U.S. patent application number 11/490033 was filed with the patent office on 2007-01-11 for novel haptotactic peptides.
This patent application is currently assigned to Hadasit Medical Research Services & Development Ltd.. Invention is credited to Raphael Gorodetsky, Gerard Marx.
Application Number | 20070009571 11/490033 |
Document ID | / |
Family ID | 37085898 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009571 |
Kind Code |
A1 |
Gorodetsky; Raphael ; et
al. |
January 11, 2007 |
Novel haptotactic peptides
Abstract
A group of novel peptides having a sequence selected from the
group consisting of KGSWYSMRKMSMKIRPFFPQQ (SEQ ID NO:1),
KTRWYSMKKTTMKIIPFNRL (SEQ ID NO:2) and RGADYSLRAVRMKIRPLVTQ (SEQ ID
NO:3) are provided. Compositions and methods of use of these
peptides are also provided.
Inventors: |
Gorodetsky; Raphael;
(Jerusalem, IL) ; Marx; Gerard; (New York,
NY) |
Correspondence
Address: |
WINSTON & STRAWN LLP;PATENT DEPARTMENT
1700 K STREET, N.W.
WASHINGTON
DC
20006
US
|
Assignee: |
Hadasit Medical Research Services
& Development Ltd.
Jerusalem
IL
|
Family ID: |
37085898 |
Appl. No.: |
11/490033 |
Filed: |
July 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09487790 |
Jan 20, 2000 |
7122620 |
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11490033 |
Jul 21, 2006 |
|
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09084371 |
May 27, 1998 |
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09487790 |
Jan 20, 2000 |
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Current U.S.
Class: |
424/423 ;
435/320.1; 435/325; 435/69.1; 514/16.7; 514/17.1; 514/19.3;
514/21.4; 530/350 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/75 20130101 |
Class at
Publication: |
424/423 ;
530/350; 514/012; 435/069.1; 435/320.1; 435/325 |
International
Class: |
A61K 38/18 20070101
A61K038/18; C07K 14/47 20070101 C07K014/47; C12P 21/06 20060101
C12P021/06; A61K 38/00 20060101 A61K038/00 |
Claims
1. An isolated polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO:2, SEQ ID NO:3, or a functional analogue thereof
that has haptotactic activity.
2. The isolated polypeptide of claim 1, comprising an amino acid
sequence as set forth in SEQ ID NO:2 and functional analogues
thereof having haptotactic activity.
3. The isolated polypeptide of claim 1, comprising an amino acid
sequence as set forth in SEQ ID NO:3 and functional analogues
thereof having haptotactic activity.
4. The isolated polypeptide of claim 1, comprising an amino acid
sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
5. An isolated nucleic acid comprising a polynucleotide encoding a
polypeptide as set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID
NO:3.
6. The isolated nucleic acid of claim 5, comprising a
polynucleotide encoding a polypeptide as set forth in SEQ ID
NO:1.
7. The isolated nucleic acid of claim 5, comprising a
polynucleotide encoding a polypeptide as set forth in SEQ ID
NO:2.
8. The isolated nucleic acid of claim 5, comprising a
polynucleotide encoding a polypeptide as set forth in SEQ ID
NO:3.
9. A composition comprising an isolated peptide according to claim
1 and a pharmaceutically acceptable carrier.
10. The composition of claim 9, further comprising a biological
agent.
11. A composition comprising the isolated polypeptide of claim 1 or
a haptotactic peptide having a sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, wherein the
isolated or haptotactic peptide is attached to the surface of a
prosthetic device, a bead, or a matrix or is present in combination
with cells.
12. The composition of claim 11, wherein the haptotactic peptide is
attached to a bead.
13. The composition of claim 11, wherein the haptotactic peptide is
attached to a matrix.
14. The composition of claim 11, wherein the haptotactic peptide is
present in combination with cells selected from the group
consisting of fibroblasts, endothelial cells, chondrocytes,
osteoblasts, neuroblastroma cells, kidney cells, liver cells,
pancreatic cells, thyroid cells, glial cells, nerve cells, smooth
muscle cells, mouse mammary carcinoma cells, bone or cartilage
forming cells and combinations thereof.
15. A polymer composition, comprising: (a) a plurality of subunits,
each featuring at least one isolated polypeptide according to claim
1 or a haptotactic peptide selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and (b) a plurality of
linker moieties for attaching each of the plurality of subunits to
another of the plurality of subunits to form the polymer.
16. The polymer composition of claim 15, wherein each of the
plurality of subunits is comprised of the at least one haptotactic
peptide, such that the polymer is a peptide polymer.
17. The polymer composition of claim 15, wherein the at least one
haptotactic peptide is attached to the subunit, such that the
polymer is a co-polymer.
18. A cell structure, comprising: (a) the isolated polypeptide of
claim 1 or a haptotactic peptide having a sequence selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;
(b) a cell bound to the haptotactic peptide; and (c) a structure
for supporting the cell, the haptotactic peptide being attached to
the structure such that the cell is supported by the structure.
19. The cell structure of claim 18, wherein the cell is selected
from the group consisting of fibroblasts, endothelial cells,
chondrocytes, osteoblasts, neuroblastroma cells, kidney cells,
liver cells, pancreatic cells, thyroid cells, glial cells, nerve
cells, smooth muscle cells, mouse mammary carcinoma cells, bone or
cartilage forming cells and combinations thereof.
20. The cell structure of claim 18, wherein said structure is a
biomedical device.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/487,790 filed Jan. 20, 2000, which is a continuation-in-part
of U.S. application Ser. No. 09/084,371 filed May 27, 1998, now
abandoned, the content of each of which is incorporated herein by
reference thereto.
FIELD AND BACKGROUND
[0002] The present invention relates to novel haptotactic peptides,
and in particular, to novel peptides which are homologous to a
portion of the carboxy termini of fibrinogen chains, as well as to
potential uses for these peptides.
[0003] Fibrinogen is the plasma protein which forms the clot when
blood coagulates. Many studies have been conducted on the amino
acid sequences and structure of fibrinogen (Mosesson, M. and
Doolittle, R. (Eds.) "The biology of fibrinogen and fibrin", Ann.
N.Y. Acad. Sci., 408, 1983, Henschen, A. et al., "Structure of
fibrinogen", Ann. N.Y. Acad. Sci., 408, 1983, Spraggon, G. et al.,
"Crystal structure of fragment D from human fibrinogen and its
crosslinked counterpart from fibrin", Nature, 389:455-462, 1997,
Murakawa, M. et al., "Diversity of primary structures of the
carboxy-terminal regions of mammalian fibrinogen A.alpha.-chains",
Thromb. & Haemostat., 69:351-360, 1993). Normally, fibrinogen
itself has a molecular weight of 340 kDa and is constructed from
two sets of three peptide chains, named .alpha., .beta. and
.gamma.. The constituent chains of fibrinogen are highly conserved
between species. Recent work has also described a fibrinogen
protein with a longer a chain, called .alpha.E fibrinogen, which
has a concomitantly higher molecular weight of 420 kDa and which
may play a role in development (Fu, Y. and Grieninger, G. "Fib420:
A normal human variant of fibrinogen with two extended .alpha.
chains", Proc. Natl. Acad. Sci. USA, 91: 2625-2628, (1994), Fu, Y,
et al., "Carboxy-terminal-extended variant of the human fibrinogen
.alpha. subunit: A novel exon conferring marked homology to .beta.
and .gamma. subunits" Biochem., 31:11968-11972, (1992)). Thus,
these four types of fibrinogen chains, .alpha., .beta., .gamma. and
.alpha.E, have 610, 410, 391 and 1096 amino acids, respectively
(numbering based on the Gene-bank data base at
info@ncbi.nlm.nih.gov).
[0004] Fibrin clots are formed in vivo at the sites of tissue
injury based upon the reaction of fibrinogen and thrombin in the
presence of calcium ions. These clots have a major role in
hemostasis. After clot formation, fibrin serves a provisional
matrix for cell recruitment into the wound bed. Normally, the
earliest cells mobilized into the wound bed are inflammatory, such
as leukocytes and particularly macrophages. Concomitant with their
penetration into the fibrin, these inflammatory cells participate
in lysing the fibrin by generating plasmin, metallo-proteinases
(MIPs) and/or free radicals. Thus, the wound bed contains
substantial quantities of peptides A and B (FPA and FPB) released
by thrombin during the onset of coagulation and numerous fibrin
breakdown products are venerated by lytic enzymes or free-radicals
(Gray, A. J., Reeves, J. T., Harrison, N. K., Winlove, P. and
Laurent, G. J., "Growth factors for human fibroblasts in the solute
remaining after clot formation", J. Cell Sci., 96: 271-274, (1990),
Marx G. "Immunological monitoring of Fenton fragmentation of
fibrinogen", Free Radicals Res. Comm. 12: 517-520 (1991), Francis,
C. W., Marder, V. J. and Barlow, G. H., "Plasmic degradation of
crosslinked fibrin", J. Clin Invest., 66: 1033-1043, (1980),
Cottrell, B. A. and Doolittle, R. F. "The amino acid sequence of a
27-residue peptide released from .alpha.-chain carboxy-terminus
during the plasmic digestion of human fibrinogen", Acad. Press.,
71: 754-766, (1976)).
[0005] Subsequently, the inflammatory cells are followed by the
migration of cells of the mesenchymal cell lineage such as
fibroblasts which further digest fibrin, replacing it with
extracellular matrix (ECM). Endothelial cells also infiltrate the
wound bed and generate microcapillary structures. Ultimately these
cells replace the provisional fibrin matrix with granulation tissue
populated by parenchymal cells and vasculature within the newly
synthesized ECM.
[0006] The attachment and migratory responses of cells to matrix
were proposed to be controlled mainly by specific receptors
(integrins) or by intercellular adhesion molecules (ICAM) that
interact with cell membrane receptors which subsequently induce
either migratory reactions or cell adhesion to matrix. These
interactions may trigger other regulatory mechanisms of cell
activity, such as shape change or proliferation. Growth factors and
cytokines activate such cell receptors by binding to them, and
thus, trigger cellular responses (Ruslahti, E. (1996) RGD and other
recognition sequences for integrins. Ann. Rev. Cell Dev. Biol. 12,
697-715 and Hynes, R. O. (1992) Integrins: Versatility, modulation
and signaling in cell adhesion. Cell 69, 11-25).
[0007] Cytokines of different classes regulate cellular activity
and responses, control cell survival, growth and differentiation.
Excluding classical endocrine hormones, cytokines encompass those
families of cell regulators variously known as growth factors,
interleukins, lymphokines and interferons.
[0008] All previously described cytokines are composed of more than
50 amino acids (aa); most are over 100 aa long. Based on X-ray
crystallography, cytokines exhibit 8 structural groups (Nathan C.
& Sporn M., "Cytokines in context", J. Cell. Biol. 113: 981-986
(1991)) and bind to a variety of cellular receptors such as
integrins or interferon receptors. Binding to cell receptors
triggers a cascade of events leading to intra-cellular
phosphorylation of proteins, which is transduced into gene
expression, cell proliferation, cell differentiation, changes in
cell shape, motility and apoptosis. Thus, cytokines play an
important role in physiological processes such as development and
wound healing.
[0009] Human fibroblasts are the major cellular entities
responsible for the regeneration of the extracellular matrix within
the wound bed. Human fibroblasts also express specific membrane
receptors to fibrinogen and thrombin. In the case of skin wounds,
human fibroblasts reform the matrix of the dermis. For example,
during the course of healing of an incisional skin wound, human
fibroblasts are mobilized from the surrounding tissue and enter
into the fibrin clot, help to dissolve tile clot, and then generate
as well as reform the collagen in the extracellular matrix. Based
upon these properties of human fibroblasts, fibroblast implants
have been suggested to supplement the process of healing in damaged
skin (Gorodetsky, R. et al., Radiat. Res., 125:181-186, 1991).
[0010] One material used for this purpose is benzoylated hyaluronic
acid (HA) sheets containing holes or pores as a carrier for
fibroblasts and keratinocytes for wound healing (Andreassi, L., et
al., Wounds, 3:116-126, 1991). Specifically, HA sheets were
cultured with such cells which grow within the pore structure. The
HA sheets were then affixed to the site of the burn injury, where
the cells migrated out of the sheet and ultimately accelerated the
rate of wound re-granulation. A major problem with implanted HA
sheets, however, is that they are not metabolized by tissue, are
mechanically cumbersome to administer, and may cause undesired
immunological effects in the long term.
[0011] Another material used for prosthetic tissue engineering is
collagen from pig or beef sources. However, collagen has several
mechanical limitations and may reduce the new collagen synthesis by
cells that are incorporated in it. There is also concern regarding
the safety of animal collagen products for medical implantation and
its use has been severely limited in Europe.
[0012] Fibrin microbeads (FMB) have been disclosed as possessing
both chemotactic and proliferative effects for certain types of
cells in U.S. application Ser. No. 08/934,283, filed in Sep. 19,
1997 and Gorodetsky, R., Vexler A., Shamir M., An J., Levdansky L.,
and Marx G. (1999). J. Invest. Dermatol. 112, 866-872 (1999)). The
cells that are attracted to FMB include fibroblasts and smooth
muscle endothelial cells, but typically not keratinocytes. The
cells were shown to migrate into these FMB by chemotaxis, attach to
them (haptotaxis) and then to proliferate on the FMB. Furthermore,
the cells were shown to remain stable for prolonged periods of time
when cultured within the FMB. Thus, the disclosed FMB appeared to
stimulate both cell chemotaxis, haptotaxis and cell growth.
[0013] However, the fibrin microbeads themselves have certain
inherent limitations. For example, the FMB are particularly useful
only as three-dimensional micro-structures. If other structures
were desired, and in particular if the lack of such was desired,
FMB would not be particularly useful. Furthermore, FMB would not be
particularly useful for avoiding the use of blood plasma
proteins.
[0014] A more useful approach would identify the epitopes of
fibrin(ogen) responsible for its chemotactic and haptotactic
properties. Attempts have been made to find these small epitopes
within the larger fibrin(ogen) molecule. A voluminous literature
exists which describes the binding of fibrinogen (.gamma.400-411)
to platelets through the GPIIb/IIIa receptor (see for example
Savage B., Bottini E. & Ruggeri Z M., "Interaction of integrin
alpha IIb beta with multiple fibrinogen domains during platelet
adhesion", J. Biol. Chem. 270: 28812-7 (1995)), and the aggregation
activity of the amino B.beta. 15-42 terminus which is exposed after
release of fibrinopeptide B. In addition, a peptide containing the
16 amino acids of the sequence of the .gamma.-carboxy terminus of
fibrinogen was synthesized and was found to bind to platelet
integrin (D'Souza, S. E. et al., J. Biol. Chem., 265:3440-3446.
1990). However, the biological activities of only a few other
fibrinogen breakdown products have been investigated with cells and
the activity of these different breakdown products seems to be
widely variable.
[0015] In another example, fibrinogen fragment E was reported to
exhibit angiogenic properties and to inhibit endothelial cell
migration in a Boyden chamber chemotactic assay (Thompson, W. D.,
Smith E. B., Stirk, C. M., Marshall, F. I., Stout, A. J. and
Kocchar, A., "Angiogenic activity of fibrin degradation products is
located in fibrin fragment E", J. Pathol, 168: 47-53 (1992)).
Fragment D was reported to cause detachment of cultured endothelial
cells from the extracellular matrix (ECM) substratum in a process
which was both concentration and time dependent (Savage B., Bottini
E. & Ruggeri Z M., "Interaction of integrin alpha IIb beta with
multiple fibrinogen domains during platelet adhesion", J Biol.
Chem. 270: 28812-7 (1995)). Isolated constituent chains of
fibrinogen (A.alpha.1, A.alpha.2 and B.beta.) released upon
activation of the fibrinogen by thrombin were observed to stimulate
fibroblast proliferation by 23-31% above controls, whereas isolated
.gamma. chain had no effect (Gray, A. J., Bishop, J. E., Reeves, J.
T. and Laurent, G. J.; "A.alpha. and B.beta. Chains of fibrinogen
stimulate proliferation of human fibroblasts", J. Cell Sci., 104:
409-413, (1993)). Human polymorphonuclear leukocytes (PMN) were
shown to bind to fibrin(ogen) coated surfaces via a type 3
(CD11b/CD18) complement receptor homologous to the GPIIb/IIIa
receptor through a decamer of the y chain carboxy terminus
(LGGAKQAGDV). Vasoactive peptides corresponding to residues 43-47
of the B.beta. chain and 220-230 of the A.alpha. chain were
identified (Gray., A. J., Bishop, J. E., Reeves, J. T. and Laurent,
G. J.; "A.alpha. and B.beta. Chains of fibrinogen stimulate
proliferation of human fibroblasts", J. Cell Sci., 104: 409-413,
(1993)).
[0016] The biological activities of only few other fibrinogen
breakdown products have been investigated, whose cellular activity
seems to be widely variable (Saldeen T: Vasoactive peptides derived
from degradation of Fibrinogen and fibrin. Proc. NY Acad Sci USA,
408: 424-431 (1983)).
[0017] Fibrinogen itself, when bound to sepharose beads, did not
significantly affect cell proliferation, but elicited
haplotactic/attachment reactions from human (HF) or mouse (MF)
fibroblasts, endothelial cells (EC) and smooth muscle (SMC) cells.
Thrombin treatment of fibrinogen-sepharose beads (SB-fib), which
would not affect the carboxy termini of the molecule, did not alter
cellular responses, though plasmin, which clips off gamma carboxy
termini and digests D-domain sequences, nearly totally abrogated
the cell-attractant properties of SB-fib (Gorodetsky R., Vexler A.,
An J., Mou X, Marx G. (1998) J. Lab. Clin. Med. 131: 269-280).
[0018] Specific epitopes on fibrinogen have been hypothesized to
express cell binding (haptotactic) properties. However, the amino
acid sequence(s) of such putative haptotactic epitopes have not yet
been specified. The identification of such epitopes would have a
number of applications, enabling more specific intervention in the
wound healing process and in the development of novel therapeutic
compositions or devices. Furthermore, novel diagnostic tests for
testing cellular haptotactic responses could potentially be
developed. Thus, the identification of these specific epitopes or
peptides exhibiting cellular activity would have great utility.
[0019] There is thus a recognized need for, and it would be highly
advantageous to have, a peptide or peptides with specifically
determined cellular effects, such as cell proliferative or
chemotactic or haptotactic properties, which do not require the
presence of the entirety of the fibrin molecule to exert cellular
effects.
SUMMARY OF THE INVENTION
[0020] It is one object of the present invention to provide
haptotactic peptides with novel amino acid sequences which are
featured within the carboxy termini of fibrinogen.
[0021] It is another object of the present invention to provide
such peptides useful for pharmaceutical compositions.
[0022] It is still another object of the present invention to
provide such haptotactic peptides useful for cell culture and cell
separation.
[0023] It is yet another object of the present invention to provide
such haptotactic peptides useful for novel cell structures,
including biomedical devices.
[0024] These and other objects of the present invention are
explained in greater detail in the descriptions, Figures and claims
below.
[0025] The novel synthetic peptide sequences of the present
invention are homologous to selected regions present within the
fibrin molecule, yet retain certain derived properties of the
entire molecule, such as cell adhesive effects, for example. The
specific sequences of these haptotactic peptides are
KGSWYSKMSMKIRPFFPQQ (peptide-C.beta. (code name--(09), hereinafter
referred to as `peptide-C.beta.`, (SEQ ID NO: 1)),
KTRWYSMKKTTMIIPFNRL (peptide preC.gamma., (code name 70a,
hereinafter referred to as `peptide preC.gamma.`, (SEQ ID NO:2))
and RGADYSLRAVRMKIRPLVTQ (peptide-C.alpha.E, (code name (71),
hereinafter referred to as peptide-C.alpha.E, (SEQ ID NO:3)).
[0026] According to the teachings of the present invention, there
is provided a synthetic polypeptide, comprising an amino acid
sequence as set forth in SEQ ID NO:1. There is also provided a
synthetic polypeptide, comprising an amino acid sequence as set
forth in SEQ ID NO:2. There is also provided a synthetic
polypeptide, comprising an amino acid sequence as set forth in SEQ
ID NO:3. In addition, there is provided a polypeptide, comprising
an amino acid sequence as set forth in SEQ ID NO:1 and functional
analogues thereof having at least one amino acid substitution into
a naturally occurring or non-naturally occurring amino acid and
having a haptotactic activity. There is provided a polypeptide,
comprising an amino acid sequence as set forth in SEQ ID NO:2 and
functional analogues thereof having at least one amino acid
substitution into a naturally occurring or non-naturally occurring
amino acid and having a haptotactic activity. There is also
provided a polypeptide, comprising an amino acid sequence as set
forth in SEQ ID NO:3 and functional analogues thereof having at
least one amino acid substitution into a naturally occurring or
non-naturally occurring amino acid and having a haptotactic
activity.
[0027] According to another embodiment there is provided an
isolated nucleic acid comprising a polynucleotide encoding a
polypeptide as set forth in SEQ ID NO:1. There is also provided an
isolated nucleic acid comprising a polynucleotide encoding a
polypetide as set forth in SEQ ID NO:2. Additionally, there is
provided an isolated nucleic acid comprising a polynucleotide
encoding a polypetide as set forth in SEQ ID NO:3.
[0028] According to another embodiment of the present invention,
there is provided a composition, comprising a haptotactic peptide
having a sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2 and SEQ ID NO:3. Preferably, the composition
further comprises a pharmaceutically acceptable carrier. Also
preferably, the composition further comprises a biological agent
selected from the group consisting of drugs, vitamins, vitamin
derivatives, growth factors, glucocorticosteroids, steroids,
antibiotics, toxins, enzymes, enzyme inhibitors, immunomodulators,
immunoglobulins and fragments thereof, fatty acid derivatives,
polysaccharides, cell receptor binding molecules,
anti-inflammatories, nucleic acids, and polynucleotides.
[0029] In a preferred embodiment the haptotactic peptide is
attached to the surface of a prosthetic device.
[0030] In a preferred embodiment the haptotactic peptide is
attached to a bead.
[0031] In a preferred embodiment the haptotactic peptide is
attached to a matrix.
[0032] According to preferred embodiments of the present invention,
the composition further comprises a cell selected from the group
consisting of fibroblasts, endothelial cells, chondrocytes,
osteoblasts, neuroblastoma cells, kidney cells, liver cells,
pancreatic cells, thyroid cells, glial cells, nerve cells, smooth
muscle cells, mouse mammary carcinoma cells, bone or cartilage
forming cells, and combinations thereof.
[0033] According to yet another embodiment of the present
invention, there is provided a cell structure, comprising: (a) a
peptide having a sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; (b) a cell bound to the
peptide; and (c) a structure for supporting the cell, the peptide
being attached to the structure such that the cell is supported by
the structure. Preferably, the structure is a biomedical
device.
[0034] According to another embodiment of the present invention,
there is provided a polymer composition, comprising: (a) a
plurality of subunits, each subunit featuring at least one
haptotactic peptide selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2 and SEQ ID NO:3; and (b) a plurality of linker
moieties for attaching each of the plurality subunits to another of
the plurality of subunits to form the polymer. Preferably, the
subunit is comprised of the at least one haptotactic peptide, such
that the polymer is a peptide polymer. Alternatively and
preferably, the at least one haptotactic peptide is attached to the
subunit, such that the polymer is a co-polymer.
[0035] Hereinafter, the term "wound-healing cells" refers to those
cells which promote healing of a wound, including, but not limited
to fibroblasts, smooth muscle endothelial cells osteoblasts and
chondrocytes.
[0036] The term "fibrin(ogen)" is known in the art as a mixture of
fibrin and fibrinogen, and is referred to herein according to this
definition. Hereinafter, the term "biologically active" refers to
molecules, or complexes thereof, which are capable of exerting an
effect in a biological system. Hereinafter, the term "fragment"
refers to a portion of a molecule or a complex thereof, in which
the portion includes substantially less than the entirety of the
molecule or the complex thereof.
[0037] Hereinafter, the term "amino acid" refers to both natural
and synthetic molecules which are capable of forming a peptidic
bond with another such molecule. Hereinafter, the term "natural
amino acid" refers to all naturally occurring amino acids,
including both regular and non-regular natural amino acids.
Hereinafter, the term "regular natural amino acid" refers to those
amino acids which are normally used as components of a protein.
Hereinafter, the term "non-regular natural amino acid" refers to
naturally occurring amino acids, produced by mammalian or
non-mammalian eukaryotes, or by prokaryotes, which are not usually
used as a component of a protein by eukaryotes or prokaryotes.
Hereinafter, the term "synthetic amino acid" refers to all
molecules which are artificially produced and which do not occur
naturally in eukaryotes or prokaryotes, but which fulfill the
required characteristics of an amino acid as defined above.
Hereinafter, the term "peptide" includes both a chain of a sequence
of amino acids, and analogues and mimetics having substantially
similar or identical functionality thereof, including analogues
having synthetic and natural amino acids. As shown in Table 1
below, peptide-C.alpha. (code name peptide (07, hereinafter
referred to as `peptide-C.alpha.`, SEQ ID NO:4) has the amino acid
sequence of the C-terminus of the alpha chain of fibrinogen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The invention is herein described, by way of example only,
with reference to the accompanying drawings, wherein:
[0039] FIG. 1 shows the haptotactic responses of cells to sepharose
beads (SB) coated with active C-terminal fibrinopeptides (either
C.beta. (SEQ ID NO: 1), preC.gamma. (SEQ ID NO:2) and C.alpha.E
(SEQ ID NO:3)) in comparison with positive controls fibrinogen
(Fib) or fibronectin (FN) and in contrary to non-active C.alpha.
peptide (SEQ ID NO:4) and the minor negligible activity of the
C.gamma. peptide (SEQ ID NO:5). The ligands covalently-bound to SB
were added to near confluent cell culture of SMC, HF and BAEC in
12-well plates. The rate of attachment was monitored visually by
counting the % of attached SB over time. The C.alpha. (SEQ ID NO:4)
was inactive, whereas C.beta. (SEQ ID NO:1), preC.gamma. (SEQ ID
NO:2) and to a lesser degree C.alpha.E (SEQ ID NO:3) were highly
haptotactic, with response kinetics equivalent to fibrinogen and
somewhat less than with fibronectin.
[0040] FIG. 2A shows the attachment and mounting of BAEC onto
SB-C.beta. (SEQ ID NO:1) visualized by light microscopy after 2
days of incubation with a nearly confluent culture. The cells on
Sepharose beads (SB) were fixed and stained by Giemsa (objective
x.times.40). The attraction and adhesive response of the cells that
mounted on the SB-C.beta. (SEQ ID NO:1) is evident.
[0041] FIG. 2B shows phagocytosis of small chopped fragments of
SB-C.beta..sup.FITC (SEQ ID NO:1) (approx. 1-5 .mu.m in diameter)
as viewed in an intracellular planar section in the middle of the
cells by fluorescent confocal microscopy and Numarski optics. This
shows that neutral materials can be rendered so attractive by the
peptides that they become engulfed (phagocytosis) by the cell.
[0042] FIG. 3 shows binding and internalization of dissolved
C.beta..sup.FITC (SEQ ID NO:1) and C.alpha.E.sup.FITC (SEQ ID NO:3)
to BAEC and HF as viewed by fluorescent microscopy (objective
.times.40). HF (A and B) and BAEC (C and D) were incubated with 100
.mu.g/ml of C.alpha.E.sup.FITC (SEQ ID NO:3) for 30 min (A and C
respectively) and 60 min (C and D respectively) and
C.beta..sup.FITC (SEQ ID NO:1) (C and D respectively). The arrow
points to the cell nuclei. Confocal microscopy showed the FITC
peptides distributed over the cell surface and in the cytoplasm
with little penetration into the nucleus. Cells initially
accumulated tagged peptides in the cell membrane and after a
prolonged exposure distributed within the cytoplasm to the
perinuclear area and into granular bodies.
[0043] FIG. 4 shows FACS analysis of FITC-labeled
fibrinopeptides-C.beta. (SEQ ID NO:1) (FIGS. 4A-4C), C.alpha.E (SEQ
ID NO:3) (FIG. 4D), C.gamma. (SEQ ID NO:5) (FIG. 4E) and C.alpha.
(SEQ ID NO:4) (FIG. 4F). Control represents the auto-fluorescence
of cells not exposed to FITC-peptides. While C.beta. (SEQ ID NO:1)
and to a lesser degree C.alpha.E (SEQ ID NO:3) clearly bound to
cells, C.alpha. (SEQ ID NO:4) served as a negative control and did
not bind to them.
[0044] FIGS. 5A, 5B and 5C show the concentration dependence of
haptotactic peptide uptake to cells as determined by FACS analysis.
FIG. 5A shows the rate of binding of C.beta..sup.FITC (SEQ ID NO:1)
and C.alpha.E.sup.FITC (SEQ ID NO:3) showing fast equilibrium
within 5 minutes. FIG. 5B shows a dose response of C.beta..sup.FITC
(SEQ ID NO:1) and C.alpha.E.sup.FITC (SEQ ID NO:3) showing binding
to HF and BAEC in concentrations of up to 40 .mu.M, assayed after
incubation for 1 hr with peptides at 4.degree. C. FIG. 5C shows the
effect of elevated concentrations of unlabeled C.beta. (SEQ ID
NO:1) on the uptake of C.beta..sup.FITC.
[0045] FIG. 6 shows the chemotaxis of BAEC to C.beta. (SEQ ID NO:1)
gradient in multi-chamber micro-Boyden assay. Similar results were
obtained with HF. Cells that transversed through the membrane were
stained and counted. C.alpha. (SEQ ID NO:4) elicited no response
relative to negative control with no additions, whereas conditioned
medium (CM) served as positive chemotactic control that induced
100% migration. The values and errors represent over 5 repeated
experiments for each of the agents.
[0046] FIG. 7 shows the lack or proliferative response of HF
exposed to the active C.beta. (SEQ ID NO:1) panel C, C.alpha.E (SEQ
ID NO:3) panel B, and pre C.gamma. (SEQ ID NO:2) panel E, peptides
versus the less active C.alpha. (SEQ ID NO:4) panel A and C.gamma.
(SEQ ID NO:5) panel D peptides that served as negative controls.
The peptides were tested in a concentration of 0.4 nM-4 .mu.M in 96
well plates with the MTS proliferation assay. Clearly none of these
peptides stimulated or inhibited proliferation at any
concentration.
BRIEF DESCRIPTION OF THE INVENTION
[0047] The present invention relates to novel peptides, and in
particular, to their corresponding novel peptide amino acid
sequences, as well as to potential uses for these sequences. For
example, these peptide sequences have potential medical uses, such
as for therapeutic and diagnostic uses. The synthetic peptide
sequences are homologous to regions of the fibrin molecule, yet
retain certain desired properties of the entire molecule, such as
cell adhesive effects, for example.
[0048] In particular, these haptotactic peptides are composed of a
sequence homologous to 19-21 amino acids sequence at the carboxy
terminus of the .beta. chain (termed C.beta. or code 09, (SEQ ID
NO:1)) and a sequence (termed C.alpha.E or code 71, (SEQ ID NO:3))
homologous to the C-terminus sequence of the recently discovered
.alpha.E chain, the so-called extended .alpha.E segment (.alpha.E)
(Fu, Y. and Grieninger, G. "Fib.sub.420: A normal human variant of
fibrinogen with two extended .alpha. chains," Proc. Natl. Acad.
Sci. USA, 91:2625-2628, (1994)). Additionally, is included
preC.gamma. (70A) (SEQ ID NO:2), a 20 mer peptide homologous to the
internal 7-chain fibrinogen chain sequence at address
.gamma.373-392 (411 total) (termed preC.gamma. or code 70A). Two
other 19-2 1-mer peptides homologous to the C-termini of the
.alpha. and the .gamma. chains (termed C.alpha. or code 07 (SEQ ID
NO:4) and C.gamma. or code 71 (SEQ ID NO:5) respectively) are
described and used as controls for haptotactic tests. Sequences of
these peptides are given in Table 1 below.
[0049] Table 1 shows the names, codes and sequences of five
peptides synthesized and tested, of which only C.beta. (SEQ ID
NO:1), C.alpha.E (SEQ ID NO:3) and PreC.gamma. (SEQ ID NO:2)
elicited significant haptotactic responses from cells. The
haptotactic C.beta. (SEQ ID NO: 1), C.alpha.E (SEQ ID NO:3) and
PreC.gamma. (SEQ ID NO:2) peptides are homologous to each other as
shown in bold (sequence numbering according to the database of the
Swiss Gene bank); TABLE-US-00001 TABLE 1 Synthetic peptides
corresponding to the carboxy termini of fibrinogen. Name Code #
Chain Address Sequence C.alpha. (07) .alpha. 591-610
EADHEGTHSTKRGHAKSRP (SEQ ID NO:4) C.beta. (09) .beta. 441-461
KGSWYSMRKMSMKIRPFFPQQ (SEQ ID NO:1) C.gamma. (70) .gamma. 392-411
LTIGEGQQHHLGGAKQAGDV (SEQ ID NO:5) PreC.gamma. (70A) .gamma.
373-392 KTRWYSMKKTTMKIIPFNRL (SEQ ID NO:2) C.alpha.E (71) .alpha.E
828-847 RGADYSLRAVRMKIRPLVTQ (SEQ ID NO:3)
[0050] Hereinafter, the term "haptotactic peptide" refers to
peptides-C.beta. (SEQ ID NO:1), C.alpha.E (SEQ ID NO:3) or
preC.gamma. (SEQ ID NO:2), having a sequence selected from the
group consisting of: KGSWYSMRKMSMKIRPFFPQQ (SEQ ID NO:1),
KTRWYSMKKTTMKIIPFNRL (SEQ ID NO:2) or RGADYSLRAVRMKIRPLVTQ (SEQ ID
NO:3); as well as to analogues, derivatives, equivalents or
peptido-mimetics thereof, displaying substantially identical or
similar functional activity as one of the above-listed sequences.
Peptides C.beta. (SEQ ID NO:1) and preC.gamma. (SEQ ID NO:2)
elicited the greatest haptotactic activity, followed by peptide
C.alpha.E (SEQ ID NO:3).
[0051] The mitogenic effects of these peptides were tested in cell
culture systems. No effects on cell number were observed with any
of these peptides. These peptides were also evaluated for their
cell adhesive properties when bound to sepharose beads (SB) placed
on nearly confluent cell cultures. Specifically, peptides C.beta.
(SEQ ID NO:1) and preC.gamma. (SEQ ID NO:2) elicited the greatest
haptotactic response to EC, followed by the C.alpha.E peptide (SEQ
ID NO:3) (FIG. 2, Table 3). The relative haptotactic activity of
these peptides varies with the cell types. By contrast,
peptide-C.alpha. (SEQ ID NO:4) elicited no cell adhesive effects
and C.gamma. (SEQ ID NO:5) showed only negligible effect with EC
that varied with the number of cell passages.
[0052] Two of the most potent peptides, peptides C.beta. (SEQ ID
NO:1) and C.alpha.E (SEQ ID NO:3), share an underlined sequence of
YSXRXXMKIRPXXXQ (SEQ ID NO:10). The shared sequence itself,
possibly with the addition of a spacer moiety or moieties for
proper geometrical configuration, is also contemplated as a peptide
of the present invention.
[0053] The two active peptides, peptides C.beta. (SEQ ID NO:1) and
preC.gamma. (SEQ ID NO:2) show marked homology and share an
underlined sequence of KXXWYSMXKXXMKIXPFXXX (SEQ ID NO.11) as is
shown in Table 1.
[0054] Three synthetic peptides, homologues of the C-termini chain
sequences of fibrinogen, C.beta. (SEQ ID NO:1), C.alpha.E (SEQ ID
NO:3) and preC.gamma. (SEQ ID NO:2) were active towards cells and
are well conserved in evolution (Table 2). Other sequences with
this same homology are also potentially active. Without wishing to
be limited by a single mechanism, functional cell attachment
features of fibrinogen chains are critical to the normal
development and wound healing of all species.
[0055] Table 2 shows interspecies homology and sequence
conservation of the active C-termini sequences of the fibrinogen
C.beta. (SEQ ID NO:1), the C.alpha.E (SEQ ID NO:3) and the
PreC.gamma. (SEQ ID NO:2). Shaded areas represent the homologous
sequences. It has been shown that there is a chain of amino acids
having a fibrinogen C.beta. consensus sequence of
++GVVW++++G+-YS+R-+-MKIRP---Q (SEQ ID NO:6). The + sign denotes
similar amino acids and the - sign indicates dissimilar amino
acids. In addition there is a chain of amino acids having a
fibrinogen C.beta. homology sequence of
D+G++W--WK--WK--WYSM+K-+MKI-PF---- (SEQ ID NO:12). The high
statistical significance of these conserved sequences suggests that
these sequences confer haptotactic activity to fibrinogen in all
species. TABLE-US-00002 TABLE 2 ##STR1## ##STR2## ##STR3## ##STR4##
##STR5## ##STR6##
[0056] The DNA and RNA sequences that code for the amino acids of
the haptotactic peptides were deduced. Without wishing to be
limited, one example of DNA sequences that code for the amino acids
of the laptotactic peptides is as follows: TABLE-US-00003
C.beta.-DNA AAGGGGTCATGGTACTCAATGAGGAAGATGAGTATGA (SEQ ID NO:7)
AGATCAGGCCCTTCTTCCCACAGCAA TAG C.alpha.E-DNA
AGAGGGGCAGATTATTCCCTCAGGGCTGTTCGCATGA (SEQ ID NO:8)
AAATTAGGCCCCTTGTGACCCAA TAG PreC.gamma.-DNA (SEQ ID NO:9)
AAAACCCGGTGGTATTCCATGAAGAAAACCACTATGA
AGATAATCCCATTCAACAGACTCACA
[0057] The amino acids of the haptotactic peptides can be encoded
by other DNA sequences.
[0058] The DNA and RNA sequences that code for the amino acids of
the haptotactic peptides can be used for medical as well as
diagnostic purposes. The haptotactic peptides of the present
invention are contemplated for many different uses. Therapeutic
uses include, but are not limited to, treatment of a wound bed.
Methods for treatment of the wound bed with the peptides of the
present invention are given in greater detail in Example 7 below.
In addition, therapeutic compositions which include the peptides of
the present invention are given in greater detail in Example 6
below.
[0059] Additional uses of the haptotactic peptides of the present
invention include, but are not limited to, the growth and transport
of cells in cell culturing systems, the separation of different
types of cells from mixed cell cultures, and the implantation of
peptide-coated prosthetic devices. These uses are explained in
greater detail in Example 6 below. Furthermore, as explained in
greater detail in Example 6 below, the haptotactic peptides of the
present invention can also be used as tools for biological analysis
and for further research and development.
[0060] These contemplated compositions, composites and uses of the
peptides of the present invention are outlined in the examples
below and are intended as illustrations only and are not meant to
be limiting in any way.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0061] The present invention is drawn towards novel peptidic
sequences of fibrin. Methods of using these peptidic sequences are
also contemplated, including methods for the promotion of wound
healing as well as diagnostic methods. These peptidic sequences
retain desirable properties exhibited by the entire fibrin
molecule, such as cell haptotaxis.
[0062] The principles and operation of such peptidic amino acid
sequences of fibrin and related sequences according to the present
invention may be better understood with reference to the
non-limiting illustrative examples below.
[0063] The peptides of the present invention (C.alpha.E (SEQ ID
NO:3), C.beta. (SEQ ID NO:1). PreC.gamma. (SEQ ID NO:2)) as well as
control peptides (C.alpha. (SEQ ID NO:4) and C.gamma. (SEQ ID
NO:5)) were synthesized and tested in cell culture systems as
described below. The experimental procedure is described in the
section entitled "Experimental Procedure". The results are given in
the section entitled "Results".
[0064] Essentially, designated peptides peptide-C.alpha. (SEQ ID
NO:4), peptide-C.beta. (SEQ ID NO:1), peptide preC.gamma. (SEQ ID
NO:2) and peptide-C.alpha.E (SEQ ID NO:3) were synthesized and
covalently attached to sepharose beads, to form SB-C.alpha.,
SB-C.beta. (SEQ ID NO:1), SB-preC.gamma. (SEQ ID NO:2) and
SB-C.alpha.E (SEQ ID NO:3), respectively. Fibrinogen was also
covalently attached to sepharose beads, to form SB-Fib. The
SB-ligand combination was then incubated with cultured cells. The
data are shown in the "Results" section below (Table 3). In
summary, SB-C.beta. (SEQ ID NO:1) and preC.gamma. (SEQ ID NO:2)
appeared to be the most potent for cell binding, showing even
greater potency than SB-Fib for all cell lines which bind
fibrin(ogen). All tested cell lines bound to SB-C.beta. (SEQ ID
NO:1) under these conditions, with the exception of OV-1063,
keratinocytes and cells derived from a leucocytic lineage. The next
most potent peptide/bead combination was SB-C.alpha.E (SEQ ID
NO:3), as strong binding was observed for SB-C.alpha.E (SEQ ID
NO:3) for the following cell lines: HF, MF, EC and EMT-6. Weak
attachment of SMC cells to SB-C.alpha.E (SEQ ID NO:3) was also
observed. Variable haptotaxis of SB-C.gamma. (SEQ ID NO:5) to EC
was observed. No cell binding was observed to SB-C.alpha. (SEQ ID
NO:4).
[0065] Furthermore, FITC-labeled peptides C.beta. (SEQ ID NO:1) and
C.alpha.E (SEQ ID NO:3) were clearly able to bind to the cell
membrane, and after a prolonged exposure, accumulated in the
cytoplasm, and migrated to the perinuclear area and granular
bodies.
Experimental Procedures
Preparation of Peptides
[0066] Peptidic analogues of the carboxy termini fibrinogen were
synthesized according to standard techniques (Synthetic Peptide
Corp., Dublin, Calif., USA). The amino acid sequences of these
analogues are given in Table 1 above. Each peptide was labeled with
a fluorescent label which was either fluorescein or EACA (epsilon
amino caproic acid). The labeling was performed according to well
known methods in the art, for example during the synthesis of the
peptide itself.
Preparation of sepharose Beads With Bound Peptides or Proteins.
[0067] Peptides or fibrinogen were covalently bound to CNBr
activated sepharose beads (SB) (Pharmacia, Piscataway, N.J.) using
techniques previously used to bind albumin, fibrinogen and
thrombin. Briefly, CNBr-activated sepharose 4B (Pharmacia) was
washed with 1N HCl, and suspended in the coupling buffer (pH 8.3),
following the protocol supplied by the manufacturer. The peptides
were dialyzed against the coupling buffer to remove Tris. Peptides
(1 mg) were not highly water soluble. The peptides were dissolved
in 1 mL 70% ethanol or dimethylformamide, and were then mixed into
the coupling buffer containing 1 mL CNBr-activated sepharose. The
suspension was gently agitated overnight, then centrifuged
500.times.g to pack the beads. This procedure covalently binds more
than 95% of proteins or peptides including fibrinogen or
peptides-C.alpha.E (SEQ ID NO:3), C.beta. (SEQ ID NO:1) and
preC.gamma. (SEQ ID NO:2) to the beads determined by Abs.sub.280
readings of the solutions.
[0068] Concentrations of peptides bound to SB were: peptide
C.alpha. (SEQ ID NO:4) was 7 mg/mL; peptide C.beta. (SEQ ID NO:1)
was 5.2 mg/mL; peptide C.alpha.E (SEQ ID NO:3) was 6.0 mg/mL;
peptide preC.gamma. (SEQ ID NO:2) was 5.5 mg/mL. The coated beads
were washed with Tris-saline buffer and stored at 4.degree. C. with
0.1% azide. Before use, the beads were washed 3 times in sterile
saline to remove all traces of azide.
[0069] After incubation with cells, samples of beads were prepared
for scanning electron microscopy (SEM) by fixing with 4%
glutaraldehyde, critical point dried, and coated with osmium
tetroxide, sputter coated with Au/Pd and then examined with a
Hitachi S-530 Scanning Microscope.
Preparation of Cell Cultures.
[0070] All cell cultures were prepared as previously described.
Normal human skin fibroblasts (HF) were isolated from skin biopsies
of young human subjects. The dermal layer of skin was chopped and
digested for 1 hour by 0.25% trypsin/versen. The isolated cells
were washed and plated on plastic Petri dishes with DMEM
supplemented by 10% fetal calf serum (FCS), antibiotics, and
glutamine. The plates were washed after 10 hrs to select for the
better attached fibroblasts. At the 3-4 passage, the cells
consisted of homogeneous populations of fibroblasts, as judged by
microscopy. Immunohistology with monoclonal
anti-human-fibroblast-surface proteins confirm that this procedure
yields homogeneous fibroblast culture (Ronnov-Jessen L, Celis J E,
Van-Deurs B, Petersen O W: "A fibroblast-associated antigen:
characterization in fibroblasts and immunoreactivity in smooth
muscle differentiated stromal cells", J. Histochem. Cytochem. 40:
475-486 (1992)). Normal murine fibroblasts (MF) were isolated from
the skin of 2-3 days old neonate C3H mice by 3 step digestion, each
for 2 hrs, with trypsin/versen. The use of neonate mice with low
cross linking of collagen served to enhance the high yield of cells
during the proteolytic digestion. The details of the rest of the
protocol are similar to those used for the isolation and growth of
HF. These cells could be grown for at least 12-14 passages before
any decrease of the rate of proliferation occurred. Cells were used
from the fourth passage to the tenth passage.
[0071] Porcine smooth muscle cells (SMC) were isolated from
thoracic aortas of young animals and kept in culture with twice
weekly medium change and splitting once in 1-2 week. Cells of up to
10 passages were used. The purity of the SMC culture was verified
by immunohistology with monoclonal anti muscle-specific-actin
HHF-35 (Bar-Shavit R, Benezra M, Eldor A, Hy-Am E, Fenton J W,
Wilner G D & Vlodavsky I: "Thrombin immobilized to
extracellular matrix is a potent mitogen for vascular smooth muscle
cells: nonenzymatic mode of action", Cell Regul. 1: 453-463, 1990).
Other cell lines were obtained from different sources and cultured
in their standard conditions as described in the following
references: murine fibroblast line (3T3) and normal human
keratinocytes (Ben-Bassat H, Eldad A, Chaouat M. Livotf A, Ron N,
Neeman Z, and Wexler M R: "Structural and functional evaluation of
modifications in the composite skin graft: cryopreserved dermis and
cultured keratinocytes", Plastic & reconstructive Surgery 89:
510-520 (1992)); murine mast cells (MC-9) (Razin E, and Marx G.,
"Thrombin-induced degranulation of cultured bone marrow-derived
mast cells", J. Immunol. 133: 3282-3285 (1984)); normal bovine
aortic endothelial cells (BAEC) (Vlodavsky I, Greenburg G, Johnson
L K and Gospodarowicz D: "Vascular endothelial cells maintained in
the absence of fibroblast growth factor undergo structural and
functional alterations that are incompatible with their in vivo
differentiated properties", J Cell Biol. 83:468-486, 1979); porcine
smooth muscle cells, isolated and cultured as previously described
(Bar-Shavit R, Benezra M, Eldor A, Hy-Am E, Fenton J W, Wilner G D
& Vlodavsky I: "Thrombin immobilized to extracellular matrix is
a potent mitogen for vascular smooth muscle cells; nonenzymatic
mode of action", Cell Regul. 1: 453-463, 1990); murine leukemic
cells (P-388) (Ramu A, Ramu N. & Gorodestsky R. "Reduced oubain
resistant potassium entry as a possible mechanism of
multidrug-resistance in p388 cells", Biochem. Parmacol., 42:
1699-1704 (1992)); human ovarian carcinoma cells (OV-1063) were
isolated from a primary tumor and then maintained as previously
described (Horowitz A T, Treves A J, Voss R, Okon E, Fuks Z,
Davidson L, and Biran S., "A new human carcinoma cell line:
establishment and analysis of tumor associated markers", Oncology
42: 332-337 (1985)); murine mammary adenocarcinoma cells (EMT-6)
were grown under the standard conditions (Rockwell S., "Cytotoxic
and radiosensitizing effects of hypoxic cell sensitizers on EMT6
mouse mammary tumor cells in vivo and in vitro", Br J Cancer 37:
212-215 (1978)); and the murine macrophage-like cells (J774.2)
(Ringel R, Roy S N, and Horwitz S B., "A phosphoglycoprotein
associated with taxol resistance in J774.2 cells", Cancer Res. 45:
3856-3863 (1985)).
[0072] All culture medium components were purchased from Biological
Industries (Beit-HaEmek, Israel) and fetal calf serum was supplied
by GIBCO (Grand Island, New York, N.Y., USA). The cell cultures
were maintained at 37.degree. C. in a water-jacketed CO.sub.2
incubators, and were harvested by trypsin/versen solution with 1-2
passages per week in a split ratio of 1:10 for fast proliferating
transformed cells and 1:4 for normal cell types.
Assays for Cell Proliferation
[0073] Cell proliferation was evaluated by measuring cell density
as a function of time by two different colorimetric assays. The MTS
colorimetric assay (CellTitre 96 Aqueous Assay by Promega) is based
on dehydrogenase conversion of MTS (methyl tetrazolium salt) by
viable cells to colored tetrazolium salt (Ge M. Tang, G., Ryan. T.
J. and Malik. A. B., "Fibrinogen degradation product fragment D
induces endothelial cell detachment by activation of cell-mediated
fibrinolysis", J. Clin. Inves., 90:2508-2516 (1992)); the methylene
blue (MB) assay is based on the staining of monolayer cells after
their fixation, and reading the absorbence of the absorbed dye.
[0074] The MTS assay for viable cells was performed as follows: 30
.mu.L of freshly prepared MTS/PMS were added to each well;
following 2 hrs incubation at 37.degree. C., the plates were placed
on a computer driven microplate reader (Anthos HT-II, Salzburg,
Austria), programmed to shake the plate for 1 minute before reading
the optical density (OD) of the dye at 490 nm. The measurements
were repeated following 4 and 6 hours of incubation.
Haptotaxis Activity Assay for Monitoring Cell Adhesion to Peptides
Bound to Sepharose Beads (SB).
[0075] The migratory/adhesion response of cells to proteins (such
as fibrinogen) or peptides covalently bound to SB was measured as
follows. Cells were grown in 6 or 12 well plates to near confluence
or in suspension until they covered about 1/2 to 2/3 of the plate
surface. At that point, about 20-150 mL of a suspension containing
50% v/v SB coated with the test protein or peptide were added to
the plate and dispersed by gentle shaking for 1 min. The plate was
then returned to the incubator and examined at different times by
inverted phase microscopy.
[0076] The SB (naked or ligand bound) sedimented onto the nearly
confluent cell layer and occasionally made physical contact with
cell membranes on the plate (ascribed to Brownian motion or
micro-convection currents). In a positively responding system, this
resulted in the tethering of SB to the cell layer, which could be
detected by visual inspection at different time points. Typically,
300 beads (but not less then 200) were counted in each well and the
ratio of SB bound to the cells relative to total number SB could be
calculated (SB bound/SB total). Counting the percentage of SB
attached to the cell surface at different time intervals provided a
quantitative assay of the kinetics of the attachment of coated
beads to cells. Negative control with uncoated SB or positive
controls with SB-fibrinogen were employed with at least 3 wells
measured for each. The statistical error was calculated from the
square root of the total counts.
[0077] After incubation with cells, samples of beads were prepared
for scanning electron microscopy (SEM) by fixing with 4%
glutaraldehyde, critical point dried and coated with osmium
tetroxide, sputter coated with Au/Pd and then examined with an
Hitachi S-5390 Scanning Microscope.
Electron Microscopy
[0078] Samples of the cells with attached sepharose beads were
fixed as described previously, and were then examined with a
Hitachi S-530 Scanning Microscope (SEM).
Fluorescence Microscopy
[0079] The cells examined were grown in 6-well plates on cover
slips to reach near confluence. At the time of examination, the
cover slips were inverted and put on a microscope slide supported
by 2 thin spacers so that a thin gap (.about.2 mm) was left between
the cells on the coverslip and the slide. This was filled with
culture medium. To follow the uptake, 10 .mu.g/mL FITC-labeled
peptide C.beta. (SEQ ID NO:1) or C.alpha.E (SEQ ID NO:3) was added
into the culture medium in the gap. At different time points,
medium was replaced with fresh medium and the fluorescence was
viewed and photographed, using an Olympus fluorescent microscope
system.
Cell Migration (Chemotaxis) Assay
[0080] Chemotaxis was evaluated using 48-well plastic Microtaxis
micro-Boyden chambers. Polycarbonate filters with pore size of 8
.mu.m were coated with fibronectin for 2 hours at 37.degree. C.
Conditioned medium from 3T3 cells served as a positive control.
Test solution (26 .mu.l) containing the tested peptides or protein
or medium with no additions that served as control were placed in
the lower chamber, covered with the filter. 50 .mu.l cell
suspension was introduced to the upper chamber of each well. The
number of cells/well varied in the range of 5,000-10,000 in
different experiments. The device loaded with cells was incubated
at 37.degree. C. for 4-5 hours. The filter was then removed and the
non-migrating cells on its upper part were carefully wiped off with
a damp cotton swab. The filter was fixed in methanol and stained
with Accustain (modified Wright stain solution Sigma Diagnostics:
St. Lois, Mo.). Cells that crossed or were retained within the
filter were counted microscopically at low magnification so that
the viewing field corresponded to the whole area of each well. Each
experimental variant was performed in duplicated or triplicates and
the data from, three successful experiments were normalized to the
values of the positive control and averaged.
Results
EXAMPLE 1
Haptotactic Effect of the Peptides
[0081] The peptides of the present invention were synthesized and
tested in cell culture systems as described above. Essentially,
peptides were covalently attached to sepharose beads to form
SB-C.alpha. (SEQ ID NO:4), SB-C.beta. (SEQ ID NO:1), SB-PreC.gamma.
(SEQ ID NO:2), SB-C.gamma. (SEQ ID NO:5) and SB-C.alpha.E (SEQ ID
NO:3), respectively, as was fibrinogen to form SB-Fib. The
SB-peptide combination was then incubated with cultured cells.
Under these conditions all tested cell lines bound to SB-C.beta.
(SEQ ID NO:1) and its homologues SB-PreC.gamma. (SEQ ID NO:2) and
SB-C.alpha.E (SEQ ID NO:3), with the exception of OV-1063,
keratinocytes and cells derived from a leucocytic lineage. The
haptotactic potency of the peptides is summarized in Table 3 with
the percentage of SB-Fib/peptide attached to cells (by day 4) given
in columns 2-5 of Table 3.
[0082] Three homologues of fibrinogen carboxy termini were active
in attracting and binding to most of the mesenchymal cell lines
tested, as shown FIG. 1. Fo example, SB-C.beta. (SEQ ID NO:1) bound
to 5 out of 7 cell lines with higher efficacy than SB-Fib.
Sepharose beads coated with peptide C.alpha.E (SEQ ID NO:3
(SB-C.alpha.E) bound to 5 out of 7 tested cell lines. Sepharose
beads coated with PreC.gamma. (SEQ ID NO:2) (SB-PreC.gamma.) bound
to HF, SMC and EC cells, whereas Sepharose beads coated with
peptide C.gamma. (SEQ ID NO:5) bound variably only to EC, and
Sepharose beads coated with peptide C.alpha. (SEQ ID NO:4)
(SB-C.alpha.) bound to none. TABLE-US-00004 TABLE 3 % Binding of
peptide-coated SB to normal cultured cells. SB-Ligand -C.alpha.
-C.beta. PreC.gamma. C.gamma. -C.alpha.E (SEQ (SEQ (SEQ (SEQ (SEQ
Control ID ID ID ID ID Cell Line -Fib NO: 4) NO: 1) NO: 2) NO: 5)
NO: 3) HF 71 0 100 100 2 52 MF 81 0 89 ND 0 61 SMC 74 0 100 98 1 8
EC 70 0 94 100 2 to 93* 43 EMT-6 1 99 ND 2 64 Keratinocyte 5 0 0 ND
ND 0 OV-1063 0 4 ND 0 0 *Response varied with different cell
batches. **ND--Not determined
[0083] FIG. 1 shows the kinetics of the haptotactic effect
(attachment) of the SB-peptides of the present invention for
various cell types.
[0084] Thus, these tests demonstrate the haptotactic activity of 3
short ( 19-21 mer) peptides, homologues of fibrinogen carboxy
terminal regions. FIG. 2 also slows the ability of these peptides
to render otherwise inactive material, such as Sepharose into a
haptotactic material, and illustrates the utility of SB coated with
these peptides.
EXAMPLE 2
Effect of the Haptotactic Peptides on Cell Proliferation
[0085] Peptides (C.alpha. (SEQ ID NO:4), C.alpha. (SEQ ID NO:1),
PreC.gamma. (SEQ ID NO:2) and C.alpha.E (SEQ ID NO:3)), up to 1
.mu.g/mL or 100 .mu.g/mL fibrinogen or thrombin (final
concentrations) were added to the culture medium and cell numbers
were assayed by day 3 using the MTS assay. The change in cell
number was compared with that observed versus saline control. Of
the 5 peptides tested at dosages up to 10 .mu.g/mL (approximately
equimolar to 1 .mu.g/mL fibrinogen), none of the peptides exerted
significant effect on cell proliferation (FIG. 7).
EXAMPLE 3
Uptake of FITC-C.beta. and FITC-C.alpha.E by Cells by Fluorescence
Microscopy
[0086] Exposure of cultured human fibroblast cells to a solution of
10 .mu.M peptide FITC-C.alpha.E (SEQ ID NO:3) resulted in uptake by
human fibroblasts as shown by fluoro-microscopy (FIG. 3).
Accumulation of the FITC-peptide in the cytoplasm and around the
nucleus was clearly observed. Exposure of cultured HF, EC and SMC
cells to solutions of 10 micromolar peptide FITC-C.alpha. (SEQ ID
NO:1) resulted in significant uptake similar to that seen with
peptide-C.alpha.E (SEQ ID NO:3). After a short exposure of the
cells to 10 .mu.M of peptide FITC-C.beta. (SEQ ID NO:1): the
FITC-peptide was observed to bind to the cell membrane. After a
longer exposure of more than 1 hour or with fixed cells,
accumulation of the FITC-peptide in the cytoplasm and around the
nucleus was clearly observed (data not shown). In most cases, the
fluorescence became concentrated in discrete cytoplasmic
vesicles.
EXAMPLE 4
FACS Analysis and Uptake of FITC-Labeled Peptides-C.beta. (SEQ ID
NO:1) and C.alpha.E (SEQ ID NO:3)
[0087] EC and HF cell monolayers were washed and then incubated
with trypsin-versene. Cells were then washed with growth medium and
resuspended in medium with 0.1% albumin. FITC-labeled
peptide-C.beta. (SEQ ID NO:1) was incubated with 5.times.10.sup.5
EC cells (10 .mu.g/ml or 100 .mu.g/ml) or with 2.5.times.10.sup.5
HF cells (100 .mu.g/ml) in medium with 0.1% albumin. FITC-labeled
peptide-C.alpha.E (SEQ ID NO:3) and C.alpha. (SEQ ID NO:4) (10
.mu.g/ml) was incubated with 5.times.10.sup.5 EC cells in medium
with 0.1% albumin. Cells were then washed with PBS and 1% albumin.
Cells were resuspended in PBS and 1% albumin and then filtered
through a mesh for FACS analysis, in which the FITC tluorescence
was measured for each cell.
[0088] FIG. 4 shows FACS analysis of the binding of soluble
FITC-labeled peptides-C.beta. (SEQ ID NO:1) (FIGS. 4A-4C),
C.alpha.E (SEQ ID NO:3) (FIG. 4D), C.gamma. (SEQ ID NO:5) (FIG. 4E)
and C.alpha. (SEQ ID NO:4) (FIG. 4F). The x-axis shows fluorescence
in arbitrary units and the y-axis shows number of cells. The
control is without FITC-labeled peptide (background fluorescence).
Similar binding was seen to EC and HF cells with 100 .mu.g/ml
FITC-labeled peptide-C.beta. (SEQ ID NO:1). FITC-labeled
peptide-C.alpha.E (SEQ ID NO:3) also was taken up by cells. By
contrast, FITC-labeled peptide C.alpha. (SEQ ID NO:4) and C.gamma.
(SEQ ID NO:5) bound only slightly (non-specifically) to cells. It
is expected that the haptotactic preC.gamma. (SEQ ID NO:2) would
act in a similar way to C.alpha. (SEQ ID NO:1) (results not
shown).
[0089] Kinetics FACS experiments with FITC-peptides showed that
binding of C.beta. (SEQ ID NO:1) and its C.alpha.E (SEQ ID NO:3)
analogue by cells such as HF or BAEC is fast and can reach a
maximum within 2-5 minutes (FIG. 5A). Similar kinetics binding of
C.beta. and C.alpha.E was observed for both BAEC and HF. The
affinity of cells to peptide was higher in BAEC than HF and in both
cell types, the uptake of C.beta. (SEQ ID NO:1) was higher than
that of C.alpha.E (SEQ ID NO:3). In competition experiments, cells
were incubated with various concentrations of unlabeled C.beta.
(SEQ ID NO:1) for 15 mins, an aliquot of C.beta..sup.FITC (4 mM)
was added and the sample was subjected to FACS analysis.
Paradoxically, rather than competing with C.beta..sup.FITC,
elevated concentrations of cold peptide increased the level of
total C.beta. (SEQ ID NO:1) uptake by cells (FIG. 5). The
C.beta..sup.FITC (SEQ ID NO:1) uptake experiments best fitted a
linear-quadratic curve-fit. Consequently, an attempt to plot a
Scatchard curve from 4he data could not yield a K.sub.D associated
with saturation of binding. Rather, the data indicate cooperative
binding kinetics, possibly associated with receptor clustering or
peptide endocytosis.
EXAMPLE 5
Chemotactic Effect of Peptides on Migration of Bovine Endothelial
Cells
[0090] The chemotactic response of BAEC to a soluble gradient of
C.alpha. (SEQ ID NO:1), the most haptotactic of the peptides
studied was as follows. The results can be seen in FIG. 6. At
concentrations of up to 40 .mu.g/ml CD (SEQ ID NO:1) induced highly
significant chemotaxis, similar to that induced by conditioned
medium (CM), which served as a positive control (100% chemotaxis).
This response was significantly higher than that of fibrinogen and
3 times higher than the results obtained with the negative control
of 1% BSA serum-free medium. A similar, though more moderate,
chemotaxis response to C.beta. (SEQ ID NO:1) was obtained with HF.
At concentrations of up to 40 .mu.M PreC.gamma. (SEQ ID NO:2) also
induced significant chemotaxis. The results suggest that the
cellular haptotaxis is also associated with chemotactic
properties.
EXAMPLE 6
Structured Cell Systems Using the Haptotactic Peptides of the
Present Invention
[0091] The peptides of the present invention could be used as part
of structured cell systems, for example as for tissue engineering.
The cell system of the present invention includes at least one type
of cell bound to at least one haptotactic peptide of the present
invention. Suitable types of cells include any cells which are
capable of binding to at least one peptide of the present
invention. Examples of such cells may include, but are not limited
to, fibroblasts, endothelial cells, chondrocytes, neuroblastoma
cells, melanoma cells, kidney cells, liver cells, pancreatic cells,
thyroid cells, glial cells, smooth muscle cells, mouse mammary
carcinoma cells, bone or cartilage forming cells, and combinations
thereof.
[0092] The cell system of the present invention could also be used
to culture cells as part of a cell culture system. At least one
type of cell would be allowed to bind to the peptide of the present
invention. The cells would then be grown in a culture medium under
suitable conditions for cell culture. The advantage of such a cell
culture system is that the peptide of the present invention could
be attached to a suitable structure, such as a sepharose bead or
glass or collagen or any other geometrically and biologically
suitable structure, thereby rendering it attractive to select cell
types. Hereinafter, the term "structure" includes but is not
limited to the term "matrix".
[0093] The cells would then attach and grow on that structure,
rather than on a conventional Petri dish. Therefore, when ready for
implantation or for moving to another cell growth system, the
cell-coated structure would be removed from the culture medium
without trauma to the cells. By contrast, conventional methods for
removing cells from culture medium often require trypsinization,
which may damage certain receptors on the cells and otherwise cause
trauma to the cells. The ability to transfer cells from one
environment to another by moving the structure or matrix on which
cells are attached, also enables the cells to be re-seeded into
fresh culture medium with minimal damage to the cells.
[0094] Such a cell culture system which incorporates the peptides
of the present invention could also be used to culture the cells at
a higher density than conventional cell cultures. Such high density
cell cultures are particularly useful for the production of
recombinant proteins and for other types of cell culture products.
The cell culture system of the present invention could be used to
transfect cells with various vectors, viruses, nucleic acids and
the like, in order to facilitate the production of the cell culture
products or to genetically modify the cells themselves.
[0095] The peptides of the present invention could also be used to
separate cells which are capable of binding to one or more of these
peptides from those cells which are not capable of binding to such
peptide(s). For example, a peptide of the present invention could
be attached to a matrix or structure as described previously. This
structure or matrix could then be incubated with a mixture of cells
in solution, under suitable conditions to enable binding of those
cells which are capable of binding to the peptide. The structure or
matrix could then be removed, and with it the bound cells. Cells
which are not capable of binding to the peptide, whether because
these cells are of a type which does not bind or because the cells
have been damaged in some way, would remain behind. As an
additional example, a peptide of the present invention could be
attached to a substantially immobile support (such as the surface
of a prosthetic device), and the solution of cells could be placed
in contact with it. Those cells which are capable of binding to the
peptide would remain bound to the support, while the other cells
would be removed. Thus, the peptides of the present invention could
be used for separation of cells and for coating the surface of such
a device.
[0096] Such separation may also prove useful for diagnostic
purposes. For example, the presence or absence of a certain type of
cell, or of a certain cell function, could be determined by
examining whether the cell bound to the peptide. The presence or
absence of substantially any cell type which binds to one or more
of the peptides of the present invention could be determined in
such an assay (such as by fluorescent cell sorter analysis).
Examples of cell functions which can be diagnosed include, but are
not limited to, the ability to respond to a chemotactic or an
attachment signal.
[0097] These functions would be assessed by the determination of
cell binding to the peptide-coated structure. The determination of
cell binding could be performed by the ability to separate the cell
from a solution or mixture of different cell types, as described
previously.
[0098] Alternatively and preferably, the peptide could be labeled
with a reporter, such as a fluorescent or radioactive moiety. The
reporter would be used to determine if the peptide had bound to any
of the cells, thus enabling the presence or absence of the cell
type, or of a certain cell function, to be determined.
[0099] Examples of suitable fluorescent moieties include, but are
not limited to, FITC (fluorescein), rhodamine and Texas red.
Examples of suitable radioactive moieties include, but are not
limited to, phosphorous 32, iodine 131 and tritium. The reporter
could be attached to the peptide during synthesis or alternatively
post-synthesis, according to well known methods in the art.
[0100] The haptotactic peptides of the present invention are also
contemplated as being useful for the formation of a therapeutic
structure of cells. Examples of the therapeutic structure include,
but are not limited to, a gel, a prosthetic device, and a collagen
sheet. At least one peptide of the present invention would be
attached to the therapeutic structure, for example by a covalent
bond formed with a chemical cross-linking reagent as is well known
in the art, or with fibrin glue. The cells would then be allowed to
attach to the peptide on the therapeutic structure, for example
through cell culture or by the separation methods described above.
The choice of cells will depend upon the type of tissue being
contacted and the desired therapeutic structure, and could
potentially include any cell type which is capable of binding to at
least one peptide of the present invention.
EXAMPLE 7
Methods for Treatment with the Haptotactic Peptides of the Present
Invention
[0101] The haptotactic peptides of the present invention are
contemplated as being useful for treatment of a subject with a
disease condition, in which the condition can be ameliorated or
cured, at least in part, through cell chemotaxis or proliferation,
or by transplantation of cells. Examples of such a condition
include, but are not limited to, the presence of a wound and
diseases characterized by an absence of a cell product. The term
"wound" includes any disruption of the normal integrity of an organ
of the subject. Examples of such an organ include, but are not
limited to, the skin, the abdominal cavity, the intestine, the
heart, the lungs, any blood vessel, any bone, the pancreas, the
liver, a kidney, the reproductive organs or the stomach.
[0102] The wound may be present as the result of a surgical
intervention or as the result of a non-surgical intervention. The
surgical intervention could be either planned or as the result of a
medical emergency. The non-surgical intervention could be a bum, an
ulcer, a laceration or any type of accidental injury or trauma.
[0103] Methods of treatment with the haptotactic peptides of the
present invention for surgical intervention could include placing
one or more of the peptides at the site of the surgical
intervention, in order to increase the efficiency of the wound
healing process. The one or more peptides could be placed at the
site of the surgical intervention before surgery, particularly for
emergency surgery, during surgery or after surgery. The one or more
peptides could be included in a therapeutic composition, as
described in Example 8 below.
[0104] Methods of treatment of non-surgical interventions would
include placing one or more of the peptides at the site of the
non-surgical intervention, in order to increase the efficiency of
the wound healing process. The one or more peptides could also be
included in a therapeutic composition, as described in Example 8
below.
[0105] The one or more peptides of the present invention could be
placed at the site of the surgical or non-surgical intervention
once, or repeatedly, depending upon the type and gravity of the
wound which was sustained. The concentration and rate of treatment,
if repeated, could easily be determined by one of ordinary skill in
the art.
[0106] Examples of diseases characterized by an absence of a cell
product include, but are not limited to, diabetes mellitus,
hemophilia A (factor VIII deficiency), hemophilia B (factor IX)
deficiency and Parkinson's disease. These diseases could be
ameliorated or cured by introducing cells which produce the
necessary cell metabolite or product into the subject. These cells
could be prepared by introduction of a vector containing the
nucleic acid sequence coding for a protein or peptide, for example,
as is well known in the art. The peptide or protein could itself be
the desired cell product, such as insulin. Alternatively and
preferably, the protein could cause the cell to produce the desired
cell product, for example through an enzymatic reaction or
reactions. In any case, the cell would then be able to produce the
desired cell product after such preparation.
[0107] Once prepared, the cells would be attached to a haptotactic
peptide of tile present invention, which would in turn be
incorporated within a suitable cell structure as described in
Example 6. The cell structure would be administered to the subject
and would then produce the necessary cell metabolite or product.
The advantage of such a cell structure according to the present
invention is that the cells would remain substantially localized,
although the cell products could be enabled to enter the
bloodstream if desired. Thus, by using the haptotactic peptide of
the present invention, the cell structure could be used to treat
the disease condition with the necessary cell metabolites or
products.
EXAMPLE 8
Suitable Formulations for Administration of the Haptotactic
Peptides
[0108] The haptotactic peptides of the present invention can be
administered to a subject in a number of ways, which are well known
in the art. Hereinafter, the term "subject" refers to the human or
lower animal to whom the peptide was administered. For example,
administration may be done topically (including opthalmically,
vaginally, rectally, intranasally), orally, or parenterally, for
example by intravenous drip or intraperitoneal, subcutaneous, or
intramuscular injection.
[0109] Formulations for topical administration may include but are
not limited to lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable.
[0110] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous medial
sachets, capsules or tablets. Thickeners, diluents, flavorings,
dispersing aids, emulsifiers or binders may be desirable.
[0111] Formulations for parenteral administration may include but
are not limited to sterile aqueous solutions which may also contain
buffers diluents and other suitable additives.
[0112] The haptotactic peptides of the present invention may be
placed on the wound bed as part of a composition for wound
treatment. The composition for wound treatment can include a
suitable pharmaceutically acceptable carrier. For example, the
haptotactic peptide could be incorporated into lasered or heated
albumin to accelerate wound healing, minimize scarring, accelerate
the rate of deposition of new extracellular matrix and augment
angiogenesis.
[0113] As another example, a polymer could be made of subunits of
at least one of the peptides of the present invention, such that a
plurality of these peptides would be linked to form the peptide
polymer. The peptides could be linked with a chemical cross-linking
moiety, for example. More than one of the peptides of the present
invention could be used to form the polymer. Alternatively, at
least one of the peptides of the present invention could be
attached to a biologically acceptable synthetic polymer, again
through a suitable cross-linking moiety, to form a co-polymer. In
either case, the resultant peptide polymer or co-polymer could be
used to fabricate microparticles which could either be included in
a composition according to the present invention, or else could be
used form cell structures as described in Example 6 above.
[0114] The composition for wound treatment can also include at
least one bioactive agent. Suitable bioactive agents include, but
are not limited to, drugs, neurologics, vitamins, vitamin
derivatives, growth factors, glucocorticosteroids. steroids,
antibiotics, antibacterial compounds including bacteriocidal and
bacteriostatic compounds, antiparasitic compounds, tumoricidal
compounds, tumoristatic compounds, toxins, enzymes, enzyme
inhibitors, proteins, peptides, minerals, neurotransmitters,
lipoproteins, glycoproteins, immunomodulators, immunoglobulins and
fragments thereof, fatty acid derivatives, polysaccharides, cell
receptor binding molecules, anti-inflammatories, anti-glaucomic
compounds, mydriatic compounds, anesthetics, nucleic acids,
polynucleotides and the like.
[0115] The therapeutic composition could also include at least one
type of cell in a structured format, as described in Example 6
above. For example, the previously described sheet structure for
cell culture could be placed on the wound in order to both protect
the wound during the healing process, and to promote the wound
healing process itself. The structure could also be the previously
described haptotactic peptide-containing gel, which would be placed
on the wound for transplanting the cells onto the site of the
wound, and would then be able to promote the wound healing process.
Other examples of such structured cell systems could also be used
as part of the therapeutic composition of the present invention for
wound healing. When used for wound healing, suitable cell types
include, but are not limited to, fibroblasts, smooth muscle cells,
endothelial cells, chondrocytes, bone or cartilage forming cells,
and combinations thereof.
[0116] Combinations of any two or more of these different
components of therapeutic compositions are also possible as
therapeutic compositions of the present invention.
[0117] Dosing is dependent on the severity of the symptoms and on
the responsiveness of the subject to the peptide or fragments of
the present invention. Persons of ordinary skill in the art can
easily determine optimum dosages, dosing methodologies and
repetition rates.
EXAMPLE 9
Analysis Performed with the Haptotactic Peptides of the Present
Invention
[0118] The peptides of the present invention are also contemplated
as tools for performing analysis of other systems, and for further
research and development. For example, the haptotactic peptides
could be used to identify and isolate cell receptors. As described
previously, the peptide could be labeled with a reporter, such as a
fluorescent or radioactive moiety. The reporter would be used to
determine if the peptide had bound to any of the cells, thus
enabling the presence or absence of the cell type, or of a certain
cell function, to be determined.
[0119] Examples of suitable fluorescent moieties include, but are
not limited to, FITC (fluorescein), rhodamine and Texas red.
Examples of suitable radioactive moieties include, but are not
limited to, phosphorous 32, iodine 131 and tritium. The reporter
could be attached to the peptide during synthesis or alternatively
post-synthesis, according to well known methods in the art. Thus,
the ability of the peptide to bind to a novel receptor or other
protein could be determined according to a binding assay.
[0120] In addition, the peptides of the present invention could be
used to design analogues, such as non-peptide mimetics, of these
peptides. Such non-peptide mimetics could be used for therapeutic
purposes, for example. Non-peptide compounds are potentially easier
to administer, since peptides are preferably administered nasally
or parenterally, for example, while non-peptide compounds could
potentially be administered orally. Furthermore, particular
properties of each peptide could be selected or augmented by
designing a specific analogue. Thus, the peptides of the present
invention could potentially yield many new and different types of
therapeutic medicaments.
[0121] While the invention has been described with respect to a
limited number of embodiments, it will be appreciated that many
variations, modifications and other applications of the invention
may be made.
Sequence CWU 1
1
12 1 21 PRT Artificial Equivalent to the c-terminus of fibrinogen
beta-chain 1 Lys Gly Ser Trp Tyr Ser Met Arg Lys Met Ser Met Lys
Ile Arg Pro 1 5 10 15 Phe Phe Pro Gln Gln 20 2 20 PRT Artificial
Equivalent to an internal sequence of fibrinogen gamma-chain 2 Lys
Thr Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro 1 5 10
15 Phe Asn Arg Leu 20 3 20 PRT Artificial Equivalent to the
c-erminus of fibrinogen alpha-E-chain 3 Arg Gly Ala Asp Tyr Ser Leu
Arg Ala Val Arg Met Lys Ile Arg Pro 1 5 10 15 Leu Val Thr Gln 20 4
19 PRT Artificial Equivalent to the c-terminus of fibrinogen
alpha-chain 4 Glu Ala Asp His Glu Gly Thr His Ser Thr Lys Arg Gly
His Ala Lys 1 5 10 15 Ser Arg Pro 5 20 PRT Artificial Equivalent to
the c-terminus of fibrinogen gamma-chain 5 Leu Thr Ile Gly Glu Gly
Gln Gln His His Leu Gly Gly Ala Lys Gln 1 5 10 15 Ala Gly Asp Val
20 6 29 PRT Artificial Fibrinogen C-beta consensus sequence
MISC_FEATURE (1)..(2) Xaa = Hydrophilic amino acid selected from
Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr MISC_FEATURE (7)..(7)
Xaa = Hydrophobic amino acid selected from Ala, Phe, Ile, Leu, Met,
Pro, Val, Trp, Tyr MISC_FEATURE (8)..(8) Xaa = Hydrophilic amino
acid selected from Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr
MISC_FEATURE (9)..(9) Xaa = Hydrophobic amino acid selected from
Ala, Phe, Ile, Leu, Met, Pro, Val, Trp, Tyr MISC_FEATURE (10)..(10)
Xaa = Hydrophilic amino acid selected from Asp, Glu, His, Lys, Asn,
Gln, Arg, Ser, Thr MISC_FEATURE (12)..(12) Xaa = Ala or Ser
MISC_FEATURE (13)..(13) Xaa = Any amino acid MISC_FEATURE
(16)..(16) Xaa = Hydrophobic amino acid selected from Ala, Phe,
Ile, Leu, Met, Pro, Val, Trp, Tyr MISC_FEATURE (18)..(18) Xaa = Any
amino acid MISC_FEATURE (19)..(19) Xaa = Hydrophobic amino acid
selected from Ala, Phe, Ile, Leu, Met, Pro, Val, Trp, Tyr
MISC_FEATURE (20)..(20) Xaa = Any amino acid MISC_FEATURE
(26)..(28) Xaa = Any amino acid 6 Xaa Xaa Gly Val Val Trp Xaa Xaa
Xaa Xaa Gly Xaa Xaa Tyr Ser Xaa 1 5 10 15 Arg Xaa Xaa Xaa Met Lys
Ile Arg Pro Xaa Xaa Xaa Gln 20 25 7 65 DNA Artificial Encodig
haptotactic peptide C-beta 7 aaggggtcat ggtatcaatg aggaagatga
gtatgaagat caggcccttc ttcccacagc 60 aatag 65 8 63 DNA Artificial
Encoding haptotactic peptide C-alpha-E 8 agaggggcag attattccct
cagggctgtt cgcatgaaaa ttaggcccct tgtgacccaa 60 tag 63 9 63 DNA
Artificial Encoding haptotactic peptide Pre-C-gamma 9 aaaacccggt
ggtattccat gaagaaaacc actatgaaga taatcccatt caacagactc 60 aca 63 10
15 PRT Artificial Haptotactic consensus sequence MISC_FEATURE
(3)..(3) Xaa is absent or hydrophobic amino acid selected from Ala,
Phe, Ile, Leu, Met, Pro, Val, Trp, Tyr. MISC_FEATURE (5)..(5) Xaa =
Any amino acid or absent MISC_FEATURE (6)..(6) Xaa is absent or
hydrophobic amino acid selected from Ala, Phe, Ile, Leu, Met, Pro,
Val, Trp, Tyr MISC_FEATURE (12)..(14) Xaa = Any amino acid or
absent 10 Tyr Ser Xaa Arg Xaa Xaa Met Lys Ile Arg Pro Xaa Xaa Xaa
Gln 1 5 10 15 11 20 PRT Artificial Haptotactic consensus sequence
MISC_FEATURE (2)..(3) Xaa = Any amino acid or absent MISC_FEATURE
(8)..(8) Xaa is absent or hydrophilic amino acid selected from Asp,
Glu, His, Lys, Asn, Gln, Arg, Ser, Thr MISC_FEATURE (10)..(10) Xaa
= Any amino acid or absent MISC_FEATURE (11)..(11) Xaa is absent or
hydrophilic amino acid selected from Asp, Glu, His, Lys, Asn, Gln,
Arg, Ser, Thr MISC_FEATURE (15)..(15) Xaa = Any amino acid or
absent MISC_FEATURE (18)..(20) Xaa = Any amino acid or absent 11
Lys Xaa Xaa Trp Tyr Ser Met Xaa Lys Xaa Xaa Met Lys Ile Xaa Pro 1 5
10 15 Phe Xaa Xaa Xaa 20 12 30 PRT Artificial Fibrinogen C beta
homology sequence MISC_FEATURE (2)..(2) Xaa = Hydrophilic amino
acid selected from Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr
MISC_FEATURE (4)..(5) Xaa = Hydrophobic amino acid selected from
Ala, Phe, Ile, Leu, Met, Pro, Val, Trp, Tyr MISC_FEATURE (7)..(8)
Xaa = Any amino acid MISC_FEATURE (11)..(12) Xaa = Any amino acid
MISC_FEATURE (17)..(17) Xaa = Hydrophilic amino acid selected from
Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr MISC_FEATURE (19)..(19)
Xaa = Any amino acid MISC_FEATURE (20)..(20) Xaa = Hydrophilic
amino acid selected from Asp, Glu, His, Lys, Asn, Gln, Arg, Ser,
Thr MISC_FEATURE (24)..(24) Xaa = Any amino acid MISC_FEATURE
(27)..(30) Xaa = Any amino acid 12 Asp Xaa Gly Xaa Xaa Trp Xaa Xaa
Trp Lys Xaa Xaa Trp Tyr Ser Met 1 5 10 15 Xaa Lys Xaa Xaa Met Lys
Ile Xaa Pro Phe Xaa Xaa Xaa Xaa 20 25 30
* * * * *