TWEAK as a therapeutic target for treating central nervous system diseases
associated with cerebral edema and cell death
United States Patent: 7,939,490
Issued: May 10, 2011
Inventors: Winkles; Jeffrey
A. (Frederick, MD), Yepes; Manuel S. (Atlanta, GA)
Assignee: University of
Maryland, Baltimore (Baltimore, MD)
Appl. No.: 11/718,786
Filed: November 8, 2005
PCT Filed: November 08,
PCT No.: PCT/US2005/040360
371(c)(1),(2),(4) Date: May
PCT Pub. No.: WO2006/052926
PCT Pub. Date: May 18, 2006
Pharm Bus Intell
& Healthcare Studies
The present invention is directed to
compositions and methods for treating cerebral edema and cell death in
neurological conditions characterized by disruption of the architecture of
the neurovascular unit with increase in the permeability of the NVU,
particularly for treating stroke. One aspect of the present invention
relates to a composition comprising an agent that interferes with a
TWEAK-mediated signaling pathway. Another aspect of the present invention
relates to a method of using a composition which comprises an agent that
inhibits Fn14 activity or Fn14 expression for the prevention and/or
treatment of cerebral edema and cell death occurring in certain
neurological conditions such as cerebral ischemia.
Description of the
One aspect of the present invention relates to a method for treating a
condition associated with an increase in blood brain barrier (BBB)
permeability, the method comprising administering to a subject in need
thereof an effective amount of an agent that either disrupts interaction
between a TWEAK protein and a Fn14 receptor, or interferes a Fn14 signal
transduction pathway, Fn14 expression, or TWEAK expression.
In one embodiment, the agent comprises an Fn14 decoy receptor. In another
embodiment, the agent comprises an anti-TWEAK antibody or anti-Fn14
antibody. In yet another embodiment, the agent comprises an antisense
polynucleotide or an RNAi molecule. In yet another embodiment; the method
further comprises co-administering to the subject an inhibitor of a NF-.kappa.B-regulated
biomolecule, such as MMP-9. In yet another embodiment, the method further
comprises co-administering to the subject a tissue plasminogen activator (tPA).
The methods of the present invention may be used to treat stroke and other
diseases associated with increased BBB permeability, such as, for example,
head trauma, seizures, meningitis, encephalitis, primary brain tumors,
brain metastasis, brain abscesses, hemorrhagic stroke, septic
encephalopathy, HIV-induced dementia, multiple sclerosis, and/or
DETAILED DESCRIPTION OF THE INVENTION
Applicants disclose herein a therapeutic strategy aimed at attenuating the
disruption of the BBB, and therefore, ameliorating the severity of
vasogenic edema and significantly reducing the morbidity and mortality of
patients with neurological diseases associated with increased permeability
of the NVU. The practice of the present invention will employ, unless
otherwise indicated, conventional methods of histology, virology,
microbiology, immunology, and molecular biology within the skill of the
art. Such techniques are explained fully in the literature. All
publications, patents and patent applications cited herein, whether supra
or infra, are hereby incorporated by reference in their entirety.
As disclosed herein, NF-.kappa.B signaling and NF-.kappa.B-inducible gene
products have been directly implicated in increased BBB permeability and
cell death during cerebral ischemia (Bolton, S. J. et al., Neuroscience
86:1245-1257 (1998); Matsumoto, T. et al., Lab. Invest. 77:119-125; 45-48
(1997); Asahi, M et al., J. Cereb. Blood Flow Metab. 20:1681-1689 (2000);
Asahi, M, J. Neurosci. 21:7724-7732(2001); Schneider, A., et al., Nat.
Med. 5:554-559 (1999); Xu, L. et al., Biochem. Biophys. Res. Commun.
TWEAK and Fn14 expression have been associated with ischemia in both in
vitro and in vivo models. For example, Potrovita et al. (Potrovita, I., J.
Neurosci. 24:8237-8244 (2004)) reported that TWEAK and Fn14 mRNA levels
increased when murine cortical neurons were subjected to oxygen glucose
deprivation (an in vitro model of ischemia). The same researchers also
found that TWEAK treatment of cortical neurons promoted NF-.kappa.B
pathway activation and a .about.2-fold increase in the number of cells
with apoptotic features. Gene profiling (microarray) experiments revealed
that Fn14 expression is up-regulated in two distinct in vivo models of
axonal regeneration, sciatic nerve transection (Tanabe, K., J. Neurosci.
23:9675-9686 (2003)) and optic nerve injury (Fischer, D., J. Neurosci.
24:8726-8740 (2004)). In addition, Fn14 mRNA expression is induced in the
ischemic hemisphere following MCAO in the mouse (Potrovita, I., J.
Neurosci. 24:8237-8244 (2004); Trendelenburg, G., J. Neurosci.
22:5879-5888 (2002)). It was also found that the TWEAK mRNA levels
increased slightly in response to cerebral ischemia and that
intraperitoneal administration of a neutralizing anti-TWEAK monoclonal
antibody reduced cerebral in (Potrovita, I., Supra (2004)).
Applicants have demonstrated that intracerebroventricular injection of a
soluble Fn14-Fc decoy receptor immediately after MCAO significantly
reduces the volume of the ischemic lesion and the extent of microglial
cell activation and apoptotic cell death in the ischemic penumbra.
Moreover, TWEAK alters BBB permeability by activating the NF-.kappa.B
signaling pathway, which results in upregulation of MMP-9 (FIG. 21 (see Original Patent)).
These results indicate that the TWEAK-Fn14 signaling system contributes to
cerebral ischemia-mediated brain damage in the mouse.
One aspect of the present invention relates to a method for treating
neurological diseases associated with increase in the permeability of the
BBB using an agent that interferes with TWEAK-Fn14 slanting. In one
embodiment, the method comprises administering to a subject in need
thereof an effective amount of an agent that inhibits Fn14 activity or
Fn14 expression. In a preferred embodiment, the agent comprises an Fn14-Fc
decoy receptor. In another embodiment, the agent comprises a neutralizing
antibody to TWEAK or Fn14. In another embodiment, the agent comprises an
antisense polynucleotide or an RNAi that inhibits TWEAK or Fn14
expression. In another embodiment the method comprises administering to a
subject in need thereof an effective amount of an agent that inhibits Fn14
signal transduction. In yet another embodiment, the method further
comprises co-administering to the subject an inhibitor of NF-.kappa.B-regulated
biomolecule. In yet another embodiment, the method fiber comprises
co-administering to the subject an effective amount of tPA. As used
herein, a biomolecule is a naturally-occurring or synthetic molecule
having bioactivity in a subject, such as a protein, a peptide, a
saccharide, a polysaccharide, a nucleotide, a polynucleotide and a lipid.
As used herein, the term "inhibit" refers to a substantial reduction of
bioactivity or level of expression. For example, an agent inhibits the
activity or expression of a biomolecule if the activity or expression of
the biomolecule is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%
or 100% in the presence of the agent.
As is known in the art, a "decoy receptor" is a mutated or modified
receptor that is capable of binding to an agonist or antagonist as the
wild-type receptor, but lacks the ability to perform certain biological
functions. Decoy receptors have been successfully used to inhibit the
activity of various proteins. For example, a TNF receptor-Fc decoy that
functions as a TNF-.alpha. antagonist is used for treatment of patients
with rheumatoid arthritis (Olsen, N. et al., N. Eng. J. Med. 350:2167-2179
(2004)). In one embodiment the decoy Fn14 receptor of the present
invention comprises the extracellular, ligand-binding domain of Fn14. In a
specific embodiment, the Fn-14 decoy receptor of the present invention
comprises a fusion protein comprising the extracellular, lid-binding
domain of Fn14 fused to the Fc portion and hinge region of the IgG1 heavy
As is known in the art an "antisense polynucleotide" comprises a
nucleotide sequence, which is complementary to a "sense polynucleotide"
encoding a protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
Accordingly, an antisense polynucleotide can hydrogen bond to a sense
polynucleotide. The antisense polynucleotide can be complementary to an
entire coding strand of a gene of the invention or to only a portion
thereof. In one embodiment, an antisense polynucleotide molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence of the invention. The term "coding region" includes the region of
the nucleotide sequence comprising codons, which are translated into amino
acid. In another embodiment the antisense polynucleotide molecule is
antisense to a "noncoding region" of the coding strand of a nucleotide
sequence of the invention.
Antisense polynucleotides of the present invention can be designed
according to the rules of Watson and Crick base pairing. The antisense
polynucleotide molecule can be complementary to the entire coding region
of an mRNA corresponding to a gene of the invention, but more preferably
is an oligonucleotide, which is antisense to only a portion of the coding
or noncoding region. An antisense oligonucleotide can be, for example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense polynucleotide of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense polynucleotide (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally occurring
nucleotides or variously modified nucleotides designed to increase the
biological stability of the molecules or to increase the physical
stability of the duplex formed between the antisense and sense
polynucleotides, e.g., phosphorothioate derivatives and acridine
substituted nucleotides can be used. Examples of modified nucleotides
which can be used to generate the antisense polynucleotide include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xantine, 4-acetylcytosine, 5-(carboxyhydroxyhnehyl) uracil,
dihydrouracil beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,
2-methylthio-N6-isopentenyladen4exine, unacil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thio-aracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense polynucleotide can be
produced biologically using an expression Vector into which a
polynucleotide has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted polynucleotide will be of an antisense
orientation to a target polynucleotide of interest, described further in
the following subsection).
The antisense polynucleotide molecules of the present invention are
typically administered to a subject or generated in situ such that they
hybridize with or bind to cellular mRNA and/or genomic DNA encoding the
TWEAK and/or Fn14 protein to thereby inhibit expression of the protein,
e.g., by inhibiting transcription and/or translation. The hybridization
can be by conventional nucleotide complementarity to form a stable duplex,
or, for example, in the cases of an antisense polynucleotide molecule
which binds to DNA duplexes, through specific interactions in the major
groove of the double helix. An example of a route of administration of
antisense polynucleotide molecules of the invention is direct injection at
a tissue site (e.g., intestine or blood). Alternatively, antisense
polynucleotide molecules can be modified to target selected cells and then
administrated systemically. The antisense polynucleotide molecules can
also be delivered to cells using the vectors described herein. To achieve
sufficient intracellular concentrations of the antisense molecules, vector
constructs in which the antisense polynucleotide molecule is placed under
the control of a strong promoter are preferred.
RNA inference ("RNA.sub.i") is a phenomenon of the introduction of
double-stranded RNA (dsRNA) into certain organisms and cell types causing
degradation of the homologous mRNA. RNAi was first discovered in the
nematode Caenorhabditis elegans, and it has since been found to operate in
a wide range of organisms. In recent years, RNAi has becomes an
endogenous, efficient and potent gene-specific silencing technique that
uses double-stranded RNAs (dsRNA) to mark a particular transcript for
degradation in vivo. RNA.sub.i technology is disclosed, for example, in
U.S. Pat. No. 5,919,619 and PCT Publication Nos. WO99/14346 and
Briefly, dsRNAs 21-25 nucleotides long, called short interfering RNAs (siRNA),
are introduced into the cell. SiRNAs may also be produced endogenously by
degradation of long sRNA molecules by an RNAse III-related nuclease called
Dicer. Once formed, the siRNAs assemble with protein components into an
RNA-induced silencing complex (RISC). An ATP-generated unwinding of the
siRNA activates the RISC, which in turn targets the homologous mRNA
transcript by Watson-Crick base-pairing and cleaves the mRNA. This
sequence specific degradation of mRNA results in gene silencing.
Antibodies suitable to the present invention include isolated polyclonal
and monoclonal antibodies. Preferably for therapeutic use in a subject,
the antibodies are humanized, as per the description of antibodies
A target antigen (e.g., TWEAK or Fn14), or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind the antigen
using standard techniques for polyclonal and monoclonal antibody
preparation. The immunogen should be an antigenic peptide comprising at
least 8 amino acid residues, and encompasses an epitope of the target
antigen such that an antibody raised against the peptide forms a specific
immune complex with the target antigen. Preferably, the antigenic peptide
comprises at least 8 amino acid residues, more preferably at least 12
amino acid residues, even more preferably at least 16 amino acid residues,
and most preferably at least 20 amino acid residues.
Immunogenic portions (epitopes) may generally be identified using
well-known techniques. Such techniques include screening polypeptides for
the ability to react with antigen-specific antibodies, antisera and/or
T-cell lines or clones. As used herein, antisera and antibodies are
"antigen-specific" if they bind to an antigen with an affinity of 10.sup.5
M.sup.-1 or greater. Such antisera and antibodies may be prepared as
described herein, and using well-known techniques. An epitope of an
antigen is a portion that reacts with such antisera and/or T-cells at a
level that is not substantially less than the reactivity of the
full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity
assay). Such epitopes may react within such assays at a level that is
similar to or greater than the reactivity of the full-length polypeptide.
Such screens may generally be performed using methods well known to those
of ordinary skill in the art. For example, a polypeptide may be
immobilized on a solid support and contacted with patient sera to allow
binding of antibodies within the sera to the immobilized polypeptide.
Unbound sera may then be removed and bound antibodies detected using, for
example, .sup.125I-labeled Protein A.
Preferred epitopes encompassed by the antigenic peptide are regions of a
target antigen that are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity.
An immunogen typically is used to prepare antibodies by immunizing a
suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the
immunogen. An appropriate immunogenic preparation can contain, for
example, recombinantly expressed antigen or a chemically synthesized
antigen. The preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or similar immunostimulatory agent
immunization of a suitable subject with an immunogenic peptide preparation
induces a polyclonal antibody response. Techniques for preparing,
isolating and using antibodies are well known in the art.
The antibodies of the present invention include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibodies with an enzyme
such as pepsin. The antibodies also include "single-chain Fv" or "scFv"
antibody fragments. The scFv fragments comprise the V.sub.H and V.sub.L
domains of antibody, wherein these domains are present in a single
polypeptide chain Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains, which enables
the scFv to form the desired structure for antigen binding.
In certain specific embodiments, the antibody of the present invention is
an Fc portion and/or hinge region of an IgG1 heavy chain.
Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which
can be made using standard recombinant DNA techniques, are within the
scope of the invention. Such chimeric and humanized monoclonal antibodies
can be produced by recombinant DNA techniques known in the art.
Humanized antibodies are particularly desirable for therapeutic treatment
of human subjects. Humanized forms of non-human (e.g. murine) antibodies
are chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal sequence
derived from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues forming a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and capacity. In
some instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies may
also comprise residues, which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general the humanized
antibody will comprise substantially au of at least one, and typically
two, variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and an or
substantially all of the constant regions being those of a human
immunoglobulin consensus sequence. The humanized antibody will preferably
also comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. In certain embodiments, the Fc
portion the IgG1 heavy chain is employed in the present invention.
Such humanized antibodies can be produced using transgenic mice which are
incapable of expressing endogenous immunoglobulin heavy and light: chain
genes, but which can express human heavy and light chain genes. The
transgenic mice are immunized in the normal fashion with a selected
antigen, e.g., all or a portion of a polypeptide corresponding to TWEAK or
Fn14. Monoclonal antibodies directed against the antigen can be obtained
using conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and somatic
mutation. Thus, using such a technique, it is possible to produce
therapeutically useful IgG, IgA and IgE antibodies.
Humanized antibodies, which recognize a selected epitope, can be generated
using a technique referred to as "guided selection." In this approach a
selected non-human monoclonal antibody, e.g., a murine antibody, is used
to guide the selection of a humanized antibody recognizing the same
In a preferred embodiment, the antibodies to a target antigen are capable
of reducing or eliminating the biological function of the target antigen,
as is described below. That is, the addition of the anti-target antigen
antibodies (either polyclonal or preferably monoclonal) to the target
antigen may reduce or eliminate the bioactivity of the target antigen.
Generally, at least a 25% decrease in activity is preferred, with at least
about 50% being particularly preferred and about a 95-100% decrease being
The expression vectors of the present invention pertains to vectors
capable of in vitro and/or in vivo expression of a polynucleotide or a
polypeptide that directly or indirectly inhibits the activity of TWEAK,
Fn14 or other components of the TWEAK/Fn14 signaling pathway. One type of
vector is a "plasmid," which includes a circular double stranded DNA loop
into which additional DNA segments can be ligated. In the present
specification, "plasmid" and "expression vector" can be used
interchangeably as the plasmid is the most commonly used form of vector.
However, the invention is intended to include such other forms of
expression vectors, such as viral vectors.
The expression vectors of the present invention comprise one or more
regulatory sequences, which is operatively linked to the polynucleotide
sequence to be expressed. The expression vectors of the invention can be
introduced into a target tissue to thereby produce proteins or peptides,
including fusion proteins or peptides, in the target tissue.
The term "regulatory sequence" refers collectively to promoter sequences,
polyadenylation signals, transcription termination sequences, upstream
regulatory domains, origins of replication, internal ribosome entry sites
("IRES"), enhancers, and the like, which collectively provide for the
replication, ascription and translation of a coding sequence in a
recipient cell. Not all of these control sequences need always be present
so long as the selected coding sequence is capable of being replicated,
transcribed and translated in an appropriate host cell.
The term "promoter" is used herein in its ordinary sense to refer to a,
DNA regulatory sequence that are sufficient for RNA polymerase
recognition, binding and transcription initiation. Additionally, a
promoter includes sequences that modulate the recognition, binding and
transcription initiation activity of RNA polymerase. Such sequences may be
cis acting or may be responsive to transacting factors. Depending upon the
nature of the regulation, promoters may be constitutive or regulated.
Examples of promoters are SP6, T4, T7, SV40 early promoter,
cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)
steroid-inducible promoter, Moloney marine leukemia virus (MMLV) promoter,
phosphoglycerate kinase (PGK) promoter, muscle creatine kinase (MCK)
promoter, myosin promoter, .alpha.-actin promoter and the like.
The term "operably linked" refers to an arrangement of elements wherein
the components so described are configured so as to perform their usual
function. Thus, control elements operably linked to a coding sequence are
capable of effecting the expression of the coding sequence. The control
elements need not be contiguous with the coding sequence, so long as the
function to direct the expression thereof. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a promoter
sequence and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
In another embodiment the expression vector is capable of directing
expression of the polynucleotide preferentially in a particular cell type
(e.g., tissue-specific regulatory elements are used to express the
polynucleotide). Tissue-specific regulatory elements are known in the art
and may include epithelial cell-specific promoters. Other non-limiting
examples of suitable tissue-specific promoters include neuron-specific
promoters (e.g., the neurofilament promoter).
The invention provides a recombinant expression vector comprising a
polynucleotide encoding a target protein cloned into the expression vector
in an antisense orientation. That is, the DNA molecule is operatively
linked to a regulatory sequence in a manner, which allows for expression
(by transcription of the DNA molecule) of an RNA molecule which is
antisense to mRNA corresponding to the target protein. Regulatory
sequences operatively linked to a polynucleotide cloned in the antisense
orientation can be chosen which direct the continuous expression of the
antisense RNA molecule in a variety of cell types, for instance viral
promoters and/or enhancers, or regulatory sequences can be chosen which
direct constitutive, tissue specific or cell type specific expression of
antisense RNA. The antisense expression vector can be in the form of a
recombinant plasmid, phagemid or attenuated virus in which antisense
polynucleotides are produced under the control of a high efficiency
regulatory region, the activity of which can be determined by the cell
type into which the vector is introduced.
The invention further provides viral vectors and, more preferably, a
retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), herpes
virus, or alphavirus vectors. The viral vector can also be an astrovirus,
coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus,
picornavirus, poxvirus, togavirus vector.
In addition to virus-mediated gene delivery, other delivery methods and
media may be employed such as, for example, nucleic acid expression
vectors, polycationic condensed DNA linked or unlinked to killed
adenovirus alone, for example see Curiel, Hum Gene Ther 3:147-154 (1992)
ligand linked DNA, for example, see Wu, J. Biol. Chem 264:16985-16987
(1989), eukaryotic cell delivery vehicles cells, for example see U.S. Ser.
No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796,
deposition of photopolymerized hydrogel materials, handheld gene transfer
particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation
as described in U.S. Pat. No. 5,206,152 and in PCT Patent Publication No.
WO 92/11033, nucleic charge neutralization or fusion with cell membranes.
Additional approaches are described in Philip, Mol. Cell. Biol.
14:2411-2418 (1994) and in Woffendin, Proc. Natl. Acad Sci. 191:581-585
(1994). Particle mediated gene transfer may be employed, for example see
U.S. provisional application Ser. No. 60/023,867. Briefly, the sequence
can be inserted into conventional vectors that contain conventional
control sequences for high level expression, and then be incubated with
synthetic gene transfer molecules such as polymeric DNA-binding cations
like polylysine, protamine, and albumin, linked to cell targeting ligands
such as asialoorosomucoid, as described in Wu, J. Biol. Chem 262:4429-4432
(1987), insulin as described in Hucked, Biochem. Pharmacol. 40:253-263
(1990), galactose as described in Plank Bioconjugate Chem 3:533-539
(1992), lactose or transferrin Naked plasmid DNA may also be employed.
Exemplary naked DNA introduction methods are described in PCT Patent
Publication No, WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency
may be improved using biodegradable latex beads. DNA coated latex beads
are efficiently transported into cells after endocytosis initiation by the
beads. The method may be improved further by treatment of the beads to
increase hydrophobicity and thereby facilitate disruption of the endosome
and release of the DNA no the cytoplasm. Liposomes that can act as gene
delivery vehicles are described in U.S. Pat. No. 5,422,120, PCT Patent
Publication Nos. WO 95/13796, WO 94/23697, and WO 91/144445, and EP No.
The invention also provides chimeric or fusion proteins. In a preferred
embodiment a fusion protein comprises at least one biologically active
portion of a component of the TWEAK/Fn14 signaling pathway (hereinafter
the "TWEAK/Fn14-related polypeptide"). Within the fusion protein, the term
"operatively linked" is intended to indicate that the TWEAK/Fn14-related
polypeptide and the non-TWEAK/Fn14-related polypeptide are fused in-frame
to each other. The non-TWEAK/Fn14-related polypeptide can be fused to the
N-terminus or C-terminus of the TWEAK/Fn14-related polypeptide.
A peptide linker sequence may be employed to separate the
TWEAK/Fn14-related polypeptide from non-TWEAK/Fn14-related polypeptide
components by a distance sufficient to ensure that each polypeptide folds
into its secondary and tertiary structures. Such a peptide linker sequence
is incorporated into the fusion protein using standard techniques well
known in the art. Suitable peptide linker sequences may be chosen based on
the following factors: (1) their ability to adopt a flexible extended
conformation; (2) their inability to adopt a secondary structure that
could interact with functional epitopes on the TWEAK/Fn14-related
polypeptide and non-TWEAK/Fn14-related polypeptide, and (3) the lack of
hydrophobic or charged residues that might react with the polypeptide
frictional epitopes. Preferred peptide linker sequences contain Gly, Asn
and Ser residues. Other near neutral amino acids, such as Thr and Ala may
also be used in the linker sequence. Amino acid sequences which may be
usefully employed as linkers include those disclosed in Maratea et al.,
Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA
83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linker
sequence may generally be from 1 to about 50 amino acids in length. Linker
sequences are not required when the TWEAK/Fn14-related polypeptide and
non-TWBAW/Fn14-related polypeptide have non-essential N-terminal amino
acid regions that can be used to separate the functional domains and
prevent steric interference.
For example, in one embodiment, the fusion protein is the extracellular,
ligand-binding domain of Fn14 fused to the Fc portion and hinge region of
the IgG1 heavy chain (termed "Fn14/IgG1 Fc fusion protein". In another
embodiment the Fn14/IgG1 Fc fusion protein her contains a heterologous
signal sequence at its N-terminus. In certain host cells (e.g., mammalian
host cells), expression and/or secretion of the fusion protein can be
increased through use of a heterologous signal sequence. Signal sequences
are typically characterized by a core of hydrophobic amino acids, which
are generally cleaved from the mature protein during secretion in one or
more cleavage events. Such signal peptides contain processing sites that
allow cleavage of the signal sequence from the mature proteins as they
pass through the secretory pathway. Thus, the invention pertains to the
described polypeptides having a signal sequence, as well as to
polypeptides from which the signal sequence has been proteolytically
cleaved (i.e., the cleavage products). In one embodiment a polynucleotide
sequence encoding a signal sequence can be operably linked in an
expression vector to a protein of interest, such as a protein, which is
ordinarily not secreted or is otherwise difficult to isolate. The signal
sequence directs secretion of the protein, such as from a eukaryotic host
into which the expression vector is transformed, and the signal sequence
is subsequently or concurrently cleaved. The protein can then be readily
purified from the extracellular medium by art-recognized methods.
Alternatively, the signal sequence can be linked to the protein of
interest using a sequence, which facilitates purification, such as with a
The TWEAK/Fn14 fusion proteins of the present invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo, as described herein. The fusion proteins can be used to
affect the bioavailability or to facilitate the purification of the
The TWEAK/Fn14 fusion proteins of the present invention can be used as
immunogens to produce antibodies. The TWEAK/Fn14-fusion proteins used as
immunogens may comprise a non-TWEAK/Fn14 immunogenic protein. Preferably
the immunogenic protein is capable of eliciting a recall response.
Preferably, a TWEAK/Fn14-chimeric or fusion protein of the invention is
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are ligated
together in-frame in accordance with conventional techniques, for example
by employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini filling of
cohesive ends as appropriate, alkaline phosphatase treatment to avoid
undesirable joining, and enzymatic ligation. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene
fragments can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments which can
subsequently be annealed and reamplified to generate a chimeric gene
sequence. Moreover, many expression vectors are commercially available,
that already encode a fusion moiety (e.g., a GST polypeptide). A
TWEAK/Fn14 polypeptide-encoding polynucleotide can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to the
TWEAK/Fn14 polypeptide-encoding polynucleotide.
Expression of proteins in prokaryotes is most often carried out in E. coli
with vectors containing constitutive or inducible promoters directing the
expression of either fusion or non-fusion proteins. Fusion vectors add a
number of amino acids to a protein encoded therein, usually to the ammo
terminus of the recombinant protein. Such fusion vectors typically serve
three purposes: 1) to increase expression of recombinant protein; 2) to
increase the solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by awing as a ligand in affinity
purification. Often, in fusion expression vectors, a proteolytic cleavage
site is introduced at the junction of the fusion moiety and the
recombinant protein to enable separation of the recombinant protein from
the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and their cognate recognition sequences, include Factor Xa,
thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmaci, a Piscataway, N.J.), pMAL (New England Biolabs, Beverly, Mass.)
and pRITS (Pharmacia, Piscataway, N.J.) which fuse glutathione S
transferase (GST), maltose B binding protein, or protein A, respectively,
to the target recombinant protein.
One strategy to maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein. Another strategy is to
alter the polynucleotide sequence of the polynucleotide to be insert into
an expression vector so that the individual codons for each amino acid are
those preferentially utilized in E. coli. Such alteration of
polynucleotide sequences of the invention can be carried out by standard
DNA synthesis techniques.
The expression vector of the present invention can be a yeast expression
vector or a baculovirus expression vector. Examples of vectors for
expression in yeast S. cerevisiae include pYepSec1, pMFa, pJRY88, pYES2
(In Vitrogen Corporation, San Diego, Calif.), and picZ (In Vitrogen Corp,
San Diego, Calif.). Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series
and the pVL series.
For purposes of this invention, an effective amount of an agent that
inhibits Fn14 activity or Fn14 expression is an amount that is sufficient
to palliate, ameliorate, stabilize, reverse, slow or delay the progression
of the ischemic-inducted BBB breakdown or brain injury. Some individuals
are refractory to these treatments, and it is understood that the methods
encompass administration to these individuals. The amount to be given will
be determined by the condition of the individual, the extent of disease,
the route of administration, how many doses will be administered, and the
Assessment of the efficacy of a particular treatment regimen may be
determined by any of the techniques known in the art, including diagnostic
methods such as imaging techniques, biopsy, and/or an evaluation of the
presence, absence or amelioration of ischemia-associated symptoms. It will
be understood that a given treatment regime may be modified, as
appropriate, to maximize efficacy.
Another aspect of the present invention relates to a composition for
preventing the development of cerebral edema in several neurological
diseases including acute cerebral ischemia. In one embodiment; the
composition comprises an agent that inhibits Fn14 activity or Fn14
expression and a pharmacologically acceptable carrier. In a preferred
embodiment, the agent comprises an Fn14-Fc decoy receptor. In another
embodiment, the agent comprises an inhibitor of TWEAK or Fn14 expression.
In another embodiment, the agent is an antibody to TWEAK or Fn14 that
prevents TWEAK-Fn14 interactions. In another embodiment, the composition
further comprises an inhibitor of NF-.kappa.B-regulated proteins such as
IL-1, IL-6, IL-8 and MMP-9. In yet another embodiment, the composition
further comprises tPA.
As used herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, solubilizers, fillers,
stabilizers, binders, absorbents, bases, buffering agents, lubricants,
controlled release vehicles, diluents, emulsifying agents, humectants,
lubricants, dispersion media, coatings, antibacterial or antifungal
agents, isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well-known in the art. See e.g., A.
H. Kibbe Handbook of Pharmaceutical Excipients, 3rd ed. Pharmaceutical
Press, London, UK (2000). Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary agents can also be
incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes
of administration include intravenous, intradermal subcutaneous, oral, and
transmucosal administration. Solutions or suspensions used for
intravenous, intradermal, or subcutaneous application can include the
following components: a sterile diluent such as water for injection,
saline solution, fixed oils, polyethylene glycols, glycerine; propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
In all cases, the injectable composition should be sterile and should be
fluid to the extent that easy syringability exists. It should be stable
under the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms such as bacteria and
fungi. The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about
by including in the composition an agent which delays absorption, for
example, aluminum monostearte and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound or agent (e.g., an inhibitor of Fn14) in the required amount in
an appropriate solvent with one or a combination of ingredients enumerated
above, as rued, followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the active compound into a sterile vehicle
which contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a powder
of the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
For administration by inhalation, the compounds are delivered in the form
of an aerosol spray from pressured container or dispenser which contains a
suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants appropriate to
the battier to be permeated are used in the formulation. Such penetrants
are generally known in the art, and include, for example, for transmucosal
administration, detergents, bile salts, and fusidic acid derivatives.
Transmucosal administration can be accomplished through the use of nasal
sprays or suppositories. For transdermal administration, the bioactive
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
In one embodiment the therapeutic moieties, which may contain a bioactive
compound, are prepared with carriers that will protect the compound
against rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will be
apparent to those skilled in the art. The materials can also be obtained
commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared according to
methods known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions
in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein includes physically discrete units suited
as unitary dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of the
active compound and the particular therapeutic effect to be achieved, and
the limitations inherent in the at of compounding such an active compound
for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects is the
therapeutic index and it can be expressed as the ratio LD50/ED50.
Compounds which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should be
taken to design a delivery system &at targets such compounds to the site
of affected tissue in order to minims potential damage to uninfected cells
and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage of
such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed
and the route of administration utilized. For any compound used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range that
includes the IC50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in cell
culture. Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured for example, by high
performance liquid chromatography.
Pharmaceutical compositions appropriate for clinical applications must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such as
bacteria and fungi.
Claim 1 of 26 Claims
1. A method for treating a condition
associated with an increase in blood brain barrier permeability selected
from the group consisting of cerebral ischemia and a stroke, said method
comprising: administering to a subject suffering from cerebral ischemia or
a stroke an effective amount of an agent that disrupts interaction between
the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) protein
and the fibroblast growth factor-inducible 14 (Fn14) receptor; wherein the
agent is an Fn14 decoy receptor, an anti-TWEAK antibody, or an anti-Fn14
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