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WO2016078672A1 - Inhibiteur du tnf-alpha pour le traitement d'un accident vasculaire cérébral - Google Patents

Inhibiteur du tnf-alpha pour le traitement d'un accident vasculaire cérébral Download PDF

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Publication number
WO2016078672A1
WO2016078672A1 PCT/DK2015/050358 DK2015050358W WO2016078672A1 WO 2016078672 A1 WO2016078672 A1 WO 2016078672A1 DK 2015050358 W DK2015050358 W DK 2015050358W WO 2016078672 A1 WO2016078672 A1 WO 2016078672A1
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tnf
mice
dominant negative
inhibitor
variant
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Kate Lykke LAMBERTSEN
Bettina Hjelm CLAUSEN
Morten MEYER
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Syddansk Universitet
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Syddansk Universitet
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    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates generally to the administration of a TNF-a inhibitor, preferably a dominant negative inhibitor of soluble TNF-a for the prevention or treatment of symptoms associated with stroke.
  • Stroke is a leading cause of death and disability and a growing problem to global healthcare.
  • over 700,000 people per year suffer a major stroke. Even more disturbing, this already troubling situation is expected to worsen as the "baby boomer" population reaches advanced age. Of those who survive a stroke, approximately 90% will have long-term impairment of movement, sensation, memory or reasoning, ranging from mild to severe.
  • the total cost to the US healthcare system is estimated to be over $50 billion per year.
  • Strokes may be caused by a rupture of a cerebral artery ("hemorrhagic stroke") or a blockage in a cerebral artery due to a thromboembolism ("ischemic stroke").
  • ischemic stroke treatment may be accomplished via pharmacological elimination of the thromboembolism and/or mechanical elimination of the thromboembolism.
  • Pharmacological elimination may be accomplished via the administration of thombolytics (e.g., streptokinase, urokinase, tissue plasminogen activator (TPA» and/or anticoagulant drugs (e.g., heparin, warfarin) designed to dissolve and prevent further growth of the thromboembolism.
  • thombolytics e.g., streptokinase, urokinase, tissue plasminogen activator (TPA» and/or anticoagulant drugs (e.g., heparin, warfarin) designed to dissolve and prevent further growth of the thromboembolism.
  • thombolytics e.g., streptokinase, urokinase, tissue plasminogen activator (TPA» and/or anticoagulant drugs (
  • principles of the present disclosure provide a method of treating stroke or symptoms associated with stroke or focal cerebral ischemia comprising administering a therapeutically effective amount of a dominant negative TNF-a inhibitor to a subject in need thereof, whereby said symptoms are improved in said subject.
  • the dominant negative inhibitor is XProl595. Phrased in another way, the present disclosure is directed to a method of treating stroke and/or symptoms associated with stroke by
  • DN-TNF polypeptide that inhibits the activity of soluble TNF- but not transmembrane TNF-a.
  • FIG. 1A shows the nucleic acid sequence of human TNF-a (SEQ ID NO: 1). An additional six histidine codons, located between the start codon and the first amino acid, are underlined.
  • FIG. IB shows the amino acid sequence of human TNF-a (SEQ ID NO: 2) with an additional 6 histidines (underlined) between the start codon and the first amino acid. Amino acids changed in exemplary TNF-a variants are shown in bold.
  • FIG. 2 shows the positions and amino acid changes in certain TNF-a variants.
  • FIG. 3 Genetic ablation of soluble TNF does not change microglial cell density and morphology or behavioral phenotype under naive conditions.
  • A,B To assess exploratory behavior, the total number of arm entries in the Y-maze (A) and spontaneous alternation (B) were measured.
  • C-E To assess spontaneous locomotor activity and anxiety-related behavior, total distance travelled (C), center/perimeter ratio (D) and number of rearings (vertical activity) (E) were measured in the open field.
  • FIG. 4 Genetic ablation of solTNF reduces infarct volumes after pMCAO with no changes in locomotor- activity and anxiety-related behaviors assessed in the open field test.
  • A Toluidine blue staining of brain sections from mTNFwt/wt and mTNFA/A 1 and 5 days after pMCAO. Ctx, cortex; IF, infarct; Str, striatum.
  • FIG. 5 Rung walk, rotarod and grip strength assessments of mTNFA/A and mTNFwt/wt mice after pMCAO.
  • Motor coordination and asymmetry by rung walk analysis was assessed 2 days after pMCAO and expressed as total number of missteps of the contralateral, right front limb compared to the unaffected left limb (paired Student's t-test) (A).
  • Motor coordination with the rotarod test was assessed 3 and 5 days after pMCAO as time spent on the rod (Student's t- test) (B).
  • FIG 8 Systemic anti-TNF therapy does not affect infarct volume after focal cerebral ischemia.
  • A Toluidine blue staining of brain sections from mice treated with either saline, XProl 595 or etanercept and allowed either 6h, 24h or 5d survival. IF, infarct; Str, striatum. Scale bar: 1 mm.
  • FIG 9 Anti-TNF therapy improves functional outcomes after focal cerebral ischemia.
  • A Neuromuscular function presented as grip strength (g), showing post-surgical weakness in both left and right front paws in saline- and XPro 1595 -treated mice 3 and 5d after pMCAO compared to baseline grip strength.
  • Etanercept-treated mice showed no loss of grip strength on the left paw but a significant reduction in grip strength 3 and 5d after pMCAO.
  • FIG. 10 Analysis of brain microglial and macrophage responses after anti-TNF therapy.
  • Gate 1 comprising leukocytes, monocytes and granolocytes.
  • Gate 2 was defined to include singlet cells using a FSC-A/FSC-H dot plot, and Gate 3 to ensure that only live cells were included in the further analysis.
  • CD1 lb+CD45dimGrl- microglia (upper left graph) after pMCAO showed that the number of microglia was significantly increased at 6h and 24h in saline-, Xprol595- and etanercept-treated mice compared to the respective unlesioned control mice.
  • the number of microglia was significantly increased in XProl 595-treated mice compared to saline-treated mice at 24h after pMCAO.
  • MFI for CD45 in microglia (lower left graph) was significantly increased at 24h in XProl 595- and etanercept-treated mice compared to saline-treated mice.
  • CD1 lb+ cells appeared to be similar in saline-, XProl 595- and etanercept-treated mice at all three time points investigated (shown for saline-treated mice only). Scale bar: 100 ⁇ .
  • cc corpus callosum, IF: infarct.
  • D Brain CD1 lb and iNOS mRNA levels, analysed by qPCR, showed that anti-TNF therapy did not affect CD1 lb or iNOS mRNA levels in the brain after pMCAO.
  • Brain IL-1 ⁇ mRNA levels were found to be significantly increased at 24h in etanercept-treated mice, compared to 6h and 5d and compared to saline- and XPro 1595 -treated mice at 24h.
  • Argl mRNA levels were found to be significantly increased in saline-treated mice at 24h compared to 6h and 5d and in XProl 595-treated mice at 24h compared to 5d.
  • FIG 11 Liver and brain TNF expression following anti-TNF therapy.
  • TNF mRNA+ cells (arrows) were located in the infarct and peri-infarct at 6h, 24h and 5d after pMCAO (shown for saline-treated mice). Scale bar: 30 ⁇ .
  • 6h TNF protein was present in all mice; however, expression was less in XProl 595- and etanercept-treated mice.
  • FIG 12 The effect of anti-TNF therapy on the number of granulocytes in the infarct.
  • A Representative photomicrographs of TB-stained brain sections from saline-, XProl 595-, and etanercept-treated mice allowed 24h survival after focal cerebral ischemia demonstrating infiltration of polymorphonucleated cells into the ischemic infarct. Colocalization of polymorphonucleated cells in TB-stained sections with a granulocyte (Grl) marker was verified using immunohistochemistry in saline-treated mice allowed 24h survival. Scale bars: left 30 ⁇ and right 10 ⁇ .
  • FIG 13 The effect of anti-TNF on the APR after focal cerebral ischemia.
  • A-D Changes in liver
  • A CXCL10, CXCL1 CCL2, IL-1 ⁇ , SAA2 and SAP mRNA levels in saline-,
  • the number of T cells were decreased in etanercept-treated mice compared to saline-treated mice.
  • the number of monocytes (CD1 lb+CD45highGrl-) was found to change significantly only in XProl 595-treated mice with at significant increase at 6h and a significant decrease at 24h compared to unlesioned XProl 595-treated mice. No change was found in saline- and etanercept-treated mice.
  • CD1 lb+CD45highGrl+ was found to increase significantly at 6h in all groups compared to unlesioned control mice and to significantly decrease at 24h in saline- and XProl 595-treated mice at 24h compared to unlesioned control mice.
  • C Flow cytometry analysis of blood samples showed that the number of T cells significantly increased at 6h in saline-treated, but not in Xprol595- and etanercept- treated, mice compared to unlesioned control mice. Furthermore, the total number of T cells was significantly increased in saline-treated mice compared to XProl595- and etanercept-treated mice at 6h.
  • FIG. 14 The effect of anti-TNF therapy on microvesicle counts and size.
  • A Estimations of the total numbers of microvesicles after focal cerebral ischemia showing altered counts with anti- TNF therapy.
  • B Estimation of the mean diameter of microvesicles after focal cerebral ischemia.
  • Figure 15 shows thermal stimulation using the Hargreave's test, showing a significant increase in latency time to withdraw paws between in saline-treated mice probably as a consequence of increased injury in the ipsilateral sensory cortex.
  • XProl 595-treated mice showed a decrease in latency time, whereas etanercept-treated mice showed no change (*P ⁇ 0.05).
  • an aspect of the invention relates to a method of treating and/or preventing symptoms associated with stroke comprising administering a therapeutically effective amount of a dominant negative TNF-a inhibitor to a subject in need thereof, whereby said symptoms are improved in said subject.
  • the said administering comprises administration of a DN-TNF polypeptide.
  • said dominant negative TNF-a inhibitor comprises a variant sequence relative to wild-type TNF-a.
  • said dominant negative TNF-a inhibitor inhibits soluble TNF-a but does not inhibit signaling by transmembrane TNF-a.
  • said variant comprises the amino acid substitutions A145R/I97T or VI M/R31 C/C69V/Y87H/C 101 /A 145R.
  • said dominant negative TNF-a inhibitor is PEGylated.
  • said dominant negative TNF-a inhibitor is XProl595.
  • said symptoms are selected from the group consisting of sensory- motor functions including numbness, weakness or paralysis in the face, arm or leg, especially on one side of the body, loss of balance and/or coordination, speech, vision and cognitive functions including impairment in learning and memory.
  • said administering comprises intraventricular injection, epidural injection, oral administration, intravenous administration and/or topical administration.
  • said symptoms are improved to a greater extent when said dominant negative TNF-a inhibitor, than when a non-selective inhibitor of TNF- a is administered.
  • said stroke is ischemic and/or hemorrhagic stroke.
  • the invention relates to a the method of treating and/or preventing stroke comprising administering a therapeutically effective amount of a dominant negative TNF-a inhibitor to a subject.
  • the invention relates to a dominant negative TNF-a inhibitor for use in the treatment and/or prevention of symptoms associated with stroke.
  • said dominant negative TNF-a inhibitor is a DN-TNF polypeptide.
  • said dominant negative TNF-a inhibitor comprises a variant sequence relative to wild-type TNF-a.
  • said dominant negative TNF-a inhibitor inhibits soluble TNF-a but does not inhibit signaling by transmembrane TNF-a.
  • said dominant negative TNF-a inhibitor inhibits soluble TNF-a but does not inhibit signaling by transmembrane TNF-a.
  • said variant comprises the amino acid substitutions Al 45R/I97T or VI M/R31 C/C69V/Y87H/C 101 /A 145R.
  • said dominant negative TNF-a inhibitor is PEGylated.
  • said dominant negative TNF-a inhibitor is XProl595.
  • said symptoms are selected from the group consisting of sensory- motor functions including numbness, weakness or paralysis in the face, arm or leg, especially on one side of the body, loss of balance and/or coordination, speech, vision and cognitive functions including impairment in learning and memory.
  • said inhibitor is administered by a route selected from the group consisting of intraventricular injection, epidural injection, oral administration, intravenous administration and/or topical administration.
  • said stroke is ischemic and/or hemorrhagic stroke.
  • the invention relates to a composition comprising a dominant negative TNF- ⁇ inhibitor according to the invention.
  • the invention relates to a dominant negative TNF-a inhibitor for use in the treatment and/or prevention of stroke.
  • said stroke is ischemic and/or hemorrhagic stroke.
  • Tumor necrosis factor is a pleiotropic cytokine important in the regulation of numerous physiological and pathological processes such as inflammation, autoimmunity, neurodegeneration, neuroprotection, demyelination and remyelination.
  • TNF Tumor necrosis factor
  • TNFRl has a death domain and signaling through this receptor has been implicated in both neuronal and oligodendrocyte death whereas signaling through TNFR2 has been implicated in neuroprotection and remyelination. It has recently been demonstrated that systemic delivery of a selective inhibitor of solTNF, XProl595, which binds solTNF forming inactive heterodimers, significantly improves functional recovery, reduces axonal damage and promotes remyelination in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.
  • EAE experimental autoimmune encephalomyelitis
  • treatment with TNF inhibitors effectively improved functional outcome in a stroke model.
  • selective soluble TNF- a inhibitors and non-selective TNF- a inhibitors both improved functional outcome in a stroke model.
  • the two classes of inhibitors provided for differential effects and functional outcomes in the stroke model. - In one embodiment, no increase in TNF producing microglia at 6 hours after (experimental) stroke ( Figure 4E, Clausen et al., in press).
  • compositions and methods for treating symptoms associated with stroke comprise administering to a patient in need thereof an inhibitor of TNF-a.
  • the methods comprise administering to a patient in need thereof an inhibitor of TNF-a that inhibits signaling of soluble TNF-a but not transmembrane TNF-a.
  • the inhibitor is a dominant negative inhibitor of soluble TNF-a.
  • the dominant negative inhibitor of TNF-a is
  • stroke is meant when the blood supply to the brain is interrupted, usually because a blood vessel bursts or is blocked by a clot. This cuts off the supply of oxygen and nutrients, causing damage to the brain tissue.
  • symptoms associated with or caused by stroke is meant sudden weakness or numbness of the face, arm or leg, most often on one side of the body.
  • Other symptoms include: confusion, difficulty speaking or understanding speech; difficulty seeing with one or both eyes; difficulty walking, dizziness, loss of balance or coordination; severe headache with no known cause; fainting or unconsciousness.
  • inhibitors of TNFa may be non-selective inhibitors, such as, but not limited to etanercept, infliximab, adalimumab and the like.
  • Preferred inhibitors of TNFa may be dominant negative TNFa proteins, referred to herein as "DNTNF-a,” “DN-TNF-a proteins,” “TNFa variants,” “TNFa variant proteins,” “variant TNF-a,” “variant TNF-a,” and the like.
  • variant TNF-a or “TNF-a proteins” is meant TNFa or TNF-a proteins that differ from the corresponding wild type protein by at least 1 amino acid.
  • a variant of human TNF-a is compared to SEQ ID NO: 1 DN-TNF- ⁇ proteins are disclosed in detail in U.S. Patent No. 7,446,174, which is incorporated herein in its entirety by reference.
  • variant TNF-a or TNF-a proteins include TNF-a monomers, dimers or trimers. Included within the definition of "variant TNF-a” are competitive inhibitor TNF-a variants. While certain variants as described herein, one of skill in the art will understand that other variants may be made while retaining the function of inhibiting soluble but not transmembrane TNF-a.
  • the proteins of the invention are antagonists of wild type TNF-a.
  • antagonists of wild type TNF-a is meant that the variant TNF-a protein inhibits or significantly decreases at least one biological activity of wild-type TNF-a.
  • the variant is antagonist of soluble TNF-a, but does not significantly antagonize transmembrane TNF-a, e.g., DN- TNF-a protein as disclosed herein inhibits signaling by soluble TNF-a, but not transmembrane TNF-a.
  • inhibits the activity of TNF-a and grammatical equivalents is meant at least a 10% reduction in wild-type, soluble TNF-a, more preferably at least a 50% reduction in wild-type, soluble TNF-a activity, and even more preferably, at least 90% reduction in wild-type, soluble TNF-a activity.
  • soluble TNF-a there is an inhibition in wild-type soluble TNF-a activity in the absence of reduced signaling by transmembrane TNF-a.
  • the activity of soluble TNF-a is inhibited while the activity of transmembrane TNF-a is substantially and preferably completely maintained.
  • the TNF proteins of the invention have modulated activity as compared to wild type proteins.
  • variant TNF-a proteins exhibit decreased biological activity (e.g. antagonism) as compared to wild type TNF-a, including but not limited to, decreased binding to a receptor (p55, p75 or both), decreased activation and/or ultimately a loss of cytotoxic activity.
  • cytotoxic activity herein refers to the ability of a TNF-a variant to selectively kill or inhibit cells.
  • Variant TNF-a proteins that exhibit less than 50% biological activity as compared to wild type are preferred.
  • variant TNF-a proteins that exhibit less than 25%, even more preferred are variant proteins that exhibit less than 15%, and most preferred are variant TNF-a proteins that exhibit less than 10% of a biological activity of wild-type TNF-a.
  • Suitable assays include, but are not limited to, caspase assays, TNF-a cytotoxicity assays, DNA binding assays, transcription assays (using reporter constructs), size exclusion chromatography assays and radiolabeling/immuno-precipitation,), and stability assays (including the use of circular dichroism (CD) assays and equilibrium studies), according to methods know in the art.
  • At least one property critical for binding affinity of the variant TNF-a proteins is altered when compared to the same property of wild type TNF-a and in particular, variant TNF-a proteins with altered receptor affinity are preferred. Particularly preferred are variants of TNF-a with altered affinity toward oligomerization to wild type TNF-a.
  • the invention provides variant TNF-a proteins with altered binding affinities such that the variant TNF-a proteins will preferentially oligomerize with wild type TNF-a, but do not substantially interact with wild type TNF receptors, i.e., p55, p75.
  • “Preferentially” in this case means that given equal amounts of variant TNF-a monomers and wild type TNF-a monomers, at least 25% of the resulting trimers are mixed trimers of variant and wild type TNF-a, with at least about 50% being preferred, and at least about 80-90% being particularly preferred.
  • the variant TNF-a proteins of the invention have greater affinity for wild type TNF-a protein as compared to wild type TNF-a proteins.
  • do not substantially interact with TNF receptors is meant that the variant TNF-a proteins will not be able to associate with either the p55 or p75 receptors to significantly activate the receptor and initiate the TNF signaling pathway(s).
  • at least a 50% decrease in receptor activation is seen, with greater than 50%, 76%, 80-90% being preferred.
  • the variants of the invention are antagonists of both soluble and transmembrane TNF-a.
  • preferred variant TNF-a proteins are antagonists of the activity of soluble TNF-a but do not substantially affect the activity of transmembrane TNF-a.
  • a reduction of activity of the heterotrimers for soluble TNF-a is as outlined above, with reductions in biological activity of at least 10%, 25, 50, 75, 80, 90, 95, 99 or 100% all being preferred.
  • some of the variants outlined herein comprise selective inhibition; that is, they inhibit soluble TNF-a activity but do not substantially inhibit transmembrane TNF-a.
  • transmembrane TNF-a activity it is preferred that at least 80%, 85, 90, 95, 98, 99 or 100% of the transmembrane TNF-a activity is maintained. This may also be expressed as a ratio; that is, selective inhibition can include a ratio of inhibition of soluble to transmembrane TNF- ⁇ . For example, variants that result in at least a 10: 1 selective inhibition of soluble to transmembrane TNF-a activity are preferred, with 50: 1, 100:1 , 200: 1, 500: 1, 1000: 1 or higher find particular use in the invention.
  • one embodiment utilizes variants, such as double mutants at positions 87/145 as outlined herein, that substantially inhibit or eliminate soluble TNF-a activity (for example by exchanging with homotrimeric wild-type to form heterotrimers that do not bind to TNF-a receptors or that bind but do not activate receptor signaling) but do not significantly affect (and preferably do not alter at all) transmembrane TNF-a activity.
  • the variants exhibiting such differential inhibition allow the decrease of inflammation without a corresponding loss in immune response, or when in the context of the appropriate cell, without a corresponding demyelination of neurons.
  • the affected biological activity of the variants is the activation of receptor signaling by wild type TNF-a proteins.
  • the variant TNF-a protein interacts with the wild type TNF-a protein such that the complex comprising the variant TNF-a and wild type TNF-a has reduced capacity to activate (as outlined above for "substantial inhibition"), and in preferred embodiments is incapable of activating, one or both of the TNF receptors, i.e. p55 TNF-R or p75 TNF-R.
  • the variant TNF-a protein is a variant TNF-a protein which functions as an antagonist of wild type TNF-a.
  • the variant TNF-a protein preferentially interacts with wild type TNF-a to form mixed trimers with the wild type protein such that receptor binding does not significantly occur and/or TNF-a signaling is not initiated.
  • mixed trimers is meant that monomers of wild type and variant TNF-a proteins interact to form heterotrimeric TNF-a.
  • Mixed trimers may comprise 1 variant TNF-a protein: 2 wild type TNF-a proteins, 2 variant TNF-a proteins: 1 wild type TNF-a protein.
  • trimers may be formed comprising only variant TNF-a proteins.
  • variant TNF-a antagonist proteins of the invention are highly specific for TNF-a antagonism relative to TNF-beta antagonism. Additional characteristics include improved stability, pharmacokinetics, and high affinity for wild type TNF-a. Variants with higher affinity toward wild type TNF-a may be generated from variants exhibiting TNF-a antagonism as outlined above. [0064]Similarly, variant TNF-a proteins, for example are experimentally tested and validated in in vivo and in in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays.
  • TNF-a activity assays such as detecting apoptosis via caspase activity can be used to screen for TNF-a variants that are antagonists of wild type TNF-a.
  • Other assays include using the Sytox green nucleic acid stain to detect TNF-induced cell permeability in an Actinomycin-D sensitized cell line. As this stain is excluded from live cells, but penetrates dying cells, this assay also can be used to detect TNF-a variants that are agonists of wild-type TNF-a.
  • agonists of "wild type TNF-a” is meant that the variant TNF-a protein enhances the activation of receptor signaling by wild type TNF-a proteins.
  • variant TNF-a proteins that function as agonists of wild type TNF-a are not preferred. However, in some embodiments, variant TNF-a proteins that function as agonists of wild type TNF-a protein are preferred.
  • An example of an NF kappaB assay is presented in Example 7 of U.S. Patent 7,446,174, which is expressly incorporated herein by reference.
  • binding affinities of variant TNF-a proteins as compared to wild type TNF-a proteins for naturally occurring TNF-a and TNF receptor proteins such as p55 and p75 are determined.
  • Suitable assays include, but are not limited to, e.g., quantitative comparisons comparing kinetic and equilibrium binding constants, as are known in the art. Examples of binding assays are described in Example 6 of U.S. Patent 7,446,174, which is expressly incorporated herein by reference.
  • the variant TNF-a protein has an amino acid sequence that differs from a wild type TNF-a sequence by at least 1 amino acid, with from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 amino acids all contemplated, or higher.
  • the variant TNF-a proteins of the invention preferably are greater than 90% identical to wild-type, with greater than 95, 97, 98 and 99% all being contemplated.
  • variant TNF-a proteins based on the human TNF-a sequence of FIG. IB (SEQ ID NO: 2), variant TNF-a proteins have at least about 1 residue that differs from the human TNF-a sequence, with at least about 2, 3, 4, 5, 6, 6 or 8 different residues.
  • Preferred variant TNF-a proteins have 3 to 8 different residues.
  • sequence in FIG IB includes an N-terminal 6 His tag.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • TNF-a proteins may be fused to, for example, to other therapeutic proteins or to other proteins such as Fc or serum albumin for therapeutic or pharmacokinetic purposes.
  • a TNF-a protein of the present invention is operably linked to a fusion partner.
  • the fusion partner may be any moiety that provides an intended therapeutic or pharmacokinetic effect. Examples of fusion partners include but are not limited to Human Serum Albumin, a therapeutic agent, a cytotoxic or cytotoxic molecule, radionucleotide, and an Fc, etc.
  • an Fc fusion is synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52- 60; Ashkenazi et al, 1997, Curr Opin Immunol 9:195-200, both incorporated by reference).
  • An Fc fusion combines the Fc region of an immunoglobulin with the target-binding region of a TNF-a protein, for example. See for example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which are incorporated by reference.
  • the variant TNF-a proteins comprise variant residues selected from the following positions 21, 23, 30, 31, 32, 33, 34, 35, 57, 65, 66, 67, 69, 75, 84, 86, 87, 91, 97, 101, 111, 112, 115, 140, 143, 144, 145, 146, and 147.
  • Preferred amino acids for each position, including the human TNF-a residues, are shown in FIG. 3.
  • preferred amino acids are Glu, Asn, Gin, Ser, Arg, and Lys; etc.
  • Preferred changes include: VIM, Q21 C, Q21 R, E23C, R31C, N34E, V91E, Q21R, N30D, R31C, R31I, R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C, L57F, L57W, L57Y, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, C69V, L75E, L75K, L75Q, A84V, S86Q, S86R, Y87H, Y87R, V91E, I97R, I97T, CI 01 A, Al 11R, Al 1 IE, Kl 12D, Kl 12E, Yl 15D, Yl
  • the invention provides TNF-a variants selected from the group consisting of XENP268 XENP344, XENP345, XENP346, XENP550, XENP551, XENP557, XENP1593, XENP1594, and XENP1595 as outlined in Example 3 OF U.S. PATENT
  • the invention provides methods of forming a TNF-a heterotrimer in vivo in a mammal comprising administering to the mammal a variant TNF-a molecule as compared to the corresponding wild-type mammalian TNF-a, wherein said TNF-a variant is substantially free of agonistic activity.
  • the invention provides methods of screening for selective inhibitors comprising contacting a candidate agent with a soluble TNF-a protein and assaying for TNF-a biological activity; contacting a candidate agent with a transmembrane TNF-a protein and assaying for TNF-a biological activity, and determining whether the agent is a selective inhibitor.
  • the agent may be a protein (including peptides and antibodies, as described herein) or small molecules.
  • the invention provides variant TNF-a proteins that interact with the wild type TNF-a to form mixed trimers incapable of activating receptor signaling.
  • variant TNF-a proteins with 1, 2, 3, 4, 5, 6 and 7 amino acid changes are used as compared to wild type TNF-a protein.
  • these changes are selected from positions 1, 21, 23, 30, 31, 32, 33, 34, 35, 57, 65, 66, 67, 69, 75, 84, 86, 87, 91, 97, 101, 111, 112, 115, 140, 143, 144, 145, 146 and 147.
  • the non-naturally occurring variant TNF- ⁇ proteins have substitutions selected from the group of substitutions consisting of V1M,Q21C, Q21R, E23C, N34E, V91E, Q21R, N30D, R31C, R311, R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C, L57F, L57W, L57Y, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, C69V, L75E, L75K, L75Q, A84V, S86Q, S86R, Y87H, Y87R, V91E, I97R, I97T, ClOl A, Al 11R, Al 1 IE
  • substitutions may be made either individually or in combination, with any combination being possible.
  • Preferred embodiments utilize at least one, and preferably more, positions in each variant TNF-a protein. For example, substitutions at positions 31, 57, 69, 75, 86, 87, 97, 101, 115, 143, 145, and 146 may be combined to form double variants. In addition triple, quadruple, quintuple and the like, point variants may be generated.
  • the invention provides TNF-a variants comprising the amino acid substitutions A145R/I97T.
  • the invention provides TNF-a variants comprising the amino acid substitutions VIM, R31C, C69V, Y87H, ClOl, and A145R.
  • this variant is PEGylated.
  • the variant is XProl 595, a PEGylated protein comprising VIM, R31C, C69V, Y87H, ClOl, and A145R mutations relative to the wild type human sequence.
  • the areas of the wild type or naturally occurring TNF-a molecule to be modified are selected from the group consisting of the Large Domain
  • the Large Domain, the Small Domain and the DE loop are the receptor interaction domains. The modifications may be made solely in one of these areas or in any combination of these areas.
  • the Large Domain preferred positions to be varied include: 21, 30, 31, 32, 33, 35, 65, 66, 67, 111, 112, 115, 140, 143, 144, 145, 146 and/or 147.
  • the preferred positions to be modified are 75 and/or 97.
  • the preferred position modifications are 84, 86, 87 and/or 91.
  • the Trimer Interface has preferred double variants including positions 34 and 91 as well as at position 57.
  • substitutions at multiple receptor interaction and/or trimerization domains may be combined. Examples include, but are not limited to, simultaneous substitution of amino acids at the large and small domains (e.g. A145R and I97T), large domain and DE loop (A145R and Y87H), and large domain and trimerization domain (A145R and L57F). Additional examples include any and all combinations, e.g., I97T and Y87H (small domain and DE loop). More specifically, theses variants may be in the form of single point variants, for example Kl 12D, Yl 15K, Yl 151, Yl 15T, A145E or A145R. These single point variants may be combined, for example, Yl 151 and A145E, or Yl 151 and A145R, or Yl 15T and A145R or Yl 151 and A145E; or any other combination.
  • A145R and I97T large domain and DE loop
  • A145R and Y87H large domain and trimerization domain
  • Preferred double point variant positions include 57, 75, 86, 87, 97, 115, 143, 145, and 146; in any combination.
  • double point variants may be generated including L57F and one of Yl 151, Y115Q, Y115T, D143K, D143R, D143E, A145E, A145R, E146K or E146R.
  • triple point variants may be generated. Preferred positions include 34, 75, 87, 91, 115, 143, 145 and 146. Examples of triple point variants include V91 E, N34E and one of Y115I, Y115T, D143K, D143R, A145R, A145E E146K, and E146R. Other triple point variants include L75E and Y87H and at least one of Y115Q, A145R, Also, L75K, Y87H and Y115Q. More preferred are the triple point variants V91E, N34E and either A145R or A145E.
  • variant TNF-a proteins may also be identified as being encoded by variant TNF-a nucleic acids.
  • nucleic acid the overall homology of the nucleic acid sequence is commensurate with amino acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence, with lower homology being preferred.
  • a variant TNF-a nucleic acid encodes a variant TNF-a protein.
  • nucleic acids may be made, all of which encode the variant TNF-a proteins of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the variant TNF-a.
  • the nucleic acid homology is determined through hybridization studies.
  • nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in FIG. IB (SEQ ID NO: 2) or its complement and encode a variant TNF- ⁇ protein is considered a variant TNF-a gene.
  • High stringency conditions are known in the art; see for example Maniatis et al, Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in
  • Tm thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.
  • nucleic acid variants encode TNF-a protein variants comprising the amino acid substitutions described herein.
  • the TNF-a variant encodes a polypeptide variant comprising the amino acid substitutions A145R/I97T.
  • nucleic acid variant encodes a polypeptide comprising the amino acid substitutions VIM, R31C, C69V, Y87H, ClOl, and A145R, or any 1, 2, 3, 4 or 5 of these variant amino acids.
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides.
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids.
  • Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half-life of such molecules in physiological environments.
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
  • FIG. 1A SEQ ID NO: 1
  • recombinant nucleic acid nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature.
  • an isolated variant TNF-a nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention.
  • JBy "vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild-type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a variant TNF-a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • all of the variant TNF-a proteins outlined herein are in a form not normally found in nature, as they contain amino acid substitutions, insertions and deletions, with substitutions being preferred, as discussed below.
  • variant TNF-a proteins of the present invention are amino acid sequence variants of the variant TNF-a sequences outlined herein and shown in the Figures. That is, the variant TNF-a proteins may contain additional variable positions as compared to human TNF-a. These variants fall into one or more of three classes: substitutional, insertional or deletional variants.
  • amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the variant TNF-a protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • a replacement of the naturally occurring secretory leader sequence is desired.
  • an unrelated secretory leader sequence is operably linked to a variant TNF-a encoding nucleic acid leading to increased protein secretion.
  • any secretory leader sequence resulting in enhanced secretion of the variant TNF-a protein when compared to the secretion of TNF-a and its secretory sequence, is desired.
  • Suitable secretory leader sequences that lead to the secretion of a protein are known in the art.
  • a secretory leader sequence of a naturally occurring protein or a protein is removed by techniques known in the art and subsequent expression results in intracellular accumulation of the recombinant protein.
  • operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the fusion protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the fusion protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element.
  • the expression vector may comprise additional elements.
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT US97/01019 and PCT US97/01048, both of which are hereby incorporated by reference.
  • the expression vector comprises the components described above and a gene encoding a variant TNF-a protein. As will be appreciated by those in the art, all combinations are possible and accordingly, as used herein, the combination of components, comprised by one or more vectors, which may be retroviral or not, is referred to herein as a "vector composition".
  • retroviral systems are known and generally employ packaging lines which have an integrated defective provirus (the "helper") that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence.
  • helper an integrated defective provirus
  • the cell line produces empty viral shells.
  • Producer lines can be derived from the packaging lines which, in addition to the helper, contain a viral vector which includes sequences required in cis for replication and packaging of the virus, known as the long terminal repeats (LTRs).
  • LTRs long terminal repeats
  • retroviral vectors include but are not limited to vectors such as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740, incorporated herein by reference in its entirety, as well as derivatives of these vectors.
  • Retroviral vectors can be constructed using techniques well known in the art. See, e.g., U.S. Pat. No. 5,219,740; Mann et al. (1983) Cell 33: 153-159.
  • Adenovirus based systems have been developed for gene delivery and are suitable for delivery according to the methods described herein.
  • Human adenoviruses are double-stranded DNA viruses, which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro.
  • adenoviruses infect quiescent as well as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis. The virus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses cause only low level morbidity and are not associated with human malignancies. Accordingly, adenovirus vectors have been developed which make use of these advantages. For a description of adenovirus vectors and their uses see, e.g., Haj-Ahmad and Graham (1986) J. Virol.
  • the viral vectors used in the subject methods are AAV vectors.
  • AAV vector is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • Typical AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
  • An AAV vector includes at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • AAV serotypes see for example Cearley et al, Molecular Therapy, 16: 1710-1718, 2008, which is expressly incorporated herein in its entirety by reference.
  • AAV expression vectors may be constructed using known techniques to provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region.
  • the control elements are selected to be functional in a thalamic and/or cortical neuron.
  • AAV ITRs adeno-associated virus inverted terminal repeats
  • AAV ITRs the art- recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus.
  • AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
  • the nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M.
  • an "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
  • the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.
  • Suitable DNA molecules for use in AAV vectors will include, for example, a gene that encodes a protein that is defective or missing from a recipient subject or a gene that encodes a protein having a desired biological or therapeutic effect (e.g., an enzyme, or a neurotrophic factor).
  • a desired biological or therapeutic effect e.g., an enzyme, or a neurotrophic factor.
  • the artisan of reasonable skill will be able to determine which factor is appropriate based on the neurological disorder being treated.
  • the selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
  • control elements can comprise control sequences normally associated with the selected gene.
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the S V40 early promoter, mouse mammary tumor virus LTR promoter;
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • the TNF-a protein may be covalently modified.
  • a preferred type of covalent modification of variant TNF-a comprises linking the variant TNF-a
  • polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, incorporated by reference.
  • PEG polyethylene glycol
  • These nonproteinaceous polymers may also be used to enhance the variant TNF-a's ability to disrupt receptor binding, and/or in vivo stability.
  • cysteines are designed into variant or wild type TNF-a in order to incorporate (a) labeling sites for characterization and (b) incorporate PEGylation sites.
  • labels that may be used are well known in the art and include but are not limited to biotin, tag and fluorescent labels (e.g. fluorescein). These labels may be used in various assays as are also well known in the art to achieve characterization.
  • a variety of coupling chemistries may be used to achieve PEGylation, as is well known in the art. Examples include but are not limited to, the technologies of
  • Shearwater and Enzon which allow modification at primary amines, including but not limited to, lysine groups and the N-terminus. See, Kinstler et al, Advanced Drug Deliveries Reviews, 54, 477-485 (2002) and M J Roberts et al, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both hereby incorporated by reference.
  • the optimal chemical modification sites are 21, 23, 31 and 45, taken alone or in any combination.
  • a TNF-a variant of the present invention includes the R31 C mutation.
  • the variant TNF-a protein is purified or isolated after expression.
  • Variant TNF-a proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • treatment include amelioration or elimination of a disease or condition once it has been established or alleviation of the characteristic symptoms of such disease or condition.
  • a method as disclosed herein may also be used to, depending on the condition of the patient, prevent the onset of a disease or condition or of symptoms associated with a disease or condition, including reducing the severity of a disease or condition or symptoms associated therewith prior to affliction with said disease or condition.
  • prevention or reduction prior to affliction refers to administration of the compound or composition of the invention to a patient that is not at the time of administration afflicted with the disease or condition.
  • Treatments that currently are available for stroke are limited and include an acute ischemic stroke is treated in a hospital with intravenous/intraarterial thrombolysis in 4.5-6 hours or by thrombectomy, but the effect of such invasive approaches is limited, major adverse events including death may follow, and there is a controversy regarding the effectivity of intraarterial thrombolysis and thrombectomy.
  • OJPrevention of stroke in case of risk factors may involve the administration of antiplatelet drugs, such as aspirin and dipyridamole to interfere with platelet aggregation on existing vascular plaques and reduce the risk of local thrombosis and embolisation. This may be combined with controlling other risk factors e.g. reduction of high blood pressure, the use of statins to decrease serum cholesterol, or treatment of diabetes.
  • antiplatelet drugs such as aspirin and dipyridamole to interfere with platelet aggregation on existing vascular plaques and reduce the risk of local thrombosis and embolisation. This may be combined with controlling other risk factors e.g. reduction of high blood pressure, the use of statins to decrease serum cholesterol, or treatment of diabetes.
  • thrombolytic agents are selected from the group consisting of tissue plasminogen activators includingreteplase, and tenecteplase, anistreplase, streptokinase, and urokinase), fibrinolytic agents, anti-thrombotic agents, anti-platelet aggregation agents (anti-platelet aggregation agents are selected from the group consisting of Irreversible cyclooxygenase inhibitors including Aspirin and Triflusal, Adenosine diphosphate (ADP) receptor inhibitors including Clopidogrel, Prasugrel, Ticagrelor, Ticlopidine, Phosphodiesterase inhibitors including Cilostazol, Protease-activated receptor- 1 (PAR-1) antagonists including Vorapaxar, Glycoprotein IIB/IIIA inhibitors including Abciximab, Eptifibatide, Tirofiban, Adenosine
  • Rosiglitazone anti-dyslipidemia agents, cholesterol reducing agents, statins (statins are selected from the group consisting of atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, bezafibrat, and gemfibrozil).
  • statins are selected from the group consisting of atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, bezafibrat, and gemfibrozil.
  • Hemorrhagic stroke may damage brain tissue due to bleeding into the brain parenchyma or the subarachnoid/subdural space.
  • DN-TNFs that inhibit soluble but not transmembrane TNF-a find use in treating stroke or symptoms associated with stroke. These molecules find particular use when combined with currently available stroke therapies as known in the art and as described herein. For instance, DN-TNFs, such as XProl 595 may be combined in a therapeutic regimen with other molecules. DN-TNFs as described herein may also be used following surgery to alleviate symptoms or treat stroke.
  • treatment of the DN-TNF in a therapeutic regimen in combination with the co-therapies as described herein results in synergistic efficacy as compared to either of the treatments alone.
  • synergistic is meant that efficacy is more than the result of additive efficacy of the two treatments alone.
  • treatment of the DN-TNF in a therapeutic regimen includes the combination of steroidal anti-inflammatory molecules, such as but not limited to dexamethasone and the like or non-steroidal anti-inflammatory molecules.
  • the pharmaceutical composition may be formulated in a variety of ways.
  • concentration of the therapeutically active variant TNF-a protein in the formulation may vary from about 0.1 to 100 weight %.
  • the concentration of the variant TNF-a protein is in the range of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram of body weight being preferred.
  • compositions of the present invention comprise a variant TNF-a protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as
  • pharmaceutically acceptable salts which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, gly colic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In a preferred embodiment the formulation is as described in U.S.
  • the formulation comprises between 5 mg/ml and 500 mg/ml of a TNF inhibitor polypeptide; between 10 mM and 25 mM of a phosphate or citrate buffer; between 5% and 10% of a carbohydrate; and optionally NaCl, wherein the combined ionic strength of the buffer and the optional salt is an equivalent ionic strength of between 0.1M and 0.2M NaCl, wherein the formulation has a pH of between 6 and 7, is fluid at room temperature and at 37°, and has a viscosity of 10 centipoise or less at room temperature.
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents coloring agents
  • polyethylene glycol polyethylene glycol.
  • Additives are well known in the art, and are used in a variety of formulations.
  • the variant TNF-a proteins are added in a micellular formulation; see U.S. Pat. No. 5,833,948, hereby incorporated by reference.
  • liposomes may be employed with the TNF-a proteins to effectively deliver the protein.
  • Combinations of pharmaceutical compositions may be administered.
  • antibodies including but not limited to monoclonal and polyclonal antibodies, are raised against variant TNF-a proteins using methods known in the art. In a preferred embodiment, these anti-variant TNF-a antibodies are used for
  • immunotherapy is meant treatment of a TNF-a related disorders with an antibody raised against a variant TNF-a protein.
  • immunotherapy can be passive or active.
  • Passive immunotherapy is the passive transfer of antibody to a recipient (patient).
  • Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient).
  • Induction of an immune response can be the consequence of providing the recipient with a variant TNF-a protein antigen to which antibodies are raised.
  • the variant TNF-a protein antigen may be provided by injecting a variant TNF-a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a variant TNF-a protein encoding nucleic acid, capable of expressing the variant TNF-a protein antigen, under conditions for expression of the variant TNF-a protein antigen.
  • variant TNF-a proteins are administered as therapeutic agents, and can be formulated as outlined above.
  • variant TNF-a genes (including both the full-length sequence, partial sequences, or regulatory sequences of the variant TNF-a coding regions) may be administered in gene therapy applications, as is known in the art.
  • variant TNF-a genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • the nucleic acid encoding the variant TNF-a proteins may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense
  • oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 83:4143-4146 (1986), incorporated by reference).
  • the oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al, Trends in Biotechnology 11 :205-210 (1993), incorporated by reference).
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.
  • variant TNF-a genes are administered as DNA vaccines, either single genes or combinations of variant TNF-a genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998). Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a variant TNF-a gene or portion of a variant TNF-a gene under the control of a promoter for expression in a patient in need of treatment.
  • the variant TNF-a gene used for DNA vaccines can encode full-length variant TNF-a proteins, but more preferably encodes portions of the variant TNF-a proteins including peptides derived from the variant TNF-a protein.
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant TNF-a gene.
  • a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant TNF-a gene.
  • expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing TNF-a proteins.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the variant TNF-a polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
  • compositions are contemplated wherein a TNF-a variant of the present invention and one or more therapeutically active agents are formulated.
  • Formulations of the present invention are prepared for storage by mixing TNF-a variant having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, incorporated entirely by reference), in the form of lyophilized formulations or aqueous solutions. Lyophilization is well known in the art, see, e.g., U.S. Pat. No. 5,215,743, incorporated entirely by reference.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as histidine, phosphate, citrate, acetate, and other organic acids;
  • antioxidants including ascorbic acid and methionine; preservatives (such as statin), statin, statin, statin
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorb
  • the pharmaceutical composition that comprises the TNF-a variant of the present invention may be in a water-soluble form.
  • the TNF- ⁇ variant may be present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, gly colic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, gly colic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as
  • the formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • any of a number of delivery systems are known in the art and may be used to administer TNF-a variants of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g. PLA/PGA microspheres), and the like.
  • an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used.
  • Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®, and poly-D-(-)-3-hydroxyburyric acid. It is also possible to administer a nucleic acid encoding the TNF-a of the current invention, for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents. In all cases, controlled release systems may be used to release the TNF-a at or close to the desired location of action.
  • a nucleic acid encoding the TNF-a of the current invention for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents.
  • controlled release systems may be used to release the TNF-
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, com and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, com and other starches
  • binding agents such as microcrystalline cellulose, lactose, com and other starches
  • sweeteners and other flavoring agents coloring agents
  • polyethylene glycol polyethylene glycol.
  • Additives are well known in the art, and are used in a variety of formulations.
  • the variant TNF-a proteins are added in a micellular formulation; see U.S. Pat. No. 5,833,948, incorporated entirely by reference.
  • liposomes may be employed with the TNF-a proteins to effectively deliver the protein. Combinations of pharmaceutical compositions may be administered.
  • the TNF-a compositions of the present invention may be administered in combination with other therapeutics, either substantially simultaneously or co-administered, or serially, as the need may be.
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents coloring agents
  • polyethylene glycol polyethylene glycol
  • liposomes may be employed with the TNF-a proteins to effectively deliver the protein.
  • Combinations of pharmaceutical compositions may be administered.
  • the TNF-a compositions of the present invention may be administered in combination with other therapeutics, either substantially simultaneously or co-administered, or serially, as the need may be.
  • Dosage forms for the topical or transdermal administration of a DN-TNF -protein disclosed herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the DN-TNF -protein may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to the DN-TNF-protein, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to the DN-TNF-protein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • administration of the variant TNF-a proteins of the present invention may be done in any number of ways but is preferably administered centrally, directly into the spinal cord.
  • administration may be done peripherally, i.e., not intracranially, in a variety of ways including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally,
  • the variant TNF-a protein may be directly applied as a solution, salve, cream or spray.
  • the TNF-a molecules of the present may also be delivered by bacterial or fungal expression into the human system (e.g., WO 04046346 A2, hereby incorporated by reference).
  • variant TNF-a proteins are administered as therapeutic agents, and can be formulated as outlined above.
  • variant TNF-a genes (including both the full-length sequence, partial sequences, or regulatory sequences of the variant TNF-a coding regions) may be administered in gene therapy applications, as is known in the art.
  • variant TNF-a genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • the nucleic acid encoding the variant TNF-a proteins may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense
  • oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 83:4143-4146 (1986), incorporated entirely by reference).
  • the oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • Dosage may be determined depending on the disorder treated and mechanism of delivery.
  • an effective amount of the compositions of the present invention sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 2000 mg per kilogram body weight per day.
  • An exemplary treatment regime entails administration once every day or once a week or once a month.
  • a DN-TNF protein may be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly.
  • a DN-TNF protein may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the agent in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • an effective amount e.g., dose
  • a DN-TNF protein described herein will provide therapeutic benefit without causing substantial toxicity to the subject.
  • Toxicity of the agent described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the agent described herein lies suitably within a range of circulating concentrations that include the effective dose with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. See, e.g., Fingl et al, In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).
  • mice- Homozygous mTNF AM (Ruuls et al. 2001) breeders were obtained from the Department of Biochemistry, University of Lusanne, kindly provided by Dr. Tacchini-Cottier and established as heterozygous mTNF A/wt breeding colonies at the animal facility of the Biomedical Laboratory, University of Southern Denmark. All experiments were performed on age-matched (8-12 weeks old) male, homozygous mTNFA/A mice and mTNF wt/wt littermates. Animals were housed in ventilated cages at a 12 h light/dark cycle, under controlled temperature and humidity, and with free access to food and water.
  • mice that did not meet these criteria were excluded.
  • the rotarod test was conducted with accelerating speed from 4 to 40 rpm over a 5 min period, with each mouse performing in total 4 trials. After each trial, mice were transferred back to their cage and allowed to rest for 20 min to prevent exhaustion or stress. The time spent on the rod before fall was automatically recorded and the total time spent on the rod was calculated (Bach et al. 2012).
  • Rung walk test Rung walk analysis was performed as described by Novrup et al. (2014) 2 days after induction of focal cerebral ischemia. Mice were allowed to transverse the rungs and filmed using a handheld GoPro HD camera with 48 fps. Data were evaluated frame by frame using VLC Mediaplayer (2.1.2, Rincewind). Left and right scores were calculated as follows: 6, complete miss; 5, touching the rung, but sliding off and losing balance; 4, touch, miss but no loss of balance; 3, replacement, mouse placed paw on rung but quickly removed it; 2, recorrection, aimed for rung but changed direction; 1, anterior or posterior placement; 0, perfect step. The total number of mistakes on each paw was plotted for analysis of asymmetry. Prior to surgery, mice were pre-trained in the rung walk test and no asymmetry was observed under baseline conditions (data not shown).
  • the underlying superior pole of the parotid gland and the upper part of the temporal muscle were pushed in the distal direction after partial resection.
  • a craniotomy was performed directly above the distal part of the MCA using a 0.8 mm high-speed microdrill.
  • the bone was removed and the dura carefully opened.
  • the distal part of the MCA was coagulated using bipolar forceps coupled to an electrosurgical unit (ICC 50, Erbe) ensuring a restricted cortical infarct.
  • Post- operative treatment consisted of supplying the mice with physiological saline (0.9% NaCl) and Temgesic® (Buprenorphinum 0.3 mg/ml, RB Pharmaceuticals) three times during the first 24 h. The mice were allowed to survive for 1 or 5 days.
  • mice were anesthetized with Pentobarbital (200 mg/ml)/Lidocainhydrochlorid (20 mg/ml) and transcardially perfused with 4%
  • Immunolabeled Ibal + cells were counted at a 60x magnification in the right hemisphere of 10-14 sections from each mouse by the use of CAST1 (Visiopharm).
  • the number of Ibal + positive cells was recorded by systematically counting cells using a counting frame of 1,012.6 ⁇ 2 and a X-Y step size of 350 ⁇ resulting in an area (a(step)) of 122,500 ⁇ 2 to ensure that a representative sample consisting of a minimum of 100-200 cells were counted.
  • the number of microglial cells per areal unit was calculated as previously described and expressed Ibal + cells/mm 2 . The person performing the cell counting was blinded to the genotype.
  • RNA isolation and real time RT-PCR- Total RNA was extracted from one series of brain tissue with TRIZOL® according to manufacturer's instructions (Invitrogen). To ensure complete elimination of genomic DNA, RNA was further purified with RNeasy MinElute Cleanup Kit (Qiagen) in combination with DNA digestion using RNase-free DNase (Qiagen). Reverse transcription was performed with Omniscript (Qiagen), according to manufacturer's protocols. cDNA equal to 10-50 ng of initial total RNA was used as a template in each PCR reaction. Real time PCR was performed in the Rotor-Gene 3000 Real Time Cycler (Corbett Life Science) with QuantiTect SYBR Green PCR MasterMix (Qiagen). Relative expression was calculated by comparison with a standard curve, after normalization to ⁇ -actin gene expression.
  • phosphatase inhibitor cocktail 1 (Sigma). The total protein concentration was quantified utilizing the Lowry assay. Proteins were resolved by SDS-PAGE on 8-11% gels, transferred to nitrocellulose and blocked in 5% non-fat milk in TBS-T. Membranes were probed overnight with antibodies against myelin associated glycoprotein (MAG) (mouse, 1 : 1,000, Santa Cruz Biotechnology), myelin basic protein (MBP) (mouse, 1 :5,000, Santa Cruz Biotechnology), TNFR1 (mouse, 1 :500, Santa Cruz Biotechnology), and ⁇ -tubulin (mouse, 1 :5,000, Sigma), followed by HRP-conjugated secondary antibodies (GE Healthcare/Amersham).
  • MAG myelin associated glycoprotein
  • MBP myelin basic protein
  • TNFR1 mouse, 1 :500, Santa Cruz Biotechnology
  • ⁇ -tubulin (mouse, 1 :5,000, Sigma)
  • Proteins were visualized with Super Signal West Pico chemiluminescent substrate (Thermo Scientific) and bands quantified with Quantity One software (Biorad). Data were normalized against ⁇ -tubulin and expressed as percent of naive mTNFwt/wt.
  • JStatistics- Real-time PCR was analyzed with one-way ANOVA followed by Tukey's test for multiple comparisons. For single comparisons, Student's t-test was applied. P values ⁇ 0.05 were considered statistically significant. Data are presented as mean ⁇ SEM.
  • periphery e.g. neutrophils, macrophages
  • solTNF is neuroprotective following focal cerebral ischemia.
  • selective TNF inhibitors such as XProl595
  • that selectively target only solTNF may be prove beneficial in reducing lesion volume, inflammation and improving functional outcome following stroke.
  • Example 2 Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia
  • mice were injected subcutaneously with 1 ml of 0.9% saline and allowed to recover in a 25°C controlled environment. Mice surviving for 5d were returned to the conventional animal facility after 24 hours (h). For post-surgical analgesia, mice were treated with Temgesic (0.001 mg/20 g buprenorphinum; Reckitt & Colman) three times at 8-hour intervals starting immediately prior to surgery.
  • [00170]XProl 595 or etanercept (Enbrel, Amgen-Wyeth) were administered once, intravenously (i.v.) at a dose of 10 mg/kg, 30min after surgery. Saline was used as vehicle. Mice subjected to sham surgery were given an i.v. injection of saline 30min after surgery. The peak concentration in serum (Cmax) after murine i.v. dosing of XProl595 at 10 mg/kg was 945.7 Dg/mL and the terminal half-life was 19.1 hr (data not shown).
  • mice were placed on the centre of a horizontal rod, located 80 cm above the floor. Mice were allowed to explore and walk the rod for 3min. The frequency of right and left hindlimb slips was recorded and the total distance travelled was tracked using the SMART video tracking software (Panlab).
  • rotarod test (LE8200, Panlab Harvard Apparatus). The test comprised a pre-training part prior to surgery (30 sec at 4 rotations per minute (rpm)) and a trial part consisting of 4 trials (Tl - T4) 1 or 5d after surgery. Mice were placed on the rotarod set in accelerating mode. Speed of the rotor was accelerated from 4 to 40 rpm over 5 min. Time spent on the rotarod in each trial for each mouse was recorded.
  • Fresh frozen tissue Mice were killed by cervical dislocation, and brains and livers were quickly removed, frozen in C02 and stored at -80°C until further processing. Blood samples were collected in EDTA-coated Eppendorf tubes, spun 2x lOmin at 3,000g, 4°C, and stored at - 80°C until further processing. Brains were cut coronally in 6 parallel series of 30 ⁇ and liver samples into 30 ⁇ cryostat sections and stored at -80 °C until further processing. [00180]Perfusion fixed tissue: Mice were deeply anesthetized with an overdose of pentobarbital containing lidocaine and perfused through the left ventricle using 4% paraformaldehyde (PFA) as previously described. Brains were cut coronally in 6 parallel series as free-floating 60 ⁇ thick sections and stored in a cryoprotective solution at -12°C or into 12 parallel series as 20 ⁇ thick cryostat sections and stored at -20°C until further processing.
  • PFA paraformal
  • Liver and brain mRNAs were extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
  • cDNA was prepared as previously described and qPCR analysis perfomed using the following conditions: 5min primer extension at 25°C, followed by 25min reverse trancription at 55 °C and finally 5min enzyme inactivation at 95°C as previously described. Samples were run against standard curves generated from serially diluted cDNA from liver samples obtained from mice subjected to pMCAO. Primer sets were designed by
  • SAA2 forward: TTCATTTATTGGGGAGGCTT (SEQ ID NO: 3)
  • CCL2 forward: TGAAGTTGACCCGTAAATCTGAA (SEQ ID NO: 7)
  • IL-1 ⁇ (forward: TGTAATGAAGACGGCACAC (SEQ ID NO: 9) and reverse:
  • CXCL1 forward: GCTGGGATTCACCTCAAGAAC (SEQ ID NO: 11) and reverse: TGTGGCTATGACTTCGGTTTG (SEQ ID NO: 12)
  • CXCL10 forward: CATCCCGAGCCAACCTTCC (SEQ ID NO: 13) and reverse: CACTCAGACCCAGCAGGAT (SEQ ID NO : 14)
  • Argl primers (Mm00475988_ml) were purchased from Life technologies. Liver results were reported relative to the expression of the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) [20]. All data were normalized to the corresponding sham group, which at all time points represented a mean value of 1. Brain TNF, IL-1 ⁇ and CD1 lb mRNA qPCR analyses were performed as previously described.
  • Total protein was extracted in 1% lysis buffer (RIP A, Merck Millipore) containing a soluble protease inhibitor cocktail (Roche Diagnostics) according to Lambertsen et al. Protein concentrations were estimated using the Bradford Protein Quantification method.
  • Western blotting analysis for TNF (Abeam, 1 :2,000) was performed using 20 ⁇ g protein extract separated on bis/tris 4-12% SDS-PAGE gels (Nupage, Invitrogen) essentially as previously described. SeeBlue Plus2 prestained standard (Invitrogen) was used as a molecular weight marker and 0.5ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included as a positive control.
  • TNF+ microglia [CDl lb+CD45dim]
  • TNF+ macrophages [CDl lb+CD45highGrl-]
  • TNF+ granulocytes [CD1 lb+CD45highGrl+] were identified as previously detailed.
  • Control mice and mice allowed 6 and 24h survival after pMCAO were treated i.v. with either saline, XProl595 or etanercept 30min after surgery.
  • Nanoparticle Tracking Analysis using a NS500 analyser equipped with a 488 nm laser and NTA software (Nanosight Ltd) as previously described. Analysis settings were standardised using lOOnm colloidal silica microspheres (100, 150, 300 and 400 nm; Polysciences) and these data were used to verify size measurements and calibrate concentration measurements. Five 30- second videos were made for each sample. The sample was advanced with a 5-second delay between each recording using the script control facility. The videos were analysed in batch process mode using automatic blur and minimum expected particle size with an automatic detection threshold level 10, after visually checking that the five size profiles on the screen were in concordance.
  • mice subject to sham surgery were also included.
  • a post-surgical weakness of both the left (L) and right (R) front paws in saline- and XPro 1595 -treated mice 3 and 5 days after pMCAO compared to normal baseline grip strength (Figure 9A) was detected.
  • Etanercept-treated mice showed no difference on the left paw, however a significant reduction on the right paw ( Figure 9A).
  • XProl 595- and etanercept-treated mice performed significantly better on day 3 compared to saline-treated mice ( Figure 9A).
  • grip strength analysis showed significant pMC AO-induced front paw asymmetry in saline-treated mice 24h and 5d after pMCAO as compared to sham mice and pre-treatment baseline grip strengths (represented by delta ( ⁇ ) values) (Figure 9B). Minor asymmetry was observed in XProl 595- treated mice at 24h, but not at 5d ( Figure 9B). No asymmetry was observed in etanercept-treated mice.
  • the grip strength data indicate that anti-TNF therapy ameliorates neuromuscular asymmetry normally caused by pMCAO.
  • mice displayed a significant drop in weight from baseline to day 3 (PO.0001 for all groups) and day 5 (P ⁇ 0.01 for saline, XProl 595 and etanercept); however the weight drop at day 5 was less in the sham group (PO.05).
  • CD1 lb+CD45dim cells CD1 lb+CD45dim cells
  • macrophages CD1 lb+CD45highGrl -cells
  • CD1 lb+CD45dim microglia was found to be significantly increased in all treatment groups at 6h and 24h after pMCAO compared to unlesioned control mice.
  • CD1 lb+CD45dim microglial population was evaluated in mice treated with either XProl595 or etanercept and allowed 24h survival, the estimated number of CD1 lb+CD45dim microglia in the lesioned cortex had increased in etanercept-treated mice, though not quite significant, and significantly in XPro 1595 -treated mice as compared to saline treated mice ( Figure 10B, upper left graph).
  • TNF mRNA+ cells were observed within the infarct and peri-infarct in all groups after pMCAO ( Figure 11A, shown for saline only). Cells were most numerous at 24h, with very few cells observed at 5d. These findings were confirmed by qPCR, showing a transient increase in TNF mRNA at 24h ( Figure 11 A). As part of the APR to brain injury, TNF mRNA+ cells were also found in the liver primarily 6h after pMCAO, as supported by qPCR analysis ( Figure 1 IB). By 24h, there was a significant reduction in TNF mRNA transcription in the liver in saline- and XPro 1595 -treated mice, consistent with findings of very few TNF mRNA+ cells.
  • TNF+CDl lb+CD45dim microglia had increased significantly in all treatment groups compared to unlesioned control mice with no differences between treatment groups ( Figure 1 IE).
  • TNF+ macrophages TNF+CDl lb+CD45highGrl - cells
  • TNF+ granulocytes TNF+ granulocytes
  • Anti-TNF therapy affects granulocyte infiltration into the infarct 24h after focal cerebral ischemia
  • Anti-TNF therapy affects the liver acute phase response after focal cerebral ischemia
  • Hepatic CCL2 a chemokine primarily referred to as monocyte chemotactic protein
  • mRNA levels were only affected in XPro 1595 -treated mice, which displayed a transient increase 24h after pMCAO compared to both 6h and 5d ( Figure 13 A), suggesting that solTNF plays a role in recruitment of monocytes into the liver.
  • Anti-TNF therapy impacts microvesicle size and number after focal cerebral ischemia
  • microvesicle number and infarct size have been shown to correlate and possibly be an indicator of inflammation
  • microvesicle number and size after pMCAO was analyzed. Comparisons showed similar numbers in saline- (2.7 ⁇ 0.5xl010/mL) and anti-TNF -treated control mice (XProl 595: 3.3 ⁇ 0.2xl010/mL and etanercept 3.2 ⁇ 0.8x1010/mL).
  • XPro 1595 3.3 ⁇ 0.2xl010/mL and etanercept 3.2 ⁇ 0.8x1010/mL
  • Figure 14A we found significantly more microvesicles in XPro 1595 -treated mice compared to saline-treated mice
  • Figure 14A an effect which was observed in both XProl 595- and etanercept-treated mice 5d after pMCAO.
  • significant increases were observed in microvesicle numbers in all groups 5d after pMCAO compared to 6h survival.
  • the aim of the study is to investigate whether direct administration of XProl 595 to the brain of mice with experimentally induced stroke will reduce lesion size and improve functional recovery.
  • the experimental stroke model comprises occlusion of a major blood vessel of the brain (the middle cerebral artery) in mice, and it is the inventors hypothesis that XProl 595 will reduce lesion size and improve functional outcome in experimental stroke model and that XProl 595 is superior to etanercept in doing so.
  • mice suffering from the experimentally induced stroke were treated with XProl 595, etanercept or placebo either into the interconnected cavities in the brain or directly onto the brain tissue supplied by the middle cerebral artery (using micro-osmotic pumps).
  • Drugs were administered topically using micro-osmotic pumps (2.5 mg/mL concentration/1 mL/hour) placed immediately above the middle cerebral artery on the surface of the brain after induction of experimental stroke allowing for direct administration to the infarct for 3 days or administered into the ventricles (10 mg/kg) 30 minutes after experimental stroke using stereotactic surgery.
  • mice were induced with experimental stroke and treated. Groups consisted of 20 mice, which were based on a long-standing experience with this experimental model and power analyses. Surgeries were performed by a group of 3-4 people and blinded to the surgeon and the person administrating the drugs.
  • Group a intraventricular XProl 595 treatment (10 mg/kg)
  • Group b topical XProl595 treatment (2.5 mg/mL concentration/1 mL/hour)
  • Group c intraventricular etanercept treatment (10 mg/kg)
  • Group d topical etanercept treatment (2.5 mg/mL concentration/1 mL/hour)
  • Group e controls receiving intraventricular placebo (saline) treatment
  • Group f controls receiving topical placebo (saline) treatment
  • Brains were processed so that series of parallel tissue can be used for volume estimation and molecular and biological analyses. Work package 4 - Molecular and biological analyses
  • Intracerebroventricular injections in animals with 1 day survival after pMCAO resulted in an average 22% decrease in infarct size in XProl 595 treated animals and an average 23% decrease in Etancept-treated animals compared to saline.
  • Figure 15 shows that thermal stimulation using the Hargreave's test resulted in a significant increase in latency time to withdraw paws between in saline-treated mice probably as a consequence of increased injury in the ipsilateral sensory cortex.
  • XProl 595-treated mice showed a decrease in latency time, whereas etanercept-treated mice showed no change (*P ⁇ 0.05).
  • tmTNF present at the lesion site is necessary for neuroprotection (decreased IFV and improved Hargreave's in Xprol 595-pump treated mice and increased IFV in etanercept- treated), whereas the ICV studies pomts towards a beneficial effect of removing solTNF overall (decrease in IFV in both groups).
  • anti-TNF therapy is beneficial, but removing tmTNF at the lesion site is detrimental.

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Abstract

La présente invention concerne une méthode de traitement d'un accident vasculaire cérébral et/ou de symptômes associés à un accident vasculaire cérébral par administration à un sujet, ayant besoin d'un tel traitement, d'un polypeptide de DN-TNF qui inhibe l'activité du TNF soluble mais pas celle du TNF-alpha transmembranaire.
PCT/DK2015/050358 2014-11-21 2015-11-20 Inhibiteur du tnf-alpha pour le traitement d'un accident vasculaire cérébral Ceased WO2016078672A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050265962A1 (en) * 2001-03-02 2005-12-01 Xencor, Inc. Protein based TNF-alpha variants for the treatment of TNF-alpha related disorders
WO2012061289A2 (fr) * 2010-11-01 2012-05-10 Tact Ip Llc Méthodes de traitement de lésions cérébrales à l'aide d'agents biologiques
WO2014040076A1 (fr) * 2012-09-10 2014-03-13 Xencor Procédés de traitement de maladies neurologiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050265962A1 (en) * 2001-03-02 2005-12-01 Xencor, Inc. Protein based TNF-alpha variants for the treatment of TNF-alpha related disorders
WO2012061289A2 (fr) * 2010-11-01 2012-05-10 Tact Ip Llc Méthodes de traitement de lésions cérébrales à l'aide d'agents biologiques
WO2014040076A1 (fr) * 2012-09-10 2014-03-13 Xencor Procédés de traitement de maladies neurologiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIANA M. SAMA ET AL: "Inhibition of Soluble Tumor Necrosis Factor Ameliorates Synaptic Alterations and Ca2+ Dysregulation in Aged Rats", PLOS ONE, vol. 7, no. 5, 1 January 2012 (2012-01-01), pages e38170 - e38170, XP055085799, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0038170 *
FILIP VAN HAUWERMEIREN ET AL: "Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1", CYTOKINE AND GROWTH FACTOR REVIEWS, vol. 22, no. 5, 14 September 2011 (2011-09-14), pages 311 - 319, XP028337283, ISSN: 1359-6101, [retrieved on 20110914], DOI: 10.1016/J.CYTOGFR.2011.09.004 *
MCCOY MELISSA K ET AL: "TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease", JOURNAL OF NEUROINFLAMMATION, BIOMED CENTRAL LTD., LONDON, GB, vol. 5, no. 1, 17 October 2008 (2008-10-17), pages 45, XP021044894, ISSN: 1742-2094, DOI: 10.1186/1742-2094-5-45 *

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