WO2006052926A2 - Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and and cell death - Google Patents
Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and and cell death Download PDFInfo
- Publication number
- WO2006052926A2 WO2006052926A2 PCT/US2005/040360 US2005040360W WO2006052926A2 WO 2006052926 A2 WO2006052926 A2 WO 2006052926A2 US 2005040360 W US2005040360 W US 2005040360W WO 2006052926 A2 WO2006052926 A2 WO 2006052926A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tweak
- fnl4
- receptor
- bbb
- permeability
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/49—Urokinase; Tissue plasminogen activator
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the disclosure generally relates to the field of Neurology. More particularly, it relates to compositions and methods for treating neurological diseases associated with an increase in the permeability of the blood brain barrier (BBB), cerebral edema and/or cell death.
- BBB blood brain barrier
- Stroke is the second leading cause of death and a major cause of disability in the world. This clinical syndrome is characterized by rapidly developing symptoms and/or signs of focal or global loss of cerebral function with no apparent cause other than that of vascular origin.
- Blood brain barrier (BBB) breakdown and brain (cerebral) edema with subsequent increase in intracranial pressure is the major cause of death in stroke patients.
- the BBB consists of endothelial cells, pericytes, astrocytes, and the vascular basement membrane. Its main function is to form a tight barrier that regulates the entry of selected molecules from the blood into the central nervous system (CNS), and to prevent the passage of potentially harmful substances into the brain.
- CNS central nervous system
- TWEAK Tumor necrosis factor-like weak inducer of apoptosis
- TWEAK is initially synthesized as a type II transmembrane protein but can be cleaved to generate a 17-kDa soluble factor with biological activity (see Figure 1 and Chicheportiche, Y. et al., J Biol Chem. 272:32401-32410 (1997)). Soluble TWEAK induces various cellular responses when it is added to cells in culture. TWEAK activity is mediated via binding to fibroblast growth factor-inducible 14 (Fn 14), a member of the TNF receptor superfamily (see . e.g., Wiley, S. R. et al., Cytokine Growth Factor Rev. 14:241-249 (2003)).
- Fn 14 fibroblast growth factor-inducible 14
- the Fnl4 gene is expressed in a variety of cell and tissue types (supra). In addition, Fnl4 gene expression is up-regulated following growth factor stimulation of quiescent cell cultures and after injury to the blood vessel wall or the liver (Wiley, S. R. et al., Immunity 15:837-846 (2001); Feng, S. L. et al., Am. J. Pathol. 156:1253-1261(2000)). [006] It has been shown that TWEAK binding to Fn 14 activates the NF- ⁇ B
- the neurovascular unit is a dynamic structure consisting of endothelial cells, the basal lamina, the astrocytic foot processes, the pericyte and the neuron.
- NVU neurovascular unit
- NF-KB is expressed at low levels in the CNS (Kaltschmidt C. et al., MoI. Cell Biol. 14:3981-3992 (1994)).
- NF- ⁇ B activity is significantly increased in animal models of ischemic stroke and data obtained from mice deficient in the NF- ⁇ B p50 subunit indicates thatNF- ⁇ B activation enhances ischemic neuronal death (Schneider, A. et al., Nat. Med. 5:554-559 (1999)).
- NF- KB signaling and NF- KB -inducible gene products have been implicated in increased BBB permeability and cell death during cerebral ischemia (Xu, L. et al., Biochem. Biophys. Res. Commun. 299:14-17 (2002); Nadjar A. et al., J. Neurochem. 87:1024-1036 (2003)).
- TWEAK-inducible genes such as IL-8, IL-6, MCP-I, GM-
- CSF, MMP-9, ICAM-I and RANTES are known to be regulated via the NF- ⁇ B pathway (see e.g., Lynch, C. N. et al., J. Biol. Chem. 274:8455-8459 (1999); Kim, S. H. et al., Circ. J. 68:396-3991 (2004); Jin, L. et al., J. Invest. Dermatol. 122:1175- 1179 (2004); Mattson, M. P. and Carnandola, S. J. Clin. Invest. 107:247-254 (2001).
- IL-6 and MMP-9 play a central role in the development of cerebral edema and the increase of permeability of the BBB, respectively (Pantoni, L. et al., Arterioscler. Thromb. Vase. Biol. 18:503-513 (1998); Romanic, A. M. and Madri, J. A., Brain Pathol. 4: 145-156 (1994)). It has also been reported that inhibition of IL-I and IL-8 during cerebral ischemia results in a significant decrease in cerebral edema (see e.g., Rothwell, N. et al., J. Clin. Invest. 100:2648-2652 (1997); Matsumoto, T. et al., Lab. Invest. 77:119-125 (1997)).
- 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 Fn 14 receptor, or interferes a Fnl4 signal transduction pathway, Fnl4 expression, or TWEAK expression.
- the agent comprises an Fn 14 decoy receptor.
- the agent comprises an anti-TWEAK antibody or anti-Fnl4 antibody.
- the agent comprises an antisense polynucleotide or an RNAi molecule.
- the method further comprises co ⁇ administering to the subject an inhibitor of a NF- ⁇ 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).
- tPA tissue plasminogen activator
- 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.
- 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.
- FIG. 1 is a diagram showing structural features of the TWEAK and
- TWEAK is a 249-amino acid type II transmembrane glycoprotein that can be proteolytically processed by the enzyme furin into a soluble 156-amino acid monomeric cytokine.
- the TWEAK receptor, Fnl4 is a 129-amino acid type I transmembrane protein that is processed by signal peptidase into a mature 102-amino acid plasma membrane-anchored receptor.
- FIG. 2 is a schematic drawing showing the TWEAK-Fnl4 signaling system.
- Soluble TWEAK monomers associate into trimers and then bind to cell surface Fnl4 receptors. Receptor trimerization recruits TRAF adaptor proteins to the
- TRAF binding site (TBS) on the Fn 14 cytoplasmic tail, leading to signal transduction pathway activation and cellular responses.
- Figure 3 shows MMP-9 activity in rat brain following cerebral ischemia.
- Lane 1 is purified human proMMP-9 and all other lanes are brain extracts. Lanes 2, 4 and 6 are ipsilateral to the ischemic area and lanes 3, 5 and 7 are contralateral.
- Figure 4 shows immunohistochemical analysis of TWEAK and FnH expression in normal mouse brain.
- Panels A-B show TWEAK staining in pial and intracerebral blood vessels surrounded by cells with morphological features of astrocytes (arrows).
- Panels C-D show Fnl4 staining in cortical neurons and cerebellar Purkinje cells.
- Figure 5 shows FnH mRNA expression in human CNS specimens.
- RNA dot blot containing normalized loading levels of poly (A)+RNA isolated from various tissues was hybridized to an Fnl4 cDNA probe.
- Grid coordinates for tissue types are as follows: Al, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; Bl, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord.
- Figure 6 shows immunohistochemical staining of TWEAK and Fnl4 in a normal human brain.
- Panel A shows TWEAK staining in endothelial cells of a blood vessel of medium caliber.
- Panel B shows TWEAK staining in astrocytes (thin arrows) and cells with morphological features of perivascular microglia (thick arrows) surrounding a blood vessel (BV).
- Panel C shows Fnl4 staining in a cortical neuron.
- Panel D shows FnH staining in neurons (thick arrows) and cells with morphological features of perivascular microglia (thin arrows) surrounding a blood vessel (BV).
- Magnification in A is X 40, and in B 5 C 5 D is X 100.
- Figure 7 shows real-time quantitative RT-PCR analysis of TWEAK and Fn 14 mRNA expression in a mouse brain after cerebral infarction.
- Mice were subjected to MCAO and then sacrificed at the indicated time points.
- RNA was isolated from the ipsilateral hemisphere (IH) and contralateral hemisphere (CH) regions dissected from the same brain specimen and real-time RT- PCR was performed to analyze TWEAK mRNA (panel A) or Fnl4 mRNA (panel B) expression.
- TWEAK and Fnl4 mRNA expression levels were normalized to GAPDH mRNA expression, and then the IH and CH 24, 48, and 72 hour values were normalized to the corresponding 0 hour value.
- Figure 8 shows immunohistochemical analysis of TWEAK expression in a mouse brain at 48 hours after cerebral infarction.
- mice were subjected to MCAO and sacrificed 48 hours later. Tissue sections were prepared from the area surrounding the necrotic core in the ipsilateral hemisphere (panels A and C) and from a corresponding area in the healthy, non ⁇ ischemic contralateral hemisphere (panels B and D) and stained with anti-TWEAK antibodies.
- IP ischemic penumbra
- HT healthy tissue.
- Magnification is 2OX in A and B and 4OX in C and D.
- Figure 9 shows immunohistochemical analysis of Fn 14 expression in a mouse brain at 48 hours after cerebral infarction.
- mice were subjected to MCAO and sacrificed 48 hours later. Tissue sections were prepared from the area surrounding the necrotic core in the ipsilateral hemisphere (panels A and C) and from a corresponding area in the healthy, nonischemic contralateral hemisphere (panels B and D) and stained with anti-Fnl4 antibodies.
- IP ischemic penumbra
- HT healthy tissue.
- Magnification is 2OX in A and B and 4OX in C and D.
- Figure 10 shows indirect immunofluorescence analysis of TWEAK and Fnl4 expression in mouse cerebral cortex-derived cell cultures.
- Astrocyte enriched cell cultures were incubated with anti-TWEAK antibodies in combination with anti-GFAP antibodies. TWEAK staining is shown in panel A, GFAP staining is shown in panel B, and a merged image is shown in panel
- Fnl4 antibodies in combination with anti-NeuN or anti-Mac-1 antibodies are shown in panels D and G, NeuN staining is shown in panel E and Mac-1 in panel H; merged images are shown in panels F & I. Magnification is X 60 in Panels
- Figure 11 shows structural properties of the murine Fnl4-Fc fusion protein.
- a schematic depiction of the Fn 14-Fc decoy receptor is shown.
- the cysteine-rich domains are represented by loops and the bars represent cysteine residues.
- Figure 12 shows effect of the Fnl4-Fc decoy receptor on TWEAK-
- Fnl4-Fc protein purified from the conditioned medium of stably transfected human 293 T cells was incubated with either nonreducing (NR) or reducing (R) gel loading buffer and then subjected to SDS PAGE. The samples were run in duplicate. One portion of the gel was stained with Coomassie Blue (CS; Coomassie-stained) and the other portion was transferred to nitrocellulose for subsequent Western blot analysis using anti-Fnl4 antiserum (WB; Western blot).
- CS Coomassie Blue
- WB Western blot
- B) TWEAK binding to immobilized FnH-Fc protein was quantified using an ELISA.
- OPG-Fc was used as a control for non-specific binding.
- C) EC were seeded at low density, placed into growth factor-reduced medium for one day and then either left unstimulated (NA; no addition) or stimulated with 50 ng/ml TWEAK (T), 2.5 ug/ml Fnl4-Fc decoy receptor (D; decoy), or 2.5 ug/ml mouse IgG isotype control (I; IgG) alone or in combinations for three days.
- D) EC were treated as above and then either left unstimulated (NA; no addition) or stimulated with 10 ng/ml FGF-2 (F), 30 ng/ml VEGF-A (V), 2.5 ug/ml FnH-Fc decoy receptor (D), or 2.5 ug/ml mouse IgG control (I) alone or in combinations for three days.
- Figure 13 shows the effect of Fn 14-Fc or Fc administration on infarct volume.
- mice were subjected to MCAO and treated with either PBS (panels A and B) or with the FnH-Fc decoy receptor (panels C and D) and sacrificed 72 hours later.
- Tissue sections were prepared from the area surrounding the necrotic core in the ipsilateral hemisphere (panels A and C) and from a corresponding area in the healthy, nonischemic contralateral hemisphere (panels B and D) and stained with anti- Mac- 1 antibodies. Magnification is 4OX.
- Figure 15 shows the effect of Fnl4-Fc administration on ischemia- induced apoptotic cell death in the ischemic penumbra.
- mice were subjected to MCAO and treated with either PBS (panels A and B) or with the Fn 14-Fc decoy receptor (panels C and D) and sacrificed 72 hours later.
- Tissue sections were prepared from the area surrounding the necrotic core in the ipsilateral hemisphere (panels A and C) and from a corresponding area in the healthy, non-ischemic contralateral hemisphere (panels B and D) and stained for apoptotic cells using a TUNEL-based fluorescence assay.
- IP ischemic penumbra
- HT healthy tissue.
- Magnification is 2OX.
- Figure 16 shows that TWEAK induces a dose-dependent increase in
- Panels A & B Evans blue dye extravasation following the intracerebral injection of 2 ⁇ g of TWEAK (A) or PBS (B). Blue is DAPI and red is Evans blue dye. Magnification X 100.
- the asterisks show fluid-fill spaces indicative of developing edema in the TWEAK-treated brain (panels B & D).
- the arrows indicate places in the neurovascular unit with disruption of the glia limitans and detachment of the astrocytic processes.
- BV blood vessel;
- A astrocytic processes.
- Figure 18 shows that TWEAK increases MMP-9 activity in the brain.
- Lane 1 is purified murine MMP-9 and all other lanes show MMP-9 activity after injection of either TWEAK (lanes 2, 3 & 4) or PBS (lanes 5, 6 & 7). Brains were extracted at 6 (lanes 2 & 5), 12 (lanes 3 & 6), or 24 hours (lanes 4 & &) after the intracerebral injections and gelatin zymography was performed.
- Figure 19 shows the effect of TWEAK administration on NF- ⁇ B activation in the brain.
- Panels A and B Western blot analysis of I ⁇ B ⁇ phosphorylation and total I ⁇ B ⁇ expression in brain extracts 0, 1, 3 and 6 hours following the intracerebral injection of TWEAK (Panel A) or PBS (Panel B). Actin expression levels were also assayed as a control for protein loading.
- Figure 20 shows that TWEAK treatment of murine astrocytes increases
- Lane 1 is purified murine MMP-9 and all other lanes show MMP-9 activity after exposure to either TWEAK (lanes 2 & 3) or PBS (lanes 3 & 5). Lanes 2 & 3 represent MMP-9 activity in cell lysates and lanes 4 & 5 depict MMP-9 activity in the corresponding media.
- Figure 21 shows that the NF- KB signaling pathway mediates
- Panel A Gelatin zymography of brains treated with either TWEAK or
- Lane 1 is purified murine MMP-9. Lanes 2 through 5 show MMP-9 activity in wild-type mice injected with either TWEAK (lanes 2 & 3) or PBS (lanes 4 & 5). Lanes 6 through 9 show MMP-9 activity in p50 "A mice injected with either TWEAK (lanes 6 & 7) or PBS (lanes 8 & 9). Brains were harvested 24 hours after the injection of TWEAK or PBS.
- Panel B Quantification of Evans blue dye extravasation 24 hours after the intracerebral injection of TWEAK in wild-type (WT) or p50 deficient (p50 "A ) animals. Bars denote mean value (n-6) and error bars describe standard deviation of the mean. *p ⁇ 0.005 compared to wild-type mice.
- Figure 22 shows the effect of endogenous TWEAK on the permeability of the BBB and the integrity of the NVU.
- Panel A Evans blue dye extravasation 72 hours after MCAO and the intraventricular injection of either PBS, Fnl4-Fc or Fc. The blue staining denotes the areas with increased BBB permeability.
- Panel B Quantification of Evans blue dye extravasation following MCAO and treatment with either PBS, FnH-Fc or Fc. *p ⁇ 0.05 compared to PBS- or Fc-treated brains (n-4).
- Panel C Electron microscopy of cerebral capillaries of mouse brains subjected to MCAO and intraventricular administration of either PBS (top) or Fn 14-Fc decoy (bottom). The asterisks show fluid-filled spaces indicative of developing edema in the PBS-treated brain. BV blood vessel. Magnification X 5000.
- Figure 23 shows effect of TWEAK on NF- ⁇ B pathway activation in astrocytes, neurons, and microglia.
- TWEAK( ⁇ -C, G-I, M-O) or PBS (D-F, J-L,P-R) are shown.
- Red is GFAP staining in A and D, neuronal (NeuN) staining in G and J, and MAC-I staining in M and P.
- Blue is 4',6'-diamidino-2-phenylindole.
- Green is p65 staining in B, E, H, K, M, N, and Q, C, F, I, L, O, and R represent the corresponding merged images.
- Thick arrows indicate cells with nuclear translocation of the p65 subunit.
- Thin arrows indicate the cytoplasmic location of the p65 subunit of the inactive NF- KB complex.
- Figure 24 shows that Fnl4-Fc administration decreases cerebral edema and improves locomotor activity following MCAO.
- A left panel; Evans blue dye extravasation 48 hours after MCAO and treatment with either control Fc protein (black bar) or Fn 14-Fc decoy protein (white bar) immediately after the onset of ischemia.
- 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.
- NF- ⁇ B signaling and NF- ⁇ B-inducible gene products have been directly implicated in increased BBB permeability and cell death during cerebral ischemia (Bolton,SJ. 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 Fnl4 expression have been associated with ischemia in both in vitro and in vivo models.
- Potrovita et al. (Potrovita, L, J. Neurosci. 24:8237-8244 (2004)) reported that TWEAK and Fnl4 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- ⁇ B pathway activation and a ⁇ 2-fold increase in the number of cells with apoptotic features.
- Fnl4 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)).
- Fnl4 mRNA expression is induced in the ischemic hemisphere following MCAO in the mouse (Potrovita, L, 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 infarct (Potrovita, L, Supra (2004)).
- Applicants have demonstrated that intracerebroventricular injection of a soluble Fn 14-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- KB signaling pathway, which results in upregulation of MMP-9 ( Figure 21). These results indicate that the TWEAK-Fnl4 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-FnH signaling.
- the method comprises administering to a subject in need thereof an effective amount of an agent that inhibits Fnl4 activity or Fnl4 expression.
- the agent comprises an Fn 14-Fc decoy receptor.
- the agent comprises a neutralizing antibody to TWEAK or FnI 4.
- the agent comprises an antisense polynucleotide or an RNAi that inhibits TWEAK or Fnl4 expression.
- the method comprises administering to a subject in need thereof an effective amount of an agent that inhibits Fnl4 signal transduction. In yet another embodiment, the method further comprises co ⁇ administering to the subject an inhibitor of NF- KB -regulated biomolecule. In yet another embodiment, the method further comprises co-administering to the subject an effective amount of tPA.
- 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.
- the term “inhibit” refers to a substantial reduction of bioactivity or level of expression.
- 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.
- 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- ⁇ antagonist is used for treatment of patients with rheumatoid arthritis (Olsen, N. et al., N. Engl. J. Med. 350:2167-2179 (2004)).
- the decoy Fn 14 receptor of the present invention comprises the extracellular, ligand-binding domain of Fnl4.
- the Fn 14 decoy receptor of the present invention comprises a fusion protein comprising the extracellular, ligand-binding domain of Fn 14 fused to the Fc portion and hinge region of the IgGl heavy chain.
- 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.
- 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.
- the antisense polynucleotide molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of the invention.
- 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.
- an antisense polynucleotide e.g., an antisense oligonucleotide
- an antisense polynucleotide 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.
- 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- (carboxyhydroxyhnethyl) 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'-
- 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).
- 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 Fn 14 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.
- antisense polynucleotide molecules of the invention is direct injection at a tissue site (e.g., intestine or blood).
- tissue site e.g., intestine or blood
- antisense polynucleotide molecules can be modified to target selected cells and then administered systemically.
- the antisense polynucleotide molecules can also be delivered to cells using the vectors described herein.
- vector constructs in which the antisense polynucleotide molecule is placed under the control of a strong promoter are preferred.
- RNA interference is a phenomenon of the introduction of double-stranded RNA (dsRNA) into certain organisms and cell types causing degradation of the homologous mRNA.
- dsRNA double-stranded RNA
- 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.
- siRNAs are introduced into the cell. SiRNAs may also be produced endogenously by degradation of long dsRNA molecules by an RNAse Ill-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.
- RISC RNA- induced silencing complex
- 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 Fnl4
- 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.
- 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 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 5 M "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.
- 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, 125 I-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') 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 H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired structure for antigen binding.
- the antibody of the present invention is an Fc portion and/or hinge region of an IgGl heavy chain.
- 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.
- 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') 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.
- CDR complementary determining region
- 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.
- the humanized antibody will comprise substantially all 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 all 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.
- the Fc portion the IgGl 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 Fnl4.
- 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.
- 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.
- 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, Fn 14 or other components of the TWEAK/Fnl4 signaling pathway.
- a vector is a "plasmid,” which includes a circular double stranded DNA loop into which additional DNA segments can be ligated.
- plasmid and "expression vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
- 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.
- regulatory sequences 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, transcription 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.
- promoter is used herein in its ordinary sense to refer to a
- 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.
- promoters examples include SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid- inducible promoter, Moloney murine leukemia virus (MMLV) promoter, phosphoglycerate kinase (PGK) promoter, muscle creatine kinase (MCK) promoter, myosin promoter, ⁇ -actin promoter and the like.
- CMV cytomegalovirus
- MMTV mouse mammary tumor virus
- PGK phosphoglycerate kinase
- MCK muscle creatine kinase
- myosin promoter examples include myosin promoter, ⁇ -actin promoter and the like.
- operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- a fusion protein comprises at least one biologically active portion of a component of the TWEAK/Fnl4 signaling pathway (hereinafter the "TWEAK/Fnl4- related polypeptide").
- TWEAK/Fnl4-related polypeptide a component of the TWEAK/Fnl4 signaling pathway
- the term "operatively linked” is intended to indicate that the TWEAK/Fnl4-related polypeptide and the non- TWEAK/Fnl4-related polypeptide are fused in-frame to each other.
- the non- TWEAK/Fnl4-related polypeptide can be fused to the N-terminus or C-terminus of the TWEAK/Fnl4-related polypeptide.
- a peptide linker sequence may be employed to separate the
- TWEAK/Fnl4-related polypeptide from non- TWEAK/Fnl4-related polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
- 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/Fnl4-related polypeptide and non- TWEAK/Fnl4-related polypeptide; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
- Preferred peptide linker sequences contain GIy, 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. ScL USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 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/Fnl4-related polypeptide and non- TWEAK/Fnl4-related polypeptide have non-essential N- terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
- the fusion protein is the extracellular, ligand-binding domain of Fn 14 fused to the Fc portion and hinge region of the IgGl heavy chain (termed “Fnl4/IgGl Fc fusion protein").
- the Fnl4/IgGl Fc fusion protein further contains a heterologous signal sequence at its N-terminus.
- 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.
- 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 (Le., the cleavage products).
- 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.
- 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/Fnl4 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/Fnl4-related polypeptide.
- the TWEAK/Fnl4 fusion proteins of the present invention can be used as immunogens to produce antibodies.
- the TWEAK/Fnl4-fusion proteins used as immunogens may comprise a non-TWEAK/Fnl4 immunogenic protein.
- the immunogenic protein is capable of eliciting a recall response.
- a TWEAK/Fnl4-chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
- 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-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- 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.
- 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.
- many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
- a TWEAK/Fnl4 polypeptide-encoding polynucleotide can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TWEAK/Fnl4 polypeptide-encoding polynucleotide.
- Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino 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 acting as a ligand in affinity purification.
- 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.
- enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
- Typical fusion expression vectors include pGEX (Pharmaci, a Piscataway, NJ), pMAL (New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
- GST glutathione S transferase
- 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 inserted 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.
- yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl, pMFa, pJRY88, pYES2 (In Vitrogen Corporation, San Diego, CA), and picZ (In Vitrogen Corp, San Diego, CA).
- Baculovirus vectors available for expression of proteins in cultured insect cells ⁇ e.g., Sf9 cells) include the pAc series and the pVL series.
- an effective amount of an agent that inhibits Fnl4 activity or Fnl4 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.
- 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.
- the composition comprises an agent that inhibits Fnl4 activity or Fnl4 expression and a pharmacologically acceptable carrier.
- the agent comprises an Fnl4-Fc decoy receptor.
- the agent comprises an inhibitor of TWEAK or Fnl4 expression.
- the agent is an antibody to TWEAK or Fnl4 that prevents TWEAK-Fnl4 interactions.
- the composition further comprises an inhibitor of NF- KB -regulated proteins such as IL- 1, IL-6, IL-8 and MMP-9.
- the composition further comprises tPA.
- 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.
- 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.
- 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.
- 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 monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound or agent (e.g., an inhibitor of Fnl4) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- active compound or agent e.g., an inhibitor of Fnl4
- 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.
- 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.
- 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.
- a suitable propellant e.g., a, gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be by transmucosal or transdermal means.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- 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.
- the bioactive compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
- 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.
- 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.
- Dosage unit form 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 art 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 that targets such compounds to the site of affected tissue in order to minimize 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.
- 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.
- IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
- 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.
- present invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables are incorporated herein by reference.
- EXAMPLE 1 Animal Models of Cerebral Ischemia.
- Model 1 Middle cerebral artery occlusion (MCAO) without reperfusion in mice
- the right common carotid artery (CCA), the right external carotid artery (ECA) and the right internal carotid artery (ICA) are isolated and carefully separated from the adjacent vagus nerve.
- a 6-0 suture is loosely tied at the origin of the ECA and a second suture is ligated at the distal end of the ECA.
- the CCA and the ICA are then temporarily clamped using a curved microvascular clip.
- a modified PE-50 catheter (0.15-0.18 mm diameter) containing a clot is attached to a 10 ⁇ l Hamilton syringe and introduced into the ECA lumen through a small puncture. The suture around the origin of the ECA is tightened around the intraluminal catheter to prevent bleeding, and the microvascular clip is removed.
- An 8-mm length catheter is then advanced from the ECA into the lumen of the ICA.
- the intraluminal catheter is at 1-1.5 mm from the origin of the middle cerebral artery.
- the embolus is then gently injected through the catheter followed by immediate extraction of the modified PE-50 catheter.
- This method yields a cortical and subcortical area of ischemia.
- This model of focal ischemia and reperfusion permits study of the effect of thrombolytics in acute cerebral ischemia, and is very similar to the situation observed in humans with acute embolic stroke.
- Animals are then allowed to recover and after different time points brains are fixed by transcardiac perfusion with paraformaldehyde (4%), and then, surgically removed and rapidly frozen.
- EXAMPLE 2 TWEAK and Fnl4 Expression in the Normal Central Nervous System.
- TWEAK and Fnl4 Expression in the Normal Central Nervous System.
- the expression of TWEAK and Fnl4 in normal mouse brain tissue was observed using immunohistochemical analysis.
- TWEAK immunoreactivity was detected mainly in endothelial cells of medium and small caliber blood vessels, and in cells with morphological features of astrocytes that were located mainly in the periphery of the blood vessels.
- Fnl4 immunoreactivity was detected primarily in neurons in the frontal, parietal and occipital cortex, and in Purkinje cells in the cerebellum ( Figure 4).
- RNA dot blot containing normalized loading levels of poly (A)+ RNA isolated from different anatomical regions of the normal brain was obtained from Clontech Inc. and hybridized to an Fnl4 cDNA probe.
- Our results showed Fnl4 mRNA expression in different areas of the normal human CNS mainly in the occipital lobe, caudate nucleus, temporal lobe, putamen, substantia nigra, and spinal cord ( Figure 5).
- EXAMPLE 3 Effect of Cerebral Ischemia on TWEAK and Fnl4 Gene Expression
- Real-time quantitative RT-PCR analysis revealed that TWEAK mRNA levels in both hemispheres remained relatively constant during the course of the experiment ( Figure 7A).
- a statistically significant increase was observed in Fnl4 mRNA levels in the ipsilateral, ischemic hemisphere at all three time points, with peak induction at 48 hours (2.3-fold increase over baseline) (Figure 7B).
- TWEAK and Fnl4 protein expression levels and distribution in the ipsilateral and contralateral hemispheres were compared by immunohistochemical staining of mice brains at 0, 24, 48 and 72 hours following permanent MCAO. TWEAK immunoreactivity was detected in the ipsilateral hemisphere in the region surrounding the necrotic core (i.e., the ischemic penumbra). This staining was most intense at 48 hours following MCAO and much lower in the corresponding region of the non-ischemic contralateral hemisphere ( Figure 8). An increase in TWEAK protein expression but not TWEAK mRNA expression was observed under these experimental conditions.
- TWEAK mRNA expression increased and then rapidly returned to baseline levels by 24 hours, which was the first time point that the mRNA level was examined.
- Fnl4 immunoreactivity was also detected in the ipsilateral hemisphere and the staining was most intense in the ischemic penumbra at 48 hours following MCAO and significantly lower in the corresponding region of the contralateral hemisphere ( Figure 9). Staining of the ischemic penumbra region was not detected when control rabbit IgG was used instead of anti-TWEAK or anti-Fnl4 IgG as the primary immunological reagent. Together, these results demonstrate that cerebral ischemia promotes an increase in TWEAK and Fn 14 expression in the area of the ischemic penumbra.
- gene expression levels are determined in the different cellular elements of the neurovascular unit, under both non-ischemic and ischemic conditions in vivo.
- confocal immunofluorescent microscopy is performed using sections from both non-ischemic and ischemic brains. Mice undergo MCAO and brains will be extracted and embedded in paraffin 0, 24, 48 and 72 hours later.
- Sections (5 ⁇ m cuts) are stained for TWEAK and Fn 14 and co-stained with either rabbit anti-von Willebrand factor (VWF) antibodies (marker for endothelial cells; purchased from BioDesign), rabbit anti-glial fibrillary acidic protein (GFAP) antibodies (marker for astrocytes; Dako), mouse anti-Mac- 1 monoclonal antibodies (marker for microglial cells), or mouse anti-neuronspecific nuclear protein (NeuN) monoclonal antibodies (marker for neurons; Chemicon).
- VWF rabbit anti-von Willebrand factor
- GFAP rabbit anti-glial fibrillary acidic protein
- GFAP rabbit anti-glial fibrillary acidic protein
- GFAP rabbit anti-glial fibrillary acidic protein
- mouse anti-Mac- 1 monoclonal antibodies marker for microglial cells
- NeN mouse anti-neuronspecific nuclear protein
- Goat anti-rabbit secondary antibodies conjugated to Alexa 488 (Molecular Probes) and donkey anti-mouse or anti-goat secondary antibodies conjugated to Rhodamine Red-X (Jackson ImmunoResearch) may be used as secondary antibodies.
- the cells are counterstained with DAPI (Molecular Probes).
- the ipsilateral, ischemic hemisphere (necrotic core as well as in the area of ischemic penumbra) are compared to the contralateral, non-ischemic hemisphere and to brain cuts obtained from non-ischemic brains.
- TWEAK and Fnl4 were prepared and stained for TWEAK and Fn 14 expression.
- Microglial cells were also cultured using the protocol of McCarthy and de Vellis (McCarthy,K.D. et al, J. Cell Biol 85:890-902 (1980)) and identified by the presence of Mac- 1 immunoreactivity using a monoclonal antibody kindly provided by Dr Li Zhang (University of Maryland School of Medicine). Neurons, astrocytes and microglial cells expressed both TWEAK and Fnl4.
- NF- ⁇ B pathway (Donohue, PJ. et al., Arterioscler. Thromb. Vase. Biol, 23:594-600 (2003); Kim, S.H. et al., Circ. J., 68:396-399 (2004); Han, S. et al., Biochem. Biophys. Res. Commun., 305:789-796 (2003); and Jin, L. et al., J. Invest. Dermatol, 122:1175- 1179 (2004)) and induces the expression of various NE- ⁇ B-regulated genes (e.g., IL- 6, IL-8, MMP-9, ICAM-I) (Chicheportiche, Y.
- NE- ⁇ B-regulated genes e.g., IL- 6, IL-8, MMP-9, ICAM-I
- TWEAK treatment of marine cortical neurons stimulates NF- ⁇ B activation (Potrovita, I. et al., J Neuroscl, 24:8237-8244 (2004)) but the effect on microglial cells has not been reported.
- TWEAK was able to activate the NF- ⁇ B pathway when added to murine cortex- derived microglial cells were cultured as described above and incubated with TWEAK (100 ng/ml) for 0, 30, 60 or 120 minutes.
- EXAMPLE 6 Production and Characterization of a Soluble Fn 14-Fc Decoy Receptor
- An Fnl4-Fc fusion protein was prepared, in which the extracellular, ligand-binding domain of Fnl4 is fused to the Fc portion and hinge region of the IgGl heavy chain ( Figure 11). Briefly, a plasmid pSecTag2/Fnl4-Fc was constructed and a stably-transfected human embryonic kidney (HEK) 293T clonal cell line was isolated as described (Donohue PJ, et al., 2003, Arterioscler. Thromb. Vase. Biol, 23: 594- 600).
- HEK human embryonic kidney
- the plasmid pSecTag2/Fc was digested with Sfil to release the OPG cDNA insert. The DNA ends were filled in using T4 DNA Polymerase and then the plasmid was self-ligated using T4 DNA Ligase. DNA sequence analysis was performed to confirm the identity of the construct.
- This plasmid was transfected into HEK293T cells and a stably-transfected cell line (a pooled population) was isolated by drug selection as described by Donohue PJ et e ⁇ .(supra).
- the soluble Fnl4-Fc protein was purified from conditioned medium by affinity chromatography as described by Donohue PJ et al.(supra). Briefly, stably-transfected human 293T cells secreting the Fn 14-Fc protein were cultured in growth medium containing 10% Ultra-low IgG FBS (Invitrogen) until they reached confluency and then conditioned medium was collected. Cells were removed by centrifugation and then the conditioned medium was incubated with protein A-Sepharose beads for 30 minutes at room temperature with end-over-end mixing. The beads were collected by a brief centrifugation and washed two times with PBS, once with 0.5 M NaCl in PBS, and once with PBS.
- Ultra-low IgG FBS Invitrogen
- the bound Fnl4-Fc protein was eluted with 25 mM citrate buffer (pH 2.7) and then the eluate was neutralized with 1.5 M Tris/HCl pH 8.8, dialyzed against PBS, and concentrated in a Centricon-30 (Amicon).
- Fnl4-Fc The purity of the Fnl4-Fc preparation was assessed by SDS-PAGE using 4-12% Bis-Tris NuPage gels (Invitrogen). Samples were either suspended in 2X gel loading buffer containing 10% 2-mercaptothanol (ME) and heated at 95 0 C prior to loading (reducing conditions) or resuspended in 2X gel loading buffer without 2-ME and then loaded directly into the gel lanes (non-reducing conditions). Fnl4-Fc was detected by staining the gel with 0.05% Coomassie Brilliant Blue R-250 (Kodak) and also by Western blot analysis.
- ME 2-mercaptothanol
- Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and then visualized using Ponceau S Stain (Sigma, St. Louis, MO). The membranes were blocked for 1 hour at 37 0 C in TBST (25 mmol/L Tris/HCl pH 7.5, 150 mmol/L NaCl, 0.1% Tween- 20) containing 5% nonfat dry milk and then incubated for 1 hour at room temperature in TBST containing 5% BSA and a 1:500 dilution of anti-myc monoclonal antibody 9E10 (gift of Sue Robinson, UMSOM).
- TBST 25 mmol/L Tris/HCl pH 7.5, 150 mmol/L NaCl, 0.1% Tween- 20
- TWEAK binding to the Fnl4-Fc fusion protein was demonstrated using an ELISA. Briefly, an ELISA plate was coated with either purified Fn 14-Fc protein or, as a control for non-specific binding, purified OPG-Fc protein (OPG is a natural decoy receptor for the TNF superfamily members RANKL and TRAIL) and then serial dilutions of FLAG epitope-tagged TWEAK were applied to the wells. This assay demonstrated that TWEAK specifically binds to the Fn 14-Fc protein with an affinity constant (Kd) of -1.1 nM (Figure 12B).
- OPG-Fc protein OPG is a natural decoy receptor for the TNF superfamily members RANKL and TRAIL
- TWEAK increased EC growth 1.6-fold under these experimental conditions, and this effect were significantly inhibited if the cytokine was first pre-incubated with the Fnl4-Fc soluble receptor prior to its addition to the culture medium (Figure 12C). This same concentration of Fn 14-Fc protein had no significant affect on FGF-2- or VEGF-A- stimulated EC proliferation ( Figure 12D).
- EXAMPLE 8 Effect of Fn 14-Fc Decoy Receptor Administration on Ischemia- Induced Microglial Cell Activation in the Area of Ischemic Penumbra [00136]
- Microglial cells are resident brain macrophages in the CNS that respond to pathological events. In the first 24 hours following the onset of cerebral ischemia there is an increase in microglial cells in the area of the ischemic penumbra that persists for several days (Kato, H., et al., Brain Res., 734:203-212 (1996); Lehrmann, E. et al., GUa, 24:437-448 (1998); and Zhang, Z. et al., Brain Res., 744:189-198 (1997)).
- mice were placed in a stereotaxic frame and either PBS vehicle or the soluble Fnl4-Fc decoy receptor were each administered via intraventricular injection at the same coordinates described above. Mice were sacrificed 72 hours later and immunohistochemistry was performed using an antibody that recognizes the integrin Mac-1 ( ⁇ MB2), a marker for microglial cells (Lee, Y .B. et al., J Neurosci. Res., 69:94-103(2002)). Treatment with the Fnl4-Fc decoy receptor resulted in a significant decrease in the number of resident microglial cells in the ischemic penumbra following MCAO ( Figure 14). Applicants postulate that the protective effect observed after administration of Fn 14-Fc decoy receptor may be due, at least in part, to inhibition of the effects of TWEAK on microglial cells.
- mice underwent MCAO followed by the intraventricular injection of either PBS vehicle or Fn 14-Fc decoy receptor at the same coordinates described above. Animals were sacrificed 72 hours later and immunofluorescent TUNEL staining was performed. As previously reported by others (Garcia, et al., Acta Neuropathol. (Berl), 43:85-95 (1978), Aggoun-Zouaoui, D. et al., Apoptosis, 3:133-141 (1998), and Ruan, Y. W.
- EXAMPLE 10 Effect of Fn 14-Fc Decoy Receptor Administration on the Ischemia- Induced Increase in the Permeability of the Neurovascular Unit
- the neurovascular unit is composed of endothelial cells, the vascular basement membrane and astrocytes, and is in direct contact with perivascular microglia and neurons.
- perivascular microglia and neurons During cerebral ischemia there is an increase in the permeability of the NVU with passage of potentially harmful substances from the intravascular space into the brain and resultant vasogenic edema and cell death.
- TWEAK induces IL-8 (Chicheportiche, Y. et al., J. Biol. Chem., 272:32401-32410 (1997); Lynch, CN. et al., J. Biol. Chem., 274:8455- 8459 (1999); Harada, N. et al., Biochem. Biophys. Res. Commun., 299:488-493 (2002); Nakayama, M. et al., J. Immunol., 168:734-743 (2002); Nakayama, M. et al., J.
- Applicants disclose herein a therapeutic composition and method of using same for attenuating and/or preventing the opening of the BBB in neurological diseases associated with increase in the permeability of the NVU such as cerebral ischemia.
- the present invention also contemplates that TWEAK or Fn 14 deficiency is protective in cerebral ischemia and that TWEAK inhibition by the administration of Fn 14-Fc decoy receptor during the acute phase of cerebral ischemia is a therapeutic strategy aimed at preserving the integrity of the neurovascular unit and protecting neurons from the deleterious effects of cerebral ischemia.
- Brain capillary endothelial cells are isolated by a modification of a previously described technique for bovine brains (Carson, M.P. et al., In Vitro Cell Dev. Biol., 22:344-354 (1986)) that has also been successfully adapted for mice (Chen, Z., et al., Lab. Invest., 78:353-363 (1998)).
- the cerebral cortex of mice brains is aspirated and centrifuged. The tissue is homogenized, passed through nylon meshes, suspended after overnight digestion and placed on dishes coated with 1% gelatin.
- Endothelial cells are washed, fed with fresh Endothelial Cell Growth Media (Cell Applications Inc.) and treated several times with trypsin-EDTA, which results in selective release of endothelial cells.
- Endothelial cells are maintained on culture dishes in a humidified 5% CO 2 - 95% air incubator at 37 0 C and passaged twice weekly using trypsin-EDTA.
- Experiments are performed on endothelial cells between passages 5 and 15.
- methanol-fixed frozen slides are incubated with rabbit anti-VWF antibodies. Some slides are incubated with normal rabbit IgG as a control for nonspecific staining.
- Neonatal mouse glial cells are cultured from 1- to 2-day-old mice according to previously published procedures (McCarthy, K.D. et al, J. Cell Biol, 85:890-902 (1980)). In brief, cerebral hemispheres are removed and serially sieved through meshes and the filtrate is centrifuged and re-suspended in DMEM. Cells are plated at a density of one brain/30 cm 2 and maintained in DMEM with 10% fetal bovine serum (FBS; Invitrogen) in a humidified 5% CO 2 - 95% air incubator at 37° C. At confluence, oligodendroglia arc removed by orbital shaking.
- FBS fetal bovine serum
- Glial cells are used between 20-40 days after birth. To confirm the presence of astrocytes in our system, methanol-fixed frozen slides will be incubated with rabbit anti-GFAP antibodies. A monoclonal antibody directed against the Mac-1 integrin is used to detect microglial cells in these cultures. To detect active microglia, an antibody directed against EDl (phagolysosomes) (Serotec Inc.) (Flaris, N.A. et al., GHa, 7:34-40 (1993)) is used. [00145] Primary neuronal cultures: Primary neuronal cells are from mouse cortex at embryonic day 15 as described elsewhere (Bacskai, B. J. et al., PNAS U.S.
- the brain cortices are isolated in Ca +2-free PBS and homogenized in neurobasal medium containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen).
- the cells are plated onto polylysine-coated coverslips in 12-well tissue culture plates. After 60 minutes at 37°C, the medium is removed and attached cells are incubated in neurobasal medium. To confirm the presence of neurons in our cultures, the cells are stained with antiNeuN antibody (Chemicon).
- Modified protocol is used for co-culture of cerebral endothelial cells and astrocytes (Minakawa, T. et al., Lab. Invest., 65:32-40 (1991)).
- glass slides are coated with 1% gelatin and then add 4 x 10 4 endothelial cells/chamber in 1.0 mL DMEM with 2.5% equine serum. After 24 hours of incubation the cells are washed and 0.4 mL of a solution containing 80% type I collagen is added to the sub-confluent monolayer. The slides are incubated for 10 minutes at 37°C and culture medium is added.
- astrocytes (4 x 10 4 cells/chamber), are added to the capillary preparations. It has been reported that astrocyte-endothelial cell co-cultures exhibit morphological features of the BBB seven days after co-culture initiation (Tran, N.D. et al., Stroke, 27:2304-2310 (1996)). Consequently, immunohistochemical analysis of gamma-glutamyl transpeptidase (GGTP), a putative marker of the BBB (Tran, N.D. et al., J. Cereb. Blood Flow Metab., 18:1316-1324 (1998)), is monitored. GGTP staining is evident only when endothelial cell-astrocyte co-cultures develop features of the BBB (Tran, N.D. et al., Stroke, 27:2304-2310 (1996)).
- GGTP gamma-glutamyl transpeptidase
- the slides are fixed with 80% ethanol, then incubated at 37°C for 90 minutes in a saline solution containing 0.25% DMSO, 1.2 mmol/L fast blue BB (Sigma), 0.125 mg/mL gamma-glutamyl-4-methoxy-2- napthylamide (Vega Biotechnologies), 20 mmol/L glycylglycine (Sigma), 2.5 mmol/L NaOH, and 25 mmol/L phosphate buffer. After incubation, the slides are washed in saline for 2 minutes, rinsed in 0.1 mol/L CuSO 4 for 2 minutes, washed again in saline for 2 minutes, and mounted. GGTP activity was evident as a brown stain.
- GGTP activity is also measured using a standard assay kit (Sigma).
- GGTP activity is quantified using a L-gamma-glutamyl-3-carboxy-4- nitroanilide (a colorless donor) and glycylglycine.
- GGTP catalyzes the transfer of the glutamyl group to yield 5-amino-2-nitrobenzoate 5 which absorbs light at 405 nm.
- the rate of absorbance is directly proportional to GGTP activity in the sample and absorbance is measured at 405 nm using a spectrophotometer. Units of GGTP activity are then normalized to cell number determined from parallel cultures in triplicate.
- rabbit anti-VWF factor and rabbit anti-GFAP antibodies is used to detect vascular endothelial cells and astrocytes, respectively. Experiments are carried out on day 12 of co-culture, 5 days after GGTP staining is apparent.
- TWEAK and Fnl4 gene promoters may contain HIF-I binding sites, and consequently the expression of these two genes may be regulated by hypoxia in vitro.
- Standard methods are available to determine the gene expression levels of TWEAK and Fnl4, such as, for example, real-time quantitative RT-PCR, Western blot and ELISA assays.
- cells are placed in serum-lightened medium containing either 0.2 g/L or 1 g/L glucose and incubated under normal cell culture conditions (i.e., in a humidifred 5% CO 2 incubator). After each time point the cells are harvested, culture medium is collected, and TWEAK and Fnl4 expression analysis are performed.
- RNA is isolated from additional preparations obtained at similar time points and real-time RT-PCR analysis is performed to measure TWEAK and Fnl4 mRNA levels as described below. Each sample is processed in triplicate and results are compared to normoxic controls obtained at each time point, as well as to results obtained from experiments performed in monoculture systems.
- RNA Stat- 60 Tel-Test
- Applied Biosystems TaqMan Reverse Transcription Reagents
- Each PCR reaction is performed in triplicate using an ABI Prism 7900 IHT Sequence Detector System. The reactions comprise 5 ⁇ l of each cDNA, IX TaqMan Universal PCR Master Mix, and murine TWEAK-, murine Fn 14-, or rodent GAPDH-specific primers and fluorescent labeled probes (Applied Biosystems Assay-On-Demand Products) in 100 ⁇ l total volume.
- Threshold cycle (Ct) is obtained from the PCR reaction curves and TWEAK and Fn 14 mRNA levels are quantitated using the comparative Ct method with GAPDH mRNA serving as the reference. Statistical significance of the differences between each sample and its respective control is evaluated with a Wilcoxon-two- sample rank sum test.
- Western blot analysis Western blot analysis is performed as previously described (Meighan-Mantha, R. L., et al., J. Biol. Chem., 274:33166-33176 (1999)). In brief, cells are harvested at the appropriate time points and lysed in 20 mM Tris-HCI pH 7.4, 150 mM NaCI, 1% Triton X-100, 2 mM EDTA, 1 mM DTT for 30 minutes on ice and spun at 13,000 rpm in a microcentrifuge for 5 minutes at 4° C. Protein concentrations are determined using the BCA protein assay kit (Pierce). Proteins are separated using SDS-PAGE and transferred to nitrocellulose membranes.
- ELISA TWEAK levels in the culture medium are measured by
- Biotinylated anti-TWEAK antibody is added to each well and the plates incubated at room temperature for 2 hours. After washing, bound antibody is detected with avidin peroxidase and 2,20-AZINO-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate (Sigma). Color development is measured at 405 nm with a microtiter plate reader (Bio-Rad Laboratories).
- EXAMPLE 15 Effects of TWEAK Treatment on the Cellular Components of the Neurovascular Unit and on the Integrity of the Blood Brain Barrier
- the present invention contemplates that following the onset of cerebral ischemia, TWEAK secreted from astrocytes interacts with Fn 14 on microglial cells, and that this interaction results in NF- ⁇ B pathway activation with subsequent release of inflammatory cytokines and MMP-9 and resultant increases in BBB permeability and cell death.
- TWEAK treatment of microglial cells induced activation of the NF- ⁇ B pathway ( Figure 23) and injection of TWEAK directly into the cerebral cortex of healthy, non-ischemic animals resulted in increased permeability of the BBB ( Figure 16). Furthermore, this effect was greatly enhanced in the presence of an ischemic signal. Accordingly, the following exemplary methods are contemplated for determining if exposure to TWEAK results inNF- ⁇ B pathway activation, expression of inflammatory cytokines and MMP-9, increase in BBB permeability and cell death.
- TWEAK TWEAK treatment of glial cells and neurons induce NF- ⁇ B pathway activation
- expression of inflammatory cytokines and MMP-9, and cell death in vitro monocultures of astrocytes, microglial cells, and neurons are prepared as described above.
- the monocultures are incubated with either PBS or recombinant TWEAK (0, 30, 100, 300 ng/ml; endotoxin-free; purchased from R&D Systems) in the presence of normoxic, hypoxic or hypoxic/glucose deprivation conditions.
- Cells are harvested after 0, 10, 30, 60 or 120 minutes and Western blot analysis is performed using antibodies that specifically recognize the phosphorylated form of I ⁇ Ba as described (Donohue, P. J.
- RNA isolated and real-time RT-PCR analysis for IL-I, IL-6, IL-8, and MMP-9 mRNA expression is performed using TaqMan primers and reagents purchased from Applied Biosystems.
- the conditioned media is collected from these cells and can used for gelatin zymography (to measure MMP-9 activity) and ELISA assays (to measure IL-I, IL-6 and IL-8 protein levels) using commercially available kits from R & D Systems.
- a third group of cells is fixed at 0, 24, 48 or 72 hours and fluorescent TUNEL staining is performed (Chemicon) and caspase-3 activation will be evaluated using a FITC-conjugated anti-active caspase-3 antibody (BD PharMingen). The percentage of cells that are TUNEL positive or activated caspase-3 positive is calculated.
- TWEAK treatment may induce degradation of TJ proteins ZO- I 5 ZO-
- mice cortex-derived endothelial cell cultures is prepared and incubated in the absence or presence of TWEAK under either normoxic, hypoxic or hypoxic/glucose deprivation conditions.
- TWEAK vascular endothelial
- mice cortex-derived endothelial cell cultures is prepared and incubated in the absence or presence of TWEAK under either normoxic, hypoxic or hypoxic/glucose deprivation conditions.
- TWEAK vascular endothelial
- Cells were transferred to wells of a 24-well plate with coverslips containing either collagen or 0.1% gelatin to a final concentration of 1.2 X 10 5 cells per well. After confluence they were fixed with 10% buffered formalin and then stained with rabbit polyclonal anti-ZO-I, anti ZO-2, antioccludin antibodies (all three from Zymed Laboratories) and anti-VE-cadherin (Cayman Chemical Co.) antibodies. Goat anti-rabbit IgG (Alexa 488; Molecular Probes) was used as a secondary antibody. Control experiments were performed with similar conditions but using only the secondary antibody. Strong staining was observed for each of these proteins.
- Endothelial cell cultures fixed with formalin were also successfully stained for VE-cadherin and actin.
- cultures are stained for actin (with phalloidin) and viewed with a confocal scanning laser microscope. A fluorescent intensity histogram of a representative area is recorded, and the total intensity (gray scale value times the number of pixels that have that value) will be determined.
- EXAMPLE 17 TWEAK Injection Induces NF- ⁇ B Activation, Expression of Rro- inflammatory Cytokines and MMP-9, Opening of the BBB and Cell Death In Vivo [00165] Results indicate that injection of TWEAK directly into the healthy, nonischemic cortex results in an increase in BBB permeability ( Figure 16). To further characterize this effect, a dose-response curve for TWEAK-induced increase in BBB permeability is generated. Age- and sex-matched mice undergo an intracortical injection of either PBS or TWEAK (0, 2, 4, 10 ⁇ g) followed by intravenous administration of Evans blue dye. Mice are sacrificed 24 hours later and brains are analyzed for Evans blue dye extravasation.
- TWEAK TWEAK on NF- ⁇ B activation
- MCAO non-ischemic or ischemic
- brains are extracted at the same time points and gelatin zymographic assays for MMP-9 activity are performed (Figure 3) (Yepes, M. et al. s J. Clin. Invest, 112:1533-1540 (2003)).
- I ⁇ B ⁇ phosphorylation an indicator of NF- KB pathway activation, was analyzed at 0, 1, 3 or 6 hours after the intracerebral injection of the cytokine. Brain lysates were prepared and Western blot analysis performed using appropriate antibodies. I ⁇ B ⁇ phosphorylation was found as early as one hour after treatment with TWEAK with a progressive decrease of total I ⁇ B ⁇ levels ( Figure 19a). [00167] To determine what cell types in the brain were responding to TWEAK administration, TWEAK was injected into mouse brain.
- MMP-9 activity was analyzed 24 hours after the intracerebral injection of TWEAK or PBS in wild-type and p50 deficient (p50 "A ) mice.
- An increase in MMP-9 activity was found in wild-type animals treated with TWEAK ( Figure 21a, lanes 2 & 3); in contrast, no MMP-9 activity was found in either wild-type mice treated with PBS ( Figure 21a, lanes 4 & 5) or in p50 " ⁇ animals treated with TWEAK ( Figure 21a, lanes 6 & 7) or PBS ( Figure 21a, lanes 8 & 9).
- mice were placed on a locomotor activity box with an infrared device connected to a computer which detects and quantifies the vertical and horizontal movement of each animal (number of movements and total distance for each movement). Animals were sacrificed following the last evaluation on locomotor activity (48 hours after MCAO) and Evans blue dye extravasation was quantitated as described above. The results showed that treatment with Fnl4-Fc decoy receptor resulted in significant decrease in cerebral ischemia-induced increase in BBB permeability ( Figure 24A, left panel).
- Additional experiments will be performed, involving injection of either PBS or TWEAK directly into the cerebral cortex of wild-type or Fnl4-def ⁇ cient (Fnl4 -/-) mice immediately after MCAO. Brains are extracted after 72 hours and the volume of the ischemic lesion is quantified. A different set of animals can undergo similar experimental conditions but followed by the injection of Evans blue dye. After 72 hours, animals are sacrificed and Evans blue dye extravasation in the ischemic hemisphere is quantified as previously described (87). Optionally, a third set of mice is sacrificed 72 hours after MCAO and immunostaining for TUNEL and caspase-3 activation is performed. Statistical analysis for all quantitative experiments will be performed.
- EXAMPLE 18 Genetic Deficiency of TWEAK or Fnl4 is Protective In Cerebral Ischemia
- the brain inflammatory response to cerebral ischemia is characterized by the activation and proliferation of microglial cells, increased permeability of the neurovascular unit and cell death.
- Initial Fn 14-Fc decoy experiments demonstrated that inhibition of TWEAK activity during cerebral ischemia is associated with a decrease in the volume of the ischemic lesion, attenuation of the infiltration of microglial cells into the area of ischemic penumbra, decrease in cerebral edema and inhibition of cell death.
- animals deficient in either TWEAK or Fn 14 may be used.
- TWEAK and Fnl4 knockout strains are viable, fertile and have no obvious phenotypic abnormalities.
- TWEAK -/- and Fn 14 -/- mice undergo MCAO. Brains are extracted 72 hours later and the volume of the ischemic lesion is measured. Different animal/genotype are used, such as, for example, a total of 10 animals/genotype in each group, and statistical analysis of the differences between the experimental groups is performed using the Wilcoxon-two sample sum test.
- TWEAK or Fnl4 In order to determine if genetic deficiency of TWEAK or Fnl4 is protective in cerebral ischemia-induced disruption of the BBB, two different approaches are contemplated. In one approach, Evans Blue dye extravasation is quantified following cerebral ischemia in wild-type, TWEAK -/- or Fnl4-/- mice. In the second approach, the integrity of tight junction/adherens junction/cytoskeletal proteins during cerebral ischemia is examined. In order to observe the effect of genetic deficiency of TWEAK or Fnl4 on Evans Blue dye extravasation following MCAO, wild-type, TWEAK -/- or Fn 14 -/- mice are subjected to MCAO followed by the intravenous injection of Evans Blue dye (2%).
- Brains are extracted after 6, 24, 48 and 72 hours, and Evans Blue dye extravasation is quantified as previously described (Yepes, M. et al., J Clin. Invest, 112:1533-1540 (2003)). For example, a total of 10 animals/genotype may used in each group and statistical analysis of the differences between the experimental groups will be performed using, for example, the Wilcoxon-two sample sum test.
- results are compared to those obtained in either a corresponding area in the contralateral, nonischemic hemisphere, or to the ischemic area in wild-type mice. In each case, results are given as a ratio of total microglia/active microglial cells.
- results are given as a ratio of total microglia/active microglial cells.
- TUNEL and caspase-3 positive cells are counted at the same anatomic level and the results compared to the number of stained cells in the contralateral, non-ischemic hemisphere and the ischemic area of wild-type animals. For example, a total of 5 animals/genotype may be used in each experimental group and statistical analysis of the differences observed between groups is performed with the Wilcoxon-two sample sum test.
- EXAMPLE 19 Mechanisms responsible For the Protective Effect of Fnl4 Fc Decoy Receptor and Combined Administration With tPA
- mice undergo MCAO followed by the intraventricular injection of either PBS vehicle or different doses (2 ⁇ g, 4 ⁇ g, 8 ⁇ g or 10 ⁇ g) of Fn 14-Fc decoy receptor or the control Fc protein in a total volume of 2 ⁇ l.
- the injections are performed on a stereotaxic frame at the coordinates described above (Paxinos, G et al., Academic Press Inc., San Diego, CA. 1-93 pp (2001)). Brains are extracted at 72 hours and the volume of the ischemic lesion is measured as previously described (Yepes, M. et al., Blood, 96:569-576 (2000)).
- a different group of animals undergo MCAO followed by the administration of this dose of Fnl4-Fc decoy receptor (and the control Fc protein) through either an intravenous (tail vein) or a subcutaneous (back) route.
- Animals are sacrificed at 72 hours and the volume of the ischemic lesion will be determined as described above. For example, a total of 5 or more animals may be used in each experimental group.
- mice undergo MCAO followed by the administration of PBS, FnH-Fc, or Fc as above. Animals are sacrificed at 0, 6, 24, 48 or 72 hours and RNA isolated from the non-ischemic or ischemic hemisphere are used for real-time RT-PCR experiments (IL-I, IL-6, IL-8, MMP-9). A different group of animals may be subjected to MCAO and treated as described above; brains extracted 0, 6, 24, 48 or 72 hours later and Evans Blue dye extravasation quantified as described elsewhere (Yepes, M. et al., J Clin.
- animals undergo administration of either PBS, Fn 14-Fc, or control Fc protein at a predetermined dose via predetermined delivery route found to be effective.
- tPA Transcutaneous sarcoma
- animals are treated with an intravenous injection of tPA (Genentech Inc.) at a dose of 10 mg/kg (10% bolus and the remainder in a 30 minute interval).
- DWI diffusion- weighted images
- Gd-DTPA contrast multislice Tlweighted images.
- the progression of the ischemic lesion and the area of penumbra are evaluated by the relation between cerebral blood flow and apparent diffusion coefficient values as explained previously (Meng, X. et al., Ann. Neurol., 55:207-212 (2004)).
- the volume of the ischemic lesion is measured from MRI parameters of DWI as described previously (Zhang, Z. et al., Circulation, 106:740-745 (2002)), and the leakage of the BBB is calculated from Gd-DTPA contrast MRI. Seventy-two hours later brains are extracted, and the volume of the ischemic lesion is measured as described previously (Yepes, M. et al., Blood, 96:569- 576 (2000)).
- a subset of brains are embedded in paraffin and stained with hematoxylin & eosin to observe the presence of hemorrhagic transformation in the ischemic area. These results are compared to the data obtained with Gd-DTPA enhanced images.
- the skilled artisan is aware of methods and materials to determine experimental design such that the exemplary embodiments disclosed herein do not limit the scope of the present invention.
- the administration of tPA can be 6 hours after the onset of the ischemic lesion, and an MRI will be performed 2 hours later.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002587018A CA2587018A1 (en) | 2004-11-08 | 2005-11-08 | Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and cell death |
US11/718,786 US7939490B2 (en) | 2004-12-13 | 2005-11-08 | TWEAK as a therapeutic target for treating central nervous system diseases associated with cerebral edema and cell death |
EP05848068A EP1811838A4 (en) | 2004-11-08 | 2005-11-08 | Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and and cell death |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62605404P | 2004-11-08 | 2004-11-08 | |
US60/626,054 | 2004-11-08 | ||
US63602404P | 2004-12-13 | 2004-12-13 | |
US60/636,024 | 2004-12-13 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2006052926A2 true WO2006052926A2 (en) | 2006-05-18 |
WO2006052926A3 WO2006052926A3 (en) | 2006-08-03 |
Family
ID=36337123
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/040360 WO2006052926A2 (en) | 2004-11-08 | 2005-11-08 | Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and and cell death |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1811838A4 (en) |
CA (1) | CA2587018A1 (en) |
WO (1) | WO2006052926A2 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006096487A3 (en) * | 2005-03-07 | 2007-09-13 | Genentech Inc | Methods and compositions for modulating tweak and fn14 activity |
WO2010115555A2 (en) | 2009-04-02 | 2010-10-14 | F. Hoffmann-La Roche Ag | Antibodies against human tweak and uses thereof |
US8048635B2 (en) | 2005-06-13 | 2011-11-01 | Biogen Idec Ma Inc. | Measurement of soluble Tweak levels for evaluation of lupus patients |
US8048422B2 (en) | 2005-05-27 | 2011-11-01 | Biogen Idec Ma Inc. | Tweak binding antibodies |
WO2012045671A1 (en) | 2010-10-05 | 2012-04-12 | F. Hoffmann-La Roche Ag | Antibodies against human tweak and uses thereof |
US8440189B2 (en) | 1999-01-15 | 2013-05-14 | Biogen Idec Ma Inc. | Antagonists of TWEAK and of TWEAK receptor and their use to treat immunological disorders |
WO2013150043A1 (en) | 2012-04-05 | 2013-10-10 | F. Hoffmann-La Roche Ag | Bispecific antibodies against human tweak and human il17 and uses thereof |
US8728475B2 (en) | 2005-05-10 | 2014-05-20 | Biogen Idec Ma Inc. | Methods for treating inflammatory bowel disease |
US9011859B2 (en) | 2002-04-09 | 2015-04-21 | Biogen Idec Ma Inc. | Methods for treating TWEAK-related conditions |
US9775899B2 (en) | 2005-02-17 | 2017-10-03 | Biogen Ma Inc. | Treating neurological disorders |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5550799B2 (en) * | 1999-01-15 | 2014-07-16 | バイオジェン・アイデック・エムエイ・インコーポレイテッド | TWEAK antagonists and TWEAK receptor antagonists and their use to treat immunological disorders |
ES2507069T3 (en) * | 2005-05-27 | 2014-10-14 | Biogen Idec Ma Inc. | TWEAK binding antibodies |
-
2005
- 2005-11-08 WO PCT/US2005/040360 patent/WO2006052926A2/en active Application Filing
- 2005-11-08 CA CA002587018A patent/CA2587018A1/en not_active Abandoned
- 2005-11-08 EP EP05848068A patent/EP1811838A4/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of EP1811838A4 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8440189B2 (en) | 1999-01-15 | 2013-05-14 | Biogen Idec Ma Inc. | Antagonists of TWEAK and of TWEAK receptor and their use to treat immunological disorders |
US9011859B2 (en) | 2002-04-09 | 2015-04-21 | Biogen Idec Ma Inc. | Methods for treating TWEAK-related conditions |
US9775899B2 (en) | 2005-02-17 | 2017-10-03 | Biogen Ma Inc. | Treating neurological disorders |
WO2006096487A3 (en) * | 2005-03-07 | 2007-09-13 | Genentech Inc | Methods and compositions for modulating tweak and fn14 activity |
US8728475B2 (en) | 2005-05-10 | 2014-05-20 | Biogen Idec Ma Inc. | Methods for treating inflammatory bowel disease |
US8048422B2 (en) | 2005-05-27 | 2011-11-01 | Biogen Idec Ma Inc. | Tweak binding antibodies |
US9730947B2 (en) | 2005-06-13 | 2017-08-15 | Biogen Ma Inc. | Method of treating lupus nephritis |
US8048635B2 (en) | 2005-06-13 | 2011-11-01 | Biogen Idec Ma Inc. | Measurement of soluble Tweak levels for evaluation of lupus patients |
WO2010115555A2 (en) | 2009-04-02 | 2010-10-14 | F. Hoffmann-La Roche Ag | Antibodies against human tweak and uses thereof |
EP2597104A1 (en) | 2009-04-02 | 2013-05-29 | F. Hoffmann-La Roche AG | Antibodies against human TWEAK and uses thereof |
US8852887B2 (en) | 2009-04-02 | 2014-10-07 | Hoffmann-La Roche Inc. | Antibodies against human tweak and uses thereof |
US8883976B2 (en) | 2009-04-02 | 2014-11-11 | Hoffmann-La Roche Inc. | Antibodies against human tweak and uses thereof |
WO2012045671A1 (en) | 2010-10-05 | 2012-04-12 | F. Hoffmann-La Roche Ag | Antibodies against human tweak and uses thereof |
US9187564B2 (en) | 2010-10-05 | 2015-11-17 | Hoffmann-La Roche Inc. | Antibodies against human TWEAK and uses thereof |
US9714292B2 (en) | 2012-04-05 | 2017-07-25 | Hoffmann-La Roche Inc. | Bispecific antibodies against human TWEAK and human IL17 and uses thereof |
WO2013150043A1 (en) | 2012-04-05 | 2013-10-10 | F. Hoffmann-La Roche Ag | Bispecific antibodies against human tweak and human il17 and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP1811838A4 (en) | 2009-07-22 |
WO2006052926A3 (en) | 2006-08-03 |
CA2587018A1 (en) | 2006-05-18 |
EP1811838A2 (en) | 2007-08-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7939490B2 (en) | TWEAK as a therapeutic target for treating central nervous system diseases associated with cerebral edema and cell death | |
WO2006052926A2 (en) | Tweak as a therapeutic target for treating central nervous system diseases associated with cerebral edema and and cell death | |
AU2020201633B2 (en) | Methods for inhibiting angiogenesis in a subject in need thereof | |
US8828669B2 (en) | Methods of screening for a compound that inhibits the interaction between BAFF and BCMA | |
US7718177B2 (en) | Antibodies directed to fragments of connective tissue growth factor (CTGF) polypeptide and methods and uses thereof | |
US20190002541A1 (en) | Modulating the alternative complement pathway | |
JP2019503709A (en) | Humanized anti-CD73 antibody | |
CN117503923A (en) | Methods for inhibiting fibrosis in a subject in need thereof | |
EA016193B1 (en) | Antibodies directed against amyloid-beta peptide and methods using same | |
US10421809B2 (en) | Anti-TLR4 antibodies and uses thereof | |
JP2008523048A (en) | Combination therapies targeting toll-like receptors and uses thereof | |
CA2734694A1 (en) | Methods for inhibiting ocular angiogenesis | |
US20230112035A1 (en) | ANTI-avB8 INTEGRIN ANTIBODIES FOR USE IN TREATING KIDNEY DISEASE | |
CN115335120A (en) | Method for inhibiting MASP-2 for the treatment and/or prevention of coronavirus induced acute respiratory distress syndrome | |
US20190218287A1 (en) | Trpm4 channel inhibitors for stroke treatment | |
KR20210021317A (en) | How to use CD24 for the prevention and treatment of leukemia recurrence | |
WO2007049624A1 (en) | Preventives/remedies for cancer | |
CA2406961A1 (en) | Cd40 antagonists for use in treating psoriasis and other inflammatory skin conditions | |
UA126908C2 (en) | Methods for reducing proteinuria in a human subject suffering from immunoglobulin a nephropathy | |
WO2012101125A1 (en) | Specific antibodies against human cxcl4 and uses thereof | |
US11160873B2 (en) | Anti-metalloprotease antibody for diagnosis and treatment of cancers | |
EP3102940B1 (en) | Anti-metalloprotease antibody for diagnosis and treatment of cancers | |
EP3853250A1 (en) | Ptprs and proteoglycans in rheumatoid arthritis | |
EA045598B1 (en) | METHODS FOR INHIBITION OF ANGIOGENESIS IN A PATIENT |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 11718786 Country of ref document: US Ref document number: 2587018 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005848068 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2005848068 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 11718786 Country of ref document: US |