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  Pharmaceutical Patents  


Title:  TWEAK as a therapeutic target for treating central nervous system diseases associated with cerebral edema and cell death
United States Patent: 
May 10, 2011

 Winkles; Jeffrey A. (Frederick, MD), Yepes; Manuel S. (Atlanta, GA)
  University of Maryland, Baltimore (Baltimore, MD)
Appl. No.:
 November 8, 2005
PCT Filed:
 November 08, 2005
PCT No.:
371(c)(1),(2),(4) Date:
 May 07, 2007
PCT Pub. No.:
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 Invention


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 Alzheimer's disease.


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. 299:14-17 (2002)).

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.

Decoy Receptors

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 chain.

Antisense Polynucleotide

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, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil dihydrouracil beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluacil, 5-methoxyuracil, 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 WO01/29058.

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 described below.

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 epitope.

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 especially preferred.

Expression Vectors

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. 524,968.

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 GST domain.

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 TWEAK/Fn14-related polypeptide.

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 desired objective.

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.

Pharmaceutical Compositions

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 hydroxide.

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 antibody.

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