U.S. patent application number 16/242238 was filed with the patent office on 2019-05-02 for compositions and methods for tissue repair.
The applicant listed for this patent is The Regents of the University of California, TransTarget, Inc.. Invention is credited to Manley Huang, James W. Larrick, Randall J. Lee, Shirley Mihardja.
Application Number | 20190125891 16/242238 |
Document ID | / |
Family ID | 40094368 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190125891 |
Kind Code |
A1 |
Lee; Randall J. ; et
al. |
May 2, 2019 |
COMPOSITIONS AND METHODS FOR TISSUE REPAIR
Abstract
The present invention provides compositions and methods for
targeting an extracellular matrix derived (EMD) peptide
predominantly to an injured tissue, as opposed to an uninjured
tissue in vivo. The targeted EMD peptide facilitates the repair
and/or regeneration of the injured tissue by providing a surface
for cells to attach and grow, thereby facilitating the repair
and/or regeneration of the injured tissue.
Inventors: |
Lee; Randall J.;
(Hillsborough, CA) ; Mihardja; Shirley; (Daly
City, CA) ; Huang; Manley; (Palo Alto, CA) ;
Larrick; James W.; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TransTarget, Inc.
The Regents of the University of California |
Burlingame
Oakland |
CA
CA |
US
US |
|
|
Family ID: |
40094368 |
Appl. No.: |
16/242238 |
Filed: |
January 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15299865 |
Oct 21, 2016 |
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16242238 |
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14514331 |
Oct 14, 2014 |
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15299865 |
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12601522 |
May 24, 2010 |
8889140 |
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PCT/US2008/065303 |
May 30, 2008 |
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14514331 |
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60941051 |
May 31, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 14/4716 20130101; C07K 2319/00 20130101; C07K 14/4721
20130101; A61K 47/6811 20170801; C07K 14/70525 20130101; C07K
16/2833 20130101; C07K 14/7151 20130101; A61K 38/00 20130101; C07K
14/70553 20130101; C12N 9/6475 20130101; A61K 47/6843 20170801;
C07K 14/52 20130101; C07K 2318/00 20130101; C07K 14/70564 20130101;
A61K 47/6849 20170801; C07K 14/78 20130101; A61P 9/10 20180101;
A61K 47/68 20170801; C07K 7/08 20130101; C07K 14/70542
20130101 |
International
Class: |
A61K 47/68 20170101
A61K047/68; C07K 14/47 20060101 C07K014/47; C07K 14/78 20060101
C07K014/78; C07K 14/705 20060101 C07K014/705; C07K 14/715 20060101
C07K014/715; C07K 16/28 20060101 C07K016/28; C12N 9/64 20060101
C12N009/64; C07K 7/08 20060101 C07K007/08; C07K 14/52 20060101
C07K014/52 |
Claims
1. A composition for repair of an injured tissue, the composition
comprising an injury-associated antigen-specific binding component
conjugated to an extracellular matrix derived (EMD) peptide,
wherein the injury-associated antigen-specific binding component
comprising a molecule that specifically binds an antigen expressed
following tissue injury, and wherein the EMD peptide is a peptide
or a concatamer of a peptide that ranges in length from about 8
amino acid residues to about 100 amino acid residues, and exhibits
the ability to: (i) activate cells; (ii) attach cells; and (iii)
exhibits a chemotropic property.
2. The composition of claim 1, wherein the EMD peptide further
exhibits an angiogenic property.
3. The composition of claim 1, wherein the EMD peptide is a
concatamer.
4. The composition of claim 1, wherein the EMD peptide is selected
from the group consisting of a Hep III peptide as shown in SEQ ID
NO:2, and an RGD peptide as shown in SEQ ID NO:4.
5. The composition of claim 1, wherein the EMD peptide is derived
from a protein selected from the group consisting of collagen,
laminin, tenascin-C, fibronectin, fibrin, and elastin.
6. The composition of claim 1, wherein the injury-associated
antigen is a member selected from the group consisting of: myosin
light chain, myosin heavy chain, troponin I, VCAM-1, ICAM-1,
P-selectin, E-selectin, L-selectin, Mo1/CD18, TNF receptor-1, TNF
receptor-2, caspase-3, VAP-1, annexin, osteopontin, thrombospondin,
laminin, fibronectin, and collagen.
7. The composition of claim 1, wherein the injury-associated
antigen-specific binding component and the EMD peptide is
chemically or recombinantly conjugated.
8. The composition of claim 1, wherein the injury-associated
antigen-specific binding component is an antibody.
9. The composition of claim 8, wherein the antibody is selected
from the group consisting of: a (Fab)'2 fragment, an ScFv, a human
antibody, and a humanized antibody.
10. The composition of claim 1, wherein the injury-associated
antigen-specific binding component is an antibody mimetic.
11. The composition of claim 1, wherein the tissue is selected from
the group consisting of: cardiac, cartilage, bone, bone marrow,
dental, hepatic, neural, vascular, and renal.
12. The composition of claim 1, further comprising a
pharmaceutically acceptable excipient.
13. A method for repairing an injured tissue, the method comprising
administering to a mammalian subject a composition having an
injury-associated antigen-specific binding component conjugated to
an extracellular matrix derived (EMD) peptide, wherein the
injury-associated antigen-specific binding component comprising a
molecule that specifically binds an antigen expressed following
tissue injury, and wherein the EMD peptide is a peptide or a
concatamer of a peptide that ranges in length from about 8 amino
acid residues to about 100 amino acid residues, and exhibits the
ability to: (i) activate cells; (ii) attach cells; and (iii)
exhibits a chemotropic property and wherein said composition is
administered in an amount sufficient to facilitate repair or
regeneration of the injured tissue.
14. The method of claim 13, wherein the EMD peptide further
exhibits an angiogenic property.
15. The method of claim 13, wherein the EMD peptide is a
concatamer.
16. The method of claim 13, wherein the EMD peptide is selected
from the group consisting of a Hep III peptide as shown in SEQ ID
NO:2 and an RGD peptide as shown in SEQ ID NO:4.
17. The method of claim 13, wherein the EMD peptide is derived from
a protein selected from the group consisting of collagen, laminin,
tenascin-C, fibronectin, fibrin, and elastin.
18. The method of claim 13, wherein the injury-associated antigen
is a member selected from the group consisting of: myosin light
chain, myosin heavy chain, troponin I, caspase-3, VCAM-1, ICAM-1,
P-selectin, E-selectin, L-selectin, Mo1/CD18, TNF receptor-1, TNF
receptor-2, VAP-1, annexin, osteopontin, thrombospondin, laminin,
fibronectin, and collagen.
19. The method of claim 13, wherein the injury-associated
antigen-specific binding component is an antibody.
20. The method of claim 19, wherein the antibody is selected form
the group consisting of: a (Fab)'2 fragment, an ScFv, a human
antibody, and a humanized antibody.
21. The method of claim 13, wherein the injury-associated
antigen-specific binding component is an antibody mimetic.
22. The method of claim 13, wherein the injury-associated
antigen-specific binding component and the EMD peptide is
chemically or recombinantly conjugated.
23. The method of claim 13, wherein the tissue is selected from the
group consisting of: cardiac, cartilage, bone, bone marrow, dental,
neural, vascular, hepatic and renal.
24. The method of claim 13, wherein the mammalian subject is a
human.
25. The method of claim 24, wherein the human has been diagnosed
with ischemic cardiovascular disease.
26. The method of claim 25, wherein the ischemic cardiovascular
disease is selected from the group consisting of: myocardial
infarction, peripheral vascular disease, stroke, myocardial
conduction disorder, and congestive heart failure.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/299,865, filed Oct. 21, 2016, which is a Continuation of
U.S. application Ser. No. 14/514,331, filed Oct. 14, 2014, with is
a Divisional of U.S. application Ser. No. 12/601,522, filed May 24,
2010 (now U.S. Pat. No. 8,889,140, issued Nov. 18, 2014), as the
U.S. National Stage Entry of PCT/US2008/065303, filed May 30, 2008,
which claims benefit of U.S. Provisional Patent Application No.
60/941,051, filed on May 31, 2007, the disclosures of each are
hereby incorporated by reference.
REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE
[0002] The Sequence Listing written in file SEQTXT
088547-1120991-000940US.txt, created on Jan. 4, 2019, 1,423 bytes,
machine format IBM-PC, MS-Windows operating system, is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0003] This invention relates to compositions and methods for the
repair and/or regeneration of an injured tissue by targeting an
extracellular matrix derived (EMD) peptide to the injured
tissue.
BACKGROUND OF THE INVENTION
[0004] Organ or tissue failure remains a frequent, costly, and
serious problem in health care despite advances in medical
technology. Available treatments include organ transplantation,
surgical reconstruction, mechanical devices (e.g. pace makers and
kidney dialysis machines), drug therapy, and tissue engineering.
These treatments, however, are not perfect solutions. Organ
transplantation is limited by the availability of donors and
complications such as tissue rejection. Surgical reconstruction is
costly, highly invasive, and not always effective. Mechanical
devices cannot functionally replace an organ, for example, dialysis
machines can only help to remove some of the metabolic waste from
the body. Likewise, maintaining drug concentration levels in vivo,
comparable to the control systems of the body, are difficult to
achieve. Finally, tissue engineering while promising, is encumbered
by size limitations that typically require the assembly of the
engineered tissue in vitro, followed by surgical implantation in
vivo.
[0005] Recent advances in the medical, biological and physical
sciences have given rise to new strategies for tissue engineering.
For example, in one approach to engineering artificial skin, dermal
fibroblasts are suspended in a polymer mesh, whereas another
approach involves fibroblasts seeded in a collagen gel, which is
then coated with a layer of human epidermal cells (see, e.g.,
Vogel, et al., Ann. Rev. Biomed. Eng. 5:441-463 (2003)). The use of
extracellular matrix (ECM) molecules, such as collagen, is an area
of active research in tissue engineering as a way to facilitate the
regeneration or repair of damaged tissue in vivo.
[0006] Although many different compounds and cellular constituents
are important for repair and regeneration of injured tissue, the
ECM is of particular interest. ECM proteins play a pivotal role in
cell adhesion, cell signaling, cell proliferation, and regulating
tissue organization and differentiation. The main components of the
ECM include structural proteins such as collagen and elastin,
adhesive proteins such as laminin, fibronectin, and collagen IV,
anti-adhesive proteins such as tenascin, thrombospondin, and
osteopontin, and proteoglycans (see, e.g., Corda, et al., Heart
Failure Reviews 5:119-130 (2000)). ECM molecules also have the
ability to activate various intracellular signaling pathways,
depending on the nature of the adhesion complex formed between the
cell and the ECM protein. (see, e.g., Vogel, et al., Ann Rev.
Biomed. Eng. 5:441-463 (2003).
[0007] Cells adhere to and interact with the ECM via specific
receptors. One class of such receptors is the integrin proteins,
which make up a large family of transmembrane heterodimer receptors
composed of alpha and beta subunits. There are at least fourteen
known alpha subunits and eight known beta subunits that associate
in various combinations to form at least twenty distinct receptors.
One class of integrin receptors known to interact with ECM proteins
are integrins having the .beta.1 subunit, also known as very late
antigen (VLA) proteins. VLA proteins are expressed on a variety of
cells and serve as receptors for many ECM proteins including
collagen, fibronectin, and laminin. Binding of ECM proteins by
their cognate receptor (e.g. integrin receptors) is important for
both promoting stable interactions between the cells and their
environment, and initiating intracellular signaling pathways for a
variety of cellular processes important for tissue repair and/or
regeneration. For example, ECM-integrin signaling has been shown to
play a role in cell migration, cell survival, cellular
proliferation, and cellular differentiation. See, Vogel et al.
(2003).
[0008] The present invention fulfills a need in the area of tissue
repair and regeneration that is not currently provided for in the
art. In particular, the present invention provides for compositions
and methods useful for the repair and regeneration of injured
tissues in vivo by targeting an extracellular matrix derived (EMD)
peptide to an injured tissue where the EMD peptide can provide a
surface for cell attachment and growth, thereby facilitating the
repair and regeneration of the injured tissue.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides compositions and methods for
the repair and/or regeneration of an injured tissue, comprising a
targeted extracellular matrix derived (EMD) peptide conjugated to
an injury-associated antigen-specific binding component for the
repair and/or regeneration of an injured tissue. The targeted EMD
peptide can provide for a surface at the injured tissue whereby
cells can attach and grow, thereby facilitating the repair of the
injured tissue in vivo.
[0010] The present inventors have surprisingly discovered that EMD
peptides ranging in size from about 8 amino acids to about 100
amino acids can mimic the properties of full length proteins from
which the EMD peptides are derived, as described herein. These
short peptides are advantageous because they are easy to
manufacture, have reduced immunogenicity, lower toxicity, show
greater homogeneity and reproducibility in conjugation to an
injury-associated antigen-specific binding component, and are
easier and more cost effective to manufacture as pharmaceutical
compositions as compared to the full-length proteins or longer
peptides as disclosed herein.
[0011] One aspect of the invention is a composition comprising an
injury-associated antigen-specific binding component conjugated to
an EMD peptide. The injury-associated antigen-specific binding
component comprises a molecule that specifically binds an
injury-associated antigen that is either expressed or unmasked as a
result of tissue injury. The EMD peptide is a peptide or a
concatamer of a peptide that ranges in length from about 8 amino
acid residues to about 100 amino acid residues. The EMD peptides of
the invention exhibit: i) cell attachment; ii) cell activation; and
iii) chemotropic properties.
[0012] In some embodiments, the EMD peptide further exhibits a
positive angiogenic property. In some embodiments, the EMD peptide
is selected from the group consisting of a Hep III peptide as shown
in SEQ ID NO:2, and RGD as shown in SEQ ID NO:4. In some
embodiments, the EMD peptide is a concatamer of a peptide having a
length from about 8 amino acids to about 100 amino acids. In some
embodiments, the EMD peptide is derived from a protein selected
from the group consisting of collagen, laminin, tenascin-C,
fibronectin, fibrin, fibrinogen, and elastin.
[0013] In some embodiments, the injury-associated antigen is
selected from the group consisting of: a myosin light chain, a
myosin heavy chain, a troponin I, a caspase-3, a VCAM-1, an ICAM-1,
a P-selectin, an E-selectin, a L-selectin, a Mo1/CD18, a
TNF-.alpha., a Tumor Necrosis Factor (TNF) receptor-1, a TNF
receptor-2, a VAP-1, an annexin, an osteopontin, an osteonectin, a
thrombospondin, a laminin, a fibronectin, a membrane bound stem
cell factor, a hyaluron, an elastin, or a collagen molecule.
[0014] In some embodiments of the invention, the injury-associated
antigen-specific binding component is an antibody. In some
embodiments, the antibody is an intact antibody or a modified
antibody. In some embodiments, the antibody is selected from the
group consisting of: an IgG, IgM, IgD, IgE, a human antibody, a
humanized antibody, a mammalian antibody, a non-human mammalian
antibody, a non-mammalian antibody, and a chimeric antibody. In
some embodiments the antibody is an antibody fragment. In some
embodiments, the antibody fragment is selected from the group
consisting of: an (Fab)'2 fragment, an Fab fragment, a scFv, a
minibody, and a nanobody. In some embodiments, the
injury-associated antigen-specific binding component is an antibody
mimetic. In some embodiments the antibody mimetic is selected from
the group consisting of: a non-antibody polypeptide, an anticalin,
a polypeptide with a fibronectin type III domain and a
non-glycosylated single chain polypeptide composed of two or more
independent binding domains.
[0015] In some embodiments, the injury associated antigen-specific
binding component and the EMD peptide are linked through
recombinant or chemical means. In some embodiments, the conjugation
is completed using recombinant methodologies (e.g. fusion
proteins). In some embodiments, the conjugation is completed using
a chemical means selected from the group consisting of: covalent
bonding, disulfide bonding, hydrogen bonding, electrostatic
bonding, conformational bonding, or homobifunctional or
heterobifunctional cross-linkers.
[0016] In some embodiments, the injured tissue is selected from the
group consisting of: cardiac, cartilage, bone, bone marrow, dental,
hepatic, neural, vascular, and renal tissue. In some embodiments,
the composition of the invention further comprises a pharmaceutical
excipient.
[0017] Another aspect of the present invention provides a method
for repairing and/or regenerating an injured tissue. The method
involves administering to a subject a composition comprising an
injury-associated antigen-specific binding component conjugated to
an EMD peptide. The injury-associated antigen-specific binding
component comprises a molecule that specifically binds an
injury-associated antigen that is either expressed or unmasked as a
result of tissue injury. The EMD peptide is a peptide or a
concatamer of a peptide that ranges in length from about 8 amino
acid residues to about 100 amino acid residues. The EMD peptides of
the invention exhibit i) cell attachment; ii) cell activation; and
iii) chemotropic properties.
[0018] In some embodiments, the subject is a mammal. In some
embodiments, the mammal is a human.
[0019] In some embodiments, the tissue injury is selected from the
group consisting of: ischemic injury, perfusion/re-perfusion
injury, congestive heart failure dental decay, bone fracture,
cartilage damage, inflammation, chemotherapy, radiation injury,
thermal injury, and trauma. In some embodiments, the injury is an
ischemic cardiovascular injury. In some embodiments, the ischemic
cardiovascular injury is selected from the group consisting of:
myocardial infarction, peripheral vascular disease, stroke,
myocardial conduction disorder, and congestive heart failure.
[0020] In some embodiments, the route of administration of the
targeted EMD peptide is via injection. In some embodiments, the
route of injection is selected from the group consisting of:
intravascular, intra-muscular, intra-peritoneal, intra-ocular,
subcutaneous, intra articular, and direct injection into the
injured tissue. In some embodiments, the route of administration is
selected from the group consisting of: oral, topical, vaginal, and
rectal.
[0021] Each of the embodiments of the invention as described herein
can be combined with any aspect of the invention as described
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1C show the ability of different EMD peptides at
various concentrations to promote cell attachment in vitro. FIG. 1A
shows cell attachment of HUVEC cells on a substrate coated with Col
IV (positive control), Hep I, or Hep III at 20, 50, and 100
.mu.g/ml, compared to BSA at 2 mg/ml. The results show that the
positive control Col IV and the Hep III peptide were significantly
better at promoting cell attachment compared to the BSA control,
and that the Hep I peptide was not significantly different from
that of the BSA control at (p<0.05). These results demonstrate
that the Hep III peptide exhibits a cell attachment property. FIGS.
1B and 1C show the attachment of HUVEC cells to a substrate coated
with fibronectin (positive control) at 10 and 100 .mu.g/ml, RGD
peptide (FIG. 1B) or FCHV peptide (FIG. 1C) at 20, 50, and 100
.mu.g/ml, compared to BSA control at 2 mg/ml. The results show that
the positive control fibronectin and the RGD peptide were
significantly better at promoting cell attachment compared to the
BSA control, and that the FCHV peptide was not significantly
different from that of the BSA control. These results demonstrate
that the RGD peptide exhibits a cell attachment property.
[0023] FIGS. 2A-2C show the ability of different EMD peptides at
various concentrations to activate cells by stimulating the
proliferation of cells in vitro. FIG. 2A shows cell proliferation
of HUVEC cells cultured on substrates coated with Col IV (positive
control), Hep I, or Hep III at 20, 50, and 100 .mu.g/m1 compared to
BSA at 2 mg/ml. The results show that the positive control Col IV,
and the EMD peptides Hep I and Hep III were significantly better at
promoting cell proliferation, at each concentration tested,
compared to the BSA control at (p<0.05). These results
demonstrate that both the Hep I and the Hep III EMD peptides
exhibit a cell activation property as evidenced by their ability to
stimulate cell proliferation in vitro. FIGS. 2B and 2C show the
proliferation of HUVEC cells on substrates coated with fibronectin
(positive control) at 10 and 100 .mu.g/ml, RGD peptide (FIG. 2B)
and FCHV peptide (FIG. 2C) at 20, 50, and 100 .mu.g/ml compared to
BSA control at 2 mg/ml. The results show that the positive control
fibronectin and the RGD peptide were significantly better at
stimulating cellular proliferation compared to the BSA control,
while the FCHV peptide was not statistically different as compared
to the BSA control. These results demonstrate that the RGD peptide,
but not the FCHV peptide, exhibits a cell activation property as
evidenced by the ability of the RGD peptide to stimulate cell
proliferation compared to BSA control.
[0024] FIGS. 3A-3B show the chemotropic property of different EMD
peptides to promote haptotactic migration of HUVEC cells in vitro.
FIG. 3A shows haptotactic migration of HUVEC cells on membranes
coated with various concentrations of Col IV (positive control),
Hep I, and Hep III using a modified Boyden chamber as detailed in
Example 3. FIG. 3B shows the results for haptotactic migration of
HUVEC cells on membranes coated with fibronectin (positive
control), RGD peptide or FCHV peptide. The concentrations of
protein or peptide tested were from 500 ng/ml to 300 .mu.g/ml, and
were compared to the migration on untreated membranes. The results
show that the positive controls Col IV and fibronectin, as well as
the EMD peptides Hep I, Hep III and RGD promoted haptotactic
migration compared to that of untreated membranes at (p<0.05).
The EMD peptide FCHV did not promote haptotactic migration of HUVEC
cells compared to that of untreated membranes. These results
demonstrate that the EMD peptides Hep I, Hep III, and RGD each
exhibit a chemotropic property as evidenced by their ability to
stimulate haptotactic migration of HUVEC cells as detailed in
Example 3.
[0025] FIGS. 4A-4B illustrate the successful targeting of the EMD
peptide Hep III conjugated to an anti-myosin heavy chain antibody
to injured myocardial tissue in vivo. FIG. 4A shows the targeted
antibody (visualized by the brown staining) in the region of the
myocardial infarct. FIG. 4B is a negative control.
[0026] FIG. 5 shows the ability of the targeted Hep III EMD peptide
to induce angiogenesis in injured myocardial tissue compared to
antibody alone, or PBS control. The results demonstrate that the
targeted Hep III peptide and the targeted FCHV peptide exhibit an
angiogenic property when targeted to an injured myocardial tissue
in vivo as compared to the PBS control at (p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is based on the surprising discovery
that an EMD peptide, having a length from about 8 amino acids to
about 100 amino acids, exhibiting: i) cell attachment; ii) cell
activation; and iii) chemotropic properties, conjugated to an
injury-associated antigen-specific binding component can be
predominantly targeted to an injured tissue as opposed to uninjured
tissue in vivo for the repair and/or regeneration of the injured
tissue.
I. Definitions
[0028] As used herein, the following terms have the meanings
ascribed to them below unless otherwise specified.
[0029] The phrase "injured tissue" as used herein refers to any
alteration in a tissue that results in the loss or damage of cells
comprising that tissue, or any alteration to the tissue that
results in the organ, of which the tissue is a part, performing
less efficiently as compared to before the alteration. Exemplary
non-limiting tissue injuries may include ischemia,
perfusion/re-perfusion, cardiovascular injury, hypoxia,
hypertrophy, hyperplasia, trauma, bone fracture, inflammation,
dental decay, apoptosis, chemotherapy, radiation injury, thermal
injury and fibrosis.
[0030] The phrase "repair of an injured tissue" as used herein
refers to the process by which an EMD peptide can provide a surface
for the attachment, migration, proliferation and differentiation of
cells, thereby facilitating the repair and/or regeneration of the
injured tissue. Non-limiting examples evidencing repair and/or
regeneration of an injured tissue may include: the more rapid
healing, reduced scarring or decreased fibrosis in an injured
tissue treated with an EMD peptide, as compared to the healing of
the injured tissue in the absence of the EMD peptide.
[0031] The term "injury-associated antigen" as used herein refers
to a polypeptide, carbohydrate, or glycoprotein present on the
surface of a cell in an injured tissue. The antigen may be
predominantly expressed on the surface of a cell following tissue
injury, or it may be an antigen that is exposed or unmasked as a
result of the tissue injury. For example, injuries to the
myocardium caused by ischemic heart disease can lead to exposure of
cardiac antigens such as myosin light chain, myosin heavy chain,
and troponin I.
[0032] The term "binding component" as used herein refers to a
molecule (e.g., a polypeptide, or a nucleic acid, etc.) that
specifically binds to an injury associated antigen. As used herein,
the term "specifically" or "specific binding" typically means at
least a 2-fold increase in binding over background, preferable
greater than a 10-fold increase, and most preferred at least a
100-fold increase or greater over that of background. Specific
binding between an injury associated antigen and a binding
component generally means an affinity of 10.sup.6 M.sup.-1 or
stronger, preferable at least 10.sup.8 M.sup.-1 or stronger.
Specific binding between a protein binding component and an injury
associated antigen can be determined using any binding assay known
in the art, including but not limited to gel electrophoresis,
western blot, ELISA, flow cytometry, and immunohistochemistry.
[0033] The term "polypeptide" as used herein refers to an amino
acid polymer having at least two amino acid residues.
[0034] The term "antibody" refers to a polypeptide encoded by an
immunoglobulin gene or functional fragments thereof that
specifically binds to and recognizes an antigen (e.g., a cardiac
antigen such as myosin light chain or troponin I). The antibody is
comprised of at least one binding domain formed from the folding of
variable domains of an antibody molecule to form three-dimensional
binding spaces with an internal surface shape and charge
distribution complementary to the features of an antigenic
determinant of an antigen which allows an immunological reaction
with the antigen. An antigenic determinant is that portion of an
antigen molecule that determines the specificity of the antigen
antibody reaction. Antibodies as used herein include naturally
occurring as well as recombinant proteins comprising antigen
specific binding domains, as well as antibody fragments including
Fab, Fab', F(ab)2, F(ab')2 fragments, scFv, minibodies and
nanobodies.
[0035] The recognized immunoglobulin genes include variable region
and constant region genes. The constant region genes include kappa,
lambda, alpha, gamma, delta, epsilon, and mu. Immunoglobulin light
chains are classified as either kappa or lambda. Immunoglobulin
heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
which define the immunoglobulin classes, IgG, IgM, IgA, IgD and
IgE, respectively.
[0036] An exemplary immunoglobulin (whole antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100-110 or more amino
acids primarily responsible for antigen recognition.
[0037] The term "antibody mimetic" refers to non-antibody molecule
comprising at least one binding component that specifically binds
to an antigen. Antibody mimetics are typically polypeptides or
proteins comprising one or more regions that are amenable to
specific or random sequence variation, such that the polypeptide or
protein specifically binds to an antigen of interest (e.g. myosin
light chain, or troponin I).
[0038] The term "extracellular matrix derived (EMD) peptide" or
"EMD peptide" as used herein, refers to an amino acid polymer
having between about 8 and about 100 amino acid residues, derived
from an extracellular matrix protein. Non-limiting exemplary
extracellular matrix proteins that can be used to derive an EMD
peptide for use with the invention may include fibrinogen, fibrin,
fibronectin, collagen (types I, II, III and IV), laminin, tenascin,
elastin osteonectin, and osteopontin. In some embodiments, a
peptide as used in the invention may have about 8, 9, 10, 12, 14,
16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or about 100 amino acid residues.
[0039] The EMD peptide can be a naturally occurring fragment or
cleavage product of an extracellular matrix protein, or it may be a
peptide resulting from enzymatic digestion of a peptide using
proteases such as trypsin. An EMD peptide can also be a
recombinantly synthesized peptide corresponding to a portion of a
naturally occurring extracellular matrix protein. EMD peptides
suitable for use with the present invention can also have
conservative variations or substitutions compared to that of the
wild-type protein from which the peptide is derived. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles. Typically conservative substitutions for one
another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0040] The term "cell attachment" or "attach cells" refers to the
ability of an EMD peptide to support the adherence of a cell to a
surface coated with the EMD peptide. Cell attachment can occur
through an interaction of the EMD peptide with a receptor expressed
by the cell, (e.g., integrin receptors). Cell attachment property
of an EMD peptide can be shown by any suitable assay known in the
art. Cell attachment property of an EMD peptide can be demonstrated
by showing that cells will adhere with a detectably greater level
to a surface coated with an EMD peptide as compared to surface
coated with a control protein (e.g., BSA). An exemplary test that
can be used to determine if an EMD peptide exhibits cell attachment
property is detailed in Example 1.
[0041] The term "cell activation property" or "activate cells"
refers to the ability of an EMD peptide to stimulate cellular
proliferation. Any test suitable for detecting an increase in
cellular proliferation of a cell contacted with an EMD peptide as
compared to the cell proliferation in the absence of the peptide,
or in the presence of a control protein (e.g. BSA) can be used to
determine if an EMD peptide exhibits cell activation property. An
exemplary test for determining is an EMD peptide exhibits cell
activation property is detailed in Example 2.
[0042] The term "chemotropic property" as used herein refers to the
property of an EMD peptide to support the migration of a cell over
a surface coated with the EMD peptide. Chemotropic property of an
EMD can be demonstrated using any test suitable in the art to
demonstrate that a cell migrates over a surface coated with the EMD
peptide as compared to an untreated surface. An exemplary test to
determine if an EMD peptide exhibits chemotropic property is
detailed in Example 3.
[0043] The term "angiogenic property" refers to the ability of an
EMD peptide to stimulate angiogenesis in an injured tissue.
Angiogenesis can be measured as an increase in the vascular area of
an injured tissue contacted with an EMD peptide, compared to the
vascular area in an injured tissue not contacted with the EMD
peptide. A procedure suitable for determining whether an EMD
peptide exhibits an angiogenic property is detailed in Example
5.
[0044] The term "linked" or "conjugated" as used in the present
invention refers to the linkage between the injury-associated
antigen-specific binding component and an EMD peptide. The linkage
may be introduced through either recombinant (e.g. recombinant
fusion proteins) or chemical means. Non-limiting examples of
suitable chemical means include covalent bonding, disulfide
bonding, hydrogen bonding, electrostatic bonding, and
conformational bonding and may involve the use of homobifunctional
or heterobifunctional cross linkers. Suitable cross-linking and
conjugation methods are disclosed in U.S. Pat. No. 6,642,363 and
U.S. Pat. Pub. No. 20060002852.
[0045] The term "therapeutically effective dose" as used herein to
refers to an amount of a targeted EMD peptide sufficient to
effectuate a desired result, or an amount of targeted EMD peptide
required to facilitate the repair and/or regeneration of an injured
tissue in vivo. Such an amount will vary depending on the effect to
be achieved. The therapeutically effective dose will depend on a
variety of factors, including the type of injury, the type of
tissue, the extent of injury, the particular EMD peptide, and the
degree of repair and/or regeneration sought as an end-point.
II. Targeted EMD Peptides for Tissue Repair and Regeneration
[0046] The invention discloses compositions and methods for
targeted repair and/or regeneration of an injured tissue. As
described in more detail below, the invention is an EMD peptide
conjugated to an injury-associated antigen-specific binding
component that targets the EMD peptide to an injured tissue. The
invention is useful for the repair and/or regeneration of injured
mammalian tissue in vivo. Non-limiting exemplary tissue injuries
suitable for treatment with the present invention may include
ischemic injury, re-perfusion injury, congestive heart failure,
bone fractures, dental decay, inflammatory injury (e.g., due to
placement of pace maker electrodes, or arthritis), chemotherapy,
radiation or thermal injury, acute injuries resulting from trauma
(e.g. vascular damage due to stents and catheters), tissue
hypertrophy, tissue hyperplasia, and fibrosis. Non-limiting
exemplary tissues that can be targeted with the compositions and
methods described herein may include: cardiac, neural, hepatic,
renal, cartilage, vascular, dental, bone, and bone marrow tissues.
Non-limiting exemplary injury-associated antigens suitable for use
with the invention may include: myosin light chain, myosin heavy
chain, troponin I, caspases, VCAM-1, ICAM-1, P-selectin,
E-selectin, L-selectin, Mo1/CD18, Tumor Necrosis Factor (TNF)
receptor-1, TNF receptor-2, lectin-like oxidized low-density
lipoprotein receptor-1 (LOX-1), VAP-1, annexin, osteopontin,
thrombospondin, laminin, fibronectin, fibrinogen, fibrin, elastin,
membrane bound stem cell factor, and collagen. The
injury-associated antigen-specific binding component, as described
in more detail below, may be an antibody or antibody mimetic that
specifically binds to an injury-associated antigen with an affinity
of 10' M or stronger. Non-limiting exemplary binding components
suitable for use with the present invention may include:
antibodies, antibody fragments (e.g. Fab, Fab', F(ab)2 fragments,
scFv, minibodies and nanobodies), and antibody mimetics.
[0047] As described in more detail below, an EMD peptide suitable
for use with the present invention exhibits: (i) cell attachment,
(ii) cell activation, and (iii) chemotropic properties. Although
many wild-type intact extracellular matrix proteins may meet these
requirements, the present inventors have surprisingly discovered
peptides ranging in size from about 8 amino acid residues to about
100 amino acid residues are preferred to longer peptides of whole
proteins. In particular, peptides in this size range that exhibit
these three requirements are easy to manufacture, have reduced
immunogenicity, lower toxicity, and show greater homogeneity and
reproducibility in conjugation to an injury-associated
antigen-specific binding component. Further, the targeted EMD
peptides of the present invention are easier and more cost
effective to manufacture as pharmaceutical compositions compared to
longer peptides of full-length proteins. Non-limiting exemplary EMD
peptides suitable for use with the present invention may be derived
from full-length proteins including: fibrinogen, fibrin,
fibronectin, collagen (types I, II, III, and IV), tenascin-C,
laminin, and elastin. These and other aspects of the invention are
described in more detail below, and in the examples.
A. Injury Associated Antigen-Specific Binding Component
[0048] The injury-associated antigen-specific binding component
specifically binds an injury-associated antigen that is either
expressed or unmasked as a result of a tissue injury. Therefore, in
making the composition of the invention, an injury-associated
antigen must be identified.
[0049] An injury-associated antigen can be any molecule expressed
on the surface of a cell in an injured tissue, or an antigen that
is unmasked, or exposed, as a result of a tissue injury. The
antigen may be a carbohydrate, protein, polypeptide, nucleic acid,
lipid, proteoglycan, or any other molecule that is predominantly
expressed or exposed in a tissue following injury.
[0050] Many injury-associated antigens are predominantly expressed
following tissue injury. Alternatively, an antigen may be exposed
or unmasked following tissue injury. For example, basement membrane
antigens (e.g. laminin, fibronectin, or collagen type IV) are
frequently exposed following vascular injury as a result of
stenting procedures or percutaneous transluminating coronary
angioplasty (PTCA).
[0051] Non-limiting exemplary injury-associated antigens that are
predominantly expressed following vascular or cardiac injury
include: myosin heavy chain, myosin light chain (Yamada, et al., J
Nuc. Med 33:1501-1508 (1992)) and troponin I (Apple, F. S., et al.,
Clin. Chim. ACTA 284:151-159 (1999)); vascular cell adhesion
molecule (VCAM-1) (Kalawski, R., et al., Eur J. Cardiothorac. Surg.
14:290-295 (1998), Oguchi, et al., Arterial Thromb Vasc. Biol.
20:1729-1736 (2000)); intercellular adhesion molecule (ICAM-1)
(Sun, B., et al., J. Mol. Cell Cardiol. 33:109-119 (2001));
.alpha.4.beta.1 integrin (Lumsen, et al., J Vasc Surg 26:87-93
(2000)); Mo1/CD18 (Aversano, T. et al., J. Am Coll. Cardiol.
25:781-788 (19995)); TNF receptor-1 and TNF receptor-2 (Irwin, M.
W., et al., Circ. 99:1492-1498 (1999)); vascular adhesion protein-1
(VAP-1) (Jaakkola, K., et al., J. Am. Coll. Cardiol. 36:122-129
(2000)); and angiotensin receptors, which are expressed locally but
under a different temporal sequence following myocardial injury
(Yang, B., et al., Vasc. Med. 3:121-130 (1998)). Additional
vascular antigens suitable for use with the instant invention are
members of the selectin family, which are cell-surface
carbohydrate-binding proteins expressed by endothelial cells and
cells of the immune system. Selectins mediate a variety of
transient, Ca.sup.2+ specific cell-cell adhesion interactions with
integrins. (See, e.g., Alberts, et al., MOLECULAR BIOLOGY OF THE
CELL, 4th ed.(2002) Garlund Science, NY, N.Y.) Non-limiting
exemplary selectin antigens suitable for use with the present
invention may include: L-selectin, P-selectin (Gumina, R. J., et
al., Basic Res. Cardiol. 92:201-213 (1997)); and E-selectin (Ma, X.
L., et al., Circ. 88:649-658 (1993)).
[0052] Non-limiting exemplary antigens suitable for use with the
present invention that are exposed or expressed as a result of an
inflammatory response may include: Mo1/CD18, TNF receptor-1, TNF
receptor-2, Vascular adhesion protein-1 (VAP-1), and annexin. In
some embodiments, where the inflammation at the affected site
persists, released cytokines, such as IL-1 and TNF, will activate
endothelial cells to express other injury-associated antigens
suitable for use with the present invention, including: integrin
receptors, VCAM-1, ICAM-1, E-selectin, and L-selectin.
[0053] Non-limiting exemplary antigens that are expressed or
exposed following injury to bone tissue that are suitable for use
with the present invention may include: osteopontin,
thrombospondin, and tenascin-C.
[0054] Non-limiting exemplary antigens that are expressed or
exposed following injury to cartilage tissue that are suitable for
use with the present invention may include: collagen (e.g., types
I, II, III and IV), glycosaminoglycans (e.g., chondroitin sulfate),
and inflammatory markers (such as Mo1/CD18, TNF receptor-1, and TNF
receptor-2).
[0055] Non-limiting exemplary antigens that are expressed or
exposed following injury to hepatic or renal tissues can include:
antigens associated with an inflammatory response as discussed
herein and apoptotic markers such as caspase-3. Additional
injury-associated antigens suitable for use with the present
invention will be well known to persons of skill in the art.
1. Antigen-Specific Binding Component
[0056] Another component of the present invention is a binding
component specific to the injury-associated antigen. Binding
components suitable for use with the present invention are well
known in the art. Non-limiting exemplary binding components
suitable for use with the present invention may include: an
antibody (monoclonal or polyclonal), an antibody fragment, a single
chain variable fragment (scFv), and an antibody mimetic. Binding
components suitable for use with the present invention can be
either generated using methods well known in the art, or purchased
from commercial suppliers. Binding components purchased from
commercial suppliers (e.g. antibodies) can be modified, for
example, to generate antibody fragments (e.g. Fab, F(ab')2, scFv).
Antibody mimetics, as described in more detail below, are
non-antibody molecules. Non-limiting exemplary antibody mimetics
suitable for use with the invention may include: anticalins,
polypeptides with fibronectin type III domains, avimers, adnectins,
and non-glycosylated single chain polypeptides having two or more
binding domains.
a) Antibodies
[0057] Methods of producing monoclonal or polyclonal antibodies
that react specifically with antigens expressed on cells of injured
tissues are well known to those of skill in the art. For example,
preparation of monoclonal antibodies by immunizing mice with an
appropriate immunogen is described in, e.g., Coligan, Current
Protocols in Immunology (1991): Harlow & Lane, ANTIBODIES, A
LABORATORY MANUAL, Cold Spring harbor Publication, New York (1988);
Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986); Kohler & Milstein, Nature 256:495497 (1975). Antibody
preparation by selection of antibodies from libraries of nucleic
acids encoding recombinant antibodies packaged in phage or similar
vectors is described in, e.g., Huse, et al., Science 246:1275-1281
(1989) and Ward, et al., Nature 341:544-546 (1989). In addition,
antibodies can be produced recombinantly using methods known in the
art and described in e.g., Sambrook, et al., Molecular Cloning, A
laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994).
[0058] The production of monoclonal antibodies is well known in the
art. In general, spleen cells from an animal immunized with the
desired immunogen (e.g., a myosin light chain, myosin heavy chain,
or troponin I) are immortalized, commonly by fusion with a myeloma
cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519
(1976). Colonies arising from single immortalized cells are
screened for the production of antibodies having the desired
binding specificity and binding affinity for the particular
antigen. In some embodiments, the immunized animal is a transgenic
animal that expresses human immunoglobulin genes for the production
of human antibodies, as disclosed in U.S. Pat. No 6,833,268. In
some embodiments, the production of human or humanized antibodies
is carried out as described in U.S. Pat. No. 6,673,986, or using
methods known to a person of ordinary skill in the art.
[0059] In some embodiments, the genes encoding the heavy and light
chain immunoglobulins can be cloned from a hybridoma cell that
produces a desired monoclonal antibody specific for a particular
injury associated antigen. In some embodiments, gene libraries
encoding heavy and light chains of monoclonal antibodies are
generated. In some embodiments, random combinations of the heavy
and light chain gene products are used to generate a pool of
antibodies with differing antigenic specificities (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Nucleic acids encoding antibodies
that specifically bind to an injury associated antigen can be
isolated directly from mRNA, cDNA, or DNA libraries using methods
well known in the art, such as polymerase chain reaction (PCR) and
ligase chain reaction (LCR) (see, e.g., U.S. Pat. Nos. 4,683,195
and 4,683,202; PCR Protocols: A Guide to Methods and Applications
(Innis et al., eds, 1990)). Phage display technology can be used to
identify antibodies and Fab fragments that specifically bind to
selected antigens (see, e.g., McCafferty et al., Nature 348:552-554
(1990); Marks et al., Biotechnology 10:779-783 (1992)).
[0060] In addition to the antibodies generated using the methods
well known in the art, or described herein, antibodies specific for
injury-associated antigens suitable for use with the present
invention can be purchased from commercial sources. For example,
antibodies against myosin light chain and troponin-I can be
purchased from: Abcam Ltd., (Cambridge, U.K.); Accurate Chemical
& Scientific Corporation (Westbury, N.Y.) and Beckman Coulter
(Fullerton, Calif.). Antibodies against activated caspase 3,
fibronectin, collagen (Types I, IV, and VII), laminin, TNF-.alpha.,
TNF receptors-1 and -2, are available from Research Diagnostics
(Concord, Mass.). VCAM-1 antibodies are available from BioSource
International (Carmillo, Calif.). ICAM-1 antibodies are available
from AbCam Ltd. (Cambrige, U.K.). Osteopontin antibodies are
available from Sigma-Aldrich (St. Louis, Mo.). Thrombospondin,
Tenascin-C, and selectin antibodies are available from Chemicon,
(Temecula, Calif.). These and other commercially available
antibodies are suitable for use with the present invention.
(1) Modification of Antibodies
[0061] Once an antibody of appropriate specificity and affinity has
been obtained, the antibody can be conjugated to an EMD peptide, or
the antibody can be modified prior to conjugation. Suitable
modifications of the antibodies include, generation of antibody
fragments or humanizing, primatizing, or chimerizing the
antibody.
[0062] Antibody fragments suitable for use with the present
invention include any antibody fragment capable of specifically
binding to the specific injury-associated antigen and capable of
being conjugated to an EMD peptide. Non-limiting exemplary antibody
fragments may include: F(ab').sub.2, Fab, Fv, single chain Fv
(scFv), dsFv, V.sub.L and V.sub.H (see, e.g., Fundamental
Immunology (Paul ed., 4d ed. 1999); Bird, et al., Science 242:423
(1988); and Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879
(1988)). The antibody fragments can be obtained by a variety of
methods, including, digestion of an intact antibody with an enzyme,
such as pepsin (to generate (Fab').sub.2 fragments) or papain (to
generate Fab fragments); or de novo synthesis. Antibody fragments
can also be synthesized using recombinant DNA methodology. In some
embodiments F(ab').sub.2 fragments that specifically bind myosin
light chain, myosin heavy chain, or troponin-I are generated.
[0063] As mentioned above, humanized antibodies may be generated
for use as an injury-associated antigen-specific binding component.
Humanized antibodies are antibodies in which the antigen binding
loops, i.e., CDRs, obtained from the VH and VL regions of a
non-human antibody are grafted to a human framework sequence.
Methods for humanizing or primatizing non-human antibodies are well
known in the art. Generally, a humanized antibody has one or more
amino acid residues introduced into it from a source, which is
non-human. Humanization, i.e., substitution of non-human CDRs or
CDR sequences for the corresponding sequences of a human antibody,
can be performed following the methods described in, e.g., U.S.
Pat. Nos. 5,545,806; 5,569,825; 5,633,425; 5,661,016; Riechmann et
al., Nature 332:323-327 (1988); Marks et al., Bio/Technology
10:779-783 (1992); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996). Transgenic mice, or
other organisms such as other mammals, may also be used to express
humanized or human antibodies, as disclosed in U.S. Pat. No.
6,673,986.
b) Antibody Mimetics
[0064] In some embodiments, antibody mimetics are used as the
injury associated antigen specific binding component. Antibody
mimetics use non-immunoglobulin protein scaffolds as alternative
protein frameworks for the variable regions of antibodies. As
defined herein, antibody mimetics are polypeptides comprising one
or more regions (i.e., loop regions) that are amenable to specific
or random sequence variation such that the antibody mimetic
specifically binds to an antigen of interest (e.g., an injury
associated antigen such as myosin light chain). Non-limiting
exemplary antibody mimetics can include anticalins which are based
on lipocalins and are described in Weiss and Lowman, Chem Biol.,
7(8):177-184 (2000); Skerra, J. Biotechnol. 74(4):257-275; and
WO99/16873; polypeptides with a fibronectin type III domain and at
least one randomized loop as described in e.g., WO01/64942 and U.S.
Pat. No. 6,818,418; polypeptides with a P-sandwich structure as
described in e.g. WO 00/60070; and non-glycosylated single chain
polypeptides composed of two or more monomer domains, that can
separately bind any type of target molecule including proteins,
joined by a linker, as described in U.S. patent application Ser.
Nos. 10/133,128 and 10/871,602.
[0065] The antibody mimetics having monomer domains of
non-glycosylated single chain polypeptides described in U.S. patent
application Ser. Nos. 10/133,128 and 10/871,602 are distinct from
the complementarity-determining region (CDR) of an antibody. The
antibody mimetic polypeptides are able to fold independently, form
stable structures, and are heat stable unlike an antibody. For
example, the polypeptides are stable to 95.degree. C. for at least
10 minutes without an appreciable loss in binding affinity.
Additional characteristics of the monomer domains includes low
immunogenicity, low toxicity, small size sufficient to penetrate
skin or other tissues, and a range of in vivo half-life and
stability.
[0066] Antibody mimetics may be generated to bind an
injury-associated antigen, such as those described herein. For
example, an antibody that binds a specific injury-associated
antigen can be analyzed using methods known in the art, such as
three-dimensional crystal structure analysis of the
antibody-antigen interaction, to identify the specific residues
that are critical for antigen binding. Once these residues have
been identified, the loop regions of the antibody mimetics can be
subjected to site directed mutagenesis such that the loop forms a
binding pocket for the particular injury associated antigen. Such
modifications are described in, e.g., Vogt and Skerra, Chembiochem.
5(2):191-9 (2004).
[0067] Lipovsek et al. (U.S. Pat. Nos. 6,818,418 and 7,115,396)
discloses an antibody mimetic featuring a fibronectin or
fibronectin-like scaffold and at least one variable loop. Known as
Adnectins, these fibronectin-based antibody mimetics exhibit many
of the same desirable characteristics of natural or engineered
antibodies, including high affinity and specificity for a targeted
ligand. Further, these fibronectin-based antibody mimetics exhibit
certain benefits over antibodies and antibody fragments. For
example, these antibody mimetics do not rely on disulfide bonds for
native folding and stability, and are therefore stable under
conditions that would normally breakdown antibodies.
[0068] Beste et al. (Proc. Natl. Acad. Sci. U.S.A. (1999)
96(5):1898-1903) discloses an antibody mimetic based on a lipocalin
scaffold (ANTICALIN.TM.). Lipocalins are composed of a b-barrel
with four hypervariable loops at the terminus of the protein. Beste
(1999), subjected the loops to random mutagenesis and selected fro
binding with, for example, fluorescein. Three variants exhibited
specific binding with fluorescein, with one variant showing binding
similar to that of an anti-fluorescein antibody. Further analysis
revealed that all of the randomized positions are variable,
indicating that ANTICALIN.TM. would be suitable for use as an
alternative to an antibody. ANTICALIN.TM. are small single chain
polypeptides, typically between 160 and 180 residues in length,
which provides several advantages over antibodies, including
decreased cost of production, increased stability during storage,
and decreased immunological reaction.
[0069] Hamilton et al. (U.S. Pat. No. 5,770,380) discloses a
synthetic antibody mimetic using the rigid, non-peptide organic
scaffold of calixarene, attached with multiple variable peptide
loops as binding sites. The peptide loops all project from the same
side geometrically from the calixarene, with respect to each other.
Because of this geometric confirmation, all of the loops are
available for binding, thereby increasing the binding affinity to
the ligand. In comparison, however, to the other antibody mimetics,
the calixarene-based antibody mimetic does not consist exclusively
of polypeptide, and is therefore less susceptible to attack by
protease enzymes, is relatively stable in extreme environmental
conditions and has a long life-span. Further, due to the relatively
small size of the antibody mimetic, it is less likely to produce an
immunogenic response.
[0070] Murali et al. (Cell. Mol. Biol. (2003) 49(2):209-216)
discloses a methodology for reducing antibodies into smaller
peptidomimetics, termed "antibody-like binding peptidomimetics"
(ABiP) which may also be used as an alternative to antibodies with
the present invention.
[0071] Silverman et al. (Nat. Biotechnol. (2005) 23:1556-1561)
discloses fusion proteins that are single chain polypeptides
comprising multiple domains, termed "avimers." Developed from human
extracellular receptor domains by in vitro exon shuffling and phage
display, the avimers are a class of binding proteins somewhat
similar to antibodies in their affinities and specificities for
target molecules. These resulting multi-domain proteins may exhibit
improved affinity (sub-nanomolar in some cases) and specificity
compared to single epitope binding proteins. Additional details
concerning the construction and use of avimers can be found in U.S.
Pat. Pub.Nos:20040175756, 20050048512, 20050053973, 20050089932,
and 20050221384.
[0072] In addition to non-immunoglobulin protein frameworks,
antibody properties have also been mimicked in compounds comprising
RNA molecules and unnatural oligomers (e.g., protease inhibitors,
benzodiazepines, purine derivatives and bb-turn mimics) all of
which are suitable for use with the present invention as
injury-associated antigen-specific binding components.
B. EMD Peptide
[0073] An EMD peptide suitable for use with the present invention
has a length of about 8 amino acid residues to about 100 amino acid
residues and exhibits positive: i) cell attachment; ii) cell
activation; and iii) chemotropic properties. In some embodiments,
the EMD peptides further exhibit a positive angiogenic property. As
described in more detail below, the present inventors have
surprisingly shown that while full length proteins from which an
EMD peptide can be derived may exhibit cell attachment, cell
activation, and chemotropic properties, not all EMD peptides
derived from these proteins exhibit these properties. For example,
of the four EMD peptides tested in the Examples (SEQ ID NOs: 1-4),
only Hep III (SEQ ID NO:2) and RGD (SEQ ID NO:4) exhibited all
three of the required properties.
[0074] Non-limiting exemplary extracellular matrix proteins
suitable for deriving an EMD peptide for use with the present
invention may include: fibrinogen, fibronectin, fibrin, collagen
(Types I, II, III, and IV), laminin, elastin, and tenascin-c.
[0075] Any method known in the art suitable for use with the
present invention may be used to derive an EMD peptide from a
naturally occurring extracellular matrix protein. In some
embodiments, the protein, or longer polypeptides may be
enzymatically cleaved using a protease, such as trypsin. In some
embodiments, EMD peptides may be identified using bioinformatic
analysis and the identified peptide then produced through
recombinant means. In some embodiments, an EMD peptide of the
invention is a variant or a mutant of a peptide corresponding to
the naturally occurring wild-type protein. In some embodiments,
conservative substitutions are made to the naturally occurring
protein or peptide, to improve the functional characteristics of
the EMD peptide. Natural matrix polymers suitable for deriving an
EMD peptide for use with the instant invention can be purified from
donor sources, or purchased from commercial suppliers, for example,
Sigma-Aldrich (St. Louis, Mo.) and Abnova Corp. (Taipei,
Taiwan).
[0076] In some embodiments, an EMD peptide of the invention is
derived from a collagen protein. The collagen family contains
approximately 20 types of triple helical fibrous proteins, and
constitutes approximately one-third of the total proteins in our
body. Collagen provides structural integrity by resisting
mechanical loading forces and degradation. Type I collagen is
prevalent in a variety of tissue types including bone, skin and
various internal organs and promotes cell growth and
differentiation via binding of .alpha.1.beta.31 and
.alpha.2.beta.31 integrins (Gullberg, D., et al., EMBO J.
11:3865-3873 (1992)). Type II collagen is present in cartilage and
binds to chondrocytes through the .alpha.2.beta.1 integrin
(Tuckwell, et al., J. Cell Sci 107(4)993-1005 (1994)). Type IV
collagen is a component of the basal lamina and binds cells through
the .alpha.1.beta.1 and .alpha.2.beta.1 integrins (Vandenberg, P.,
et al., J. Cell Biol. 113:1475-1483 (1991)). Non-limiting exemplary
EMD peptides derived from collagen may include: Hep I, having the
amino acid sequence TAGSCLRKFSTMY-OH (SEQ ID NO:1) and Hep III,
having the amino acid sequence GEFYFDLRLKGDKY-OH (SEQ ID NO:2). As
shown in the examples, Hep III exhibits i) cell attachment, ii)
cell activation, and iii) cell migration properties, and is
therefore suitable for use with the present invention. As shown in
example 5, the Hep III peptide also exhibits angiogenic property.
In contrast to Hep III, the Hep I peptide (as shown in SEQ ID NO:1)
does not exhibit a cell attachment property, and therefore is not
suitable for use with the present invention. This surprisingly
shows that not all peptides derived from an extracellular matrix
molecule are suitable for use with the present invention.
[0077] In some embodiments, an EMD peptide of the invention is
derived from an elastin protein. Elastin is a principal component
of the basal lamina and the ECM. Elastin polypeptide chains are
cross-linked together to form rubber like, elastic fibers. Each
elastin molecule uncoils into a more extended conformation when the
fiber is stretched and will recoil spontaneously as soon as the
stretching force is relaxed. Elastin combines with collagen to give
tissue its shape, rigidity, and flexibility.
[0078] In some embodiments, an EMD peptide of the invention is
derived from fibrinogen, an FDA-approved commercially available
biological sealant and adhesive that acts as a provisional material
for cellular in growth during wound repair. Fibrinogen is formed by
the polymerization of fibrin monomers and is resorbed by
degradation via a fibrinolytic enzyme such as plasmin (see, Pepper,
M. S., et al., Enzyme Protein 49:138- (1996)). Fibrin contains the
arg-gly-asp (RGD) motif that binds to the .alpha.5.beta.1 and
.alpha.8.beta.1 integrin receptors (Sierra, D. H., J. Biomater Appl
7:309-(1993)).
[0079] In some embodiments, an EMD peptide of the invention is
derived from a laminin protein. Laminin is a large flexible protein
composed of three very long polypeptide chains arranged in the
shape of an asymmetric cross and held together by disulfide bonds.
Several isoforms of each type of chain can associate in different
combinations to form a large family of laminin proteins that are
useful in deriving and EMD peptide for use with the present
invention. Laminin, like type IV collagen, can self-assemble into
felt-like sheets and binds cells through .alpha.6.beta.1 and
.alpha.7.beta.1 integrin receptors.
[0080] In some embodiments, an EMD peptide for use with the present
invention is derived from a fibronectin protein. Fibronectin, is a
large glycoprotein dimer composed of two very large subunits joined
at one end by a disulfide bond. Each subunit folds into a series of
functionally distinct domains, separated by regions of flexible
polypeptide chain. Among the functional domains contained within
fibronectin molecule are a self-association domain, collagen
binding domain, heparin binding domain and a cell binding domain. A
central feature of the cell binding domain is the RGD binding
motif, which provides sites for cell attachment via the
.alpha.5.beta.1 and .alpha.8.beta.1 integrin receptors.
Non-limiting exemplary EMD peptides derived from fibronectin may
include FC/HV having the amino acid sequence WQPPRARI-OH (SEQ ID
NO:3) and RGD having the amino acid sequence GRGDSPASSPISC-OH (SEQ
ID NO:4). As shown in the examples, RGD (SEQ ID NO:4) exhibits i)
cell attachment, ii) cell activation, and iii) cell migration
properties, and is therefore suitable for use with the present
invention. In contrast to the RGD EMD peptide, the FC/HV peptide
(as shown in SEQ ID NO:3) does not exhibit cell attachment, cell
activation, or chemotropic properties, and therefore is not
suitable for use with the present invention. As with the Hep I and
Hep III peptides discussed above, the results from the FC/HV and
RGD peptides also surprisingly demonstrate that not all peptides
derived from an extracellular matrix molecule are suitable for use
with the present invention.
[0081] In some embodiments, an EMD peptide for use with the present
invention may be derived from a tenascin protein. Tenascin is
comprised of multiple repeats of short amino acid sequences that
form functional domains. Tenascin may be adhesive or non-adhesive,
depending on the cell type (Spring, et al., Cell 59:325-334
(1989)). Tenascin is known to interact with the .alpha.8.beta.1 and
.alpha.9.beta.1 integrins. Tenascin is also known to stimulate the
synthesis and secretion of proteases, which may be important in
cell migration and remodeling of tissue during repair and
regeneration (Werb, et al., Cell Differ. Dev. 32:299-306
(1990)).
a) Cell Attachment Assay
[0082] EMD peptides suitable for use with the present invention
must exhibit a cell attachment property as described herein. A cell
attachment property refers to the adherence or attachment of cells
to a substrate coated with the EMD peptide of interest.
[0083] For purposes of the present invention, an EMD peptide is
deemed to exhibit cell attachment property when the percent of
cells that adhere to a substrate coated with an EMD peptide of
interest following a short incubation period (for example 30
minutes) is statistically greater (p<0.05) than the percentage
of cells that adhere to a substrate in the absence of the EMD
peptide. The percentage of cells that adhere to the substrate
following the short incubation period can be determined using any
suitable means known in the art. Non-limiting exemplary means
include DNA quantitation, microscopic examination of stained cells,
enzymatic analysis, and the like. Suitable cell types can include
epithelial and endothelial cells. In a preferred embodiment, HUVEC
cells are used. Additional cell lines suitable for use in the cell
activation assays disclosed herein are well known to persons of
skill in the art, as long as the results obtained with the chosen
cell line are consistent with results obtained using HUVEC cells as
shown in Example 1. Cell culture substrates suitable for use with
the assay may be any substrate that can be coated with the EMD
peptide and upon which cells can be cultured. Suitable substrates
may include glass slides, plastic treated or untreated cell culture
dishes, cytodex beads, or any other suitable cell culture substrate
known to persons of skill in the art. An exemplary assay for
determining if an EMD peptide exhibits cell attachment property is
detailed in Example 1.
b) Cell Activation Assay
[0084] An EMD peptide suitable for use with the present invention
must exhibit cell activation property as described herein. A cell
activation property refers to cellular events that occur when a
cell is activated, such as proliferation, changes in ion
permeability, induction of intracellular proteins, or an increase
in phosphorylation of intracellular proteins.
[0085] For purposes of the present invention, an EMD peptide is
deemed to exhibit cell activation properties when a cell cultured
on a substrate coated with an EMD peptide, or contacted with an EMD
peptide, shows a statistically detectable increase (p<0.05) in
cellular proliferation as compared to the proliferation rate of the
cells either cultured on a substrate in the absence of the EMD
peptide or cultured on a substrate in the absence of the EMD
peptide.
[0086] In some embodiments, cell activation is measured as an
increase in proliferation rate of cells cultured on a substrate
coated with an EMD peptide of interest as compared to the
proliferation rates of cells cultured on an untreated substrate in
the absence of the EMD peptide. In some embodiments, parallel
cultures are grown on the same substrate, and then one of the
cultures is then contacted with the EMD peptide of interest, and
the proliferation rate of the two cultures are then compared. The
cell culture substrate may be any substrate that can be coated with
the EMD peptide or any substrate on which cells can be cultured,
and may include glass slides, plastic treated or untreated cell
culture dishes, cytodex beads, or any other suitable substrate
known to persons of skill in the art. In some embodiments, the
cells used for determining cell proliferation are epithelial cells
or human umbilical vascular endothelial cells (HUVEC). Additional
cell lines suitable for use in the cell activation assays disclosed
herein are well known to persons of skill in the art, as long as
the results obtained with the chosen cell line are consistent with
results obtained using HUVEC cells as shown in Example 2. In some
embodiments, the method for measuring the cell proliferation is
quantitating the amount of DNA, use for antibodies such as PCNA for
measuring cell proliferation, 3H-thymidine, BrdU, flow cytometry,
or any other method known to persons of skill in the art for
measuring the rate of cell proliferation in vitro. An exemplary
method for determining the rate of cell proliferation is detailed
in Example 2.
c) Chemotropic Assay
[0087] EMD peptides suitable for use with the present invention
must exhibit a chemotropic property as described herein. For
purposes of the present invention, an EMD peptide is deemed to
exhibit chemotropic property, if a cell (e.g. HUVEC) shows a
statistically detectable increase (p<0.05) in migration on a
surface coated with the EMD protein as compared to cell migration
on an untreated surface.
[0088] In some embodiments, a chemotropic assay as described herein
uses a modified Boyden chamber as described in Example 3. Cell
types suitable for use in a chemotropic assay for determining if an
EMD peptide exhibits a chemotropic property as described herein can
include epithelial and endothelial cells. In a preferred
embodiment, HUVEC cells are used. Additional cell lines suitable
for use in the chemotropic assays disclosed herein are well known
to persons of skill in the art, as long as the results obtained
with the chosen cell line are consistent with results obtained
using HUVEC cells as shown in Example 3. Quantification of the
migrated cells can be measured using any means known in the art,
for example, direct cell counts, DNA quantitation, enzymatic
quantitation, and spectrophotometric determination. In a preferred
embodiment, the cells on the substrate are stained and counted
under a microscope. An exemplary chemotropic assay is detailed in
Example 3.
d) Angiogenic Assay
[0089] In some embodiments, an EMD peptide suitable for use with
the invention exhibits an angiogenic property. For purposes of the
present invention, an EMD peptide is deemed to exhibit an
angiogenic property when an injured tissue contacted with the EMD
peptide of interest shows a statistically detectable increase
(p<0.05) in vascular area of the injured tissue as compared to a
control. A suitable assay for determining if an EMD peptide of
interest exhibits an angiogenic property as described herein is
detailed in Example 5.
C. Conjugation of Binding Component and Therapeutic Moiety
[0090] Once an injury-associated antigen-specific binding component
and an EMD peptide have been generated, they are conjugated to form
a targeted EMD peptide, as described herein. Methods for
conjugating an injury-associated antigen-specific binding component
to an EMD peptide are well known in the art and may include
recombinant and chemical conjugation methods.
[0091] Chemical conjugation techniques suitable for use with the
present invention include conjugation of functional chemical groups
as described in U.S. Pat. App. No. 20060002852. Chemical group
conjugation typically involves the presence of a functional
chemical group on both the binding component and on the EMD
peptide. Exemplary functional groups include carboxylic acids,
aldehydes, amines, sulfhydrals, and hydroxyl groups. The functional
groups may be conjugated by direct crosslinking using homo- or
hetero-bifunctional crosslinkers. A crosslinker suitable for use
with the present invention is any crosslinker that couples the
binding component to an EMD peptide via a chemical modification.
Non-limiting exemplary crosslinkers suitable for use in the present
invention include CDI, EDC, and glutaraldehyde.
[0092] In some embodiments, the functional groups on the binding
component and the EMD peptide are identical, and may be conjugated
in a one-step chemical cross-linking procedure using a
homobifunctional linker. Exemplary homobifunctional cross-linkers
may include amine reactive cross-linkers; amine reactive
cross-linkers with PEO/PEG spacers; 1,5-difluoro-2,4-dinitrobenzene
(DFDNB) (useful for cross-linking between small spatial distances);
sulfhydral reactive linkers (maleimides react with --SH groups at
pH 6.5-7.5, forming stable thioether linkages); and sulfhydral
reactive linkers with PEO/PEG spacers. In some embodiments,
heterobifunctional cross-linkers will be used to join two or more
different functional groups allowing for sequential conjugations
with specific functional groups of proteins while minimizing
undesirable polymerization or self-conjugation.
[0093] In some embodiments, the conjugation method involves the
activation of hydroxyl groups, on either the EMD peptide or the
binding component, with the agent, carbonyldiimidazole (CDI) in
aprotic solvents (e.g., DMSO, acetone, or THF). Activation with CDI
forms an imidazoyl carbamate complex with the hydroxyl group, which
may then be displaced by binding the free amino group on the second
component. The reaction is an N-nucleophilic substitution, which
results in a stable N-alkylcarbamate linkage of the binding
component to the EMD peptide. The coupling of the binding component
to the EMD peptide is optimal in the pH range of 9-10 and normally
requires at least 24 hours. The resulting linkage is stable and
resists hydrolysis for extended periods of time.
[0094] In some embodiments, the coupling method involves the use of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
or "water soluble CDI" in conjunction with
N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed
carboxyl group of one component to free amino groups present on the
second component in a totally aqueous environment at a
physiological pH of 7.0. Briefly, the EDC and sulfo-NHS in the
reaction increases the efficiency of the EDC coupling by a factor
of ten-fold and provides for exceptionally gentle conditions that
ensure the viability of the resultant targeted EMD peptide.
[0095] Using either of the above protocols (CDI or EDC) it is
possible to activate almost any EMD peptide containing a carboxyl
or hydroxyl group in a suitable solvent system that will not
degrade the EMD peptide.
[0096] In some embodiments, where the EMD peptide and the binding
component have free hydroxyl and carboxyl groups are conjugated
using the crosslinking agent divinylsulfone. This method is
particularly useful for attaching sugars or other hydroxylic
compounds with bioadhesive properties to other hydroxylic
compounds. Briefly, the activation involves the reaction of
divinylsulfone to the hydroxyl groups on one of the components,
forming vinylsulfonyl ethyl ether of the component. The vinyl
groups will couple to alcohols, phenols, and even amines on the
second component. Activation and coupling take place at pH 11. The
linkage is then stable through the pH range from about 1 to about
8.
[0097] In some embodiments, coupling between the binding component
and the EMD peptide is of a direct or indirect covalent nature. For
example, the coupling may be through a linker bound to one
component, or alternatively through an interaction between two
molecules such as streptavidin and biotin. The coupling interaction
may also be an electrostatic attraction. For example, the
interaction between the binding component and the EMD peptide may
be mediated by a positively charged molecule, such as
polyethyleneimine or poly-lysine, present on one component and a
negatively charged molecule present on the other component. In some
embodiments, the binding component may be conjugated to the EMD
peptide by means of UV cross-linking.
[0098] In some embodiments, the binding component and the EMD
peptide may be conjugated by recombinant means generating a DNA
construct encoding the binding component fused to the EMD peptide.
The DNA construct can then be expressed in an appropriate protein
expression system, such as a prokaryotic or eukaryotic expression
system well known to persons of skill in the art.
D. Pharmaceutical Formulation of a targeted EMD peptide
[0099] A targeted EMD peptide, comprising an injury-associated
antigen-specific binding component conjugated to an EMD peptide, as
described herein, can be formulated as a pharmaceutical composition
for use in the methods of the present invention. General details on
the techniques for formulation and administration of pharmaceutical
compositions are well known in the art. See, e.g., "REMINGTON'S
PHARMACEUTICAL SCIENCES", Maack Publishing Co., Easton, Pa.
[0100] In some embodiments, a pharmaceutical formulation of the
invention comprises a solution of the composition and an aqueous
pharmaceutically acceptable carrier. A variety of aqueous carriers
can be used, e.g., buffered saline and the like. These solutions
are sterile and generally free of undesirable matter. The
pharmaceutical formulation may also contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents,
toxicity adjusting agents and the like, for example, sodium
acetate, sodium chloride, potassium chloride, calcium chloride,
sodium lactate and the like. The concentration of the targeted EMD
peptide in the formulation can vary widely, and will be selected
primarily based on fluid volumes, viscosities, body weight,
composition of the targeted EMD peptide, target tissue, nature of
the injury, and the like in accordance with the particular mode of
administration selected and the patient's needs.
[0101] In some embodiments, he pH of the pharmaceutical formulation
is in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. In some
embodiments, the pharmaceutical formulation comprises a suitable
pharmaceutically acceptable buffer such as phosphate, tris
(hydroxymethyl) aminomethane-HCl or citrate and the like. Buffer
concentrations should be in the range of 1 to 100 mM. In some
embodiments, the pharmaceutical formulation comprises a salt, such
as sodium chloride or potassium chloride in a concentration of 50
to 150 mM. In some embodiments, the pharmaceutical formulation
comprises an effective amount of a stabilizing agent such as
albumin, a globulin, a detergent, a gelatin, a protamine or a salt
of protamine.
[0102] In some embodiments, the pharmaceutical formulation is in
the form of a sterile injectable preparation, such as sterile
injectable aqueous or oleaginous suspension. The suspension may be
formulated according to methods well known in the art using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may be a sterile injectable solution
or suspension in a nontoxic parenterally-acceptable diluent or
solvent. Non-limiting exemplary vehicles and solvents suitable for
use with the invention include: water, Ringer's solution, and an
isotonic sodium chloride solution. In some embodiments, sterile
fixed oils may be used as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed including synthetic
mono- or diglycerides, and fatty acids such as oleic acid may be
used in the preparation of injectables.
III. Administration and Dosing
[0103] The compositions described herein (i.e., a targeted EMD
peptide comprising an injury-associated antigen-specific binding
component coupled to an EMD peptide) can be administered to a
patient alone, or in conjunction with other therapies suitable for
treatment of the particular injury. For example, a targeted EMD
peptide may be administered in conjunction with angioplasty to
facilitate the repair of injured cardiac tissue. In some
embodiments, the targeted EMD peptide may be administered prior to
the angioplasty, contemporaneous with the angioplasty, or
subsequent to the angioplasty.
[0104] The targeted EMD peptides of the present invention may be
administered by any suitable means known in the art. In some
embodiments, the compositions are suitable for parenteral
administration (e.g., intravenous, intramuscular, or
intraperitoneal injection). The compositions of the invention may
also be administered subcutaneously, into vascular spaces, or into
joints, e.g., intra-articular injection. Additional routes of
administration suitable for use with the present invention include
intranasal, topical, vaginal, rectal, intrathecal, intra-arterial,
and intraocular routes, or direct injection or application to the
injured tissue. Intravascular administration is preferred.
[0105] The amount of targeted EMD peptide required to facilitate
tissue repair and/or regeneration is defined as a "therapeutically
effective dose." The dosage schedule and amounts effective for a
particular use, i.e., the "dosing regimen," will depend upon a
variety of factors, including the tissue being treated, the type of
injury, the composition of the targeted EMD peptide,
pharmacokinetics of the composition including bioavailability and
clearance rates, the patients physical status, the route of
administration, and the desired end-point sought to be achieved
shall all be taken into account. Single or multiple administrations
of the compositions may be administered depending on the dosage and
frequency as required and tolerated by the patient. Typically, a
pharmaceutical composition of the invention is administered as a
single, or multiple therapeutically effective dose over a single
day, several days, or weeks by daily or weekly intravenous
infusion. In some cases of chronic injury, such as intractable
angina, multiple doses separated by months may be required due to
the changing (worsening) pathophysiological state. In any event,
dosage and administration schedule should provide for a sufficient
quantity of targeted EMD peptide to effectively treat the
patient.
[0106] In some embodiments, a therapeutically effective dose of a
targeted EMD peptide may be in a range from about 0.05 .mu.g to
about 500 .mu.g, or about 5 .mu.g to about 400 .mu.g, or about 10
.mu.g to about 300 .mu.g, or about 25 .mu.g to about 250 .mu.g, or
about 40 .mu.gto about 100 .mu.g, or about 50 .mu.gof targeted EMD
peptide per dose. In some embodiments, the amount of targeted EMD
peptide is administered based on body weight. In some embodiments,
the amount of targeted EMD peptide administered is from about 0.05
ng to about 500 ng, or about 5 ng to about 400 ng, or about 10 ng
to about 300 ng, or about 25 ng to about 250 ng, or about 40 ng to
about 100 ng, or about 50ng of targeted EMD peptide per kg of body
weight. In some embodiments, the dose is based on patient surface
area. For example, in some embodiments, a targeted EMD peptide may
be administered in a dose range from about 50mg to about 400 mg/
m.sup.2 of surface area, or about 100mg to about 300mg /m.sup.2 of
surface area, or about 150 mg to about 250 mg/ m.sup.2 of surface
area. In some embodiments, the dose administered is about 250
mg/m.sup.2 of surface area. In some embodiments, the dose is
administered as a single cycle, while in other embodiments,
multiple cycles are administered. The exact dose and schedule of
administration will depend on a variety of factors as discussed
herein, and is well within the skill of medical professional
treating the patient.
EXAMPLES
[0107] The following examples are included for illustration
purposes only, and are not intended as a limitation on the
invention in any way. It should be appreciated by those of skill in
the art that the techniques disclosed in the examples which follow
represent techniques discovered by the inventor to function well in
the practice of the invention, and can thus be considered to
represent preferred modes for practice of the invention. However,
those skilled in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments disclosed and still obtain similar results
without departing from the spirit or scope of the invention.
Example 1: Cell Attachment Assay
Reagents
[0108] Immulon 1B, untreated 96-well plates (CoStar), and untreated
35 mm dishes (CoStar) were purchased from Fisher Scientific
(Pittsburg, PA). Human umbilical vein endothelial cells (HUVECs),
cell culture media and supplements were purchased from Lonza
(Basel, Switzerland). EMD peptides as shown in SEQ ID Nos: 1-4,
were synthesized by Commonwealth Biotechnologies Inc., (Richmond,
Va.). Amino acid analysis was performed on the peptides to verify
the amino acid sequence.
Cell Attachment Assay
[0109] For the adhesion studies 96-well Immulon 1B plates were
used. Various concentrations of the EMD peptides (SEQ ID Nos: 1-4)
were tested for their ability to bind cells, and compared to the
full-length protein, from which the EMD peptides were derived as
positive controls. Bovine Serum Albumin (BSA) treated wells were
used as a negative control. Full-length collagen IV, and the EMD
peptides Hep I, Hep III, FCHV, and RGD were tested at
concentrations of 20.mu.g/ml, 50 .mu.g/ml, and 100 .mu.g/ml. Full
length fibronectin was tested at concentrations of 10 .mu.g/ml and
100 .mu.g/ml. Negative control wells were coated with BSA at a
concentration of 2 mg/ml. Fifty .mu.L of protein or EMD peptide
solutions at the various concentrations was added separate wells
and incubated at 37.degree. C. for at least 18 hours. The protein
or peptide solution was then removed and the wells were blocked for
2 hours at 37.degree. C. with 2 mg/mL BSA solution. Following the
blocking procedure, wells were washed twice with PBS. HUVEC cells
(Lonza, Basel Switzerland) were then added to the wells at a
concentration of 5.times.10.sup.3 to 1.times.10.sup.4 cells per
well. Following a thirty-minute incubation, cell adhesion was
assessed after 30 minute incubation wells were washed and cell
adhesion was determined using an MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfoph-
enyl)-2H-tetrazolium, inner salt) tetrazolium/formazan assay
(Promega, Madison, Wis.) according to the manufacturer's
instructions.
[0110] As shown in FIG. 1A-1C, HUVEC cells in the wells coated with
either the full-length proteins Col. IV and Fibronectin, or the EMD
peptides Hep III (SEQ ID NO:2) and RGD (SEQ ID NO:4) showed
increased cell attachment compared to cell attachment in wells
coated with BSA. Notably, cell attachment in wells coated with the
EMD peptides Hep I (SEQ ID NO:1) and FCHV (SEQ ID NO:3) were not
statistically different from that of the BSA treated wells. See,
FIG. 1A-1C. The results indicate that both the Hep III (SEQ ID
NO:2) and RGD (SEQ ID NO:4) peptides exhibit cell attachment
property.
Example 2: Cell Proliferation Assay
[0111] To determine if EMD peptides can activate cells by
stimulating cellular proliferation, 96-well plates coated using the
same protocol as described above for the cell attachment assay was
used. Following treatment of the wells, HUVEC cells (Lonza, Basel
Switzerland) were then added to the wells at a concentration of
5.times.10.sup.3 to 1.times.10.sup.4 cells per well and incubated
under standard culture conditions for 1, 2, 3 or 4 days. Cell
proliferation was then determined at the end of the incubation
period using an MTS tetrazolium/formazan assay (Promega, Madison,
Wis.) according to the manufacturer's instructions.
[0112] As shown in FIG. 2A-2C, full length proteins Col IV and
fibronectin and EMD peptides Hep I, (SEQ ID NO:1) Hep III (SEQ ID
NO:2), and RGD (SEQ ID NO:4) all showed increased cellular
proliferation compared to cells cultured in wells coated with BSA.
Notably, cells cultured in wells coated with FCHV (SEQ ID NO:3) was
not different from that of cells cultured in wells coated with BSA.
The data indicates that full length proteins Col IV and
fibronectin, as well as the EMD peptides Hep I, Hep III, and RGD
all exhibit cell activation property as demonstrated by increased
proliferation. See, FIG. 2A-2C.
Example 3: Cell Migration Assay
[0113] In order to test the ability of EMD peptides to recruit
cells haptotactic migration assays with Hep I (SEQ ID NO:1), Hep
III (SEQ ID NO:2), FC/HV (SEQ ID NO:3), RGD (SEQ ID NO:4), FN, and
Col IV were conducted using a modified Boyden chamber. Haptotactic
migration was performed in triplicate and was assessed using a
modified Boyden chamber (Corning, CoStar, Acton, Mass.). The assay
was carried out as follows: the lower chamber was first blocked
with 10% BSA for at least 30 minutes at 37.degree. C. followed by 3
washings with PBS. The lower surface of the membrane on the upper
chamber was coated with approximately 10 .mu.L of protein or EMD
peptide solution having a concentration between 500 ng/mL-300
.mu.g/mL and allowed to incubate for 15-30 minutes at 37.degree. C.
and then allowed to air dry at room temperature under aseptic
conditions. At least one assay was performed for each
concentration. Basal cell media supplemented with 0.5% BSA was then
added to the lower chamber and 100 .mu.L of HUVECs
(1.times.10.sup.4 to 2.times.10.sup.4 cells) in the same cell media
was added to the upper chamber. The chambers were then incubated at
37.degree. C., 5.0% CO.sub.2 for 6 hours. Upon completion of the
incubation period, the cells on the membrane of the upper chamber
were fixed with 4% paraformaldehyde followed by removal of the
cells on the upper side of the membrane with a Q-tip. Next, the
membrane was carefully removed from the chamber, dipped in a 1:4000
dilution of Hoechst 33342 (Invitrogen, Carlsbad, Calif.) and placed
on a glass slide. Using a fluorescent microscope (Nikon Eclipse
E800) at 10.times. magnification, five random fields of view were
photographed on each membrane for determination of the area cell
density.
[0114] In order to allow for direct comparison, the area cell
density of migrated cells was normalized to the area cell density
of migrated cells on the uncoated (0 .mu.g/mL) control membrane.
See, FIG. 3A-3B. The results show that the proteins fibronectin and
Collagen IV both promoted migration. Similarly, the EMD peptides
Hep III (SEQ ID NO:2) and RGD (SEQ ID NO:4) also showed significant
migration compared to control. Hep I (SEQ ID NO:1) showed
statistically significant migration at concentrations greater than
1 .mu.g/ml, while cells on FC/HV (SEQ ID NO:3) coated membranes did
not show statistically significant migration at any of the
concentrations tested compared to control membranes.
Example 4: Conjugation of a Hep III EMD Peptide to a Myosin Heavy
Chain (MHC) Antibody and Targeting of the EMD Peptide to Injured
Myocardium In Vivo
[0115] This example demonstrates an exemplary conjugation method
for linking the EMD peptide to an injury associated antigen
specific binding component using carboiimide chemistry. A mouse
anti-rat cardiac myosin heavy chain (anti-MHC) monoclonal antibody,
was isolated from a hybridoma, ATCC deposit # HB-276, (Manassas,
Va.) by Panorama Research Inc., Mountain View, Calif. For
conjugating the Hep III peptide (SEQ ID NO:2) to the MHC antibody,
the crosslinker 1-ethyl-3[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDC, Pierce, Rockford Ill.) was used. The EDC
together with sulfo-NHS was incubated with the Hep III peptide
followed by incubation with the MHC antibody. Unconjugated Hep III
peptide was removed from the MHC-HepIII solution using a size
exclusion column. In order to maximize the conjugation of the EMD
peptide to the antibody, a molar excess of the peptide was used.
Assessment of the conjugation efficiency can be monitored using
fluorescence spectrophotometry for the EDC conjugation, where the
peptide is fluorescently labeled with fluorescein. Alternatively,
verification of the conjugation can be confirmed via mass
spectrometry and amino acid analysis.
[0116] To demonstrate in vivo targeting of the EMD peptide, the
MHC-HepIII complex or PBS (negative control) was injected into rats
following myocardial injury. All surgical procedures were approved
by the Committee for Animal Research of the University of
California San Francisco (San Francisco, Calif.). The
ischemia-reperfusion model used in this study has been extensively
tested in our lab. Sprague-Dawley rats (225-250 g) were
anesthetized with isoflurane. The chest was opened by a median
sternotomy, and a single stitch of 7-0 Ticron suture (United States
Surgical division of Tyco Healthcare, Norwalk, Conn.) was
introduced around the left anterior descending (LAD) coronary
artery and tightened to occlude for 25 min before reperfusing the
vessel. The chest was then closed and the animal was allowed to
recover. The rats were injected intravenously via the external
jugular vein one day after the myocardial infarct (MI), and then
sacrificed on Day 1, 2, 4, and 7 (N=1 per group). The hearts from
each group were excised, fresh frozen, and sectioned into 10 .mu.m
slices. The presence of the MHC-HepIII complex in the infarct
region was determined by immunostaining the heart sections using a
Mouse on RAT HRP (Horse Radish Peroxidase) Polymer Kit (Biocare
Medical, Concord, CA). The kit stains for any primary rat antibody
that was grown in mouse, as was the case for the anti-MHC. Positive
staining (brown) is seen only in the infarct zone for the heart
treated with MHC-HepIII (FIG. 4A-4B). Thus, the Hep III EMD peptide
was successfully conjugated to an injury associated antigen
specific binding component (anti MHC antibody) and targeted to an
injured tissue (the MI region).
Example 5: In Vivo Administration of a Targeted EMD Peptide for the
Repair of Injured Myocardial Tissue
[0117] This example demonstrates that the targeted EMD peptide
facilitates the repair and/or regeneration of an injured tissue.
The tissue injury was induced using the same procedure as described
above in Example 4.
[0118] Following the MI and prior to the injection of the targeted
EMD peptide, the rats were randomized to either control (PBS) or
treatment groups (MHC-HepIII, or MHC only). Each animal received a
single injection 500 .mu.l (.about.100 .mu.g of total protein) via
the external jugular vein using a 30 gauge needle 1-2 days after
MI. The rats were sacrificed 5-6 weeks post treatment.
[0119] Immediately after sacrifice, the heart was removed, rinsed
in ice-cold saline, blotted-dry and fresh frozen in Tissue Tek
O.C.T. freezing medium (Sakura Finetek, Torrance, Calif.), and
sectioned into 10 .mu.m slices. Representative slides were stained
with Masson's trichrome stain for determination of infarct
size.
[0120] Angiogenesis in the infarct was examined by
immunohistochemical staining with mouse monoclonal anti-CD31 (BD
Biosciences Pharmingen, San Diego, Calif.) to visualize capillaries
and with mouse monoclonal anti-SMA (Sigma, St. Louis, Mo.) to
detect arterioles and myofibroblasts. The staining assay was
performed with using Mouse-on-rat HRP-polymer (Biocare Medical,
Concord, Calif.), with pretreatment with peroxidase and Background
Sniper blocking agents. Capillaries in the infarct were identified
as a single layer of CD31-positive cells with flattened morphology.
Vessel density was calculated on the basis of five high
magnification fields per section that spanned the entire infarct
and averaged among five sections for each sample. Arterioles within
or bordering the infarct were identified as staining positive for
SMA and as having a visible lumen with a diameter between 10 and
100 .mu.m. Arteriole density was calculated as the average number
of arterioles in the total infarct area, out of five representative
slides per sample.
[0121] The results as shown in FIG. 5, indicate that MHC-HepIII
(p=0.0028) and MHC-FC/HV (p=0.044) showed a statistically
significant increase in angiogenesis compared to animals treated
with PBS (control) at the p<0.05 level. Therefore, we have
successfully demonstrated that the EMD peptide HepIII targeted to
the site of a myocardial infarct aids in the repair and
regeneration of the injured tissue.
[0122] All patents, patent applications, sequences, sequence
accession numbers, and other publications cited in this application
are hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
4113PRTArtificial Sequencesynthetic EMD peptide Hep I 1Thr Ala Gly
Ser Cys Leu Arg Lys Phe Ser Thr Met Tyr1 5 10214PRTArtificial
Sequencesynthetic EMD peptide Hep III 2Gly Glu Phe Tyr Phe Asp Leu
Arg Leu Lys Gly Asp Lys Tyr1 5 1038PRTArtificial Sequencesynthetic
EMD peptide FC/HV 3Trp Gln Pro Pro Arg Ala Arg Ile1
5413PRTArtificial Sequencesynthetic EMD peptide RGD 4Gly Arg Gly
Asp Ser Pro Ala Ser Ser Pro Ile Ser Cys1 5 10
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