U.S. patent application number 11/145340 was filed with the patent office on 2006-01-05 for method for promoting neovascularization.
Invention is credited to Linda Burkly, Aniela Jakubowski.
Application Number | 20060003932 11/145340 |
Document ID | / |
Family ID | 22751060 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003932 |
Kind Code |
A1 |
Jakubowski; Aniela ; et
al. |
January 5, 2006 |
Method for promoting neovascularization
Abstract
The present invention relates to a method for enhancing
angiogenic activity to promote neovascularization comprising
administering to a subject a formulation comprising a
synergistically effective amount of a TWEAK agonist and an
angiogenic factor.
Inventors: |
Jakubowski; Aniela;
(Arlington, MA) ; Burkly; Linda; (West Newton,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
22751060 |
Appl. No.: |
11/145340 |
Filed: |
June 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10275997 |
Nov 8, 2002 |
6943146 |
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PCT/US01/14545 |
May 7, 2001 |
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11145340 |
Jun 2, 2005 |
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60202738 |
May 8, 2000 |
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Current U.S.
Class: |
514/9.1 ;
514/13.3; 514/16.4; 514/9.4; 514/9.5 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61P 9/00 20180101; A61K 38/1825 20130101; A61K 38/177 20130101;
A61P 41/00 20180101; A61P 3/10 20180101; A61K 45/06 20130101; A61P
17/02 20180101; A61K 2300/00 20130101; C12N 2501/25 20130101; A61K
2300/00 20130101; A61P 9/10 20180101; C12N 5/069 20130101; C12N
2501/115 20130101; A61K 38/177 20130101; C12N 2501/165 20130101;
A61P 1/04 20180101; C12N 2501/998 20130101; A61P 43/00 20180101;
A61P 9/14 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/18 20060101
A61K038/18 |
Claims
1-6. (canceled)
7. A method for enhancing endothelial cell proliferation in an in
vitro culture comprising adding to said culture, a formulation
consisting essentially of a synergistically effective amount of a
TWEAK agonist and an angiogenic factor.
8. A method for enhancing angiogenic activity in a mammal to
promote neovascularization comprising the step of administering to
said mammal a formulation consisting essentially of a
synergistically effective amount of a TWEAK agonist and an
angiogenic factor sufficient to promote neovascularization.
9. The methods according to claims 7 or 8, wherein the angiogenic
factor is selected from the group consisting of hepatocyte growth
factor (HGF), basic fibroblast growth factor (bFGF), angiopoietin
1, angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
10. The methods according to claims 7 or 8, wherein the angiogenic
factor is bFGF.
11. The method of claim 8, wherein said method is used in the
treatment of a myocardial ischemic condition.
12. The method of claim 8, wherein said method is used to promote
wound healing.
13. The method of claim 8, wherein said method is used in the
treatment of dermal ulcers, lacerations, burns, or other dermal
trauma in said mammal.
14. The method of claim 8, wherein said method is used to promote
growth of collateral vasculature after ischemia or recovery of
erectile function in said mammal.
15. A method for in vitro culturing of mammalian cells in an in
vitro culture comprising adding to said culture a formulation
comprising a TWEAK agonist and an angiogenic factor.
16. The method of claim 15, wherein the mammalian cells are
selected from the group consisting of vascular smooth muscle cells,
fibroblasts, hematopoietic cells, muscle, myotendonous junction,
bone-derived cells, cartilage-derived cells, and other mesenchymal
cells.
17. The method of claim 15, wherein the TWEAK agonist and the
angiogenic factor are present in a synergistically effective
amount.
18. The method of claim 15, wherein the formulation consists of a
TWEAK agonist and an angiogenic factor.
19. The method of claim 15, wherein the TWEAK agonist is TWEAK.
20. The method of claim 15, wherein the angiogenic factor is
selected from the group consisting of hepatocyte growth factor
(HGF), basic fibroblast growth factor (bFGF), angiopoietin 1,
angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
21. The method of claim 15, wherein the angiogenic factor is
bFGF.
22. The method of claim 15, wherein the TWEAK agonist is TWEAK and
the angiogenic factor is bFGF.
23. A formulation comprising a TWEAK agonist and an angiogenic
factor.
24. The formulation of claim 23, wherein the formulation consists
of a TWEAK agonist and an angiogenic factor.
25. The formulation of claim 23, wherein the TWEAK agonist and the
angiogenic factor are present in a synergistically effective
amount.
26. The formulation of claim 23, wherein the TWEAK agonist is
TWEAK.
27. The formulation of claim 23, wherein the angiogenic factor is
selected from the group consisting of hepatocyte growth factor
(HGF), basic fibroblast growth factor (bFGF), angiopoietin 1,
angiopoietin 2, and monocyte chemotactic protein-1 (MCP-1).
28. The formulation of claim 23, wherein the angiogenic factor is
bFGF.
29. The formulation of claim 23, wherein the TWEAK agonist is TWEAK
and the angiogenic factor is bFGF.
30. A cell culture comprising: mammalian cells, a cell culture
medium, and a combination of a TWEAK agonist and an angiogenic
factor, wherein the combination is an amount sufficient to promote
angiogenesis.
31. The cell culture of claim 30, wherein the mammalian cells are
endothelial cells, vascular smooth muscle cells, fibroblasts,
hematopoietic cells, muscle, myotendonous junction, bone-derived
cells, cartilage-derived cells, and other mesenchymal cells
32. The cell culture of claim 30, wherein the TWEAK agonist and the
angiogenic factor are present in a synergistically effective
amount.
33. The cell culture of claim 30, wherein the TWEAK agonist is
TWEAK.
34. The cell culture of claim 30, wherein the angiogenic factor is
bFGF.
35. The cell culture of claim 30, wherein the TWEAK agonist is
TWEAK and the angiogenic factor is bFGF.
36. A method of identifying inhibitors of TWEAK and bFGF mediated
cellular proliferation, the method comprising: culturing
endothelial cells in the presence of TWEAK and bFGF; culturing
endothelial cells in the presence of TWEAK, bFGF, and a test
compound; and comparing the amount of endothelial cell
proliferation in the cell culture containing the test compound to
the amount of proliferation in the cell culture not containing with
the test compound, wherein a decrease in proliferation in the
culture treated with the test compound indicates that the test
compound is an inhibitor of proliferation.
37. The method of claim 36, wherein the test compound is an
antibody.
Description
FIELD OF INVENTION
[0001] The present invention relates to a method for enhancing
angiogenic activity to promote neovascularization.
BACKGROUND OF THE INVENTION
[0002] Growth of microvasculature, or angiogenesis, involves
endothelial cell (EC) proliferation, migration, differentiation,
and structural organization into new vessels.
[0003] Angiogenic regulators induce changes in endothelial cells
(EC) at a variety of levels, including their proliferative,
migratory, secretory, and adhesive properties, and may do so
through their action on ECs or other cell types (Kumar et al, 1998,
Int. J. Oncology 12:749-757; Bussolino et al., 1997, Trends in
Biochem, 22:251-256). Several TNF family ligands previously have
been implicated in the process of angiogenesis, namely TNF.alpha.,
FasL and TWEAK.
SUMMARY OF THE INVENTION
[0004] TWEAK, a novel member of the TNF ligand family, may promote
angiogenesis based on its ability to induce IL-8 production by
several epithelial tumor cell lines, proliferation in various human
EC and aortic smooth muscle cells under reduced growth factor
conditions, and stimulation of an angiogenic response when
implanted in rat corneas. Herein, we further characterize the
angiogenic potential of TWEAK, demonstrating that TWEAK synergizes
with Fibroblast Growth Factor (FGF) to induce proliferation and
migration of EC's. While TWEAK weakly promotes EC survival, the
synergistic effect of TWEAK and FGF on EC proliferation appears to
be due to potentiation of cell division rather than decreased cell
death. TWEAK also did not detectably alter the expression of
receptors for FGF or VEGF, or expression of the integrins
.alpha..sub.1, .alpha..sub.5, .alpha..sub.v, .beta..sub.1, or
.beta..sub.3. The ability of TWEAK to induce ECs to form
capillaries in the absence of other cell types was demonstrated in
a 3D fibrin gel matrix where, strikingly, TWEAK induced the
morphogenesis of lumens in invading, bFGF-dependent EC cords. Our
findings further distinguish TWEAK from other TNF family ligands,
demonstrating its ability to promote angiogenesis at multiple
discrete stages.
[0005] One aspect of the present invention is a method for
enhancing endothelial cell proliferation in an in vitro culture
comprising adding to said culture, a formulation consisting
essentially of a synergistically effective amount of a TWEAK
agonist and an angiogenic factor.
[0006] A second aspect of the present invention is a method for
enhancing angiogenic activity in a mammal to promote
neovascularization comprising the step of administering to said
mammal a formulation comprising a synergistically effective amount
of a TWEAK agonist and an angiogenic factor sufficient to promote
neovascularization. A preferred embodiment is the use of bFGF.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 illustrates the effect of TWEAK on bFGF-dependent
HUVEC proliferation.
[0008] FIG. 2 illustrates the effect of TWEAK on HUVEC death.
[0009] FIG. 3 illustrates the effect of TWEAK on bFGF-dependent
HUVEC migration.
[0010] FIG. 4 depicts the synergistic effect of TWEAK and bFGF on
capillary tube formation.
DETAILED DESCRIPTION
[0011] As used herein the terms, "angiogenesis,"
"revascularization," "increased collateral circulation," and
"regeneration of blood vessels" are considered as synonymous.
[0012] "Angiogenesis" is defined as any alteration of an existing
vascular bed or the formation of new vasculature which benefits
tissue perfusion. This includes the formation of new vessels by
sprouting of endothelial cells from existing blood vessels or the
remodeling of existing vessels to alter size, maturity, direction
or flow properties to improve blood perfusion of tissues.
[0013] A therapeutic is said to have "therapeutic efficacy" in
modulating angiogenesis and an amount of the therapeutic is said to
be a "angiogenic modulatory amount", if administration of that
amount of the therapeutic is sufficient to cause a significant
modulation (i.e., increase or decrease) in angiogenic activity when
administered to a subject (e.g., an animal model or human patient)
needing modulation of angiogenesis.
[0014] The term angiogenic factor refers to factors which promote
the angiogenic process, including but not limited to the following
phases of the process, ie. the degradation of the extracellular
matrix, cell proliferation, cell migration and structural
organization (Kumar et al, 1998, Int. J. Oncology 12:749-757;
Bussolino et al., 1997, Trends in Biochem, 22:251-256). Angiogenic
factors include but are not limited to fibroblast growth factor
(bFGF), acidic FGF (aFGF), FGF-5, vascular endothelial growth
factor isoforms (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2
(Ang-2), Platelet-derived endothelial cell growth factor (PD-ECGF),
hepatocyte growth factor (HGF), interleukin-8 (IL-8),
granulocyte-colony stimulating factor (G-CSF), placental growth
factor, proliferin, B61, soluble vascular cell adhesion
molecular-1, soluble E-selection, 12-hydrozyeicosatetraenoic acid,
Tat protein of HIV-1, angiogenin, TNF.alpha., FasL, Transforming
growth factor-.beta..
[0015] As used herein, the ability of TWEAK to act synergistically
with another angiogenic factor means that the combination of TWEAK
and the angiogenic factor induce a response that is greater than
the sum of the responses to either agent alone, as measured in one
or more in vitro assays which measure stages of the angiogenic
process. These include but are not limited to endothelial cell
survival, proliferation, migration, or capillary tube formation, as
described herein.
[0016] The term "pharmaceutically acceptable" when referring to a
natural or synthetic substance means that the substance has an
acceptable toxic effect in view of its much greater beneficial
effect, while the related the term, "physiologically acceptable,"
means the substance has relatively low toxicity.
[0017] As used herein, the term "antibody homolog" includes intact
antibodies consisting of immunoglobulin light and heavy chains
linked via disulfide bonds. The term "antibody homolog" is also
intended to encompass a TWEAK therapeutic comprising one or more
polypeptides selected from immunoglobulin light chains,
immunoglobulin heavy chains and antigen-binding fragments thereof
which are capable of binding to one or more antigens (i.e., TWEAK
or patched). The component polypeptides of an antibody homolog
composed of more than one polypeptide may optionally be
disulfide-bound or otherwise covalently crosslinked. Accordingly,
therefore, "antibody homologs" include intact immunoglobulins of
types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof),
wherein the light chains of the immunoglobulin may be of types
kappa or lambda or portions of intact antibodies that retain
antigen-binding specificity, for example, Fab fragments, Fab'
fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers
or dimers, light chain monomers or dimers, dimers consisting of one
heavy and one light chain, and the like.
[0018] As used herein, a "humanized antibody homolog" is an
antibody homolog, produced by recombinant DNA technology, in which
some or all of the amino acids of a human immunoglobulin light or
heavy chain that are not required for antigen binding have been
substituted for the corresponding amino acids from a nonhuman
mammalian immunoglobulin light or heavy chain. A "human antibody
homolog" is an antibody homolog in which all the amino acids of an
immunoglobulin light or heavy chain (regardless of whether or not
they are required for antigen binding) are derived from a human
source.
[0019] An "amino acid" is a monomeric unit of a peptide,
polypeptide, or protein. There are twenty amino acids found in
naturally occurring peptides, polypeptides and proteins, all of
which are L-isomers. The term also includes analogs of the amino
acids and D-isomers of the protein amino acids and their
analogs.
[0020] The term "bioavailability" refers to the ability of a
compound to be absorbed by the body after administration. For
instance, a first compound has greater bioavailability than a
second compound if, when both are administered in equal amounts,
the first compound is absorbed into the blood to a greater extent
than the second compound.
[0021] An "expression vector" is a polynucleotide, such as a DNA
plasmid or phage (among other common examples) which allows
expression of at least one gene when the expression vector is
introduced into a host cell. The vector may, or may not, be able to
replicate in a cell.
[0022] The phrase "extracellular signaling protein" means any
protein that is either secreted from a cell, or is associated with
the cell membrane, and upon binding to the receptor for that
protein on a target cell, triggers a response in the target
cell.
[0023] A "functional equivalent" of an amino acid residue is (i) an
amino acid having similar reactive properties as the amino acid
residue that was replaced by the functional equivalent; (ii) an
amino acid of a ligand of a polypeptide of the invention, the amino
acid having similar properties as the amino acid residue that was
replaced by the functional equivalent; (iii) a non-amino acid
molecule having similar properties as the amino acid residue that
was replaced by the functional equivalent.
[0024] "Heterologous promoter" as used herein is a promoter which
is not naturally associated with a gene or a purified nucleic
acid.
[0025] "Homology" and "identity" each refer to sequence similarity
between two polypeptide sequences, and both `homology and
`identity` are used interchangeably in this disclosure. Homology
can be determined by comparing a position in each sequence which
may be aligned for purposes of comparison. When a position in the
compared sequence is occupied by the same amino acid residue, then
the polypeptides can be referred to as identical at that position;
when the equivalent site is occupied by the same amino acid (e.g.,
identical) or a similar amino acid (e.g., similar in steric and/or
electronic nature), then the molecules can be refered to as
homologous at that position. A percentage of homology between
sequences is a function of the number of matching or homologous
positions shared by the sequences. An "unrelated" or
"non-homologous" sequence shares less than 40 percent identity,
though preferably less than 25 percent identity, with a sequence of
the present invention.
[0026] For instance, if 6 of 10 of the positions in two sequences
are matched or are homologous, then the two sequences are 60%
homologous. By way of example, the DNA sequences CTGACT and CAGGTT
share 50% homology (3 of the 6 total positions are matched).
Generally, a comparison is made when two sequences are aligned to
give maximum homology. Such alignment can be provided using, for
instance, the method of Needleman et al., J. Mol Biol. 48: 443-453
(1970), implemented conveniently by computer programs described in
more detail below. Homologous sequences share identical or similar
amino acid residues, where similar residues are conservative
substitutions for, or "allowed point mutations" of, corresponding
amino acid residues in an aligned reference sequence. In this
regard, a "conservative substitution" of a residue in a reference
sequence are those substitutions that are physically or
functionally similar to the corresponding reference residues, e.g.,
that have a similar size, shape, electric charge, chemical
properties, including the ability to form covalent or hydrogen
bonds, or the like. Particularly preferred conservative
substitutions are those fulfilling the criteria defined for an
"accepted point mutation" in Dayhoff et al., 5: Atlas of Protein
Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat.
Biomed. Res. Foundation, Washington, D.C. (1978).
[0027] "Percent homology/identity" of two amino acids sequences or
two nucleic acid sequences is determined using the alignment
algorithm of Karlin and Altschul (Proc. Nat. Acad. Sci., USA 87:
2264 (1990) as modified in Karlin and Altschul (Proc. Nat. Acad.
Sci., USA 90: 5873 (1993). Such an algorithm is incorporated into
the NBLAST or XBLAST programs of Altschul et al., J. Mol. Biol.
215: 403 (1990). BLAST searches are performed with the NBLAST
program, score=100, wordlength=12, to obtain nucoetide sequences
homologous to a nucleic acid of the invention. BLAST protein
searches are performed with the XBLAST program, score=50,
wordlength=3, to obtain amino acid sequences homologous to a
reference polypeptide. To obtain gapped alignments for comparisons,
gapped BLAST is used as described in Altschul et al., Nucleic Acids
Res., 25: 3389 (1997). When using BLAST and Gapped BLAST, the
default parameters of the respective programs (XBLAST and NBLAST)
are used. See http://www/ncbi.nlm.nih.gov
[0028] The term "hydrophobic" refers to the tendency of chemical
moieties with nonpolar atoms to interact with each other rather
than water or other polar atoms. Materials that are "hydrophobic"
are, for the most part, insoluble in water. Natural products with
hydrophobic properties include lipids, fatty acids, phospholipids,
sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes,
prostaglandins, thromboxanes, leukotrienes, isoprenoids, retenoids,
biotin, and hydrophobic amino acids such as tryptophan,
phenylalanine, isoleucine, leucine, valine, methionine, alanine,
proline, and tyrosine. A chemical moiety is also hydrophobic or has
hydrophobic properties if its physical properties are determined by
the presence of nonpolar atoms.
[0029] The phrase "internal amino acid" means any amino acid in a
peptide sequence that is neither the N-terminal amino acid nor the
C-terminal amino acid.
[0030] "Isolated" (used interchangeably with "substantially pure")
when applied to nucleic acid i.e., polynucleotide sequences that
encode polypeptides, means an RNA or DNA polynucleotide, portion of
genomic polynucleotide, cDNA or synthetic polynucleotide which, by
virtue of its origin or manipulation: (i) is not associated with
all of a polynucleotide with which it is associated in nature
(e.g., is present in a host cell as an expression vector, or a
portion thereof); or (ii) is linked to a nucleic acid or other
chemical moiety other than that to which it is linked in nature; or
(iii) does not occur in nature. By "isolated" it is further meant a
polynucleotide sequence that is: (i) amplified in vitro by, for
example, polymerase chain reaction (PCR); (ii) synthesized
chemically; (iii) produced recombinantly by cloning; or (iv)
purified, as by cleavage and gel separation.
[0031] "Isolated" (used interchangeably with "substantially pure")
when applied to polypeptides means a polypeptide or a portion
thereof which, by virtue of its origin or manipulation: (i) is
present in a host cell as the expression product of a portion of an
expression vector; or (ii) is linked to a protein or other chemical
moiety other than that to which it is linked in nature; or (iii)
does not occur in nature, for example, a protein that is chemically
manipulated by appending, or adding at least one hydrophobic moiety
to the protein so that the protein is in a form not found in
nature. By "isolated" it is further meant a protein that is: (i)
synthesized chemically; or (ii) expressed in a host cell and
purified away from associated and contaminating proteins. The term
generally means a polypeptide that has been separated from other
proteins and nucleic acids with which it naturally occurs.
Preferably, the polypeptide is also separated from substances such
as antibodies or gel matrices (polyacrylamide) which are used to
purify it.
[0032] A "protein" is any polymer consisting essentially of any of
the 20 amino acids. Although "polypeptide" is often used in
reference to relatively large polypeptides, and "peptide" is often
used in reference to small polypeptides, usage of these terms in
the art overlaps and is varied. The term "protein" as used herein
refers to peptides, proteins and polypeptides, unless otherwise
noted.
[0033] The terms "peptide(s)", "protein(s)" and "polypeptide(s)"
are used interchangeably herein. The terms "polynucleotide
sequence" and "nucleotide sequence" are also used interchangeably
herein.
[0034] "Recombinant," as used herein, means that a protein is
derived from recombinant, mammalian expression systems.
[0035] Thus, "substantially pure nucleic acid" is a nucleic acid
which is not immediately contiguous with one or both of the coding
sequences with which it is normally contiguous in the naturally
occurring genome of the organism from which the nucleic acid is
derived. Substantially pure DNA also includes a recombinant DNA
which is part of a hybrid gene encoding additional TWEAK
sequences.
[0036] The amounts of a TWEAK agonist and angiogenic factor
required to be effective in enhancing angiogenic activity for
promoting neovascularization will, of course, vary with the
individual being treated and is ultimately at the discretion of the
physician. The factors to be considered include the condition of
the patient being treated, the efficacy of the particular TWEAK
agonist being used, the nature of the formulation, and the
patient's body weight. While it possible to administer and a TWEAK
agonist simultaneously, it is also contemplated that angiogenic
factor can be given as a bolus before starting the infusion of the
TWEAK agonist. It is also contemplated that angiogenic factor can
be administered after the infusion of the TWEAK agonist.
[0037] TWEAK agonists include those taught in WO98/05783,
WO98/35061 and WO99/19490 all of which are incorporated herein by
reference. Such TWEAK agonists include soluble recombinant TWEAK
protein.
[0038] "Standard hybridization conditions" refer to salt and
temperature conditions substantially equivalent to 0.5.times.SSC to
about 5.times.SSC and 65.degree. C. for both hybridization and
wash. The term "standard hybridization conditions" as used herein
is therefore an operational definition and encompasses a range of
hybridization conditions. Nevertheless, for the purposes of this
present disclosure "high stringency" conditions include hybridizing
with plaque screen buffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll
400; 0.2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5); 1 M NaCl;
0.1% sodium pyrophosphate; 1% SDS); 10% dextran sulfate, and 100
.mu.g/ml denatured, sonicated salmon sperm DNA at 65.degree. C. for
12-20 hours, and washing with 75 mM NaCl/7.5 mM sodium citrate
(0.5.times.SSC)/1% SDS at 65.degree. C. "Low stringency" conditions
include hybridizing with plaque screen buffer, 10% dextran sulfate
and 110 .mu.g/ml denatured, sonicated salmon sperm DNA at
55.degree. C. for 12-20 hours, and washing with 300 mM NaCl/30 mM
sodium citrate (2.0.times.SSC)/1% SDS at 55.degree. C. See also
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
New York, Sections 6.3.1-6.3.6, (1989).
[0039] A "therapeutic composition" as used herein is defined as
comprising the therapeutics of the invention and other biologically
compatible ingredients. The therapeutic composition may contain
excipients such as water, minerals and carriers such as
protein.
[0040] "Wild type" means the naturally-occurring polynucleotide
sequence of an exon of a protein, or a portion thereof, or protein
sequence, or portion thereof, respectively, as it normally exists
in vivo.
[0041] Practice of the present invention will employ, unless
indicated otherwise, conventional techniques of cell biology, cell
culture, molecular biology, microbiology, recombinant DNA, protein
chemistry, and immunology, which are within the skill of the art.
Such techniques are described in the literature. Unless stipulated
otherwise, all references cited in the Detailed Description are
incorporated herein by reference.
A. Production of Fragments and Analogs
[0042] Fragments of an isolated protein (e.g., fragments of TWEAK)
can also be produced efficiently by recombinant methods, by
proteolytic digestion, or by chemical synthesis using methods known
to those of skill in the art. In recombinant methods, internal or
terminal fragments of a polypeptide can be generated by removing
one or more nucleotides from one end (for a terminal fragment) or
both ends (for an internal fragment) of a DNA sequence which
encodes for the isolated TWEAK polypeptide. Expression of the
mutagenized DNA produces polypeptide fragments. Digestion with "end
nibbling" endonucleases can also generate DNAs which encode an
array of fragments. DNAs which encode fragments of a protein can
also be generated by random shearing, restriction digestion, or a
combination or both. Protein fragments can be generated directly
from intact proteins. Peptides can be cleaved specifically by
proteolytic enzymes, including, but not limited to plasmin,
thrombin, trypsin, chymotrypsin, or pepsin. Each of these enzymes
is specific for the type of peptide bond it attacks. Trypsin
catalyzes the hydrolysis of peptide bonds in which the carbonyl
group is from a basic amino acid, usually arginine or lysine.
Pepsin and chymotrypsin catalyse the hydrolysis of peptide bonds
from aromatic amino acids, such as tryptophan, tyrosine, and
phenylalanine. Alternative sets of cleaved protein fragments are
generated by preventing cleavage at a site which is suceptible to a
proteolytic enzyme. For instance, reaction of the .epsilon.-amino
acid group of lysine with ethyltrifluorothioacetate in mildly basic
solution yields blocked amino acid residues whose adjacent peptide
bond is no longer susceptible to hydrolysis by trypsin. Proteins
can be modified to create peptide linkages that are susceptible to
proteolytic enzymes. For instance, alkylation of cysteine residues
with .beta.-haloethylamines yields peptide linkages that are
hydrolyzed by trypsin (Lindley, (1956) Nature 178, 647). In
addition, chemical reagents that cleave peptide chains at specific
residues can be used. For example, cyanogen bromide cleaves
peptides at methionine residues (Gross and Witkip, (1961) J. Am.
Chem. Soc. 83, 1510). Thus, by treating proteins with various
combinations of modifiers, proteolytic enzymes and/or chemical
reagents, the proteins may be divided into fragments of a desired
length with no overlap of the fragments, or divided into
overlapping fragments of a desired length.
[0043] Fragments can also be synthesized chemically using
techniques known in the art such as the Merrifield solid phase F
moc or t-Boc chemistry. Merrifield, Recent Progress in Hormone
Research 23: 451 (1967).
B. Production of Altered DNA and Peptide Sequences: Random
Methods
[0044] Amino acid sequence variants of a protein can be prepared by
random mutagenesis of DNA which encodes the protein or a particular
portion thereof. Useful methods include PCR mutagenesis and
saturation mutagenesis. A library of random amino acid sequence
variants can also be generated by the synthesis of a set of
degenerate oligonucleotide sequences. Methods of generating amino
acid sequence variants of a given protein using altered DNA and
peptides are well-known in the art. The following examples of such
methods are not intended to limit the scope of the present
invention, but merely serve to illustrate representative
techniques. Persons having ordinary skill in the art will recognize
that other methods are also useful in this regard.
PCR Mutagenesis: See, for example Leung et al., (1989) Technique 1,
11-15.
Saturation Mutagenesis: One method is described generally in Mayers
et al., (1989) Science 229, 242.
[0045] Degenerate Oligonucleotide Mutagenesis: See for example
Harang, S. A., (1983) Tetrahedron 39, 3; Itakura et al., (1984)
Ann. Rev. Biochem. 53, 323 and Itakura et al., Recombinant DNA,
Proc. 3rd Cleveland Symposium on Macromolecules, pp. 273-289 (A. G.
Walton, ed.), Elsevier, Amsterdam, 1981.
C. Production of Altered DNA and Peptide Sequences: Directed
Methods
[0046] Non-random, or directed, mutagenesis provides specific
sequences or mutations in specific portions of a polynucleotide
sequence that encodes an isolated polypeptide, to provide variants
which include deletions, insertions, or substitutions of residues
of the known amino acid sequence of the isolated polypeptide. The
mutation sites may be modified individually or in series, for
instance by: (1) substituting first with conserved amino acids and
then with more radical choices depending on the results achieved;
(2) deleting the target residue; or (3) inserting residues of the
same or a different class adjacent to the located site, or
combinations of options 1-3.
[0047] Clearly, such site-directed methods are one way in which an
N-terminal cysteine (or a functional equivalent) can be introduced
into a given polypeptide sequence to provide the attachment site
for a hydrophobic moiety.
Alanine scanning Mutagenesis: See Cunningham and Wells, (1989)
Science 244, 1081-1085).
Oligonucleotide-Mediated Mutagenesis: See, for example, Adelman et
al., (1983) DNA 2, 183.
Cassette Mutagenesis: See Wells et al., (1985) Gene 34, 315.
Combinatorial Mutagenesis: See, for example, Ladner et al.,
W088/06630
[0048] Methods of Treatment
[0049] The method of the present invention are useful as a
treatment in diseases where enhanced angiogenic activity is
desirable to promote neovascularization. Such diseases and
conditions include: myocardial ischemic conditions (e.g.,
myocardial infarction, improve blood flow in patients with coronary
artery disease suffering from myocardial ischemia or inadequate
blood flow to areas other than the heart such as in peripheral
vascular disease, where decreased blood flow is a problem,
revascularization of necrotic tissue, for example of the myocardium
after an infarction or an angioplasty, angina, heart transplants,
vascular grafts, and reopening vessels to improve vascularization,
perfusion, collagenization and organization of said lesions), wound
healing, and tissue and organ transplantations (e.g., enhancement
of autologous or heterologous microvascular transplantation).
Promotion of wound healing includes healing of incisions, bone
repair, burn healing, post-infarction repair in myocardial injury,
healing of gastric ulcers and other ulcers of the gastrointestinal
tract and generally in promoting the formation, maintenance and
repair of tissue. Neovascularization of grafted or transplanted
tissue is also contemplated, especially in subjects suffering from
vascular insufficiency, such as diabetic patients.
[0050] As a general matter, the methods of the present invention
may be utilized for any mammalian subject needing modulation of
angiogenesis. Mammalian subjects which may be treated according to
the methods of the invention include, but are not limited to, human
subjects or patients. In addition, however, the invention may be
employed in the treatment of domesticated mammals which are
maintained as human companions (e.g., dogs, cats, horses), which
have significant commercial value (e.g., dairy cows, beef cattle,
sporting animals), which have significant scientific value (e.g.,
captive or free specimens of endangered species), or which
otherwise have value. In addition, as a general matter, the
subjects for treatment with the methods of the present invention
need not present indications for treatment with the agents of the
invention other than those indications associated with need for
modulation of angiogenesis. That is, the subjects for treatment are
expected to be otherwise free of indications for treatment with the
TWEAK therapeutic agents of the invention.
[0051] One of ordinary skill in the medical or veterinary arts is
trained to recognize subjects which may need modulation of
angiogenesis. In particular, clinical and non-clinical trials, as
well as accumulated experience, relating to the presently disclosed
and other methods of treatment, are expected to inform the skilled
practitioner in deciding whether a given subject is in need of
modulation and whether any particular treatment is best suited to
the subject's needs, including treatment according to the present
invention.
[0052] Accordingly, the methods of this invention may employ TWEAK
agonists or biologically active portions thereof, and angiogenic
factors, to promote angiogenesis, such as, to repair damage of
myocardial tissue as a result of myocardial infarction. Such
methods may also include the repair of the cardiac vascular system
after ischemia including the growth of collateral vasculature.
Methods utilizing TWEAK agonists and angiogenic factors may be
employed to stimulate the growth of transplanted tissue and
collateral vasculature where coronary bypass surgery is performed.
Methods may also treat damaged vascular tissue as a result of
coronary artery disease and peripheral or central nervous system
vascular disease or ischemia.
[0053] Methods of the invention may also promote wound healing,
particularly to re-vascularize damaged tissues or stimulate
collateral blood flow during ischemia and where new capillary
angiogenesis is desired. Other methods of the invention may be
employed to treat full-thickness wounds such as dermal ulcers,
including pressure sores, venous ulcers, and diabetic ulcers. In
addition, methods employing TWEAK therapeutics may be employed to
treat full-thickness burns and injuries where a skin graft or flap
is used to repair such burns and injuries. Such TWEAK agonists and
angiogenic factors may also be employed for use in plastic surgery,
for example, for the repair of lacerations, burns, or other trauma.
In urology, methods of the invention may assist in recovery of
erectile function.
[0054] Since angiogenesis is important in keeping wounds clean and
non-infected, methods may be employed in association with surgery
and following the repair of cuts. They may also be employed for the
treatment of abdominal wounds where there is a high risk of
infection. Methods using TWEAK therapeutics described herein may be
employed for the promotion of endothelialization in vascular graft
surgery. In the case of vascular grafts using either transplanted
or synthetic material, TWEAK agonists and angiogenic factors can be
applied to the surface of the graft or at the junction to promote
the growth of vascular smooth muscle and adventitial cells in
conjunction with endothelial cells.
[0055] Methods of the invention may also be employed to coat
artificial prostheses or natural organs which are to be
transplanted in the body to minimize rejection of the transplanted
material and to stimulate vascularization of the transplanted
materials and may also be employed for vascular tissue repair, for
example, that occurring during arteriosclerosis and required
following balloon angioplasty where vascular tissues are damaged.
Specifically, methods of the invention may be employed to promote
recovery from arterial wall injury and thereby inhibit
restenosis.
[0056] Nucleic acid sequences encoding TWEAK agonists may also be
employed for in vitro purposes related to scientific research,
synthesis of DNA and manufacture of DNA vectors, and for the
production of diagnostics and therapeutics to treat human disease.
For example, methods of the invention may involve in vitro
culturing of vascular smooth muscle cells, fibroblasts,
hematopoietic cells, muscle, myotendonous junction, bone or
cartilage- derived cells and other mesenchymal cells, where a TWEAK
therapeutic is added to the conditional medium in a concentration
from 10 ng/ml to 20 ug/ml.
[0057] These therapeutic agents may be administered by any route
which is compatible with the particular agent employed. The
therapeutic agents of the invention may be provided to an
individual by any suitable means, preferably directly (e.g.,
locally, as by injection or topical administration to a tissue
locus) or systemically (e.g., parenterally or orally). Where the
agent is to be provided parenterally, such as by intravenous,
intraarterial, subcutaneous, or intramuscular, administration, the
agent preferably comprises part of an aqueous solution. The
solution is physiologically acceptable so that in addition to
delivery of the desired agent to the subject, the solution does not
otherwise adversely affect the subject's electrolyte and/or volume
balance. The aqueous medium for the therapeutic may comprise normal
physiologic saline (e.g., 9.85% NaCl, 0.15M, pH 7-7.4).
[0058] The therapeutics are preferably administered as a sterile
pharmaceutical composition containing a pharmaceutically acceptable
carrier, which may be any of the numerous well known carriers, such
as water, saline, phosphate buffered saline, dextrose, glycerol,
ethanol, and the like, or combinations thereof. The compounds of
the present invention may be used in the form of pharmaceutically
acceptable salts derived from inorganic or organic acids and bases.
Included among such acid salts are the following: acetate, adipate,
alginate, aspartate, benzoate, benzenesulfonate, bisulfate,
butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
and undecanoate. Base salts include ammonium salts, alkali metal
salts, such as sodium and potassium salts, alkaline earth metal
salts, such as calcium and magnesium salts, salts with organic
bases, such as dicyclohexylamine salts, N-methyl-D-glucamine,
tris(hydroxymethyl)methylamine and salts with amino acids such as
arginine, lysine, and so forth. Also, the basic nitrogen-containing
groups can be quatemized with such agents as lower alkyl halides,
such as methyl, ethyl, propyl, and butyl chloride, bromides and
iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and
diamyl sulfates, long chain halides such as decyl, lauryl, myristyl
and stearyl chlorides, bromides and iodides, aralkyl halides, such
as benzyl and phenethyl bromides and others. Water or oil-soluble
or dispersible products are thereby obtained.
[0059] Pharmaceutical compositions of TWEAK agonists and angiogenic
factors comprise any of the compounds of the present invention, or
pharmaceutically acceptable derivatives thereof, together with any
pharmaceutically acceptable carrier. The term "carrier" as used
herein includes acceptable adjuvants and vehicles. Pharmaceutically
acceptable carriers that may be used in the pharmaceutical
compositions of this invention include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins,
such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, potassium sorbate, partial glyceride mixtures
of saturated vegetable fatty acids, water, salts or electrolytes,
such as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0060] Injection Delivery
[0061] According to this invention, the pharmaceutical compositions
may be in the form of a sterile injectable preparation, for example
a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as do natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant.
[0062] Controlled release administration of a particular
therapeutic may be useful. For example, the therapeutic may be
administered using intravenous infusion, an implantable osmotic
pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump may be used [Langer et
al., eds., Medical Applications of Controlled Release, CRC Pres.,
Boca Raton, Fla. (1974); Sefton, CRC Crit. Ref. Biomed. Eng.,
14:201 (1987); Buchwald et al., Surgery, 88:507 (1980); Saudek et
al., N. Engl. J. Med., 321:574 (1989)]. In another embodiment,
polymeric materials can be used [see, Langer, 1974, supra; Sefton,
1987, supra; Smolen et al., eds., Controlled Drug Bioavailability,
Drug Product Design and Performance, Wiley, N.Y. (1984); Ranger et
al., J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also
Levy et al., Science, 228:190 (1985); During et al., Ann. Neurol.,
25:351 (1989); Howard et al., J. Neurosurg., 71:105 (1989)]. In yet
another embodiment, a controlled release system can be placed in
proximity of the therapeutic target, e.g., a tumor, thus requiring
only a fraction of the systemic dose [see. e.g., Goodson, in
Medical Applications of Controlled Release, vol. 2, pp. 115-138
(1984)]. Other controlled release systems are discussed in the
review by Langer, Science, 249:1527-1533 (1990). In another
embodiment, the therapeutic compound can be delivered in a vesicle,
in particular a liposome (see Langer, 1990, supra); Treat et al.,
in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365
(1989); Lopez-Berestein, pp. 317-327; see generally id.).
[0063] Oral Delivery
[0064] Contemplated for use herein are oral solid dosage forms,
which are described generally in Martin, Chapter 89, 1990, supra,
which is herein incorporated by reference. Solid dosage forms
include tablets, capsules, pills, troches or lozenges, cachets or
pellets. Also, liposomal or proteinoid encapsulation may be used to
formulate the present compositions (as, for example, proteinoid
microspheres reported in U.S. Pat. No. 4,925,673). Liposomal
encapsulation may be used and the liposomes may be derivatized with
various polymers (e.g., U.S. Pat. No. 5,013,556). A description of
possible solid dosage forms for the therapeutic is given by
Marshall, in Modern Pharmaceutics, Chapter 10, Banker and Rhodes
ed., (1979), herein incorporated by reference. In general, the
formulation will include the therapeutic (or chemically modified
form), and inert ingredients which allow for protection against the
stomach environment, and release of the biologically active
material in the intestine.
[0065] For the protein (or derivative) the location of release may
be the stomach, the small intestine (the duodenum, the jejunem, or
the ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
protein (or derivative) or by release of the biologically active
material beyond the stomach environment, such as in the intestine.
To ensure full gastric resistance, a coating impermeable to at
least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed films.
A coating or mixture of coatings can also be used on tablets, which
are not intended for protection against the stomach. This can
include sugar coatings, or coatings which make the tablet easier to
swallow. Capsules may consist of a hard shell (such as gelatin) for
delivery of dry therapeutic i.e. powder; for liquid forms, a soft
gelatin shell may be used. The shell material of cachets could be
thick starch or other edible paper. For pills, lozenges, molded
tablets or tablet triturates, moist massing techniques can be
used.
[0066] The therapeutic can be included in the formulation as fine
multiparticulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression. Colorants and flavoring agents may all be included.
For example, the protein (or derivative) may be formulated (such as
by liposome or microsphere encapsulation) and then further
contained within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents. One may dilute or
increase the volume of the therapeutic with an inert material.
These diluents could include carbohydrates, especially mannitol,
alpha-lactose, anhydrous lactose, cellulose, sucrose, modified
dextrans and starch. Certain inorganic salts may be also be used as
fillers including calcium triphosphate, magnesium carbonate and
sodium chloride. Some commercially available diluents are Fast-Flo,
Emdex, STA-Rx 1500, Emcompress and Avicell. Disintegrants may be
included in the formulation of the therapeutic into a solid dosage
form. Materials used as disintegrants include but are not limited
to starch including the commercial disintegrant based on starch,
Explotab. Sodium starch glycolate, Amberlite, sodium
carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,
orange peel, acid carboxymethyl cellulose, natural sponge and
bentonite may all be used. Another form of the disintegrants are
the insoluble cationic exchange resins. Powdered gums may be used
as disintegrants and as binders and these can include powdered gums
such as agar, Karaya or tragacanth. Alginic acid and its sodium
salt are also useful as disintegrants. Binders may be used to hold
the therapeutic agent together to form a hard tablet and include
materials from natural products such as acacia, tragacanth, starch
and gelatin. Others include methyl cellulose (MC), ethyl cellulose
(EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP)
and hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic. An antifrictional
agent may be included in the formulation of the therapeutic to
prevent sticking during the formulation process. Lubricants may be
used as a layer between the therapeutic and the die wall, and these
can include but are not limited to: stearic acid including its
magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid
paraffin, vegetable oils and waxes. Soluble lubricants may also be
used such as sodium lauryl sulfate, magnesium lauryl sulfate,
polyethylene glycol of various molecular weights, and Carbowax 4000
and 6000. Glidants that might improve the flow properties of the
drug during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0067] To aid dissolution of the therapeutic into the aqueous
environment, a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
Additives which potentially enhance uptake of the protein (or
derivative) are for instance the fatty acids oleic acid, linoleic
acid and linolenic acid.
[0068] Pulmonary Delivery
[0069] Also contemplated herein is pulmonary delivery of the
present proteins (or derivatives thereof). The protein (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the
blood-stream. Other reports of this include Adjei et al.,
Pharmaceutical Research, 7(6):565-569 (1990); Adjei et al.,
International Journal of Pharmaceutics, 63:135-144 (1990)
(leuprolide acetate); Braquet et al., Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard
et al., Annals of Internal Medicine, 3(3):206-212 (1989) (alpha
1-antitrypsin); Smith et al., J. Clin. Invest., 84:1145-1146 (1989)
(alpha 1-proteinase); Oswein et al., "Aerosolization of Proteins",
Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,
Colo., (March 1990) (recombinant human growth hormone); Debs et
al., J. Immunol., 140:3482-3488 (1988) (interferon-gamma and tumor
necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656
(granulocyte colony stimulating factor). Contemplated for use in
the practice of this invention are a wide range of mechanical
devices designed for pulmonary delivery of therapeutic products,
including but not limited to nebulizers, metered-dose inhalers, and
powder inhalers, all of which are familiar to those skilled in the
art.
[0070] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered-dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Mass. All
such devices require the use of formulations suitable for the
dispensing of protein (or derivative). Typically, each formulation
is specific to the type of device employed and may involve the use
of an appropriate propellant material, in addition to the usual
diluents, adjuvants and/or carriers useful in therapy. Also, the
use of liposomes, microcapsules or microspheres, inclusion
complexes, or other types of carriers is contemplated. Chemically
modified protein may also be prepared in different formulations
depending on the type of chemical modification or the type of
device employed.
[0071] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise protein (or derivative)
dissolved in water at a concentration of about 0.1 to 25 mg of
biologically active protein per ml of solution. The formulation may
also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation may also contain a surfactant, to reduce or prevent
surface induced aggregation of the protein caused by atomization of
the solution in forming the aerosol.
[0072] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the protein
(or derivative) suspended in a propellant with the aid of a
surfactant. The propellant may be any conventional material
employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0073] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing protein (or
derivative) and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The protein (or derivative) should most
advantageously be prepared in particulate form with an average
particle size of less than 10 mu m (or microns), most preferably
0.5 to 5 mu m, for most effective delivery to the distal lung.
[0074] Dosages
[0075] For all of the above molecules, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age and general health of the recipient, will be able to ascertain
the proper dosage. Generally, for injection or infusion, dosage
will be between 0.01 mu g of biologically active protein/kg body
weight, (calculating the mass of the protein alone, without
chemical modification), and 10 mg/kg (based on the same). The
dosing schedule may vary, depending on the circulation half-life of
the protein or derivative used, whether the polypeptide is
delivered by bolus dose or continuous infusion, and the formulation
used.
[0076] Administration with Other Compounds
[0077] For therapy associated with modulating angiogenesis, one may
administer the present TWEAK agonists (or derivatives) and
angiogenic factors in conjunction with one or more pharmaceutical
compositions used for treating other clinical complications of the
need for angiogenic modulation, such as those used for treatment of
cancer (e.g., chemotherapeutics), cachexia, high blood pressure,
high cholesterol, and other adverse conditions. Administration may
be simultaneous or may be in seriatim.
[0078] Nucleic Acid-Based Therapeutic Treatment
[0079] Nucleic acid sequences encoding a TWEAK agonist could be
introduced into human tumor or blood vessel cells to develop gene
therapy.
[0080] In one embodiment, a nucleic acid sequence encoding a TWEAK
agonist is introduced in vivo in a viral vector. Such vectors
include an attenuated or defective DNA virus, such as but not
limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr
virus (EBV), adenovirus, adeno-associated virus (AAV), and the
like. Defective viruses, which entirely or almost entirely lack
viral genes, are preferred. Defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Thus, adipose
tissue can be specifically targeted. Examples of particular vectors
include, but are not limited to, a defective herpes virus 1 (HSV1)
vector [Kaplitt et al., Molec. Cell. Neurosci., 2:320-330 (1991)],
an attenuated adenovirus vector, such as the vector described by
Stratford-Perricaudet et al., J. Clin. Invest., 90:626-630 (1992),
and a defective adeno-associted virus vector [Samulski et al., J.
Virol., 61:3096-3101 (1987); Samulski et al., J. Virol.,
63:3822-3828 (1989)]. In another embodiment, the nucleic acid can
be introduced in a retroviral vector, e.g., as described in
Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell, 33:153
(1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S.
Pat. No. 4,980,289; Markowitz et al., J. Virol., 62:1120 (1988);
Temin et al., U.S. Pat. No. 5,124,263; International Patent
Publication No. WO 95/07358, published Mar. 16, 1995, by Dougherty
et al.; and Kuo et al., Blood, 82:845 (1993). Alternatively, the
vector can be introduced in vivo by lipofection. For the past
decade, there has been increasing use of liposomes for
encapsulation and transfection of nucleic acids in vitro. Synthetic
cationic lipids designed to limit the difficulties and dangers
encountered with liposome mediated transfection can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker [Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417
(1987); see Mackey et al., Proc. Natl. Acad. Sci. USA, 85:8027-8031
(1988)]. The use of cationic lipids may promote encapsulation of
negatively charged nucleic acids, and also promote fusion with
negatively charged cell membranes [Felgner et al., Science,
337:387-388 (1989)]. The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting (see Mackey
et al., 1988, supra). Targeted peptides, e.g., hormones or
neurotransmitters, and proteins such as antibodies, or non-peptide
molecules could be coupled to liposomes chemically.
[0081] It is also possible to introduce the vector in vivo as a
naked DNA plasmid. Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in the art,
e.g., transfection, electroporation, microinjection, transduction,
cell fuision, DEAE dextran, calcium phosphate precipitation, use of
a gene gun, or use of a DNA vector transporter (see, e.g., Wu et
al., J. Biol. Chem., 267:963-967 (1992); Wu et al., J. Biol. Chem.,
263:14621-14624 (1988); Hartmut et al., Canadian Patent Application
No. 2,012,311, filed Mar. 15, 1990).
[0082] It is also possible to introduce the vector in vivo in
conjunction with a catheter or other device. See Vale et al., 1999;
Komowski et al., 2000.
EXAMPLES
[0083] The following examples illustrate aspects of the present
invention but should not be construed as limitations. The symbols
and convention used in these examples are consistent with those
used in contemporary medical and scientific literature
[0084] Experimental Procedures:
[0085] Cells--Human Umbilical Vein Endothelial Cells (HUVEC) were
obtained from Cell System Corporation (CS-C) (Kirkland, Wash.) or
Clonetics (San Diego, Calif.) and Human Pulmonary Artery
Endothelial Cells (HPAEC), Human Lung Microvascular Endothelial
cells (HMVEC-L) and Human Dermal Microvascular Endothelial cells
(HMVEC-D) were purchased from Clonetics. HUVEC were routinely
passaged in CS-C Medium and used in experiments until passage
seven. The other primary cells were routinely passaged in
Microvascular Endothelial Cell Growth Medium-2 (EGM2-MV)
(Clonetics). EC Basal Medium (EBM) containing 2% fetal bovine serum
(FBS), defined as "basal media", and EBM containing 2% FBS and
supplier growth supplements, defined as "complete media", were used
in proliferation, migration and immunofluorescent staining
experiments. EC Basal Medium 2 EBM-2) and supplier growth
supplements were used in the capillary tube formation assay as
specified.
[0086] Reagents and Antibodies--Recombinant human bFGF was obtained
as a growth supplier supplement (Clonetics), bFGF also was
purchased from R&D Systems (Minneapolis, Minn.) and Sigma
(St.Louis, Mo.). Annexin V-FITC was from Pharmingen (San Diego,
Calif.), propidium iodide (PI) from Sigma (St.Louis, Mo.), rabbit
anti-human Flk-1 and rabbit anti-Flt-1 antibodies from Research
Diagnostics Inc. (Flanders, N.J.), mouse anti-Flg monoclonal
antibody (mAb) from Chemicon (Temecula, Calif.), mouse
anti-human-.beta..sub.3, mouse anti-human-.beta..sub.1 (clone LIA
1/2) and rat anti-human .alpha..sub.v mAbs from Immunotech
(Westbrook, Me.), mouse anti-human .alpha..sub.5 from Pharmingen
(San Diego, Calif.), mouse anti-human .alpha..sub.1 (clone AJH10)
from Biogen (Gotwals P. J, et al., 1999. Biochem. 38:8280-8288),
Phycoerythrin (PE)-conjugated donkey anti-rabbit IgG, goat
anti-mouse IgG and donkey anti-rat IgG from Jackson Immunoresearch
Labs Inc. (West Groove, Pa.), biotin-conjugated anti-FLAG from
Eastman Kodak Company (New Haven, Conn.), and RPE-Streptavidin from
Southern Biotechnology Associates, Inc. (Birmingham, Ala.). Soluble
CD40L was prepared at Biogen as previously described (Karpusas, M.,
et al., 1995. Structure 3:1426-xxx).
[0087] TWEAK-specific mAbs BE.B3 and AB.D3 were generated in
Armenian hamsters by immunizing with soluble human TWEAK protein
and standard hybridoma generation procedures. The ability of AB.D3
to bind to human and murine TWEAK and BE.B3 to bind to human TWEAK
was demonstrated in an ELISA assay using recombinant soluble TWEAK
proteins immobilized on 96 well microtiter plates. The blocking
activity of AB.D3 was demonstrated by the ability of this mAb but
not BE.B3 to inhibit soluble FLAG-tagged human TWEAK binding to
HT29 cells in a FACS analysis. BE.B3 was biotinylated prepared
using ImmunoPure Biotinylation kits following the manufacturer's
protocol (Pierce, Rockford, Ill.). A hamster control Ig (clone
Ha4/8-3.1) was obtained from the American Type Culture Collection
and mAb purified from culture supernatant by Protein A Fast Flow
column (Pharmacia, Piscataway, N.J.).
[0088] Recombinant Soluble Human TWEAK protein--Soluble expression
construct for myc-tagged human TWEAK was constructed as previously
described (Chicheportiche, Y., et al. 1997. J. Biol. Chem.
272:32401-32410). Flag-tagged and nontagged forms also were made.
These soluble forms of TWEAK were expressed in yeast, Pichia
pastoris strain GS115, using standard conditions.
[0089] Proliferation Assays--HUVEC were plated in 96-well
microtiter plates at subconfluence (4000 cells per well) and
cultured overnight in CS-C Medium without addition of supplier
growth supplements. Media was replaced with complete Media, or with
basal media as defined above. Cells were cultured in basal media
with or without TWEAK (100 ng/ml), bFGF using a 1/500- 1/1000
dilution of bFGF growth supplement (Clonetics) or 1 ng/ml (R&D
Systems), VEGF (10 ng/ml) or combinations of these factors. Where
indicated, 10 .mu.g/ml anti-TWEAK mAbs AB.D3, BE.B3 or hamster
control Ig Ha4/8 also were added. Cells were incubated at
37.degree. C. with 5% CO.sub.2 for three days and proliferation was
measured by pulsing with .sup.3H-Thymidine for the last 10 hours of
culture. Cell-bound radioactivity was measured with a Betaplate.TM.
(EG&G Wallac, Gaithersburg, Md.).
[0090] Analysis of Apoptosis--HUVEC seeded in 6-well plates at a
density of 1.2.times.10.sup.5 cells per well were incubated over
night in CS-C Medium without supplier growth supplements. Media was
replaced with complete media, or with basal media with or without
TWEAK (200 ng/ml), bFGF (1 ng/ml) or combinations of these factors
and cells were cultured for 24 hours. Cells were washed with
Phosphate buffered saline (PBS) and detached by incubation with
dispase (CS-C) for 15 minutes at 37.degree. C. followed by
replacement with PBS containing 5 mM EDTA and 0.1% BSA for 15
minutes at 37.degree. C. After an additional wash in PBS, cells
were stained with FITC-Annexin-V and 5 .mu.g/ml Propidium Iodide
according to the supplier (Pharmingen). Fluorescence was analyzed
within the hour using FACStar.sup.PLUS (Becton Dickinson, San Jose,
Calif.).
[0091] Endothelial Wound Repair Assay--A standard wound repair
assay was employed as previously described (Bussolino F., et al,
1991. J. Clin. Invest. 87:986-991). In brief, a confluent monolayer
of HUVEC was grown in CS-C Medium in 35.times.10 mm cell culture
dishes with 2 mm grids (Nalge Nunc International, Naperville,
Ill.). The monolayer was wounded by two perpendicular strokes
across the diameter of the dish with a 1 mm tip (Morales D. E., et
al., 1995. Circulation 91:755-763). Dislodged cells were aspirated
and plates were rinsed with PBS. Cells were cultured in complete
media, or in fresh basal media with or without TWEAK (200 ng/ml),
bFGF ( 1/1000 or 1 ng/ml), VEGF (10 ng/ml) or combinations of these
and were incubated for 18 hours at 37.degree. C. with 5% CO.sub.2
at which time plates were fixed with 1% paraformaldehyde and
stained with Harris Hematoxylin (Sigma, St. Louis, Mo.). Wound
repair was quantified by visually counting the number of grids in
which the gap was obscured by migrating cells. This number was
divided by the total number of grids that aligned the wound and
results were expressed as mean percentage wound repair +/-SEM.
[0092] Immunofluorescent staining--HUVEC were cultured in basal
media with or without TWEAK (200 ng/ml), bFGF (1 ng/ml) or both
factors for 24 hours. Cells were detached as described above and
stained with 10 .mu.g/ml primary antibody in 200 .mu.l PBS
containing 0.1% bovine serum albumin and 0.02% NaN.sub.3 for 20
minutes at 4.degree. C. Following washes with the same buffer, the
PE-conjugated detection antibodies were added at concentrations as
specified by the manufacturer for an additional 15 minutes at
4.degree. C. Cells were analyzed for TWEAK binding by incubation
with TWEAK tagged either with flag or with myc. Binding was
detected with either biotinylated mouse anti-flag antibody or
biotinylated BE.B3 and streptavidin-PE. Cold competition was
performed with non-tagged TWEAK and blocking was performed with the
AB.D3 mAb.
[0093] Capillary tube formation assay--Capillary tube formation by
ECs was analyzed using a three-dimensional fibrin matrix gel assay
based on a method previously described (Mach, F., et al., 1999. Am.
J. Pathol. 154:229-239). Briefly, 4 mg/ml plasminogen free human
fibrinogen (Calbiochem, San Diego, Calif.) was dissolved in serum
free EBM-2 media with heparin and polymixin B both at 1 .mu.g/ml
(Sigma) as well as all of the supplier supplements except for VEGF
and bFGF. The fibrin solution was filtered-sterilized and fibrin
matrices were prepared by adding thrombin (20-50 milliunits/ml)
(Sigma) and distributing 300 ul per well in 24-well plates. ECs at
appropriate concentrations (4.times.10.sup.4 cells/cm.sup.2 for
HUVEC and HPAEC and 8.times.10.sup.4/cm.sup.2 for HMVEC-L and
HMVEC-D) were then seeded onto the gel surfaces and overlayered
with EBM-2 media as above and 5% FBS in the presence or absence of
TWEAK, bFGF, sCD40L or combinations of these factors as specified.
After 72 hours of culture, phase-contrast photomicrographs of the
gel surface were taken. Gels were transferred from original wells
to new wells and fixed with 10% ethanol for 10 minutes and then
with 4% paraformaldehyde. Gels were cross sectioned for analysis
and photographs taken.
Example 1
TWEAK Enhances bFGF-Dependent Proliferation
[0094] The effect of TWEAK on EC functions was investigated by
examining EC proliferation in cultures treated with TWEAK alone and
in combination with another angiogenic growth factors. Human
Umbilical Vein EC (HUVEC) were cultured in basal media in the
presence or absence of bFGF (FIG. 1). Addition of TWEAK induced no
significant proliferation of ECs. By contrast, cells cultured with
TWEAK and an optimal concentration of bFGF displayed a
significantly enhanced proliferative response compared to cells
cultured in the presence of bFGF alone. The degree of proliferation
achieved was comparable to or greater than that of ECs cultured in
complete media. Similar results were obtained using bFGF at 1
ng/ml. The synergistic activity of TWEAK with bFGF was completely
inhibited by anti-TWEAK mAb AB.D3 suggesting that the effect of
TWEAK was specific, whereas there was no inhibition by a anti-TWEAK
mAb BE.B3 or an irrelevant control Ig. In addition, no enhancement
was seen with recombinant soluble APRIL, another TNF ligand (data
not shown). The experimental conditions for the results shown in
FIG. 1 are described here in detail. HUVEC were cultured in
complete media or in basal media. TWEAK (100 ng/ml), bFGF ( 1/500
dilution) or combinations of these factors were added to basal
media as indicated for 3 days and proliferation measured by
.sup.3H-thymidine incorporation. In FIG. 1, data shown are the mean
value +/-SD of triplicate wells. These results are representative
of 4 independent experiments wherein proliferation in
bFGF+TWEAK-treated cultures was significantly different from that
of cultures with bFGF alone, TWEAK alone and basal media (P
values<0.05), and the difference between cultures in basal media
with and without TWEAK was not significant. In addition to growth
factors, blocking anti-TWEAKmAb AB.D3, nonblocking anti-TWEAK mAb
BE.B3, and an irrelevant hamster control Ig Ha4/8 (10 ug/ml) were
added where indicated. Results are representative of one of two
independent experiments.
Example 2
Enhancement of EC Proliferation by TWEAK with bFGF Is Not Due to
Decreased Cell Death
[0095] The apparent enhancement of HUVEC proliferation by the
TWEAK/bFGF combination could be due to increased cell division or
decreased cell death. In order to address the mechanism, HUVEC
cultured in basal media with or without TWEAK, bFGF or both were
analyzed to determine the frequency of apoptotic cells. Annexin V
staining was employed to detect cells undergoing apoptosis and
propidium iodide (PI) dye exclusion to detect viable cells.
Cultures treated with the combination of TWEAK and bFGF exhibited
percentages of viable, apoptotic and dead cells that were
comparable to those of cultures treated with bFGF alone. These
percentages are shown in FIG. 2, in quadrants 3, 4 and 2
respectively. Similar results were obtained in two additional
experiments wherein cells with subdiploid DNA content were
quantified (11% in bFGF and 11% in bFGF/TWEAK treated cultures).
Thus, the enhancement by TWEAK of bFGF-dependent proliferation is
not due to decreased cell death. Nevertheless it is noteworthy that
TWEAK alone decreased the frequency of apoptotic cells from 22% to
14%. This pattern also was observed in two independent experiments,
wherein the percentage of cells with subdiploid DNA were on average
18+/-1% and 9+/-0% in the absence and presence of TWEAK,
respectively.
[0096] The experimental conditions for the results in FIG. 2 are
described in detail here. HUVEC cultured for 24 hours in basal
media with or without TWEAK (200 ng/ml), bFGF (1 ng/ml) or both
cytokines were stained with FITC-Annexin-V (x-axis) for apoptotic
cells and by PI dye exclusion for viability (y-axis). FIG. 2 shows
the percentage of viable, apoptotic and dead cells in quadrants 3,
4, and 2, respectively.
Example 3
TWEAK Enhances bFGF-Dependent HUVEC Migration
[0097] The ability of TWEAK to effect EC migration was evaluated in
the presence and absence of other angiogenic factors. Confluent
HUVEC monolayers were wounded and EC migration was monitored within
the first 18 hours by determining the degree of wound repair.
Addition of TWEAK or bFGF to basal media induced a low level of
wound repair, however, this was not significantly greater than that
observed with basal media alone. By contrast, cultures treated with
both TWEAK and bFGF were repaired to a significantly greater degree
than cultures in basal media and with either agent alone, and were
similar to those in complete media. HUVECs were recovered from the
cultures and counted in order to determine whether or not any
increase in cell number had occurred over the course of the
experiment. In all treatment groups, cell recoveries were
comparable (data not shown) supporting that the combinatorial
effect of TWEAK and bFGF was at the level of cell migration.
[0098] The experimental conditions for the results shown in FIG. 3
are described here in detail. Confluent HUVEC monolayers treated
with TWEAK (200 ng/ml), bFGF (1 ng/ml or 1/1000 dilution), and
combinations of these factors, were wounded and repair measured
after 18 hours of culture. FIG. 3 shows the average of 4
experiments +/-SEM, with repair induced by bFGF+TWEAK significantly
different from that induced by either alone or basal media (P
values<0.05).
Example 4
Effect of TWEAK Is Not Mediated by Modulation of Growth Factor
Receptors or Integrins
[0099] Integrins, especially .alpha..sub.v.beta..sub.3,
.alpha..sub.1.beta..sub.1 and .alpha..sub.2.beta..sub.1 facilitate
cell migration through extracellular matrix and also regulate cell
survival and intracellular signaling required for the response to
angiogenic factors (Eliceiri, B. and Cheresh, D. A., 1999. J. Clin.
Invest. 103:1227-1230; Senger, D. R., et al., 1997. Proc. Natl.
Acad. Sci. 94:13612-13617). Therefore, we aimed to determine
whether or not TWEAK modulated growth factor receptors or integrins
expressed on ECs. VEGF receptors Flk-1 and Flt-1 and bFGF receptor
Flg were expressed at very low levels on HUVECs cultured in basal
media. As a positive control, these receptor-specific mAbs showed
strong staining on human dermal microvascular EC (HMVEC-D).
Consistent with the study by Lynch et al (11), we found no change
in the expression of VEGF receptors Flk-1 and Flt-1 in TWEAK
treated cultures, nor were there changes in VEGF receptor
expression in cultures treated with bFGF or the TWEAK/bFGF
combination. In addition, we found that TWEAK treatment did not
alter the level of the bFGF receptor Flg or of integrins
.alpha..sub.v, .alpha..sub.1, .alpha..sub.5, .beta..sub.1, and
.beta..sub.3.
Example 5
TWEAK Induces EC Morphogenesis
[0100] A key event in the angiogenic process is the organization of
invading ECs into capillary tubes. The effect of TWEAK on this
morphogenic step was measured with EC seeded onto the surface of
three-dimensional fibrin gels in the presence or absence of bFGF.
We found no effect of TWEAK on the EC monolayer, while an optimal
concentration of bFGF promoted cell invasion and organization of EC
into cords. The addition of TWEAK to bFBF induced clear
morphological changes in the EC monolayer. Similar results were
obtained with several different EC types, including HUVECs, human
pulmonary artery ECs (HPAEC), human lung microvascular EC (HMVEC-L)
and HMVEC-D. In addition, cross-sectional analysis of these gels
revealed that the addition of TWEAK to bFBF induced the structural
organization of invading ECs into tubes with lumens. CD40L, another
TNF member, had no effect either alone or in combination with bFGF.
Thus TWEAK synergizes with bFGF to induce the morphogenesis of
capillary lumens. The results are shown in FIG. 4.
* * * * *
References