progenitor cells. A
preferred cell of endothelial cell lineage may be used in accordance with the
present
invention to the exclusion of a cell of endothelial cell lineage other than
the preferred cell of
endothelial cell lineage.
The term "endothelialization" is used herein unless otherwise specified, for
purposes of the specification and claims, to mean one or more of the growth
(desirably
including proliferation) of endothelial cells, and differentiation of
endothelial cells, on and
over the at least one metallic surface of a medical device coated by an
effective amount of
the biofunctional coating composition according to the present invention.
Preferably, once
the cells of endothelial cell lineage are attached to the surface of a medical
device coated
by an effective amount of the biofunctional coating composition, promoted will
be
endothelial cell growth and development to provide an endothelial tissue
layer. Thus, the
term "endothelialization" can mean re-endothelialization of a vascular graft
which has lost
or been stripped of its endothelium due to any biological or mechanical
process; or it may
comprise growing new endothelial cells to cover a metallic surface of an
implanted or
implantable graft, or implanted or implantable medical device, which had not
been
previously covered by endothelial cells.
The term "medical device' is used herein, for purposes of the specification
and
claims, to refer to an intravascular device, vascular device, vascular graft,
a lead or lead tip
exposed to the vascular system (e.g., from a cardiac pacemaker or cardiac
defribillator). In
a preferred embodiment, within the scope and meaning of "medical device"
herein is a
device comprising a stent (as known in the art, a stent being a metallic
and/or polymeric
cage-like or tubular support device that is used to hold vessels (e.g., blood
vessels) open).
The terms "intravascular device" and "vascular device" are used
interchangeably herein, for
purpose of the specification and claims, to refer to a structure that is
introduced into a
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human or animal vasculature to restore function of damaged, diseased, or
blocked tissue,
and includes prosthetic devices, and vascular grafts. In a preferred
embodiment, within the
scope and meaning of "intravascular device" or "vascular device" herein is a
device
comprising a stent. The term "vascular device" as used herein also includes
device-related
materials that are associated with the device and are also introduced into a
human or
animal body in conjunction with the device. Representative vascular devices
include, but
are not limited to, heart patches, artificial heart valves, annuloplasty
rings, annular rings,
mechanical assist devices, vascular sealing devices, central venous catheters,
arterial
catheters, pacemakers, defibrillators, guidewires, embolic protection filters,
embolic
devices (e.g., coils), implantable infusion pumps, and vascular sutures.
Vascular grafts
include coronary artery bypass grafts, prosthetic heart valves, peripheral
vascular bypass
grafts, vascular access grafts, and synthetic grafts. A preferred medical
device may be
used in accordance with the present invention to the exclusion of a medical
device other
than the preferred medical device.
A medical device may be comprised of, and hence have one or more surfaces
comprised of, a variety of materials including, but not limited to, a metal, a
metal oxide, a
non-metal oxide, a ceramic, a rubber, a plastic, an acrylic, a silicone, a
polymer, and
combinations thereof. An intravascular device can be produced using any
biocompatible
material; however, because of the difficulties with biocompatibilities in the
vasculature, it is
preferred that the biocompatible material be relatively inert. Such devices
are made of a
variety of materials that are known in the art, but most typically are
biologically inert
polymers or metals. Metals used in the manufacture of medical devices are
known in the
art to include, without limitation, stainless steel, tantalum, gold, platinum,
silver, tungsten,
titanium, titanium alloys (for example, memory titanium alloys such as
nitinol), a transition
metal, alkali metals, and alkaline earth metals (each of the latter three
comprise metals
related in structure and function, as classified in the Periodic Table). Metal
alloys (e.g.,
cobalt-chrome alloy) and metal oxides of each of these groups, individually
and separately,
are included. In the present invention, a preferred surface material to which
the
biofunctional coating composition of the present invention becomes bound is a
metal, and
more preferably stainless steel. A preferred surface material of a medical
device may be
used in accordance with the present invention to the exclusion of a surface
material of the
medical device other than the preferred surface material.
When the term "surface" is used herein in conjunction with a medical device,
generally it is referring to one or more metallic surfaces of the medical
device which is or
becomes exposed to biological solutions and/or biological tissue, and
preferably comes in
contact with blood and/or is introduced into vasculature of an individual; and
hence, such
surface is susceptible to any one or more of thrombosis, neointima formation,
restenosis.
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"Metallic surface" means a surface material comprised of one or more of a
metal, metal
alloy, metal oxide, and a combination thereof.
The term "individual", as used herein, for purposes of the specification and
claims,
refers to either a human or an animal.
The term "vascular biologic", as used herein, refers to a biological substance
which
has specific biologic utility in one or more of: the repair or integration of
a vascular device
within the vascular system, especially after surgery or upon implantation of
an intravascular
device; and promotion of endothelialization. A vascular biologic may comprise
a biological
substance selected from the group consisting of a collagen (e.g., type IV
and/or type V),
vitrogen, laminin, entactin, fibronectin, glycans (e.g., proteoglycans,
glycosaminoglycans),
one or more growth factors supporting endothelial cell growth (e.g., vascular
endothelial
cell growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth
factor (basic
fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF)),
heparin-binding
epidermal-like growth factor, angiopoietin 1 (ang-1), angiopoietin 2 (ang-2),
hepatocyte
growth factor (HGF), platelet-derived endothelial cell growth factor (PD-
ECGF), LIF),
angiopoietins (e.g., ang-3, ang-4) and a combination thereof. A preferred
vascular biologic
may be used in accordance with the present invention to the exclusion of a
vascular
biologic other than the preferred vascular biologic.
The term'"surFace-binding domain", used herein for purposes of the
specification
and claims, refers to a peptide that binds specifically to a metallic surface
of a medical
device; and more particularly, has binding specificity for stainless steel of
a stent, and
consists essentially of an amino acid sequence selected from the group
consisting of SEQ
ID NOs:1-8 (see, also, Table 1). A preferred surface-binding domain (including
the type of
surface to which it binds with specificity) may be used with the present
invention to the
exclusion of a surface-binding domain other than the preferred surface-binding
domain.
The surface-binding domain in the biofunctional coating composition of the
present
invention is selected to specifically bind (e.g., typically, noncovalently,
ionically, or
electrostatically) to the metallic material of at least one surface of the
medical device
desired to be coated, wherein generally, such at least one surface becomes
exposed to a
biological tissue and/or biological fluid associated with vasculature when the
medical
device Is implanted in an Individual in need of the medical device.
The term "time sufficient for binding" generally refers to a temporal duration
sufficient for specific binding of a binding domain described herein, and a
substrate for
which the binding domain has binding specificity, as known to those skilled in
the art.
The term "endothelial-binding domain", used herein for purposes of the
specification
and claims, refers to a peptide that specifically binds to one or more cells
of endothelial cell
lineage, and wherein the peptide consists essentially of an amino acid
sequence selected
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from the group consisting of SEQ ID NOs:9-46 (see, also, Table 3). Such an
endothelial-
cell binding domain may specifically bind to a specific type of cell of
endothelial cell lineage
(e.g., endothelial cells, or endothelial progenitor cell, or endothelial cells
of a specific tissue
origin (e.g., cardiac endothelial cells)), or to more than one type of cells
of endothelial cell
lineage (e.g., sharing a common surface molecule bound by the endothelial cell-
binding
domain). Alternately, the biofunctional coating compositions of the present
invention may
be comprised of more than one type of endothelial-binding domain (e.g., two or
more
different peptides, each with binding specificity for different cells of
endothelial cell lineage).
Thus, in such case, each type of endothelial-binding domain has a binding
specificity for
cells of endothelial cell lineage that differs from the binding specificity of
another type of
endothelial-binding domain present in the biofunctional coating composition.
Excluded
from the definition "endothelial-binding domain" is an antibody, and more
particularly an
antibody having binding specificity for endothelial cells. A preferred
endothelial-binding
domain (including the type of cells of endothelial cell lineage to which it
binds with
specificity) may be used in accordance with the present invention to the
exclusion of an
endothelial-binding domain other than the preferred endothelial-binding
domain. Thus,
preferred endothelial-binding domain peptides consisting essentially of amino
acid
sequences selected from the group consisting of SEQ ID NOs:9-46, and excluded
are
endothelial-binding domain peptides consisting essentially of amino acid
sequences other
than those selected from the group consisting of SEQ ID NOs:9-46.
The terms "biofunctional coating composition" and "interfacial biomaterial"
are used
interchangeably, in reference to the present invention and for purposes of the
specification
and claims, to refer to a composition comprising at least one surface-binding
domain
comprising a peptide consisting essentially of an amino acid sequence selected
from the
group consisting of SEQ ID NOs:1-8, and at least one endothelial-binding
domain
comprising a peptide consisting essentially of an amino acid sequence selected
from the
group consisting of SEQ ID NOs:9-46, wherein the at least one surface-binding
domain
and at least one endothelial-binding domain are coupled together (e.g., by one
or more of
physically, chemically, synthetically, or biologically (e.g., via recombinant
expression)) in
such a way that each binding domain retains its respective function to bind to
the
respective molecule for which it has binding specificity (as described
herein). Such
coupling may include a multimeric molecule having two or more surface-binding
domains
coupled together, wherein an endothelial-binding domain is coupled to all or
only some of
the surface-binding domains of the multimeric molecule. For example, using
standard
reagents and methods known in the art of peptide chemistry, two binding
domains may be
coupled via a side chain-to-side chain bond (e.g., where each of the peptides
have a side
chain amine (e.g., such as the epsilon amine of lysine)), a side chain-to-N
terminal bond
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(e.g., coupling the N-terminal amine of one peptide with the side chain amine
of the other
peptide), a side chain-to-C-terminal bond (e.g., coupling the C-terminal
chemical moiety
(e.g., carboxyl)of one peptide with the side chain amine of the other
peptide), an N-
terminal-to-N-terminal bond, an N-terminal to C-terminal bond, a C-terminal to
C-terminal
bond, or a combination thereof. In synthetic or recombinant expression, a
peptide of a
surface-binding domain can be coupled directly to a peptide of an endothelial-
binding
domain by synthesizing or expressing both peptides as a single peptide. The
coupling of
surface-binding domain to an endothelial-binding domain may also be via a
linker to form a
biofunctional coating composition.
The biofunctional coating composition or interfacial biomaterial of the
present
invention comprises: (a) the at least one surface-binding domain according to
the present
invention, in an amount effective to mediate the binding of the biofunctional
coating
composition or interfacial biomaterial to the metallic surface material of the
medical device
for which the at least one surface-binding domain has binding specificity; and
(b) the at
least one endothelial-binding domain according to the present invention in an
amount
effective to confer to the coated medical device the ability to attach or
adhere to cells of
endothelial cell lineage, and more preferably and additionally, to promote
endothelialization
of the coated surface of the medical device; wherein the at least one surface-
binding
domain and the at least one endothelial-binding domain are coupled together.
In a
preferred embodiment, a linker is used to join together the at least one
surface-binding
domain and the at least one endothelial-binding domain.
-In function, when the biofunctional coating composition is applied to a
metallic
surface of a medical device (by contacting the biofunctional coating
composition with the
metal), binding of the biofunctional coating composition to the metallic
surface is mediated
primarily by a domain of the biofunctional coating composition comprising the
at least one
surface-binding domain according to the present invention; and the properties
of, or
associated with, the biofunctional coating composition as related to
attachment, adherence,
endothelialization, or a combination thereof, are mediated primarily by a
domain of the
biofunctional coating composition comprising the at least one endothelial-
binding domain
according to the present invention. Thus, when a medical device is coated with
a
biofunctional coating composition of the present invention, and then the
coated medical
device is introduced into or applied to an individual, the biofunctional
coating composition is
then the interface (hence, "interfacial biomaterial") between the medical
device and the
biological tissues and/or biological fluids of the individual. Accordingly,
provided is a
method of promoting the attachment and adherence of cells of endothelial cell
lineage to a
medical device, the method comprising coating one or more metallic surfaces of
the
medical device with a biofunctional coating composition or interfacial
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comprising at least one surface-binding, domain according to the present
invention and at
least one endothelial-binding domain according to the present invention,
wherein the at
least one surface-binding domain and the at least one endothelial binding
domain are
coupled together. In another embodiment, provided is a method of promoting
endothelialization on a metallic surface of a medical device, the metallic
surface being
suitable for contacting one or more of a. biological tissue (e.g., a blood
vessel) or biological
fluid (e.g., blood) associated with vasculature, the method comprising coating
one or more
metallic surfaces of the medical device with a biofunctional coating
composition or
interfacial biomaterial comprising at least one surface-binding domain
according to the
present invention and at least one endothelial-binding domain according to the
present
invention, wherein the at least one surface-binding domain and the at least
one endothelial-
binding domain are coupled together, and wherein the at least one endothelial-
binding
domain is bound to cells of endothelial cell lineage.
The term "linker" is used, for purposes of the specification and claims, to
refer to a
compound or moiety that acts as a molecular bridge to couple at least two
different
molecules (e.g., with respect to the present invention, coupling a surface-
binding domain to
an endothelial-binding domain, or coupling two or more surface-binding domains
in making
a multimeric molecule comprised of two or more surface-binding domains, or
coupling two
or more endothelial-binding domains in making a multimeric molecule comprised
of two or
more endothelial-binding domains). Thus, for example, one portion of the
linker binds to a
surface-binding domain according to the present invention, and another portion
of the linker
binds to an endothelial-binding domain according to the present invention. As
known to
those skilled in the art, and using methods known in the art, a surface-
binding domain and
an endothelial-binding domain may be coupled to the linker in a step-wise
manner, or may
be coupled simultaneously to the linker, to form a biofunctional coating
composition or
interfacial biomaterial according to the present invention. There is no
particular size or
content limitations for the linker so long as it can fulfill its purpose as a
molecular bridge,
and that the binding specificities of the biofunctional coating composition
are substantially
retained.
Linkers are known to those skilled in the art to include, but are not limited
to,
chemical chains, chemical compounds (e.g., reagents), and the like. The
linkers may
include, but are not limited to, homobifunctional linkers and
heterobifunctional linkers.
Heterobifunctional linkers, well known to those skilled in the art, contain
one end having a
first reactive functionality (or chemical moiety) to specifically link a first
molecule, and an
opposite end having a second reactive functionality to specifically link to a
second molecule.
It will be evident to those skilled in the art that a variety of bifunctional
or polyfunctional
reagents, both homo- and hetero-functional (such as those described in the
catalog of the
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Pierce Chemical Co., Rockford, III.), amino acid linkers (typically, a short
peptide of
between 3 and 15 amino acids, and often containing amino acids such as
glycine, and/or
serine), and polymers (e.g., polyethylene glycol) may be employed as a linker
with respect
to the present invention. In one embodiment, representative peptide linkers
comprise
multiple reactive sites to be coupled to a binding domain (e.g., polylysines,
polyornithines,
polycysteines, polyglutamic acid and polyaspartic acid) or comprise
substantially inert
peptide linkers (e.g., lipolyglycine, polyserine, polyproline, polyalanine,
and other
oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid
residues). In some
embodiments wherein amino acid linker is chosen, the biofunctional coating
composition of
the present invention may be synthesized to be a single, contiguous peptide
comprising a
surface-binding domain, a linker, and an endothelial-binding domain. Thus, the
linker
attachment is simply via the bonds of the single contiguous peptide.
Suitable polymeric linkers are known in the art, and can comprise a synthetic
polymer or a natural polymer. Representative synthetic polymers include but
are not
limited to polyethers (e.g., poly(ethylene glycol) ("PEG")), polyesters (e.g.,
polylactic acid
(PLA) and polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon),
polyurethanes,
polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic acids,
polystyrenes,
polyhexanoic acid, flexible chelators such. as EDTA, EGTA, and other synthetic
polymers
which preferably have a molecular weight of about 20 daltons to about 1,000
kilodaltons.
Representative natural polymers include but are not limited to hyaluronic
acid, alginate,
chondroitin sulfate, fibrinogen, fibronectin, albumin, coliagen, calmodulin,
and other natural
polymers which preferably have a molecular weight of about 200 daltons to
about 20,000
kilodaltons (for the constituent monomers). Polymeric linkers can comprise a
diblock
polymer, a multi-block copolymer, a comb polymer, a star polymer, a dendritic
or branched
polymer, a hybrid linear-dendritic polymer, a branched chain comprised of
lysine, or a
random copolymer. A linker can also comprise a mercapto(amido)carboxylic acid,
an
acrylamidocarboxylic acid, an acriyamido- amidotriethylene glycolic acid, 7-
aminobenzoic
acid, and derivatives thereof. Linkers are known in the art and include
linkers that can be
cleaved, and linkers that can be made reactive toward other molecular moieties
or toward
themselves, for cross-linking purposes.
Depending on such factors as the molecules to be linked, and the conditions in
which the linking is performed, the linker may vary in length and composition
for optimizing
such properties as preservation of biological function, stability, resistance
to certain
chemical and/or temperature parameters, and of sufficient stereo-selectivity
or size. For
example, the linker should not significantly interfere with the ability of a
surface-binding
domain to function in a biofunctional coating composition (i.e., to
sufficiently bind, with
appropriate avidity for the purpose, to a surface for a medical device for
which it has
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specificity according to the present invention). Likewise, the linker should
not significantly
interfere with the ability of an endothelial-binding domain to function in a
biofunctional
coating composition (i.e., to sufficiently bind, with appropriate avidity for
the purpose, to
cells of endothelial cell lineage for which it has specificity according to
the present
invention). A preferred linker may be a molecule which may have activities
which enhance
or complement the effect of the biofunctional coating composition of the
present invention.
For example, using polyethylene glycol or other polymeric molecule or protein
(e.g.,
albumin) as a linker may serve to help prevent non-specific protein and/or
undesired cell
adherence to the surface of the medical device coated with a biofunctional
coating
composition according to the present invention. A preferred linker may be used
in the
present invention to the exclusion of a linker other than the preferred
linker.
The terms "binds specifically" or "binding specificity', and like terms used
herein,
are interchangeably used, for the purposes of the specification and claims, to
refer to the
ability of a binding domain (as described herein) to have a binding affinity
that is greater
for one target molecule or surface material selected to be bound (the latter,
"target surface
material") over another molecule or surface material (other than the target
molecule or
target surface material); e.g., an affinity for a given substrate in a
heterogeneous
population of other substrates which is greater than, for example, that
attributable to non-
specific adsorption. For example, a surface-binding domain has binding
specificity for a
metallic surface, and more preferably a stainless steel surface, of a medical
device, when
the surface-binding domain demonstrates preferential binding to metal, as
compared to
binding to another component or material of the medical device (such as a
polymer). Such
preferential binding may be dependent upon the presence of a particular
conformation,
structure, and/or charge on or within the molecule or material for which the
binding domain
has binding specificity, such that it recognizes and binds to that molecule or
material rather
than to molecules or materials in general.
In some embodiments, a binding domain that binds specifically to a particular
surface, material or composition binds at least 10%, 20%, 30%, 40%, 50%, 60%,
70%,
80%, 90%, 100%, 200%, 300%, 400%, 500%, or a higher percentage, than the
binding
domain binds to an appropriate control such as, for example, a different
material or surface,
or a protein typically used for such comparisons such as bovine serum albumin.
For
example, binding specificity can determined by an assay in which quantitated
is a signal
(e.g., fluorescence, or colorimetric) representing the relative amount of
binding between a
peptide and target cells, as compared to peptide and non-target cells. Thus,
if in such an
assay, the results indicate that about 40% of the endothelial cells (as target
cells) present
in the assay are bound by a peptide, and less than 10% of the other cells
(e.g., smooth
muscle cells; "non-target cells") present in the assay are bound by the
peptide, then the
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peptide is said to have binding specificity for endothelial cells. In a
preferred embodiment,
the binding domain has binding specificity that is additionally characterized
by an EC50 of
10uM or less, and more preferably less than 1 NM. The EC50 can be determined
using any
number of methods known in the art, such as by generating a concentration
response
curve from a binding assay in which the concentration of the peptide is
titered with a known
amount of material or cells for which the peptide has binding specificity
(see, for example,
methods described in Examptes 2 and 3 herein). In such case, the EC50
represents the
concentration of peptide producing 50% of the maximal binding observed for
that peptide in
the assay.
The term "peptide" is used herein, for the purposes of the specification and
claims
to refer to an amino acid chain of no less than about 3 amino acids and no
more than about
500 amino acid residues in length, wherein the amino acid chain may include
naturally
occurring amino acids, synthetic amino acids, genetically encoded amino acids,
non-
genetically encoded amino acids, and combinations thereof; however,
specifically excluded
from the scope and definition of "peptide" herein is an antibody. Preferably,
the peptide
comprising a binding domain according to the present invention comprises a
contiguous
sequence of no less than 7 amino acids and no more than about 60 amino acids
in length.
A peptide used in accordance with the present invention may be produced by
chemical
synthesis, recombinant expression, biochemical or enzymatic fragmentation of a
larger
molecule, chemical cleavage of larger rrmolecule, a combination of the
foregoing or, in
general, made by any other method in the art, and preferably isolated. The
term "isolated"
means that the peptide is substantially free of components which have not
become part of
the integral structure of the.peptide itself; e.g., such as substantially free
of cellular material
or culture medium when produced by recombinant techniques, or substantially
free of
chemical precursors or other chemicals when chemically synthesized or produced
using
biochemical or chemical processes. A preferred peptide may be used in the
present
invention to the exclusion of a peptide other than the preferred peptide.
Peptides can include L-form amino acids, D-form amino acids, or a combination
thereof. Representative non-genetically encoded amino acids include but are
not limited to
2-aminoadipic acid; 3-aminoadipic acid; (3-aminopropionic acid; 2-aminobutyric
acid; 4-
aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic
acid; 2-
aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-
diaminobutyric acid;
desmosine; 2,2'-diaminopimelic acid; 2;3-diaminopropionic acid; N-
ethylglycine; N-
ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-
hydroxyproline;
isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-
methylisoleucine; N-
methylvaline; norvaline; norieucine; ornithine; and 3-(3,4-dihydroxyphenyl)-L-
alanine
("DOPA"). Representative derivatized amino acids include, for example, those
molecules
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in which free amino groups have been derivatized to form amine hydrochlorides,
p-toluene
sulfonyl groups,'carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl
groups or
formyl groups. Free carboxyl groups can be derivatized to form salts, methyl
and ethyl
esters or other types of esters or hydrazides. Free hydroxyl groups can be
derivatized to
form 0-acyl or 0-alkyl derivatives. The imidazole nitrogen of histidine can be
derivatized to
form N-im-benzylhistidine. In a preferred embodiment, and in a biofunctional
coating
composition according to the present invention, the at least one surface-
binding domain
comprises an N-terminal amino acid, a C-terminal amino acid, or a combination
thereof,
wherein such amino acid is a non-genetically encoded amino acid that enhances
the
binding avidity (strength of binding interactions) of the surface-binding
domain to the
surface of a medical device for which it has binding specificity. Such amino
acids can be
incorporated into a peptide comprising a surface-binding domain by standard
methods
known in the art for solid phase and/or solution phase synthesis. For example,
in one
embodiment, from about one to about four residues of DOPA , a hydroxy-amino
acid (e.g.,
one or more of hydroxylysine, allo-hydroxylysine, hydroxyproline, and the
like) or a
combination thereof, is added as terminal amino acids of an amino acid
sequence of a
peptide during synthesis, wherein the peptide comprises a surface-binding
domain used in
the biofunctional coating composition according to the present invention for
enhancing the
strength of the binding interactions (e.g., via electrostatic or ionic
interactions) between the
biofunctional coating composition and the at least one metallic surface of the
medical
device to be coated.
A peptide according to the present invention may be modified, such as by
addition
of chemical moieties, or substitutions, insertions, and deletions of amino
acids, where such
modifications provide for certain advantages in its use; provided the peptide
consists
essentially of an amino acid sequence illustrated in any one of SEQ ID NOs:1-
47. When
used herein in reference to the present invention and for purposes of the
specification and
claims, the terminology "consisting essentially of" or like terms (e.g.,
"consists essentially
of") refers to a peptide which includes the amino acid sequence of the
peptides described
herein or a peptide having at least 70% identity (and preferably at least 90%
identity)
thereto (as described in more detail herein), and may include additional amino
acids at the
carboxyl and/or amino terminal ends (e.g., from about 1 to about 20 amino
acids per
terminus), and which maintains the primary activity of the peptides as a
binding domain
described herein. In one example, a peptide consisting essentially of an amino
acid
sequence of SEQ ID NO:3 includes an amino acid sequence of SEQ ID NO:1 (the
latter
differing by having an additional 20 amino acids at the N-terminus, yet
retaining the binding
specificity for a metal surface; see e.g., Example 2 and Table 2). In another
non-limiting
example, an endothelial-binding domain comprising a peptide "consisting
essentially of'
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any one of the amino acid sequences illustrated as SEQ ID NOs:9-46 will
possess the
activity of binding cells of endothelial cell lineage with binding
specificity, as provided
herein; and will not possess any characteristics which constitutes a material
change to the
basic and novel characteristics of the peptide as an endothelial-binding
domain (e.g., thus,
in the foregoing example, a full length naturally occurring polypeptide, or a
genetically
engineered polypeptide, which has a primary activity other than as a binding
domain
described herein, and which contains the amino acid sequence of a binding
domain
comprising a peptide described in the present invention, would not constitute
a peptide
"consisting essentially of' a peptide or amino acid sequence described in the
present
invention).
Thus, the term "peptide" encompasses any of a variety of forms of peptide
derivatives including, for example, amides, conjugates with proteins, cyclone
peptides,
polymerized peptides, conservatively substituted variants, analogs, fragments,
chemically
modified peptides, and peptide mimetics. Any peptide derivative that has
desired binding
characteristics of a binding domain according to the present invention can be
used in the
practice of the present invention. For example, a chemical group, added to the
N-terminal
amino acid of a peptide to block chemical reactivity of that amino terminus of
the peptide,
comprises an N-terminal group. Such N-terminal groups for protecting the amino
terminus
of a peptide are well known in the art, and include, but are not limited to,
lower alkanoyl
groups, acyl groups, sulfonyl groups, and carbamate forming groups. Preferred
N-terminal
groups may include acetyl, Fmoc, and Boc. A chemical group, added to the C-
terminal
amino acid of a synthetic peptide to block chemical reactivity of that carboxy
terminus of
the peptide, comprises a C-terminal group. Such C-terminal groups for
protecting the
carboxy terminus of a peptide are well known in the art, and include, but are
not limited to,
an ester or amide group. Terminal modifications of a peptide are often useful
to reduce
susceptibility by proteinase digestion, and to therefore prolong a half-life
of peptides in the
presence of biological fluids where proteases can be present. Optionally, a
peptide
comprising a binding domain, as described herein, can comprise one or more
amino acids
that have been modified to contain one or more chemical groups (e.g., reactive
functionalities such as fluorine, bromine, or iodine) to facilitate linking
the peptide to a linker
molecule. As used herein, the term "peptide" also encompasses a peptide
wherein one or
more of the peptide bonds are replaced by pseudopeptide bonds including but
not limited
to a carba bond (CHz-CHZ), a depsi bond (CO-O), a hydroxyethylene bond (CHOH-
CH2), a
ketomethylene bond (CO-CHZ), a methylene-oxy bond (CH2-O), a reduced bond (CH2-
NH),
a thiomethylene bond (CH2-S), an N-modified bond (-NRCO-), and a thiopeptide
bond (CS-
NH).
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Peptides which are useful as binding domains in a biofunctional coating
composition or method of using the biofunctional coating composition according
to the
present invention also include peptides having one or more substitutions,
additions and/or
deletions of residues relative to the sequence of an exemplary peptide
disclosed in any one
or more of Tables I and 3 and SEQ ID NOs:1-47 herein, so long as the binding
properties
of the original exemplary peptide are substantially retained. Thus, binding
domain
according to the present invention includes peptides that differ from the
exemplary
sequences disclosed herein by, for example, between about 1 % to about 25% of
the amino
acid sequence of an exemplary peptide; yet substantially retain the ability of
the
corresponding exemplary sequence to bind to a particular material or to act as
a binding
domain with binding specificity as described herein (e.g., retains at least
50%, 75%, 100%
or more of the binding specificity of an exemplary sequence disclosed herein,
as measured
using an appropriate assay). That is, binding domains according to the present
invention
preferably include peptides that share sequence identity with the exemplary
sequences
disclosed herein in the range of at least 50% to about 99% or greater sequence
identity.
Sequence identity may be calculated manually or it may be calculated using a
computer
implementation of a mathematical algorithm, for example, GAP, BESTFIT, BLAST,
FASTA,
and TFASTA, or other programs or methods known in the art. Alignments using
these
programs can be performed using the default parameters.
For example, consider surface-binding domains comprising a peptide consisting
essentially of amino acid sequences identified in Table 1 as SEQ ID NOs:3 and
4. A
consensus sequence may be written (using standard single letter amino acid
designations)
as a peptide consisting essentially of the amino acid sequence illustrated as
SEQ ID NO:5.
Thus, these amino acid sequences (SEQ ID NOs:3 and 4) share significant
sequence
homology (as described herein), but share sequence identity that is less than
about 50%,
yet substantially retain binding specificity for metals, particularly
stainless steel.
A peptide having an amino acid sequence substantially identical to a sequence
of -
an exemplary peptide disclosed herein rilay have one or more different amino
acid
residues as a result of substituting an amino acid residue in the sequence of
the exemplary
peptide with a functionally similar amino acid residue (a "conservative
substitution");
provided that peptide containing a conservative substitution will
substantially retain the
binding specificity of the exemplary peptide not containing the conservative
substitution.
Examples of conservative substitutions include the substitution of one non-
polar
(hydrophobic) residue such as isoleucine, valine, leucine or methionine for
another; the
substitution of one aromatic residue such as tryptophan, tyrosine, or
phenylaianine for
another; the substitution of one polar (hydrophilic) residue for another such
as between
arginine and lysine, between glutamine and asparagine, between threonine and
serine; the
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substitution of one basic residue such as lysine, arginine or histidine for
another; or the
substitution of one acidic residue such as aspartic acid or glutamic acid for
another.
In yet another embodiment of the present invention, a binding domain may be
described herein as comprising a peptide consisting essentially of a peptide
(and/or its
amino acid sequence) useful in the present invention.
[End of formal definition section]
The present invention provides for a biofunctional coating composition (or
interfacial
biomaterial), peptides comprising endothelial-binding domains, peptides
comprising
surface-binding domains, methods for coating a medical device, methods for
manufacturing of a medical device, and a coated medical device; all relating
to a
biofunctional coating composition comprising: at least one surface-binding
domain
comprising a peptide consisting essentially of an amino acid sequence selected
from the
group consisting of SEQ ID NOs:1 -8, and a combination thereof; and at least
one
endothelial-binding domain comprising a peptide consisting essentially of an
amino acid
sequence selected from the group consisting of SEQ ID NOs:9-46, and a
combination
thereof; wherein the at least one surface-binding domain is coupled to at
least one
endothelial-binding domain. The at least one surface-binding domain is in an
amount
effective to mediate the binding of the biofunctional coating composition to
the selected
metallic surface of the medical device for which the at least one surface-
binding domain
has binding specificity; and the at least endothelial-binding domain is in an
amount
effective to render a surface of the medical device coated by a biofunctional
coating
composition according to the present invention capable of promoting one or
more of
attachrnent to, adherence of, and endothelialization with, cells of
endothelial cell lineage.
The present invention is illustrated in the following examples, which are not
intended to be
lirniting.
EXAMPLE 1
Illustrated in this example are various methods for producing a surface-
binding
domain and a endothelial-binding domain for the biofunctional coating
compositions
according to the present invention. Many of the peptides comprising the
binding domains
in the biofunctional coating composition according to the present invention
(i.e., a surface-
binding domain and an endothelial-binding domain) were developed using phage
display
technology.
Phage display technology is well-known in the art, and can be used to identify
additional peptides for use as binding domains in the interFacial binding
materials according
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to the present invention. In general, using phage display, a library of
diverse peptides can
be presented to a target substrate, and peptides that specifically bind to the
substrate can
be selected for use as binding domains. Multiple serial rounds of selection,
called
"panning," may be used. As is known in the art, any one of a variety of
libraries and
panning methods can be employed to identify a binding domain that is useful in
a
biofunctional coating composition according to the present invention. Panning
methods
can include, for example, solution phase screening, solid phase screening, or
cell-based
screening. Once a candidate binding domain is identified, directed or random
mutagenesis
of the sequence may be used to optimize the binding properties (including one
or more of
specificity and avidity) of the binding domain.
For example, a variety of different phage display libraries were screened for
peptides that bind to a selected target substrate (e.g., a substrate selected
to find a binding
domain useful in the present invention). The substrate was either bound to or
placed in
(depending on the selected substrate) the wells of a 96 well microtiter plate.
Nonspecific
binding sites on the well surface of the polystyrene microtiter plate were
blocked with a
buffer containing 1% bovine serum albumin after overnight incubation at 4 C.
The wells
were then washed 5 times with a buffer containing phosphate buffered saline
with TweenTM
("PBS-T"). Each library was diluted in PBS-T and added at a concentration of
1010
pfu/mi in a total volume of 100 l. After 3 hour of incubation at room
temperature with
20 shaking at 50 rpm, unbound phage were removed by multiple washes with PBS-
T. Bound
phage were recovered by denaturation with 0.1 M glycine buffer, pH2.2. The
eluted phage
were neutralized with phosphate buffer, and then added to E. coli cells in
growth media.
The cell and phage-containing media was cultured by incubation overnight at 37
C in a
shaker at 200 rpm. Phage-containing supernatant was harvested from the culture
after
centrifuging the culture. Second and third rounds of selection were performed
in a similar
manner to that of the first round of selection, using the amplified phage from
the previous
round as input. To detect phage that specifically bind to the selected
substrate, enzyme-
linked immunosorbent (ELISA-type) assays were performed using an anti-phage
antibody
conjugated to a detector molecule, followed by the detection and quantitation
of the amount
of detector molecule bound in the assay. The DNA sequences encoding peptides
from the
phage that specifically bind to the selected substrate were then determined;
i.e., the
sequence encoding the peptide is located as an insert in the phage genome, and
can be
sequenced to yield the corresponding amino acid sequence displayed on the
phage
surface.
As known to those skilled in the art and methods known in the art, peptides
comprising the binding domains according to the present invention may be
synthesized by
any method for peptide synthesis including, but not limited to, solid phase
synthesis,
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solution phase synthesis, and a combination thereof. For example, peptides
comprising
binding domains useful in the present invention were synthesized on a peptide
synthesizer
using standard solid-phase synthesis techniques, and using standard FMOC
peptide
chemistry. After all residues were coupled, simultaneous cleavage and side
chain
deprotection was performed using standard methods and reagents known in the
art. After
cleavage from the resin, the peptides were precipitated, and the precipitate
was lyophilized.
The peptides were then purified using reverse-phase high performance liquid
chromatography; and peptide identity was confirmed with mass spectrometry.
EXAMPLE 2
This example illustrates the discovery and characterization of surface-binding
domains comprising peptides having binding specificity for a metallic surface
of a medical
device, such as a stainless steel surface of a stent.
A. Phage screening and selections.
As a specific illustrative example, nonspecific binding sites in wells
containing
stainless steel stent material in polystyrene microtiter plates were blocked
with a buffer
containing 1 l bovine serum albumin for 2 hours at room temperature. The
wells and
stainless steel stent material were then washed three times with PBS-T. The
plate was
incubated for 1 hour at room temperature with shaking at 50 rpm. Each of 17
different
phage display libraries was diluted in PBS + 1% BSA and was added at a
concentration of
1010 pfu/ml in a total volume of 250 l. After a 1 hour incubation at room
temperature with
shaking at 50 rpm, 70 l of bovine serum was added, and then the plates were
incubated
at 37 C with shaking for 1 hour. Unbound phage were removed by washing 3 time
with
300 l of PBS-T. After the final wash, phage bound to stents were used to
infect E. coli
cells. The infected cells were incubated in 96 deep-well plates, containing 1
ml of growth
medium (e.g., 2xYT + 5 Ng/ml tetracycline) per well, at 37*C overnight with
shaking.
Amplified phage-containing supernatant from each well was harvested by
centrifugation.
Second, third, and fourth rounds of selection were performed in a similar
manner to that of
the first round, using 150 l of amplified phage supernatant from the previous
round as
input, and diluted with 150 l of PBS-T + 1% BSA. From the fourth round of
selection, 340
individual clonal phage were then isolated and tested by plating out dilutions
of phage
pools to obtain single plaques. To detect phage that specifically bound to a
metal such as
stainless steel, conventional ELISAs were performed using an anti-M13 phage
antibody
conjugated to horseradish-peroxidase, followed by the addition of chromogenic
agent
ABTS (2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid). Relative binding
strengths of
the phage were determined by testing serial dilutions of the phage for binding
to stainless
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steel in an ELISA, using an anti-M13 phage antibody conjugated to horseradish-
peroxidase,
followed by the addition of chromogenic agent ABTS, and measuring the
absorbance at
405nm. In the ELISA for determining relative binding strengths, the phage
titrations were
done in either buffer alone, or buffer containing 20% whole blood. The DNA
sequence
encoding peptides that specifically bound a metallic surface was determined.
The
sequence encoding the peptide insert was located in the phage genome and
translated to
yield the corresponding amino acid sequence displayed on the phage surface.
From the phage titration experiments, three individual phage showed a desired
relative binding specificity. Amino acid determination of the nucleic acid
inserts in these 3
individual phage revealed that the phage represented 2 different peptide
sequences (i.e., 2
of the 3 shared the same amino acid sequence), as shown in Table 1. Thus,
Table I
illustrates such surface-binding domains, having binding specificity for a
metals, such as
stainless steel of stents, and comprising peptides consisting essentially of
amino acid
sequences consisting of SEQ ID NO:1 and SEQ ID NO:2.
Table I
Binding s ecifici for a metal such as stainless steel
SEQ ID NO: Amino acid se uence (single letter code
1 SGWDAGWAEDGVSGEASRSSHRTNHKKNNPKKKNKTR
2 SVEVACVSAGGGSSDVCASRNHTISKNHKKKNKNSNKTR
3 SSHRTNHKKNNPKKKNKTR
4 NHTISKNHKKKNKNSNKTR
B. Surface binding domain characterizations and modifications
Examination of the amino acid sequences (SEQ ID NO:1 and SEQ ID NO:2) of the
two surface-binding domains revealed that in each, the C-terminal half of the
peptide is rich
in basic amino acids such as lysine and histidine. Therefore, to determine if
the binding
specificity for stainless steel is primarily due to the amino acids in the C-
terminal half of the
amino acid sequence, or if the N-terminal region also impacts binding,
peptides consisting
essentially of amino acid sequences illustrated as SEQ ID NO:3 and SEQ ID NO:4
were
synthesized. The peptides, as listed in Table 2 as SEQ ID NOs: 1, 3, and 4
were each
synthesized with a biotin tag, and then assayed for relative binding strengths
by ELISA
using similar methods as that used for determining the relative binding
strengths of phage
displaying the peptides (as previously described herein). In this assay,
serial dilutions
(ranging from 0 pM to 10 pM) of each of the peptides were incubated with the
stainless
steel stents, washed with PBS-T, and relative binding specificity was
quantitated by
detecting the colorometric signal resulting from the reaction of streptavidin-
alkaline
phosphatase (the streptavidin portion binding to the biotin-labeled peptides)
with
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chromogenic substrate. The EC50 was determined from the titration curve. As
illustrated
in Table 2, a peptide consisting essentially of an amino acid sequence of SEQ
ID NO:3 has
the strongest binding specificity (or altematively, binding affinity), as
compared to a peptide
consisting essentially of an amino acid sequence of either SEQ ID NO:1 or SEQ
ID NO:4.
Additionally, these results support that the amino acids in the C-terminal
half rich in lysine
and histidine (e.g., amino acids 21-39 of SEQ ID NO:1 which is illustrated by
SEQ ID NO:3;
and amino acids 21-39 of SEQ ID NO:2 (illustrated as SEQ ID NO:4)) are
primarily
responsible for the binding specificity of these peptides for a metal (e.g.
titanium, and other
metals having an oxide layer), and more preferentially for stainless steel.
Table 2
SEQ ID NO: EC50 expressed in nanomoles (nM)
1 <250nM
4 < 250 nM
3 <50nM
From these experiments and considering amino acids in key positions and their
contributions for mediating binding specificity to a metal such as stainless
steel, a preferred
surface-binding domain comprising a peptide variant or derivative of a peptide
having the
amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, or 4 comprises at least
one
peptide having a motif of SEQ ID NO:5, as follows.
SEQ ID NO:5: X,-H-X X-X2-X2-X2-K-X,-X,-X-K-X,-Xi-N-K; where
X is any amino acid;
X, is K, N, or S, but preferably either K or N; and
X2isK,N,orH.
Thus, a preferred surface-binding domain according to the present invention
having binding
specificity for metal such as stainless steel of a medical device comprises a
peptide
consisting essentially of an amino acid sequence illustrated by SEQ ID NO: 5.
Additional titration curves were generated from experiments in which a peptide
consisting essentially of the amino acid sequence illustrated as SEQ ID NO:3
was
subjected to ethylene oxide sterilization (642 mg/L for 2 hours). The results
show that
sterilization with ethylene oxide had minimal to no effect on the relative
binding specificity
for the peptide to stainless steel (approximately the same EC50, < 50 nm, as
from titration
curves of the same peptide without being subjected to ethylene oxide
sterilization).
A surface-binding domain comprising a peptide consisting essentially of the
amino
acid sequence illustrated as SEQ ID NO:3 was further modified to evaluate such
parameters as the effect of pH on binding specificity, and the stability in
plasma (e.g., in
presence of proteases present in the plasma). In one example, a peptide
consisting
essentially of the amino acid sequence illustrated as SEQ ID NO:3 was
synthesized with D-
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amino acids rather than L-amino acids (SEQ ID NO:6). In another example,
synthesized
were surface-binding domains comprising a multimer (a divalent version (SEQ ID
NO:7),
and a tetravalent version (SEQ ID NO:8)) of a peptide consisting essentially
of the amino
acid sequence illustrated as SEQ ID NO:3. These multimers of SEQ ID NO:3 are
illustrated as follows.
SSHRTNHKKNNPKKKNKTRC~
\ KGGK(Biotin)-NH2
SSHRTNHKKNNPKKKNKTRG~ divalent (SEQ ID NO:7)
SSHRTNHKKNNPKKKNKTRC,-,SSHRTNHKKNNPKKKNKTR \Cr'fK
\
/KGGK(Biotin)-NH2
SSHRTNHKKNNPKKKNKTRG- '
-~/K
SSHRTNHKKNNPKKKNKTRG
tetravalent (SEQ ID NO:8)
These multimers, SEQ ID NOs: 7 and 8, were synthesized as follows. Briefly,
the
multimers were built on a lysine MAP core and comprised of two and four
peptide modules,
respectively, of SEQ ID NO:3. This core matrix was used to generate dual and
tetrameric
branches of SEQ ID NO:3. The multimers were synthesized sequentially using
solid phase
chemistry on a peptide synthesizer. The synthesis was carried out at a 0.05
mmol scale
which ensures maximum coupling yields during synthesis. The biotin reporter
moiety was
placed at the C-terminus of the molecule, and was appended by a short Gly-Gly-
linker to
the lysine core. Standard Fmoc/ t-Bu chemistry was employed using
AA/HBTU/HOBt/NMM
(1:1:1:2) as the coupling reagents (AA is amino acid; HOBt is O-Pfp ester/1-
hydroxybenzo-
triazole; HBTU is N-[1H-benzotriazol-l-yl)(dimethylamino) methyiene]-N-methyl-
methanaminium hexafluorophosphate N-oxide; NMM is N-methylmorpholine). Amino
acids
were used in 5-10 fold excess in the synthesis cycles, and all residues were
doubly, triply
or even quadruply coupled depending upon the complexity of residues coupled.
The
coupling reactions were monitored by Kaiser ninhydrin test. The Fmoc
deprotection
reactions was carried out using 20% piperidine in dimethylformamide. Peptide
cleavage
from the resin was accomplished using trifluoracetic acid (TFA:
H20:Triisopropylsilane =
95: 2.5: 2.5) at room temperature for 4 hours. The crude product was
precipitated in cold
ether. The pellet obtained after centrifugation was washed thrice with cold
ether and
lyophilized to give a white solid as crude desired product. The crude products
were
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analyzed by analytical high performance liquid chromatography (HPLC) on a C-18
column
using mobile eluants (A =H20 / TFA (0.1%TFA) and B = Acetonitrile /TFA (0.1
%TFA). The
multimers were also further analyzed by mass spectrometry for before
subjecting each to
final purification by HPLC. The fractions containing the desired product were
pooled and
lyophilized to obtain a fluffy white powder (> 98% purity).
For determining the effect of pH on binding specificity, the assay for
determining
relative binding strengths (by ELISA) was performed in the presence of
different buffers,
varying in pH in a range of from pH 2 to pH 12. For example, a buffer
containing glycine
and water was adjusted to pH2 using HCI; an acetate buffer was adjusted to pH
4.0 (ionic
strength of about 0.001 M); a phosphate buffer (NaH2PO4) was adjusted to pH
6.0 (ionic
strength of about 0.012M); a phosphate buffer (NaH2PO4) was adjusted to pH 7.0
(ionic
strength of about 0.01 9M); a tris buffer was adjusted to pH 8.0 (ionic
strength of about
0.006M); an ethanolamine buffer was adjusted to pH 10.0 (ionic strength of
about 0.003M);
and a phosphate buffer (NaH2PO4) was adjusted to pH 12.0 (ionic strength of
about
0.044M). The final concentration of each peptide in this assay was 1 pM.
Tested in this
assay were surface-binding domains comprising a peptide consisting essentially
of the
amino acid sequence illustrated as SEQ ID NO:3, and the multimers thereof
(divalent
version (SEQ ID NO:7) and tetravalent version (SEQ ID NO:8)). The binding
curves
showed that all three peptides (monvalent, divalent and tetravalent of the
amino acid
sequence of SEQ ID NO:3) bind well over the range of pH values from pH 6.0 to
pH 8.0,
with the optimum pH for binding being pH 7.0; and no more than a 20% decrease
in
binding at pH 6.0 or pH 8Ø
Peptides consisting essentially of a monvalent or tetravalent version (SEQ ID
NO:8)
of the amino acid sequence of SEQ ID NO:3, and a peptide comprising the D-
amino acid
version thereof (SEQ ID NO:6) were all tested in an ELISA binding assay
essentially as
described herein, but performed with stainless steel beads in the presence of
plasma, to
assess stability (one or more of susceptibility to proteases present in
plasma, or ability to
compete with plasma components in binding to stainless steel). Both the
tetravalent
version (SEQ !D NO:8) of the amino acid sequence of SEQ ID NO:3, and a peptide
consisting essentially of the amino acid sequence illustrated in SEQ ID NO:6
showed
significantly more stability (e.g., retaining from about 1.5 to about 4 times
more peptide
bound) in the presence of plasma, including less susceptibility to degradation
by proteolytic
enzymes, than a peptide consisting essentially of the amino acid sequence
illustrated as
SEQ ID NO:3.
EXAMPLE 3
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This example illustrates the discovery and characterization of endothelial-
binding
domains comprising peptides having binding specificity for cells of
endothelial cell lineage.
A. Phage screening and selections.
Phage libraries were pooled into four groups and screened for. peptides that
bind to
human umbilical vein endothelial cells (HUVECs). The four pools were first pre-
cleared on
non-target cells (e.g., cells other than cells of endothelial cell lineage).
For each pool, 10
L of phage (1010 of phage) was added to 1 x 108 cells of each of the following
cell types:
HEK-293 cells, aortic vascular smooth muscle cells (AoSMC), and platelets. The
phage
and cells were incubated for 1 hour at room temperature. Cells and attached
phage were
pelleted by centrifugation. The phage remaining in the supernatant were used
for
subsequent selections on HUVECs. Selections on HUVECS were performed using
methods known in the art, inciuding by one or more of biopanning with the
cells including
differential centrifugation, by fluorescence-activated cell sorting (FACS),
and over cell
monolayers.
Following selection on HUVECs, cells were rinsed in buffer and centrifuged.
HUVECs with adherent phage were resuspended in 2 ml 2xYT bacterial culture
medium
and cultured with DH5aF' cells. Phage were separated from the bacterial
culture medium
and then tested on non- target and target cells using fluorescence-activated
cell sorting
(FACS) to confirm specificity. Cells and attached phage were resuspended in
medium
containing anti-M13 phage antibody conjugated to phycoerythrin. After washing,
cells were
resuspended in buffer + 1 /a BSA, and analyzed by FACS for relative
positivity. Using this
process, identified are phage displaying peptides that have specificity for
binding
endothelial cells relative to smooth muscle cells and platelets (e.g., showing
less than 10%
positivity by FACS for smooth muscle cells and platelets). The phage DNA
sequence
insert encoding peptides that specifically bound the endothelial cells was
determined, and
then translated to yield the corresponding amino acid sequence displayed on
the phage
surface (without adjoining phage sequence; e.g., SS or SR). Table 3
illustrates such
endothelial-binding domains comprising peptides consisting essentially of
amino acid
sequences consisting of SEQ ID NO:9-46. Amino acid sequences illustrated in
Table 3 as
SEQ ID NOs:39-46 were from a particular phage library favoring presence of the
amino
acid cysteine. However, from the amino acid sequences discovered from the
other phage
libraries (i.e., SEQ ID NOs: 9-38), most display relatedness in sequence
through a rich
concentration of amino acids glycine (G), valine (V), and alanine (A) (e.g.,
comprising no
less than 10% and no more than about 75% of the amino acid residues in the
sequence),
which may be an indication of structure-function relationship. In that regard,
it has been
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reported that peptides formed of glycine, alanine, valine, and aspartic acid
have tertiary
structures with potential catalytic functions.
Table 3
Amino acid sequence
SEQ ID NO: Single letter code
9 GVDEWVGSSCAGVEECY
LFSSAFVFGALAGSGAG
11 FFGADSYLGGSFASAFD
12 GDVAASFFASAASAFSV
13 LAGAGWDAVVGGEGAVG
14 AGSSSSVSFVAAAGSAV
AVFVADVLGEEFVGAVA -
16 GVGYGWYSVAASSWSA
17 PFHTGAFLWPESHSHSH
18 SEYWSVGSVFAGSS
19 FYGEVGYVGASLYAGGAS
VVESSAAYASASSFAVV
21 FEGASVASLAFAGSVAG
22 VGAVSSSSLSEEFLGSL
23 YVGSAFSAAVASSVSEG
24 WAGAGSGGVAWSADFGV
SADVSAALLVLGASEVL
26 FAVYCASLSGVCSASFE
27 AGSSAFSVVASSVSVGG
28 YFRDATPAVFGYW
29 AYEDGFYSSGWSSDWV
VSGFGFSDSGAGEGVF
31 GAWLVSALIERGVGAQW
32 VVFAASGVAADAGWSVS
33 QMRECDDCCCMVLPFTS
34 HNSPFFLDCNFDAPCL
GDLVTSTCLLGLCAERG
36 ' LSAGPLDWWSSLRSSAS
37 LFSLLPALAFLGEEQGP
38 ADSFVLASAGSVQVWA
39 EGLVASVSCYAGGSCAVSR
SCNLPACFDILFRSLDKWS
41 SCNRDYNWLDSVGHCVN
42 SCLQWSFIGAYSSLSGQPS
43 SCSLCVLPSVTFDLKLECC
44 SSRISDYVGLSACPGGCAS
SCFCAILIKIIVFLSLVFS
46 CSTALKWTC
B. Binding domain characterizations
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Several of the endothelial-binding domains listed in Table 3 with desirable
binding
specificities (e.g., SEQ ID NOs: 9-18, 23-28, 30-32, and 35) were further
characterized for
binding to various cell types in whole blood by FACS, by synthesizing the
peptides with a
C-terminal biotin tag. Cells were harvested from cell culture flasks using
trypsin/EDTA.
The cells were neutralized with complete (serum-containing) media and allowed
to recover
at 37 C for at least 20 minutes. Each cell type was labeled with its
corresponding antibody,
as set forth in Table 4, below, and incubated for 20 minutes at room
temperature.
Table 4. Antibodies to Various Cell Types
Cell T e Antibody Amount
Endothelial Anti-CD31-APC (APC is 100 pL / 10 cells
cell allophycocyanin)
(human or
Porcine)
Endothelial Rabbit anti-CD133, followed
progenitor cell by anti-rabbit-APC
Smooth muscle cell Anti-alpha actin-APC 100 L/ 10 cells
Platelet Anti-CD42b-APC 100 pL I mL blood
The antibody-labeled cells were rinsed twice by centrifugation using washing
buffer
(HBSS + 1 % BSA + 0.1 % sodium azide, sterile-filtered). One hundred (100) NL
of whole
blood was aliquotted into each well of a deep-well polypropylene plate.
Peptides were
added to each well to achieve the desired peptide concentration. Minimal
volumes of cells
(typically 10 pL of cells or approximately 50,000 cells) were added to each
well. In
experiments comparing relative binding of peptides to endothelial cells and
platelets, two
sets of samples were prepared: The first set of samples consisted of antibody-
labeled
endothelial cells in whole blood; the second set consisted of unlabeled
endothelial cells
and antibody-lalieled platelets in whole blood. The plate was covered and
incubated for 20
minutes at room temperature, with shaking. Red blood cells were lysed by
adding 'i mL of
FACS lysing solution, and the plate was covered and shaken for 15 minutes at
room
temperature. One (1) mL of room temperature washing buffer was added to each
well,
then centrifuged at less than 1500 rpm in a room temperature centrifuge. The
wells were
aspirated and filled with 1.5 mL of washing buffer, then centrifuged and
aspirated again.
Cells were re-suspended by adding 500 pL of streptavidin-AlexaFluor 532 at
1:500 dilution
in media at room temperature. The plaite was covered and incubated for 20
minutes at
room temperature, then rinsed twice by centrifugation at less than 1500 rpm
with room
temperature washing buffer. Final suspensions of cells were prepared in 250 pL
of
washing buffer + 50 pL of 4 Jo paraformaldehyde. The samples were transferred
to an
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analysis plate and analyzed for cell binding using FACS. In the analysis,
controls for each
cell type (with relevant antibody as per Table 4, but containing no peptide)
were used to
assess background fluorescence. Any signal above the "no peptide" control was
considered the percent positive population.
The results of the binding characterization show that particularly preferred
endothelial-binding domains comprise peptides consisting essentially of amino
acid
sequences of SEQ ID NOs: 10, 14-18, 23, 25-28, and 30-32. These endothelial-
binding
domains showed binding to one or more of human coronary endothelial cells,
porcine
coronary endothelial cells, and endothelial progenitor cells with binding
specificity and
selectivity approaching or exceeding 40% positivity by FACs, and less than 10%
positivity
(and often less than 5% positivity) with platelets and smooth muscle cells.
Some of these
endothelial-binding domains had binding specificity that appeared to prefer
binding to
endothelial progenitor cells as compared to endothelial cells (e.g., amino
acid sequence
SEQ ID NO:14), whereas others showed a preference for binding endothelial
cells (e.g.,
amino acid sequence SEQ ID NO:32). Additional assays characterizing binding
specificity
show one or more of these preferred endothelial-binding domains (e.g., SEQ ID
NO:19)
has an EC50 of less than 10NM.
EXAMPLE 4
As already described herein, in some instances, the binding domains comprising
peptides according to the present invention also comprised modifications;
i.e., were
blocked at the N-terminus and/or at the C-terminus, and/or were linked to
another peptide.
Using these methods, for example, a surface-binding domain having binding
specificity for
a metallic surface of a medical device may be linked to an endothelial-binding
domain, in
forming a biofunctional coating composition according to the present
invention. As
apparent to one skilled in the art, a method of preference for linking a
linker molecule to a
binding domain will vary according to the reactive groups present on each
molecule.
Protocols for covalently linking two molecules using reactive groups are well
known to one
of skill in the art. As previously described herein, using methods well known
to those
skilled In the art, two binding domains may be coupled by a linker to form a
biofunctional
coating composition according to the present invention by synthesizing a
single contiguous
peptide comprising a first binding domain (e.g., a surface-binding domain), a
linker
comprising 3 or-more amino acids (e.g., GSS), and a second binding domain
(e.g., an
endothelial-binding domain). The tenns "firsY' and "second" are only used for
purposes of
ease of description, and is not intended to be construed as to limiting the
order of the
synthesis. In other words, the first binding domain may comprise an
endothelial-binding
domain, and the second binding domain may comprise the surface-binding domain.
In an
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alternate method, at least one first binding domain having been avidinated
(using
streptavidin, avidin, or a functional derivative thereof, and methods known in
the art) may
be coupled to at least one second binding domain having been biotinylated
(using biotin,
and methods known in the art), in forming a biofunctional coating composition
according to
the present invention. In this example, the avidin-biotin molecules serve as
the linker for
coupling at least one surface-binding domain to at least one endothelial-
binding domain in
forming an interfacial biomaterial according to the present invention.
As an illustrative example of making the biofunctional coating composition
according to the present invention, at least one surface-binding domain
comprising a
peptide consisting essentially of the amino acid sequence of SEQ ID NO:3 was
linked to at
least one endothelial-binding domain comprising a peptide consisting
essentially of the
amino acid sequence of SEQ ID NO:19. In one example, the surface-binding
domain was
coupled to the endothelial-binding domain via a linker comprising a 10 unit
polyethylene
glycol linker ("PEG"), to form a biofunctional coating composition comprising
an amino acid
sequence illustrated as SEQ ID NO:47 (a biotin tag was included as part of the
PEG linker
solely to facilitate detection during characterization of the biofunctional
coating composition,
as will be described herein; and the free C-terminal amino acid was amidated).
SEQ ID NO:47
SSFYGEVGYVGASLYAGGASSRG-PEG-SSHRTNHKKNNPKKKNKTRG
Briefly, the biofunctional coating composition was synthesized on a peptide
synthesizer in linear fashion, and in the following order as one contiguous
chain: an amino
acid sequence of SEQ ID NO:3, the PEG linker, and an amino acid sequence of
SEQ ID
NO:19. Standard Fmoc/ t-Bu chemistry was employed using AA/HBTU/HOBt/ NMM
(1:1:1:2) as the coupling reagents. Amino acids were used in 5 fold excess in
the
synthesis cycles, and all residues were double coupled. The coupling reactions
were
monitored by Kaiser ninhydrin-test. In order to inhibit peptide aggregation,
pseudoproline
Fmoc-Ala-Ser(Psi Me,Me pro)-OH was employed, and was also double coupled in 5
fold
excess. Fmoc-Lys(Biotin)-OH and Fmoc-NH-(Peg)1o-COOH were double coupled
manually using the above coupling conditions in order to produce a PEG linker
with the
biotin tag. The Fmoc deprotection reactions were carried out using 20%
piperidine in DMF.
The biofunctional coating was cleaved from the resin by using Reagent K
(TFA:EDT:H20:phenol: thioanisole = 82.5:2.5:5:5:5) at room temperature for 4
hours to
yield a crude product. The crude product was precipitated in cold ether. The
pellet obtained
after centrifugation was washed thrice with cold ether, and then lyophilized
to give a white
solid as crude product. The crude product was analyzed by analytical HPLC and
by mass
spectrrometry, and then was purified by HPLC using a gradient of buffer
B(Acetonitrile
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ITFA (0.1 %TFA)). The desired product, the biofunctional coating composition,
was pooled
and lyophilized in obtaining a fluffy white powder (> 95% purity).
Using methods similar to those described in Examples 2 and 3 herein, the
biofunctional coating composition comprising the amino acid sequences of SEQ
ID NO:47
was tested in a binding specificity assay by titrating the concentrations of
the biofunctional
coating composition and measuring the relative binding to a metal comprising
stainless
steel. Briefly, stainless steel beads were blocked with buffer (PBS-T with 1%
BSA),
washed, and then incubated with the biofunctional coating composition at
concentrations
from 0 to10 pM for 1 hour at room temperature. After washing, the amount of
biofunctional
coating composition was detected with streptavidin-alkaline phosphatase (the
streptavidin
portion binding to the biotin-Iabeled biofunctional coating composition) with
chromogenic
substrate. The EC50 was determined from the titration curve. The biofunctional
coating
composition comprising an amino acid sequence illustrated as SEQ ID NO:47
bound to
stainless steel with similar binding activity (e.g., EC50) as the surface-
binding domain from
which it was made (a surface-binding domain comprising a peptide consisting
essentially of
the amino acid sequence of SEQ ID NO:3).
The biofunctional coating composition comprising an amino acid sequence
illustrated as SEQ ID NO:47 was then tested for its ability to selectively
adhere cells of
endothelial cell lineage to a metallic surface of a medical device. In this
example, stainless
steel disks were used to represent a metallic surface of a medical device. The
disks were
contacted with a buffered solution containing the biofunctional coating
composition at a
concentration of 10pM for 1 hour at room temperature. As controls for non-
specific binding,
disks were either uncoated, or coated with the surface-binding domain
comprising a
peptide consisting essentially of an amino acid sequence of SEQ ID NO:3, or an
irrelevant
peptide (having no known binding specificity for metal or stainless steel or
endothelial cells).
The disks were washed with PBS, and then 25,000 endothelial cells were added
in cell
media containing 10% bovine serum, and incubated at room temperature for 15
minutes.
The disks were washed in PBS, and the cells were then quantitated using a
commercial
luminescent cell viability assay system that measures intracellular ATP using
a luminescent
read-out. The luminescence was detected using a plate reader. The
biofunctional coating
composition comprising an amino acid sequence illustrated as SEQ ID NO:47
showed the
ability to bind endothelial cells to the metallic surface by demonstrating a
several fold
increase in the number of endothelial cells bound to disks, as compared to any
of the
controls.
EXAMPLE 5
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In this example, illustrated are methods according to the present invention:
(a) a
method for manufacturing a medical device; (b) a method of coating a metallic
surface of a
medical device so as to render the coated surface capable of adhering to cells
of
endothelial cell lineage; (c) a method for promoting endothelialization of at
least one
metallic surface of a medical device; and (d) a method for promoting the
adherence of cells
of endothelial cell lineage to a medical device. The methods comprise
contacting at least
one metallic (and more preferably stainless steel) surface of a medical device
with an
effective amount of a biofunctional coating composition under conditions
suitable to
produce a coating on the metallic surface, wherein the btofunctional coating
composition
comprises at least one surface-binding domain and at least one endothelial-
binding
domain; wherein the at least one surface-binding domain comprises a peptide
consisting
essentially of an amino acid sequence selected from the group consisting of
SEQ ID NO:1,
SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, and a combination thereof; wherein the at least one endothelial-
binding
domain comprises a peptide consisting essentially of an amino acid sequence
selected
from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, and a combination thereof; and wherein the at least one surface-
binding
domain is coupled to the at least one endothelial-binding domain. Preferably,
the at least
one surface-binding domain is covalently coupled to the at least one
endothelial- binding
domain via a linker. The at least one surface-binding domain is the component
of the
biofunctional coating composition which is primarily responsible for binding
the
biofunctional coating composition to the one or more surfaces of the medical
device to be
coated.
With respect to these methods according to the present invention, and with
respect
to a biofunctional coating composition according to the present invention, and
wherein at
least one surface of the medical device to be coated comprises more than one
metallic
material (e.g., two different metals; a metal and a metal oxide; a metal and
metal alloy; and
the like), the at least one surface-binding domain in the biofunctional
coating may comprise
= a plurality (two or more) of types of surface-binding domains, wherein each
type of
surface-binding domain has binding specificity for a different surface
material to be coated,
as compared to the other surface-binding domains of which the biofunctional
coating
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composition is comprised. Also with respect to this method according to the
present
invention, and with respect to a biofunctional coating composition according
to the present
invention, the at least one endothelial-binding domain may comprise more than
one type
(e.g., as determined by binding specificity of each type of endothelial-
binding domain; for
example, two or more different peptides, one peptide with binding specificity
for endothelial
cells, the other peptide with binding specificity for endothelial progenitor
cells).
In these methods according to the present invention, when the biofunctional
coating
composition is contacted with the at least one metallic surface of the medical
device to be
coated, either (a) the at least one endothelial-binding domain is bound to
cells of
endothelial cell lineage; or (b) the at least one endothelial-binding domain
is not yet bound
to cells of endothelial cell lineage. With respect to the latter, in a further
step of coating, the
coated surface of the medical device is then contacted with a sufficient
amount of cells of
endothelial cell lineage (in vitro or in vivo), for which the at least one
endothelial-binding
domain has binding specificity, under conditions suitable so that cells of
endothelial cell
lineage bind to the at least one endothelial-binding domain. In one example,
the medical
device may be contacted with cells of endothelial cell lineage (autologous or
from a donor
(e.g., allogeneic or xenogeneic) in vitro for the cells to bind and adhere to
the coated
surface of the device, and subsequently the device is implanted.
In another example, in a method according to the present invention for
promoting
endothelialization of a vascular device, generally one or more metallic
surfaces of the
device to be exposed to vasculature once the device is implanted in an
individual, is the
one or mores surfaces of device desired and selected to be coated by a
biofunctional
coating composition according to the present invention. The method comprises
the steps
of: (a) contacting a biofunctional coating composition according to the
present invention to
at least one surface of a vascular device desired to be endothelialized, so
that the
biofunctional coating composition becomes bound to the at least one metallic
surface, in
forming a coated surface on the device; wherein the biofunctional coating
composition
comprises at least one surface-binding domain coupled to at least one
endothelial-cell
binding domain; and (b) implanting the device into an individual in need of
the device;
wherein cells of endothelial cell lineage (produced by the individual, and
circulating in the
individual's vasculature) contact and attach to the coated surface of the
device (via the
biofunctional coating composition), wherein such contact and attachment
promotes spread
of cells of endothelial cell lineage over the coated surface of the device, in
promoting
endothelialization of the vascular device. Promoting endothelialization on the
implanted
device may further promote one or more of healing of tissue or vasculature
adjacent to the
implanted device, promote incorporation (integration) of the implanted device
into the
adjacent tissue, and reduce occurrence of thrombosis as related to the
implanted device.
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Conventional processes known in the art may be used to apply the biofunctional
coating composition according to the present invention to the one or more
metallic surfaces
of the medical device to be coated (in contacting the biofunctional coating
composition with
the one or more surfaces). Such processes are known to include, but are not
limited to,
dipping, brushing, spraying, vapor deposition, and electro-deposition.
Formulations of the
biofunctional coating composition according to the present invention may
depend on the
process used for coating the medical device. For example, a solution or
suspension
comprising the biofunctional coating composition may be applied through the
spray nozzle
of a spraying device, creating droplets that coat the metallic surface of the
medical device
to be coated. The medical device is allowed to dry, and may then be further
processed
prior to use (e.g., washed in a solution (e.g., water or isotonic buffer) to
remove excess
biofunctional coating composition; by sterilization using any one or methods
known in the
art for sterilizing medical devices; etc.). Altematively, the blofunctional
coating composition
and the medical device may all be sterilized prior to the process, and the
process
performed under sterile conditions.
In another process for applying the biofunctional coating to one or more
metallic
surfaces of a medical device to be coated, the surface of the medical device
to be coated is
dipped into a liquid (e.g., solution or suspension, aqueous or solvent)
containing the
biofunctional coating composition in an amount effective to coat the surface.
For example,
the surface is dipped or immersed into a bath containing the biofunctional
coating
composition. Suitable conditions for applying the biofunctional coating
composition include
allowing the surface to be coated to remain in contact with the liquid
containing the
biofunctional coating composition for a suitable period of time (e.g., ranging
from about 5
minutes to about 12 hours; more preferably, ranging from 15 minutes to 60
minutes), at a
suitable temperature (e.g., ranging from 10 C to about 50 C; more
preferably, ranging
from room temperature to 37 C). The coated medical device may then be further
processed, as necessary for use (washing, sterilization, and the like).
In another process for applying the biofunctional coating to one or more
metallic
surfaces of a medical device to be coated, the biofunctional coating
composition according
to the present invention is formulated in a dry powder (e.g., via air drying
or lyophilizing the
biofunctional coating composition). The powder comprising the biofunctional
coating
composition is then applied using methods known in the art for powder-coating
the surface
of the medical device to be coated. Typically, once applied, such powder
coatings are then
heat-treated (e.g., using infrared heating means) to complete the application
process.
However, these illustrative processes for applying a biofunctional coating
composition to a surface of a medical device are not exclusive, as other
coating and
stabilization methods may be employed (as one of skill in the art will be able
to select the
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compositions and methods used to fit the needs of the particular device and
purpose). For
example, where the surface of the medical device to be coated is metallic in
nature, a
hydrophilic polymer (as previously described herein in more detail) may be
used in
conjunction (either applied simultaneously, or subsequently, to application of
the
biofunctional coating composition according to the present invention) so long
as the
biofunctional coating composition on the metallic surface of the medical
device
substantially retains its function to bind to cells of endothelial cell origin
in promoting one or
more of adherence and endothelialization on the coated surface. In continuing
this
illustration, because of the elastomeric nature of the hydrophilic polymer, it
may add to the
stability of the biofunctional coating composition bound to the surface of the
medical device
should the device be subjected to mechanical forces or stress. Thus, the
methods and
compositions according to the present invention may also be used in
conjunction with drug-
eluting medical devices, or other coating technologies which provide one or
more functional
benefits to medical devices not provided by the biofunctional coating
compositions
according to the present Invention.
Additionally, in a method according to the present invention, a coat
comprising the
biofunctional coating composition may be stabilized, for example, by air
drying or by
lyophilization. However, these treatments are not exclusive, and other coating
and
stabilization methods may be employed. Suitable coating and stabilization
methods are
known in the art. For example, the at least one metallic surface of the
vascular device to
be coated with the biofunctional coating composition of the present invention
may be pre-
treated prior to the coating step so as to enhance one or more of the binding
of the surface-
binding domain to the material comprising the surface to be coated, and the
consistency
and uniformity of the coating. For example, such pretreatment may comprise
etching or
plasma treating the surface material of the device to be coated so as to make
the surface
more hydrophilic, in enhancing the binding of a surface binding domain
comprising some
hydrophobic amino acids in its amino acid sequence which interact with the
hydrophilic
moieties on the surface as part of binding specificity interactions.
In addition, or alternatively, in a further step, the at least one metallic
surface of the
vascular device coated with the biofunctional coating composition of the
present invention
may be treated, subsequent to coating but prior to implantation into an
individual, so as to
enhance endothelialization of the coated surface. For example, a matrix or
layer of a
biological substrate which supports endothelialization, and particularly
growth (including
proliferation) of endothelial cells adhering to the coated surface, may be
added to (e.g.,
overlayed and/or adsorbed onto) the coated surface (for example, prior to or
subsequent to
binding and attachment to the coated surface by cells of endothelial cell
lineage).
Components of such layer or matrix can include a vascular biologic comprising
one or
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more of collagen (e.g., type IV and/or type V), vitrogen, laminin, entactin,
fibronectin,
glycans (e.g., proteoglycans, glycosaminoglycans), and growth factors
supporting
endothelial cell growth (e.g., VEGF, EGF, FGF, heparin-binding epidermal-like
growth
factor, and the like).
Thus, in accordance with these methods of the present invention, a medical
device
may first be treated by a process which enhances binding (e.g., by increasing
the
hydrophilicity of, or the molecular adhesiveness of, the at least one metallic
surface of the
device) of the biofunctional coating composition to the at least one treated
surface of the
device; contacting the biofiunctional coating with the at least one treated
surface in binding
the biofunctional coating composition to the at least one treated surface in
forming a coated
surface. The method may further comprise contacting a vascular biologic with
the coated
surface in an amount effective to promote endothelialization on the coated
surface. The
methods may further comprise, prior to the implantation of the device, a step
of contacting
the coated device with cells of endothelial cell lineage in promoting one or
more of
attachment or adherence of the cells of the endothelial cell lineage, support
for endothelial
cell growth, and support for endothelial cell differentiation. For example,
cells of the
endothelial cell lineage may be purified and isolated using methods known in
the art. For
example, progenitor endothelial cells may be isolated from human peripheral
blood using
magnetic separation comprising magnetic beads coated with antibody to CD34. In
another
example, human umbilical vein endothelial cells may be isolated from
umbilical, cords by
collagenase treatment of the blood vessel walls to release the endothelial
cells, which may
then be cultured in suitable supporting cuiture medium known in the art.
EXAMPLE 6
It is apparent to one skilled in the art, that based on the amino acid
sequence of the
peptide comprising a preferred endothelial-binding domain and/or surface
binding domain
used in accordance with the present invention, that polynucleotides (nucleic
acid
molecules) encoding such a peptide (or variants thereof as described herein)
may be
synthesized or constructed, and that such a peptide may be produced by
recombinant DNA
technology as a means of manufacture (e.g., in culture) and/or in vivo
production by
introducing such polynucleotides in vivo. For example, it is apparent to one
skilled in the
art that more than one polynucleotide sequence can encode a peptide consisting
essentially of an amino acid sequence of SEQ ID NO:3 according to the present
invention,
and that.such polynucleotides may be synthesized on the bases of triplet
codons known to
encode the amino acids of a peptide consisting essentially of the amino acid
sequence of
SEQ ID NO:3, third base degeneracy, and selection of triplet codon usage
preferred by the
host cell, typically a prokaryotic cell or eukaryotic cell (e.g., bacterial
cells such as E. coli;
CA 02631294 2008-05-27
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yeast cells; mammalian cells; avian cells; amphibian cells; plant cells; fish
cells; and insect
cells; whether located in vitro or in vivo.) in which expression is desired.
It would be routine
for one skilled in the art to generate the degenerate variants described
above, for instance,
to optimize codon expression for a particular host (e.g., change codons in the
bacteria
mRNA to those preferred by a mammalian, plant or other bacterial host such as
E. co/1).
For purposes of illustration only, and not limitation, provided as SEQ ID
NO:48 is a
polynucleotide encoding an amino acid sequence of SEQ ID NO:3, from which, as
apparent to one skilled in the art, codon usage will generally apply to
polynucleotides
encoding a preferred surface-binding domain comprising a peptide consisting
essentially of
the amino acid sequence illustrated in SEQ ID NO:3. Also provided as SEQ ID
NO:49 is a
polynucleotide encoding an amino acid sequence of SEQ ID NO:19, from which, as
apparent to one skilled in the art, codon usage will generally apply to
polynucleotides
encoding a preferred endothelial-binding domain comprising a peptide
consisting
essentially of the amino acid sequence illustrated in SEQ ID NO:19. Thus, for
example,
using SEQ ID NO:48 in relation to SEQ ID NO:3 and SEQ ID NO:49 in relation to
SEQ ID
NO:19, one skilled in the art could readily construct a polynucleotide
encoding variants of
the amino acid sequence illustrated in SEQ ID NO:3 or SEQ ID NO:19, or
encoding any
one or more of the other amino acid sequences provided by the present
invention (e.g.,
SEQ ID NOs: 1-2, and 4-18, and 20-47).
In one illustrative embodiment, provided is a prokaryotic expression vector
containing a polynucelotide encoding a binding domain for use in accordance
with the
present invention; and its use for the recombinant production of a peptide
comprising the
binding domain. In one example, the polynucleotide may be positioned in a
prokaryotic
expression vector so that when the peptide is produced in bacterial host
cells, it is
produced as a fusion protein with other amino acid sequence (e.g., which
assist in
purification of the peptide; or as recombinantly coupled to another binding
domain
according to the present invention). For example, there are sequences known to
those
skilled in the art which, as part of a fusion protein with a peptide desired
to be expressed,
facilitates production in inclusion bodies found in the cytoplasm of the
prokaryotic cell used
for expression and/or assists in purification of fusion proteins containing
such sequence.
Inclusion bodies may be separated from other prokaryotic cellular components
by methods
known in the art to include denaturing agents, and fractionation (e.g.,
centrifugation,
column chromatography, and the like). In another example, there are
commercially
available vectors into which is inserted a desired nucleic acid sequence of
interest to be
expressed as a protein or peptide such that upon expression, the gene product
also
contains a plurality of terminal histidine residues ("His tags") that can be
utilized in the
purification of the gene product using methods standard in the art.
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It is apparent to one skilled in the art that a nucleic acid sequence encoding
a
binding domain (endothelial-binding domain, or surface-binding domain, or a
combination
thereof) comprising a peptide for use according to the present invention can
be inserted
into, and become part of a, nucleic acid molecule comprising a plasmid, or
vectors other
than plasmids; and other expression systems can be used including, but not
limited to,
bacteria transformed with a bacteriophage vector, or cosmid DNA; yeast
containing yeast
vectors; fungi containing fungal vectors; insect cell lines infected with
virus (e. g.
baculovirus); and mammalian cell lines having introduced therein (e.g.,
transfected or
electroporated with) plasmid or virai expression vectors, or infected with
recombinant virus
(e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, etc.).
Successful
expression of the peptide requires that either the recombinant nucleic acid
molecule
comprising the encoding sequence of the peptide, or the vector itself, contain
the
necessary control elements for transcription and translation which is
compatible with, and
recognized by the particular host system used for expression.
Using methods known in the art of molecular biology, including methods
described
above, various promoters and enhancers can be incorporated into the vector or
the
recombinant nucleic acid molecule comprising the encoding sequence to increase
the
expression of the peptide, provided that the increased expression of the
peptide is
compatible with (for example, non-toxic to) the particular host cell system
used. As
apparent to one skilled in the art, the selection of the promoter will depend
on the
expression system used. Promoters vary in strength, i.e., ability to
facilitate transcription.
Generally, for the purpose of expressing a cloned gene, it is desirable to use
a strong
promoter in order to obtain a high level of transcription of the gene and
expression into
gene product. For example, bacterial, phage, or plasmid promoters known in the
art from
which a high level of transcription has been observed in a host cell system
comprising E.
coli include the lac promoter, trp promoter, T7 promoter, recA promoter,
ribosomal RNA
promoter, the P<sub>R</sub> and P<sub>L</sub> promoters, IacUV5, ompF, bia, lpp, and the
like, may
be used to provide transcription of the inserted nucleotide sequence encoding
the synthetic
peptide. Commonly used mammalian promoters in expression vectors for mammalian
expression systems are the promoters from mammalian viral genes. Examples
include the
SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major
late
promoter, herpes simplex virus promoter, and the CMV promoter.
In the case where expression of the peptide may be lethal or detrimental to
the host
cells, the host cell strain/line and expression vectors may be chosen such
that the action of
the promoter is inhibited until specifically induced. For example, in certain
operons the
addition of specific inducers is necessary for efficient transcription of the
inserted DNA (e.g.,
the lac operon is induced by the addition of lactose or isopropylthio-beta-D-
galactoside
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("IPTG"); trp operon -is induced when tryptophan is absent in the growth
media; and
tetracycline can be use in mammalian expression vectors having a tet sensitive
promoter).
Thus, expression of the peptide may be controlled by culturing transformed or
transfected
cells under conditions such that the promoter controlling the expression from
the encoding
sequence is not induced, and when the cells reach a suitable density in the
growth medium,
the promoter can be induced for expression from the encoding sequence. Other
control
elements for efficient gene transcription or message translation are well
known in the art to
include enhancers, transcription or translation initiation signals,
transcription termination
and polyadenylation sequences, and the like.
The foregoing description of the specific embodiments of the present invention
have
been described in detail for purposes of illustration. In view of the
descriptions and
illustrations, others skilled in the art can, by applying, current knowledge,
readily modify
and/or adapt the present invention for various applications without departing
from the basic
concept of the present invention; and thus, such modifications and/or
adaptations are
intended to be within the meaning and scope of the appended claims.
What is claimed is:
38
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