WO2016040581A1 - Méthodes de traitement d'une lésion de la moelle épinière - Google Patents

Méthodes de traitement d'une lésion de la moelle épinière Download PDF

Info

Publication number
WO2016040581A1
WO2016040581A1 PCT/US2015/049360 US2015049360W WO2016040581A1 WO 2016040581 A1 WO2016040581 A1 WO 2016040581A1 US 2015049360 W US2015049360 W US 2015049360W WO 2016040581 A1 WO2016040581 A1 WO 2016040581A1
Authority
WO
WIPO (PCT)
Prior art keywords
tnf
protein
variant
inhibitor
sci
Prior art date
Application number
PCT/US2015/049360
Other languages
English (en)
Inventor
Kate Lykke LAMBERTSEN
Hans NOVRUP
John R. Bethea
Valerie BRACCHI-RICARD
Original Assignee
University Of Miami
University Of Southern Denmark
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Miami, University Of Southern Denmark filed Critical University Of Miami
Publication of WO2016040581A1 publication Critical patent/WO2016040581A1/fr

Links

Classifications

    • 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

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 spinal cord injury.
  • a secondary wave of neuronal and gliatoxic sequelae including a rapid increase in glutamate, immunoregulatory cytokines, toxic lipid metabolites and over time, the infiltration of peripheral blood leukocytes such as neutrophils, macrophages and T-cells (Donnelly, D.J. and P.G. Popovich, Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp Neurol, 2008. 209(2): p. 378-88.).
  • the immune system appears to play a dual role in both pathological destruction of neuronal tissue as well as in tissue repair and to some extent, functional recovery (Bracchi-Ricard, V., et al, Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury. J Neuroinflammation, 2013. 10(1): p. 92; Johnstone, J.T., et al, Inhibition of NADPH oxidase activation in oligodendrocytes reduces cytotoxicity following trauma. PLoS One, 2013. 8(11): p. e80975; Benowitz, L.I. and P.G. Popovich, Inflammation and axon regeneration.
  • principles of the present disclosure provide a method of treating SCI or symptoms associated with spinal cord injury 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 administered directly into the spinal cord.
  • the dominant negative inhibitor is XProl595.
  • FIG. 1A shows the nucleic acid sequence of human TNF-a (SEQ ID NO: l). 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.
  • FIGS. 3(A) to 3(E) show that systemically administered anti-TNF therapy does not affect motosensory functions or lesion size after SCI.
  • FIG. 3B Luxol fast blue stained thoracic spinal cord sections from mice treated with either saline, XPro 1595 or etanercept and allowed 8 weeks survival after SCI.
  • FIG. 3C Catwalk analysis showing changes in front- and hind-limb stride length and front- and hind-limb base of support (BOS) over time after SCI. No difference was observed between saline-, XPro 1595-, and etanercept-treated mice, however all mice displayed significant changes in stride length (**P ⁇ 0.01 for hind-limbs and *****p ⁇ 0.0001 for front-limbs, respectively) and BOS on the front-limbs (**P ⁇ 0.01) over time.
  • FIG. 3D Gridwalk analysis showing changes in the average number of foot falls errors and average number of foot slips errors over time after SCI.
  • FIGS. 4(A) to 4(G) show centrally administered XPro 1595 improves motor functions and decreases lesion size after SCI.
  • FIG. 4A Analysis of BMS scores in mice treated centrally for three consecutive days with either saline, XPro 1595, or etanercept showed that XPro 1595 -treated mice significantly improved their BMS score from day 3 to 35 days after SCI compared to both saline- and etanercept-treated mice (*P ⁇ 0.05 and ***P ⁇ 0.001, Two-way RM ANOVA). All groups of mice significantly improved their BMS score over time (***P ⁇ 0.001). (FIG.
  • FIG. 4B Rung walk analysis showed that XPro 1595 -treated mice significantly decreased their number of mistakes compared to saline- and etanercept-treated mice (*P ⁇ 0.05 and **P ⁇ 0.01).
  • FIG. 4C Thermal stimulation using the Hargreave's test showed no differences in latency time to withdraw paws between saline-, XProl595- and etanercept-treated mice. All mice decreased their latency to remove their paws over time after SCI (****p ⁇ 0.0001).
  • FIG. 4D Luxol fast blue stained thoracic spinal cord sections from mice treated with either saline, XProl595 or etanercept and allowed 35 days survival after SCI.
  • FIG. 4E Analysis of lesion volumes 35 days after SCI showed that the lesion size was significantly smaller in XPro 1595 -treated mice compared to both saline- and etanercept-treated mice (One-way ANOVA, followed by Tukey's test).
  • FIG. 4F Representative thoracic spinal cord sections from saline-, XPro 1595-, and etanercept-treated mice stained for anti-GFAP allowed 35 days survival. Scale bar: 100 ⁇ .
  • FIG. 4G Quantification of GFAP protein expression in spinal cord tissue of saline-, XPro 1595-, and etanercept-treated mice at 7 and 28 days after SCI.
  • FIGS. 5(A) to 5(F) show open field test analysis of SCI mice treated centrally with anti- TNF therapy for three consecutive days and allowed 35 days of survival after SCI.
  • FIGS. 5A, 5B Analysis of locomotor activity in mice treated centrally with either saline, XPro 1595 or etanercept and allowed 35 days survival after SCI showed that all mice travelled a similar distance (FIG. 5A) at comparable speeds (FIG. 5B) in the open field test.
  • FIGS. 5C, 5D Analysis of anxiety-related behavior in the open field test showed that XPro 1595 -treated mice displayed decreased anxiety-related behavior represented by increased center/peri-meter ratio (FIG.
  • FIG. 5C The number of droppings (FIG. 5E) was comparable in all groups of mice, whereas the number of groomings (FIG. 5F) was increased both in XPro 1595- and etanercept- treated mice compared to saline-treated mice.
  • FIGS. 6(A) to 6(B) show changes in Ibal protein expression following central anti-TNF treatment after SCI.
  • FIG. 6A Quantification of Ibal protein expression in spinal cord tissue of saline-, XPro 1595-, and etanercept-treated mice at 7 and 28 days after SCI. Data are normalized to ⁇ -actin protein expression. Representative experiments are shown. Results, expressed as per cent of control, are the mean ⁇ SEM of three animals per group. #P ⁇ 0.05 versus XPro 1595 and etanercept; *P ⁇ 0.05, **P ⁇ 0.01, and ***P ⁇ 0.001 versus control by One-way ANOVA with Tukey's test. (FIG.
  • FIGS. 7(A) to 7(D) show changes in MBP, TNFR2, TLR4 and GAP43 protein expression following central anti-TNF treatment after SCI.
  • FIG. 7A Quantification of MBP protein expression in spinal cord tissue of saline-, XProl595-, and etanercept-treated mice at 7 and 28 days after SCI.
  • FIG. 7B Quantification of TNFR2 protein expression in spinal cord tissue of saline-, XProl595-, and etanercept-treated mice at 7 and 28 days after SCI.
  • FIG. 7C Quantification of TLR2 protein expression in spinal cord tissue of saline-, XProl595-, and etanercept-treated mice at 7 and 28 days after SCI.
  • FIG. 7D Quantification of GAP43 protein expression in spinal cord tissue of saline-, XProl595-, and etanercept-treated mice at 7 and 28 days after SCI. Data are normalized to ⁇ -actin protein expression. Representative experiments are shown. Results, expressed as per cent of control, are the mean ⁇ SEM of three animals per group. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 and ****P ⁇ 0.0001 by One-way ANOVA with Tukey's test.
  • Tumor necrosis factor is a pleiotrophic cytokine important in the regulation of numerous physiological and pathological processes such as inflammation, autoimmunity, neurodegeneration, neuroprotection, demyelination and remyelination.
  • TNF Tumor necrosis factor
  • TNFR1 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.
  • Expression studies demonstrate that TNF is upregulated in the spinal cord within minutes to hours following injury, which coincides with elevated glutamate, suggesting that injury-induced cytotoxicity may be mediated through additive or synergistic interactions between these and other soluble factors.
  • compositions and methods for treating symptoms associated with SCI comprise administering to a patient in need thereof a selective 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 XProl595.
  • the present disclosure provides methods of selectively inhibiting solTNF-a.
  • prior art strategies attempted to non-selectively inhibit all TNF-a, including both solTNF and tmTNF-a.
  • the effects of soluble and transmembrane TNF-a are, in many instances, diametrically opposed. For instance, in the CNS, soluble TNF-a increases inflammation and causes demyelination of neurons. In contrast, transmembrane TNF-a promotes re-myelination.
  • soluble TNF-a causes inflammation, while transmembrane TNF-a promotes and maintains the immune response.
  • agents that non- selectively inhibit both soluble and transmembrane TNF-a provide for reduced inflammation, via diminished bioactive soluble TNF-a, while also reducing the beneficial effects of transmembrane TNF-a, such as an active immune response or re-myelination of neurons.
  • non-selective TNF- ⁇ inhibitors are wrought with debilitating side-effects such as demyelination of neurons and reduced immune response.
  • SCI spinal cord injury
  • symptoms associated with SCI symptoms initiated following damage to the spinal cord. These symptoms may include, but are not limited to pain, paralysis, loss of movement, loss of sensation, including the ability to feel heat, cold and touch, loss of bowel or bladder control, exaggerated reflex activities or spasms, changes in sexual function, sexual sensitivity and fertility, pain or an intense stinging sensation caused by damage to the nerve fibers in the spinal cord, and difficulty breathing, coughing or clearing secretions from the lungs, depending on the level of injury.
  • Preferred inhibitors of TNF-a may be dominant negative TNF-a proteins, referred to herein as "DN-TNF", “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 TNF-a or TNF-a proteins that differ from the corresponding wild-type protein by at least 1 amino acid.
  • SEQ ID NO: l SEQ ID NO: l .
  • DNTNF-a 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. [0023] Thus, the proteins of the invention are antagonists of wild-type TNF-a. By “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 an 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 activity, 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 Preferably 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 known 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%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 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.
  • This may also be expressed as a ratio; that is, selective inhibition can include a ratio of inhibition of soluble to transmembrane TNF-a.
  • 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 or 2 variant TNF-a proteins : 1 wild-type TNF-a protein.
  • trimers may be formed comprising only variant TNF-a proteins.
  • the 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.
  • variant TNF-a proteins 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 (Invitrogen, Grand Island, NY) nucleic acid stain to detect TNF-induced cell permeability in an Actinomycin-D sensitized cell line.
  • 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.
  • 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%, greater than 97%, greater than 98% and greater than 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, 7 or 8 different residues.
  • Preferred variant TNF-a proteins have 3 to 8 different residues.
  • the 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.
  • TNF- ⁇ proteins may be fused to, for example, other therapeutic proteins or 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 ah, 1996, Trends Biotechnol 14:52-60; Ashkenazi et ah, 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, Q21C, 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, CIOIA, A111R, Al l IE, K112D, K112E, Y115D, Y115
  • 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 7,662,367, which is incorporated herein by reference.
  • 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-a proteins have substitutions selected from the group of substitutions consisting of VIM, 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, CIOIA, A111R, Al l 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 XProl595, 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 (also known as II), Small Domain (also known as I), the DE loop, and the trimer interface.
  • 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).
  • theses variants may be in the form of single point variants, for example K112D, Y115K, Y115I, Y115T, A145E or A145R. These single point variants may be combined, for example, Y115I and A145E, or Y115I and A145R, or Yl 15T and A145R or Yl 151 and A145E; or any other combination.
  • 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 Y115I, Y115Q, Y115T, D143K, D143R, D143E, A145E, A145R, E146K or E146R.
  • Other preferred double variants are Y115Q and at least one of D143N, D143Q, A145K, A145R, or E146K; Y115M and at least one of D143N, D143Q, A145K, A145R or E146K; and L57F and at least one of A145E or 146R; K65D and either D143K or D143R, K65E and either D143K or D143R, Y115Q and any of L75Q, L57W, L57Y, L57F, I97R, I97T, S86Q, D143N, E146K, A145R and I97T, A145R and either Y87R or Y87H; N34E and V91E; L75E and Yl 15Q; L75Q
  • 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 Yl 151, 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-a 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.
  • 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.
  • the depiction of a single strand also defines the sequence of the other strand (“Crick”); thus the sequence depicted in FIG. 1A (SEQ ID NO: l) also includes the complement of the sequence.
  • recombinant nucleic acid is meant 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.
  • vector 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.
  • vector 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.
  • 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- ⁇ 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, for example, 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.
  • vector composition the combination of components, comprised by one or more vectors, which may be retroviral or not, is referred to herein as a "vector composition.”
  • a number of viral based vectors have been used for gene delivery. See for example U.S. Pat. No. 5,576,201, which is expressly incorporated herein by reference.
  • 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.
  • 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
  • the gene of interest can be inserted in the vector and packaged in the viral shells synthesized by the retroviral helper.
  • the recombinant virus can then be isolated and delivered to a subject.
  • Representative 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. Additional control elements may be included.
  • the resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.
  • 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.
  • AAV ITR regions The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, 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 SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • synthetic promoters hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif).
  • 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
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • 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 positions 21, 23, 31 and 45, taken alone or in any combination.
  • a TNF-a variant of the present invention includes the R31C 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.
  • DN- TNFs that inhibit soluble but not transmembrane TNF-a find use in treating SCI or symptoms associated with SCI. These molecules find particular use when combined with currently available SCI therapies as known in the art and as described herein. For instance, DN-TNFs, such as XProl595, may be combined in a therapeutic regimen with methylprednisolone (Medrol®) or other molecules. DN-TNFs as described herein may also be used following surgery to alleviate symptoms or treat SCI.
  • 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.
  • 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 weight % to 100 weight %.
  • concentration of the variant TNF-a protein is in the range of 0.003 molar 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, glycolic 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, glycolic acid, pyruvic 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 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 °C, and has a viscosity of 10 centipoise or less at room temperature (e.g., at 25 °C) [0082]
  • 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 poly
  • 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.
  • 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.
  • antibodies including but not limited to monoclonal and polyclonal antibodies, are raised against variant TNF-a proteins using methods known in the art.
  • these anti-variant TNF-a antibodies are used for immunotherapy.
  • methods of immunotherapy are provided.
  • 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, as defined herein, 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).
  • 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.
  • These 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.
  • 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 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. See 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 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, asparag
  • 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, glycolic 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, glycolic acid, pyruvic 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 isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • 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 trans fection 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 trans fection agents.
  • controlled release systems may be used to release the
  • 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, 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- ⁇ 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.
  • the 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. In another embodiments 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, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds, inflammation, etc., 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- ⁇ 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.
  • 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. Alternatively, 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.
  • 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.
  • 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.
  • Toxicity an effective amount (e.g., dose) of 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 LD 5 o (the dose lethal to 50% of the population) or the LDioo (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 ah, In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).
  • the window for treatment following spinal cord injury is wide. Treatment may begin immediately following injury. However, treatment may also be effective months or years following injury. In general, treatment may be administered at any time when the SCI symptons are evident.
  • mice were anaesthetized using a ketamine (100 mg/kg, VEDCO Inc., Saint Joseph, MO, USA) / xylazine (10 mg/kg, VEDCO) cocktail, laminectomized between vertebrae T8 and T10 and the impactor lowered at a predetermined impact force resulting in an approximate displacement of 500 ⁇ (moderate injury).
  • Contusion injury was induced with the mouse Infinite Horizon-0400 SCI Contusion Device (Precision Systems and Intrumentation, LLC, Fairfax Station, VA (Bracchi-Ricard, V., et al, Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury. J Neuroinflammation, 2013. 10(1): p. 92)).
  • mice were sutured and injected s.c. with 1 ml lactated Ringer's Fluid USP (B. Braun, L7502, Bethlehem, PA) to prevent dehydration and housed separately in a recovery room, where their post-surgical health status was observed during a 24-48 hour recovery period. Thereafter, mice were observed twice daily for activity level, body temperature, respiratory rate, and general physical condition. Manual bladder expression was performed twice a day until bladder function was regained. Body weight was monitored weekly. In addition, mice received s.c. prophylactic injections of antibiotic gentamicin (40 mg/kg) for 7 days following SCI to prevent urinary tract infections.
  • 1 ml lactated Ringer's Fluid USP B. Braun, L7502, Bethlehem, PA
  • mice were implanted with a micro-osmotic pump (Alzet model 1003D, Durect Corporation, Cupertino, CA), which for a period of 3 days continuously, epidurally delivered either XProl595 (2.5 mg/mL concentration; 1 ⁇ /h), etanercept (Enbrel®, Amgen, Thousand Oaks, CA; 2.5 mg/mL; 1 ⁇ /h), or saline control (0.9% physiological saline/1 ⁇ /h).
  • Treatment dose was determined based on previous publications (Bedrosian, T.A., Z.M. Weil, and R.J.
  • Treatment dose was determined based on previous publications (Brambilla, R., et al., Inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis and promotes axon preservation and remyelination. Brain, 2011. 134(Pt 9): p.
  • Time points of administration were based on previous findings of significantly elevated levels of TNF in the spinal cord within the first hour after SCI (Bethea, J.R., et al., Systemically administered inter leukin- 10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. J Neurotrauma, 1999. 16(10): p. 851-63; Pineau, I. and S. Lacroix, Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and identification of the cell types involved. J Comp Neurol, 2007. 500(2): p. 267-85.).
  • Basso Mouse Scale Functional recovery of function after SCI was determined by scoring of the locomotor hindlimb performance in the open field using the Basso Mouse Scale (BMS) system, a 0 to 9 rating system designed specifically for the mouse (Bracchi-Ricard, V., et al., Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury. J Neuroinflammation, 2013. 10(1): p. 92.; Basso, D.M., et al, Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma, 2006. 23(5): p. 635-59.).
  • BMS Basso Mouse Scale
  • mice were evaluated over a 4-min period 1 and 3 days after SCI and weekly thereafter. Only mice with a score below 2 on day 1 were included in the study. Before surgery, mice were handled and pre- trained in the open field to prevent fear and/or stress behaviors that could bias the locomotor assessment.
  • Catwalk The Catwalk test was performed, as described previously, to assess gait and motor coordination of the mice (Starkey, M.L., et al., Assessing behavioural function following a pyramidotomy lesion of the corticospinal tract in adult mice. Exp Neurol, 2005. 195(2): p. 524- 39.).
  • the Catwalk equipment and software were purchased from Noldus Information Technology (Leesburg, VA). Briefly, the mice continuously walked along a glass floor 1 m in length. Two fluorescent lamps that ran along the bottom of the floor illuminated the points of contact between the paws and floor. A video camera located beneath the glass floor recorded the gait of the mice. Each animal performed 3 runs. The following parameters were analyzed using the purchased software: the stride length, the base of support (BOS), paw contact area, and the intensity (assigned by the software) of the paw print.
  • BOS base of support
  • paw contact area the intensity (assigned by the software) of the paw print.
  • Gridwalk The grid walk was performed to analyze fine motor control.
  • the grid consisted of steel rods (2 mm in diameter) that were spaced either 5 or 10 mm apart for a total distance of 1 meter.
  • the mice continuously walked along the rods.
  • the number of slips for the hind-leg was counted.
  • One run was considered as back and forth, allowing for counting of the number of slips by both hind-legs.
  • Each mouse performed 3 runs.
  • the number of slips over the 3 runs was averaged.
  • the behavioral test was performed once a week on each animal. The placement of the rods was changed each week to prevent the mice from performing on "memory”.
  • Rung Walk In order to test stepping, interlimb coordination and balance, mice were tested on the rung walk when they reached a BMS score of 5.
  • the rung walk consisted of two plates of transparent polymer, approximately 110 cm x 20 cm, with a 2.5 cm space between them. Rungs with a 2 mm diameter were placed using a specific pattern according to (Metz, G.A. and I.Q. Whishaw, Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and co-ordination. J Neurosci Methods, 2002. 115(2): p. 169-79.).
  • mice were tested at 3, 4, and 5 weeks using a handheld GoPro HD camera with 48 fps. Data were evaluated frame by frame using QuickTime. Left and right scores were calculated as follows: 6, complete miss; 5, touching rung, but sliding off and losing balance; 4, touch, miss but no loss of balance; 3, replacement, mouse placed paw on rung but quickly moves it; 2, recorrection, aims for a rung but changes direction; 1, anterior or posterior placement; 0, perfect step. The total number of mistakes was plotted for analysis as previously described in (Metz et al, supra).
  • Thermal Hyperalgesia Thermal hyperalgesia (hindpaw withdrawal from a normally innocuous heat source) was tested with a Hargreave's heat source as described in detail by (Berrocal, Y., et al., Social and environmental enrichment improves sensory and motor recovery after severe contusive spinal cord injury in the rat. J Neurotrauma, 2007. 24(11): p. 1761-72) (systemic studies) or by using the Plantar Test apparatus (37370, Ugo Basile, Comerio VA, Italy) (central studies). For peripherally administered studies, the test was repeated on the opposite paw and repeated 2 more times. In between each run, the mouse was allowed to recover for 15 min. The latency times of 3 runs per foot were averaged. For the centrally administrated studies, each paw was tested 5 times with at least 2 min break in between. The lowest and highest reflex latency scores of each paw were discarded and the bilateral mean was calculated and plotted. The behavioral test was performed once a week on each animal.
  • Open Field The open field test was performed with a non-transparent, squared plastic box (45x45x45 cm) over a period of 10 min (Lambertsen, K.L., et al, Genetic KCa3.1 -deficiency produces locomotor hyperactivity and alterations in cerebral monoamine levels. PLoS One, 2012. 7(10): p. e47744.). Movements were tracked using the SMART video tracking software (Panlab, Barcelona, Spain) connected to a video camera (SSC-DC378P, Biosite, Swiss, Sweden). The distance travelled (m), speed (cm/sec) and the entries into the three zones (Wall, Interperiphery and Center of the box) were recorded automatically. Rearing, grooming, urination and droppings were recorded manually and are presented as number (n) of events.
  • mice were deeply anaesthetized using an overdose of pentobarbital (200 mg/ml) containing lidocaine (20 mg/ml) and perfused through the left ventricle with cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS.
  • PBS cold phosphate buffered saline
  • PFA paraformaldehyde
  • the spinal cords were quickly removed and tissue segments containing the lesion area (1 cm centered on the lesion) were paraffin-embedded and cut into 10 parallel series of 15 ⁇ thick microtome sections. Sections were stored at room temperature until further processing.
  • spinal cords were cryoprotected in 0.1 M PBS + 20% sucrose and cut into 10 series of 25 ⁇ thick cryostat sections and stored at -80 °C until further processing.
  • the volume of the injury was determined from the area of every 10th section sampled by systematic uniform random sampling.
  • the area of the lesion site was estimated essentially as described by Bethea et al. (Bethea, J.R., et al., Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. J Neurotrauma, 1999. 16(10): p. 851-63).
  • samples were homogenized in RIPA buffer (0.01M sodium phosphate pH 7.2, 0.15M NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with Roche complete protease inhibitor cocktail, mixed end-over-end at 4 °C for 30 minutes and centrifuged at 14,000 rpm for 10 min at4 °C. The supernatants were transferred to fresh tubes and stored at -80 °C.
  • RIPA buffer 0.01M sodium phosphate pH 7.2, 0.15M NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA
  • Protein quantification was performed using DC Protein Assay (Bio-Rad, Hercules, CA). [00131] Protein electrophoresis and transfer. Equal amounts of protein lysates were resolved by SDS-PAGE on 10 or 15% gels and transferred to nitrocellulose membrane (Bio-Rad).
  • Protein visualization Following blocking in 5% non-fat milk in tris buffered saline + Triton (TBS-T), membranes were probed overnight at 4 °C with one of the following antibodies: GFAP recognizing glial fibrillary acidic protein (GFAP, 1 :500, BD Pharmingen), GAP43 (Growth Associated Protein 43, 1 :5000) Ibal (Ionized calcium binding adapter molecule 1, 1 :400, Wako), MBP (Myelin Basic Protein, 1 :500, Millipore), TLR4 (Toll-like Receptor 4, 1 :200, Santa Cruz) and TNFR2 (Tumor Necrosis Factor Receptor 2, 1 :200, Santa Cruz).
  • GFAP recognizing glial fibrillary acidic protein
  • GAP43 Rowth Associated Protein 43, 1 :5000
  • Ibal Ionized calcium binding adapter molecule 1, 1 :400, Wako
  • MBP Myelin Basic Protein, 1 :500,
  • Immunostaining for macrophage/microglia-specific ionized calcium binding adapter molecule 1 was performed on paraffin-embedded sections using rabbit anti-Ibal (#019- 19741, Wako)(l :600) essentially as described in Dissing-Olesen et al. (Dissing-Olesen, L., et al, Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse. Neuroscience, 2007. 149(1): p. 112-22.). Sections were counterstained with Toluidine blue (TB). All sections were stained at the same time.
  • TIFF files were grayscaled (8 bits) using Adobe Photoshop CS5 for Mac, pictures imported as group pictures (15 sections per animal) into Image J and background subtracted. Each sections was delineated using the polygon selection tool and the densitometry measured across the section was estimated using the Logl0(mean value/255) calculation.
  • mice treated with XPro 1595 directly to the cord looked healthier and exhibited more exploratory activity and movement when in their home cages.
  • open-field analysis was performed of SCI mice treated with saline, XPro 1595 or etanercept at 35 days post-injury, with observers blinded to the treatment groups, to measure changes in general activity and anxiety. No differences were found in the total distance travelled in the open field, nor in the speed at which the mice travelled, suggesting that XPro 1595 or etancercept do not affect overall activity levels (Figure 5 A and B).
  • mice treated with XPro 1595 spent significantly more time in the center of the testing area compared to both saline- and etanercept-treated mice ( Figure 5C) and displayed significantly more zone changes (Figure 5D), suggesting less anxiety in XPro 1595 -treated mice.
  • the number of droppings between groups was not different ( Figure 5E).
  • Both XProl595- and etanercept-treated mice displayed increased numbers of groomings compared to saline-treated mice ( Figure 5F).
  • most of the XPro 1595 -treated mice were capable of rearing at least once during testing, whereas none of the etanercept- or saline-treated mice were capable of rearing (data not shown).
  • Anti-TNF therapy affects microglial/macrophage responses after SCI
  • Ibal is often used as a marker to measure microglial and macrophage activation and accumulation following injury or disease to the CNS. Therefore it was investigated whether the treatment strategies regulated Ibal expression and used this as a surrogate marker for cell activation and accumulation in the cord.
  • Western blotting for Ibal revealed a significant SCI-induced increase in Ibal expression in all treatment groups both 7 days and 28 days after SCI, likely reflecting activation of resident microglia and infiltration of macrophages. At 7 days after SCI, Ibal expression was more pronounced in saline-treated mice compared to both XPro 1595- and etanercept-treated mice, suggesting that anti-TNF therapy decreases microglial activation and/or macrophage infiltration.
  • XProl595-treatment sustains MBP expression possibly through the upregulation of TNFR2 and TLR4 expression in the lesioned spinal cord
  • TLR4 levels were significantly reduced compared to 7 days after SCI.
  • GAP43 Growth associated protein 43
  • XPro 1595 is a specific inhibitor of solTNF and therefore preferentially disrupts signaling through TNFR1, without affecting tmTNF signalling through TNFR2, whereas etanercept non-specifically inhibits both solTNF and tmTNF and thus signaling through TNFR1 and TNFR2.
  • Results show that systemic administration of either XPro 1595 or etanercept by subcutaneous injection failed to improve functional recovery and reduce tissue damage in our mouse SCI model.
  • central administration of XPro 1595 but not etanercept significantly improved functional recovery and reduced tissue damage, as shown by smaller lesion sizes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une méthode de traitement d'une lésion de la moelle épinière et/ou de symptômes associés à la moelle épinière par administration, à un sujet qui en a besoin, d'un polypeptide TNF-α négatif dominant qui inhibe l'activité de TNF-α soluble mais pas celle d'un TNF-α transmembranaire.
PCT/US2015/049360 2014-09-10 2015-09-10 Méthodes de traitement d'une lésion de la moelle épinière WO2016040581A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462048715P 2014-09-10 2014-09-10
US62/048,715 2014-09-10

Publications (1)

Publication Number Publication Date
WO2016040581A1 true WO2016040581A1 (fr) 2016-03-17

Family

ID=55459558

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/049360 WO2016040581A1 (fr) 2014-09-10 2015-09-10 Méthodes de traitement d'une lésion de la moelle épinière

Country Status (1)

Country Link
WO (1) WO2016040581A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100222272A1 (en) * 2000-03-02 2010-09-02 Xencor, Inc. Pharmaceutical Compositions for the Treatment of TNF-Alpha Related Disorders
US20120189819A1 (en) * 2011-01-25 2012-07-26 Uniroll Enterprise Co., Ltd. Reinforced Rubber Tile with Laminated Top Layer and Air Cushion Effect
US20140178474A1 (en) * 2012-12-20 2014-06-26 Pls-Design Gmbh Selective local inhibition of tnfr1-mediated functions at the site of antigen/allergen presentation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100222272A1 (en) * 2000-03-02 2010-09-02 Xencor, Inc. Pharmaceutical Compositions for the Treatment of TNF-Alpha Related Disorders
US20120189819A1 (en) * 2011-01-25 2012-07-26 Uniroll Enterprise Co., Ltd. Reinforced Rubber Tile with Laminated Top Layer and Air Cushion Effect
US20140178474A1 (en) * 2012-12-20 2014-06-26 Pls-Design Gmbh Selective local inhibition of tnfr1-mediated functions at the site of antigen/allergen presentation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NOVRUP ET AL.: "Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice.", J NEUROINFLAMMATION., vol. 11, no. 159, 10 September 2014 (2014-09-10), pages 1 - 14 *
STEED ET AL.: "Inactivation of TNF Signaling by Rationally Designed Dominant-Negative TNF Variants.", SCIENCE, vol. 301, no. 5641, 2003, pages 1895 - 8, XP002393424, DOI: doi:10.1126/science.1081297 *

Similar Documents

Publication Publication Date Title
Novrup et al. Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice
JP6473534B2 (ja) Ｃ１エステラーゼ抑制因子欠乏に関連する障害の予防及び治療のためのｃ１−ｉｎｈ組成物ならびに方法
Resche et al. Haemangioblastoma, haemangioblastomatosis, and von Hippel-Lindau disease
EP2892547B1 (fr) Inhibiteur dominant-négatif de tnf-alpha pour le traitement de troubles neurologiques du snc
CN108026533B (zh) 拮抗性pdl1适体及其在癌症治疗中的应用
Jin et al. Clinical application and evaluation of anti-TNF-alpha agents for the treatment of rheumatoid arthritis
KR20170120104A (ko) 암 또는 감염의 치료를 위한 조합 제조물
D'Haens Anti-TNF therapy for Crohn's disease
WO2019213686A1 (fr) Compositions thérapeutiques et leurs utilisations
WO2015051337A2 (fr) Méthodes de traitement de troubles gastro-intestinaux
WO2016040581A1 (fr) Méthodes de traitement d'une lésion de la moelle épinière
JP2024510636A (ja) ＴＡＣＩ－Ｆｃ融合タンパク質を用いたＩｇＡ腎症の治療方法
US11793836B2 (en) Method for treating complications related to acute or chronic hyperglycemia
Barreiro et al. Termination of antigen-specific immunity by CD95 ligand (Fas ligand) and IL-10
JP2022066353A (ja) 膵炎および疼痛をデス受容体アゴニストで処置するための組成物および方法
US10519207B2 (en) Compositions and methods for treating opioid tolerance
US20230372445A1 (en) Treatment of gulf war illness
US20200147175A1 (en) Method for treating anti-her2 therapy-resistant muc4+ her2+ cancer
WO2020097150A1 (fr) Traitement d'une lésion cérébrale traumatique
US20140296154A1 (en) Method of preventing development of psoriatic lesions
WO2022056493A1 (fr) Procédés pilotés par des biomarqueurs pour le traitement d'un trouble dépressif majeur
TWI733071B (zh) 治療或診斷ｔｎｆ相關發炎疾病的ｔｎｆ靶向適配體及其用途
US20140162958A1 (en) Copolymer-1 Composition and Methods of Use
WO2016078672A1 (fr) Inhibiteur du tnf-alpha pour le traitement d'un accident vasculaire cérébral
US20220241374A1 (en) Treatment of non-alcoholic steatohepatitis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15840165

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15840165

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 15840165

Country of ref document: EP

Kind code of ref document: A1