WO2020097150A1 - Traitement d'une lésion cérébrale traumatique - Google Patents

Traitement d'une lésion cérébrale traumatique Download PDF

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WO2020097150A1
WO2020097150A1 PCT/US2019/059981 US2019059981W WO2020097150A1 WO 2020097150 A1 WO2020097150 A1 WO 2020097150A1 US 2019059981 W US2019059981 W US 2019059981W WO 2020097150 A1 WO2020097150 A1 WO 2020097150A1
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tnf
protein
variant
tbi
mice
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Kirsty J. DIXON
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Virginia Commonwealth University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the invention is directed to a method for medical treatment of a subject patient suffering from traumatic brain injur )' (TBI).
  • the invention is directed to a method for treating traumatic brain injury (TB1) m a subject patient by administering a selective inhibitor of soluble tumor necrosis factor alpha (solTNF-a), and more particularly, wherein the selective inhibitor of solTNF-a includes a dominant negative tumor necrosis alpha (DN-TNF-a) protein or a nucleic acid encoding the DN-TNF-a protein.
  • TB1 traumatic brain injury
  • solTNF-a selective inhibitor of soluble tumor necrosis factor alpha
  • DN-TNF-a dominant negative tumor necrosis alpha
  • Traumatic brain injury is currently recognized as any disruption in the normal function of the brain that is caused by a bump, blow, or jolt to the head, or penetrating head injury'. TBI can result when the head suddenly and violently hits an object or when an object pierces the skull and enters brain tissue. Symptoms of a TBI can be mild, moderate or severe, depending on the extent of damage to the brain. Mild cases may result in a brief change in mental state or consciousness, while severe cases may result in extended periods of unconsciousness, coma or even death.
  • a computed tomography scan (CT or CAT scan) is the gold standard for the radiological assessment of a TBI patient.
  • CT scan is easy to perform and is an excellent test for detecting the presence of blood and fractures, which are the most crucial lesions to identify in medical trauma cases.
  • Plain x-rays of the skull are recommended by some as a way to evaluate patients with only mild neurological dysfunction.
  • most centers have readily available CT scanning, which is a more accurate test and therefore preferable to x-ray.
  • the solution to the aforementioned problem, and other problems that would be appreciated by one having skill in the art includes, inter alia : a method for treating a patient suffering from traumatic brain injury, comprising: administering to the patient a therapeutically effective amount of a selective inhibitor of solTNF-a, such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-ot protein, including the biologic known as XPR01595.
  • a selective inhibitor of solTNF-a such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-ot protein, including the biologic known as XPR01595.
  • the solution may include a composition comprising a DN-TNF a protein and/or a nucleic acid encoding the DN-TNF-a protein for use in a method of treating traumatic brain injury in a patient, the method comprising: administering to the patient a therapeutically effective amount of the DN-TNF a protein and/or a nucleic acid encoding the DN-TNF-a protein, whereby the patient is treated.
  • a method for treating a patient suffering from traumatic brain injur' comprising: administering to the patient a therapeutically effective amount of a selective inhibitor of soiTNF-a, such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein, may be useful, with proper regulatory approval and subject to validation in clinical trials, for application in a human subject suffering from a selective inhibitor of soiTNF-a, such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein, may be useful, with proper regulatory approval and subject to validation in clinical trials, for application in a human subject suffering from
  • FIG. 1 A shows the nuclei c acid sequence of human TNF -a (SEQ ID NO: 1). An additional six histidine codons, located between the stall 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 ammo acid. Amino acids changed in exemplary TNF-a variants are shown in bold.
  • FIG.1C shows the amino acid sequence of human TNF-a (SEQ ID O: 3).
  • FIG. 2 shows the positions and ammo acid changes in certain TNF-a variants.
  • Figures 3(A-B) show inflammatory response in the uninjured cortex two-weeks following TBI, wherein negligible GFAP protein (astrocytes) is expressed, independent of treatment.
  • Figure 3C shows inflammatory response in the mimed cortex two-weeks following TBI, wherein injur' upregulates GFAP protein (astrogliosis).
  • Figure 3D shows inflammatory response in the injured cortex two-weeks following TBI, wherein injury upregulates GFAP (astrogliosis), hut wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 3E shows inflammatory' response in the injured hippocampus two-weeks following TBI, wherein injury upregulates GFAP (astrogliosis).
  • Figure 3F shows inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates GFAP (astrogliosis), but wherein systemic treatment with XPR01595 attenuates this upregulation
  • Figures 3(G-H) show inflammatory response in the uninjured cortex two-w'eeks following TBI, wherein negligible IBA-1 protein (microglial reactivity) is expressed, independent of treatment.
  • Figure 31 shows inflammatory' response in the injured cortex two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity).
  • Figure 3J shows inflammatory response in the injured cortex two-weeks following TBI, wherein inj ury upregulates IBA-1 (microglial reactivity), hut wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 3K shows inflammatory' response in the injured hippocampus two-weeks following TBI, wherein inj ury upregulates IBA-1 (microglial reactivity).
  • Figure 3L show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity), but wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 3M shows glial reactivity (peri-lesional GFAP) in the ipsi!atera! and contralateral hemisphere following TBI.
  • Figure 3N show's glial reactivity (hippocampal GFAP) m the ipsilateral and contralateral hemisphere following TBI.
  • Figure 30 shows glial reactivity (peri-lesional IBA-1) in the ipsilateral and contralateral hemisphere following TBI.
  • Figure 3P shows glial reactivity (hippocampal IBA-1) in the ipsilateral and contralateral hemisphere following TBI.
  • Figure 4A show's the amount of cortical tissue loss after TBI.
  • Figure 4B shows systemic XPR01595 treatment reduces the amount of cortical tissue loss relative to Vehicle treatment.
  • Figure 4C shows a plot summarizing spared cortical tissue according to that of
  • Figure 4D show's a plot illustrating time to fall (seconds) pre- and post- TBI injury; wherein when assessed on the accelerating rotarod, the injured mice systemicaliy treated with XPR01595 take longer to fall off the rotating rod, and therefore have better motor- function that Vehicle-treated injured mice.
  • Figure 4E show's XPRO 1595 -treated inj ured mice also performed better than
  • V ehicle-treated inj ured mice after XPRO 1595 or Vehicle vvas infused into the lateral ventricles of mice immediately following TBI.
  • Figure 5 A shows ThyT-YFP mouse hippocampus showing dentate gyrus (DG),
  • CA1, CA2 and CAS regions CA1, CA2 and CAS regions.
  • Figure 5B show's neurons from CA1 region in naive mice show' extensive dendritic spines, whereas TBI promotes spine regression.
  • Figure 5C show's spine regression due to TBI reduced post-synaptic protein expression (PSD-95), which was rescued by XPRO 1595 treatment.
  • Figure 5D shows XPR01595 rescued LTP in hippocampal slices.
  • Figure 5E shows doubiecortin immunoreactivity reveals long, thin and straight primary dendrites, within the dentate gyrus, which degenerate (promotion of beading) in vehicle-treated injured mice, but wherein systemic treatment with XPR01595 attenuates this upregulation
  • Figure 5F show's XPR01595 treatment significantly reduced the amount of hippocampal dendritic beading, compared to vehicle-treated injured mice
  • Figure 6A shows a plot indicating cognitive abilities m the Morris w3 ⁇ 4ter maze test (latency (seconds) to find the platform) m both vehicle- and XPR01595-treated mice post- TBI (day 7 thru day 11 ). XPR01595 treatment improves learning.
  • Figure 6B shows a plot indicating cognitive abilities in the Morris water maze test (time in platform quadrant (seconds)) in both vehicle- and XPR01595-treated mice post- TBI (day 7 thru day 11). XPR01595 treatment improves learning.
  • Figure 6C show's a plot indicating cognitive abilities in the Morris water maze probe trial (time in platform quadrant (seconds)) in both vehicle- and XPROl 595-treated mice post-TBI (day 11 and day 14).
  • XPR01595 treatment has a tendency to improve learning.
  • Figure 6D shows a plot indicating cognitive abilities in the Morris water maze probe trial (number of quadrants entered (integer)) in both vehicle- and XPRO 1595 -treated mice post-TBI (day 11 and day 14).
  • Figure 6E shows a plot indicating depressive-like behavior (% sucrose preference) in vehicle- and XPROl 595-treated mice post-TBI (baseline injury, and days 3, 7, and 14).
  • XPRQ1595 treatment prevents the onset of depressive-like behavior following TBI.
  • Figure 6F shows a plot indicating hindpaw mechanical hypersensitivity
  • Figure 7 A shows a plot indicating hindpaw' mechanical hypersensitivity' (% baseline) for both vehicle- and XPROl 595-treatment (ventricle infusion) in mice both pre- injury' and post-injury (day 17).
  • Figure 7B shows a comparison of contralateral and ipsilateral images for investigating neural activation in somatosensory' cortex by labeling c-Fos protein using fluorescent IHC two weeks post-TBI in vehicle- and XPROl 595-treated mice (ventricl e infusion). DESCRIPTION OF EMBODIMENTS
  • Disclosed herein is the novel aid unexpected finding that selective neutralization of soluble tumor necrosis factor -alpha (solTNFa), by systemic administration of a DN-TNF-a protein following TBI m mice, achieved: (i) significantly reduced cortical and hippocampal inflammation, (ii) improved cortical sparing aid associated neurological outcome, (hi) mitigation of injury induced by pathophysiological changes m the hippocampus, and (iv) attenuation of cognitive impairment, pain- and depressive-like behaviors.
  • solTNFa soluble tumor necrosis factor -alpha
  • a method that applies this unexpected finding comprises the step of: administering, to a subject suffering from a traumatic brain injury, a therapeutically effective amount of a selective inhibitor of solTNF-a, such as a DN-TNF-a protein or a nucleic acid encoding the DN-TNF-a protein, for example, the DN-TNF-a protein known as XPR01595.
  • a selective inhibitor of solTNF-a such as a DN-TNF-a protein or a nucleic acid encoding the DN-TNF-a protein, for example, the DN-TNF-a protein known as XPR01595.
  • Proteins with TNF-a antagonist activity were previously discovered which function to inhibit or otherwise neutralize the soluble form of TNF-a (solTNF-a) without inhibiting transmembrane TNF-a (tmTNF-a); collectively these proteins and nucleic acids encoding these proteins are herein collectively referred to as“selecti v e inhibitors of solTNF-a”.
  • Preferred selective inhibitors of solTNF-a may be dominant negative TNF-a proteins, referred to herein as“DNTNF-a,”“DN-TNF-a proteins,”“TNFa variants,”“TNFa variant proteins,”“variant TNF-a,”“variant TNF-a,” and the like.
  • “variant TNF-a” or “TNF-a proteins” is meant TNFa or TNF-a proteins that differ from the corresponding wild type protein by at least 1 amino acid.
  • a variant of human TNF-a is compared to SEQ ID NO: !
  • DN- TNF-a proteins are disclosed in detail in U.S. Pat. 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.
  • the proteins useful in various aspects of the invention are antagonists of wild type TNF-a.
  • antagonists of wild type TNF-a is meant that the variant TNF-a protein inhibits or significantly decreases at least one biological activity of wild-type TNF-a.
  • the variant is antagonist of soluble TNF-a, but does not significantly antagonize transmembrane TNF-a, e.g., DN-TNF-a protein as disclosed herein inhibits signaling by soluble TNF-a, but not transmembrane TNF-a.
  • inhibits the activity of TNF-a and grammatical equivalents is meant at least a 10% reduction in wild-type, soluble TNF-a, more preferably at least a 50% reduction in wild-type, soluble TNF-a activity , and even more preferably, at least 90% reduction in wild-type, soluble TNF-a activity.
  • the activity 7 of soluble TNF-a is inhibited while the activity of transmembrane TNF-a is substantially and preferably completely maintained.
  • the TNF proteins useful in various embodiments 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 selecti vely kill or inhibit cells.
  • Variant TNF- a proteins that exhibit less than 50% biological activity as compared to wild type are preferred.
  • variant TNF-a proteins that exhibit less than 25%, even more preferred are variant proteins that exhibit less than 15%, and most preferred are variant TNF-a proteins that exhibit less than 10% of a biological activity of wild-type TNF-a.
  • Suitable assays include, but are not limited to, caspase assays, TNF-a cytotoxicity assays, DNA binding assays, transcription assays (using reporter constructs), size exclusion chromatography assays and radiolabeling/immuno-precipitation,), and stability assays (including the use of circular dichroism (CD) assays and equilibrium studies), according to methods know in the art.
  • At least one property' critical for binding affinity' of the variant TNF-a proteins is altered when compared to the same property' of wild type TNF-a and in particular, variant TNF-a proteins with altered receptor affinity are preferred. Particularly preferred are variant TNF-a with altered affinity toward oligomerization to wild type TNF-a.
  • the invention makes use of variant TNF-a proteins with altered binding affinities such that the variant TNF-a proteins will preferentially oligomerize with w ld 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 implemented in embodiments 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 pathw-ay(s).
  • at least a 50% decrease in receptor activation is seen, with greater than 50%, 75%, 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 biological activity of at least 10%, 25, 50, 75, 80, 90, 95, 99 or 100% all being preferred.
  • some of the variants outlined herein comprise selective inhibition; that is, they inhibit soluble TNF-a activity but do not substantially inhibit transmembrane TNF-a.
  • transmembrane TNF-a activity it is preferred that at least 80%, 85, 90, 95, 98, 99 or 100% of the transmembrane TNF-a activity is maintained.
  • This may also be expressed as a ratio; that is, selective inhibition can include a ratio of inhibition of soluble to transmembrane TNF-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 homotrimenc 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.
  • variants exhibiting such differential inhibition allow- the decrease of inflammation without a corresponding loss in immune response.
  • 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 (TNFR1) or p75 TNF-R (TNFR2).
  • the variant TNF-a protein is a variant TNF-a protein that 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 heterotnmeric TNF-a.
  • Mixed trimers may comprise 1 variant TNF-a protem:2 wild type TNF- a proteins, 2 variant TNF-a proteins: 1 wild type TNF-a protein.
  • trimers may be formed comprising only variant TNF-a proteins.
  • variant TNF-a antagonist proteins implemented in embodiments 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 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. Pat. No. 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. Pat. No. 7,446,174, which is expressly incorporated herein by reference.
  • the variant TNF-a protein has an amino acid sequence that differs from a wild type TNF-a sequence by at least 1 amino acid, with from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 amino acids all contemplated, or higher.
  • the variant TNF-a proteins of the invention preferably are greater than 90% identical to wild- type, with greater than 95, 97, 98 and 99% all being contemplated. Stated differently, based on the human TNF-a sequence of FIG. IB (SEQ ID NO:2) excluding the N- terminal 6 histidines, as shown in FIG.
  • 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.
  • 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 m 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 m the coding sequence of the cell cycle protein.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • TNF-a proteins may be fused to, for example, other therapeutic proteins or to other proteins such as Fc or serum albumin for therapeutic or pharmacokinetic purposes.
  • a TNF-a protein implemented in embodiments of the invention is operably linked to a fusion partner.
  • the fusion partner may be any moiety that provides an intended therapeutic or pharmacokinetic effect. Examples of fusion partners include but are not limited to Human Serum Albumin, a therapeutic agent, a cytotoxic or cytotoxic molecule, radionucleotide, and an Fc, etc.
  • an Fc fusion is synonymous with the terms“immunoadhesin”,“Ig fusion”,“Ig chimera”, and“receptor globulin” as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9: 195-200, both incorporated by reference).
  • An Fc fusion combines the Fc region of an immunoglobulin with the target- binding region of a TNF-a protein, for example. See for example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which are hereby 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, 1 15, 140, 143, 144, 145, 146, and 147.
  • Preferred amino acids for each position, including the human TNF-a residues, are shown in FIG. 2
  • preferred ammo acids are Glu, Asn, Gin, Ser, Arg, and Lys: etc.
  • Preferred changes include: VIM, Q21C, Q21 R, E23C, R31C, N34E, V91 E, Q21R, N30D, R31 C, 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, C101 A, A1 1 1 R, Al l IE, K1 12D, KI G2E, U ⁇ 15G),
  • 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. Pat. No. 7,662,367, which is incorporated herein by reference.
  • the invention makes use of methods of forming a ' INF
  • IX 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 makes use of 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 makes use of 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, V9IE, Q21R, N30D, R31C, R31 1, 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, C1 G1A, A111R, At 11E,
  • 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 , 1 15, 143, 145, and 146 may be combined to form double variants.
  • triple, quadruple, quintuple and the like, point variants may be generated.
  • the invention makes use of 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, C101 A, and A145R.
  • this variant is PEGylated.
  • the variant is XPR01595, a PEGylated protein comprising VIM, R31C, C69V, Y87H, C101 A, and A145R mutations relative to the wild type human sequence, also referred to as“XPro”
  • 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 11), 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, Y1 15K, Y 1 151. Y115T, A145E or A145R. These single point variants may be combined, for example, Y115I and A145E, or Y115I and A145R, or Y115T and A145R or Y115I and A145E; or any other combination.
  • Preferred double point variant positions include 57, 75, 86, 87, 97, 115, 143,
  • double point variants may be generated including L57F and one of Y1 151, Y1 15Q, Y115T, D143K, D143R, D143E, A145E, AI45R, E146K or E146R.
  • Other preferred double variants are YT15Q and at least one of D143N, D143Q, L 145 K.
  • triple point variants may be generated.
  • Preferred positions include 34,
  • triple point variants include V91 E, N34E and one of Y1 151, Y1 15T, D143K, D143R, A145R, A145E E146K, and E146R.
  • Other triple point variants include L7SE and Y87H and at least one of Y1 15Q, A145R, Also, L75K, Y87H and Yl 15Q. More preferred are the triple point variants V91E, N34E and either A145R or A145E.
  • variants may also be identified as being encoded by variants
  • TNF-a nucleic acids In the case of the nucleic acid, the o verall 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. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of w hich encode the variant TNF-a proteins of the present invention.
  • nucleic acid homology is determined through hybridization studies.
  • nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in FIG. 1 A (SEQ ID NO: 1 ) or its complement and encode a variant TNF-a protein is considered a variant TNF-a gene.
  • stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary' to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • 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.
  • nucleic acid variants encode TNF-a protein variants comprising the am o acid substitutions described herein.
  • the TNF-a variant encodes a polypeptide variant comprising the amino acid substitutions A145R/197T.
  • the nucleic acid variant encodes a polypeptide comprising the amino acid substitutions VIM, R31C, C69V, Y87H, CIOIA, 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 ceil or organism, it will replicate non-recombmantly, 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 m its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a variant TNF-a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • all of the variant TNF-a proteins outlined herein are in a form not normally found in nature, as they contain amino acid substitutions, insertions and deletions, with substitutions being preferred, as discussed below.
  • variant TNF-a proteins of the present invention are amino acid sequence variants of the variant TNF-a sequences outlined herein and shown m 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, insertiona! 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.
  • the term‘ ‘ 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 preprotem 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 m the art for a variety' of host cells.
  • the transcriptional and translational regulatory' sequences may include, but are not limited to, promoter sequences, nbosomal 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 ceils, 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 that 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-tx protein.
  • a“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 ail 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
  • 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 w3 ⁇ 4ll 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 that 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 w'eil as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insert! onal 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
  • the viral vectors used in the subject methods are
  • AAV vectors By an“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 nucleoti des, 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 are known. See, e.g., Kotin, R.
  • 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. Additionally, the AAV !TR may be derived from any of several AAV serotypes, including without limitation, AAV-l, 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 ceil 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.
  • 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. Nos. 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 m various assays as are also well known m 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 Review's, 54, 477-485 (2002) and M J Roberts et al, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both hereby incorporated by reference.
  • the optimal chemical modification sites are 21,
  • a TNF-a variant of the present invention includes the R31 C mutation.
  • the variant TNF-a protein is purified or isolated after expression.
  • Variant TNF-a proteins may be isolated or purified in a variety of ways known to those skilled in the art. depending on what other components are present in the sample.
  • the TNF-a protein is administered via gene modified autologous or allogeneic cellular therapy, wherein the gene therapy comprises mesenchymal stem cells expressing a construct of the TNF-a protein, preferably a DN-TNF-a protein, more preferably XPR01595.
  • 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.
  • Such prevention or reduction prior to affliction refers to administration of the compound or composition as described herein to a patient that is not at the time of administration afflicted with the disease or condition.
  • Preventing also encompasses preventing the recurrence or relapse-prevention of a disease or condition or of symptoms associated therewith, for instance after a period of improvement.
  • a selective inhibitor of solTNF-a as described herein is administered peripherally to a patient in need thereof to reduce inflammation, improve cortical sparing and associated neurological outcome, mitigate injury' induced by pathophysiological changes in the hippocampus, and/or atenuate cognitive impairment, pain- and depressive-like behaviors.
  • the treatment method includes administering to the patient suffering from traumatic brain injury a therapeutically effective amount of a selective inhibitor of solTNF-o, such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein, for example and without limitation, the biologic known as XPR01595, whereby the patient is treated.
  • a selective inhibitor of solTNF-o such as a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein, for example and without limitation, the biologic known as XPR01595, whereby the patient is treated.
  • the method may comprise subcutaneous injection of the selective inhibitor of solTNF-a for treatment of traumatic brain injury.
  • the method may comprise intravenous administration of the selective inhibitor of solT F-tx for treatment of traumatic brain injury.
  • the method may comprise topical administration of a selective inhibitor of solTNF-a as described herein.
  • the DN-TNF-a may be formulated as a lotion or cream.
  • the pharmaceutical composition may be formulated in a variety of ways.
  • concentration of the therapeutically active variant TNF-a protein in the formulation may vary from about 0.1 to 100 weight %.
  • the concentration of the variant TNF-a protein is in the range of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram of body weight being preferred.
  • compositions for use in embodiments of the present invention comprise a variant TNF-a protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are m 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, mandehc acid, methanesulfonic acid, ethanesulfonic acid, p-to
  • “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 tertiar' amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • 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. Lyophiiization 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 orbenzy!
  • 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 orbenzy!
  • alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cydohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, com and other starches; binding agents; additives; coloring agents; salt-forming counter-ions such as sodium; metal complexe
  • the pharmaceutical composition that comprises the TNF-a variant of the present invention may be in a water- soluble form.
  • the TNF-a 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, ethanesul ionic acid, p-toluenesul tonic 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, diethyl amine, tri ethyl amine, 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 in accordance with embodiments of the present invention. Examples include, but are not limited to, encapsulation m 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 transfect! on 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 transfect! on agents.
  • controlled release systems may be used to release
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, com and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, com and other starches
  • binding agents such as microcrystalline cellulose, lactose, com and other starches
  • sweeteners and other flavoring agents coloring agents
  • polyethylene glycol polyethylene glycol.
  • Additives are well known in the art, and are used in a variety of formulations.
  • the variant TNF-a proteins are added in a micellular formulation; see U.S. Pat. No. 5,833,948, incorporated entirely by reference.
  • liposomes may be employed with the TNF-a proteins to effectively deliver the protein.
  • Combinations of pharmaceutical compositions may be administered.
  • Dosage forms for the topical or transdermaJ administration of a DN-TNF-a protein disclosed herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the DN-TNF-a protein may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • Powders and sprays can contain, in addition to the DN-TNF-a protein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide pow'der, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofiuorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • the administration of the selective inhibitor of solTNF-a m accordance with embodiments of the present invention is done peripherally, in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, mtranasal!y, transdermally, intraperitonea!ly, intramuscularly, intrapulmonary, vaginaily, rectaliy, or mtraocularly.
  • the selective inhibitor of solTNF-a may be directly applied as a solution, salve, cream or spray.
  • the selective inhibitor of solTNF-a may also be delivered by bacterial or fungal expression into the human system (e.g., WO 04046346 A2, hereby incorporated by reference).
  • Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition.
  • Many protein therapeutics are not sufficiently potent to allow' for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate.
  • a selective inhibitor of solTNF-a may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
  • protein therapeutics are often delivered by IV infusion or bolus.
  • the selective inhibitor of solTNF-a may also be delivered using such methods.
  • administration may be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
  • Pulmonary delivery may be accomplished using an inhaler or nebulizer and a formulation comprising an aerosolizing agent.
  • a formulation comprising an aerosolizing agent For example, inha!able technology, or a pulmonary delivery system may be used.
  • the selective inhibitor of solTNF-a may be more amenable to intrapulmonary delivery.
  • the selective inhibitor of solTNF-a may also be more amenable to intrapulmonary administration due to, for example, improved solubility or altered isoelectric point.
  • the selective inhibitor of solTNF-a may be more amenable to oral delivery' due to, for example, improved stability' at gastric pH and increased resistance to proteolysis.
  • Transdermal patches may have the added advantage of providing controlled delivery' of the selective inhibitor of solTNF-a to the body. Dissolving or dispersing DN-TNF- a protein m the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of DN-TNF-a protein across the skin. Either providing a rate controlling membrane or dispersing DN-TNF-a protein in a polymer matrix or gel can control the rate of such flux.
  • Ophthalmic formulations are also contemplated as being suitable for use in embodiments of this invention.
  • the selective inhibitor of solTNF-a is administered as a therapeutic agent, 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.
  • Dosage may be determined depending on the complication being treated and mechanism of delivery.
  • an effective amount of the selective inhibitor of solTNF 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 ever) ' day or once a week or once a month.
  • a DN-TNF protein may be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly.
  • a DN-TNF protein may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the agent in the subject. The dosage and frequency of administration can vary' depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively lo dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject show's partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • 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 LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used m formulating a dosage range that is not toxic for use in human.
  • the dosage of the agent described herein lies suitably within a range of circulating concentrations that include the effective dose with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).
  • a method for treating a subject suffering from traumatic brain injury comprising: administering to the subject a therapeutically effective amount of a selective inhibitor of solTNF-a, whereby the subject is treated; for purposes herein, this shall constitute‘"the method”.
  • the selective inhibitor of solTNF-a may comprise a DN-TNF-a protein or a nucleic acid encoding the DN-TNF-ot protein.
  • the DN-TNF-a protein may comprise XPR01595.
  • the method may comprise administering XPR01595 in a dose between 0.1 mg/kg and 10.0 mg/kg.
  • the DN-TNF-a protein can be administered: intravenously; subcutaneously; orally; via aerosol; via topical application; or via gene therapy.
  • the gene therapy may comprise mesenchymal stem ceils expressing a construct of the DN-TNF-a protein.
  • the DN-TNF-a protein can be administered via gene modified autologous or allogeneic cellular therapy.
  • mice received a TBI (CCI model), followed sixty-minutes later by the administration of XPR01595 (lOmg/kg S.C., twice weekly) or vehicle.
  • FIGS 3C, E, I, K, M-P Systemic treatment with XPR01595 significantly reduced astrocytic and microglial reactivity in the cortical peri-lesiona! region and hippocampus (See for example.
  • Figures 3D, F, J, L, M-P) See for example.
  • Figures 3D, F, J, L, M-P indicating that administration of XPR01595 prevents brain inflammation following TBI
  • Figures 3(A-B) show- inflammatory response in the uninjured cortex two-weeks following TBI, wherein negligible GFAP protein (astrocytes) is expressed, independent of treatment.
  • Figure 3C show's inflammatory response in the injured cortex two-weeks following TBI, wherein injury upregulates GFAP protein (astrogiiosis).
  • Figure 3D shows inflammatory response in the injured cortex two-w'eeks following TBI, wherein injury upregulates GFAP protein (astrogiiosis), hut wherein systemic treatment with XPR01595 atenuates this upregulation.
  • Figure 3E show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates GFAP protein (astrogiiosis).
  • Figure 3F shows inflammatory response in the injured hippocampus two-weeks following TBI, wherein injur ⁇ ' upregulates GFAP protein (astrogiiosis), but wherein systemic treatment with XPR01595 attenuates this upregulation
  • Figures 3(G-H) show inflammatory response in the uninjured cortex two-weeks following TBI, wherein negligible IBA-1 protein (microglial activity) is expressed.
  • Figure 31 shows inflammatory response m the injured cortex two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity).
  • Figure 3J shows inflammatory response in the inj ured cortex two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity), but wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 3K show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity).
  • Figure 3L show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity), but wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 3M show's glial reactivity (peri-lesional GFAP) in the ipsilateral and contralateral hemisphere following TBI.
  • Figure 3N shows glial reactivity (hippocampal GFAP) in the ipsilateral and contralateral hemisphere following TBI.
  • Figure 30 show's glial reactivity (peri-lesiona) IBA-l) in the ipsilateral and contralateral hemisphere following TBI.
  • Figure 3P shows glial reactivity (hippocampal IBA-l) in the ipsilateral and contralateral hemisphere following TBI.
  • Example 2 Neutralization of solTNF-a Improves Cortical Sparing and Associated
  • solTNF-a was selectively inhibited by systematically administering XPR01595 beginning sixty -minutes following TBI for a period of two- weeks. Coronal serial brain sections forty microns (40 pm) thick were then cut and stained with the DNA dye DAPI to show the location of intact cortical tissue. Two weeks following TBI it was observed that XPR01595 therapy prevented tissue loss at the cortical injury' site, compared to vehicle-treated injured mice, resulting in a signifi cant increase in the amount of spared cortical tissue (See for example, Figures 4A-C).
  • the animals motor function was also assessed using the accelerating rotarod.
  • the mice Prior to injury, the mice can maintain their ability to walk on the accelerating rod for up to 220-240 seconds (Figure 4D), although TBI renders the mice less able to walk on the accelerating rod, resulting in them falling off sooner.
  • the XPROl 595-treated injured group do not exhibit a motor deficit following the inj ury and are significantly better able to walk on the accelerating rotarod than the vehicle-treated injured group. Collecti vely, this data suggests that treating the mice systemically with XPROl 595 improves the cortical injury site pathophysiology' leading to improved motor skills.
  • Figures 4(A-B) show images of serial sections through an injury site from individual mice, wherein it is observed that controlled cortical injury (CCI) at a velocity of 3.0m/ ' s and a depth of 0.5mm results in a small peri-!esiona! cavity.
  • CCI controlled cortical injury
  • Figure 4A shows the amount of cortical tissue loss after TBI.
  • Figure 4B shows systemic XPR01595 treatment reduces the amount of cortical tissue loss relative to V ehicle treatment
  • Figure 4C shows a plot summarizing spared cortical tissue according to that of
  • Figure 4D shows a plot illustrating time to fall (seconds) pre- and post- TBI injury; wherein when assessed on the accelerating rotarod, the injured mice systemically treated with XPR01595 take longer to fall off the rotating rod, and therefore have better motor- function that Vehicle-treated injured mice.
  • Figure 4E shows XPRO 1595 -treated injured mice also performed better than
  • V elucle-treated injured mice after XPROl 595 or Vehicle was infused into the lateral ventricles of mice immediately following TBI. This result confirms the brain-specific effect of TBI.
  • Figure 5A shows Thy 1 -YFP mouse hippocampus showing dentate gyrus (DG), CA!, CA2 and CA3 regions.
  • Figure 5B shows neurons from CA1 region in naive mice show extensive dendritic spines, whereas TBI promotes spine regression.
  • Figure 5C shows spine regression due to TBI reduced post-synaptic protein expression (PSD-95), which was rescued by XPR01595 treatment.
  • Figure 5D shows XPR01595 rescued LTP in hippocampal slices.
  • Figure 5E show's doublecortin immunoreactivity reveals long, thin and straight primary' dendrites, within the dentate gyrus, which degenerate (promotion of beading) in vehicle-treated injured mice, but wherein systemic treatment with XPR01595 attenuates this upregulation.
  • Figure 5F show's XPR01595 treatment significantly reduced the amount of hippocampal dendritic beading, compared to vehicle-treated injured mice.
  • Example 4 Neutralization of sollNF-a Improves Hippocampal-Related Neurological Outcomes ( ' attenuation of cognitive impairment, pain- and depressive-like behaviors) Following TBI in Mice
  • mice As well as cognitive impairment, depression is frequently associated with aberrant hippocampal pathology' following TBI, therefore the development of depressive-like symptoms m injured mice was assessed.
  • sucrose preference test was utilized, where mice are given free access to both water and a 2% sucrose solution overnight, and the volume of sucrose drunk is assessed as a percentage of total liquid drunk.
  • Figure 6E the naive uninjured untreated mice showed ⁇ 90% preference for the sucrose solution (Figure 6E), but this was significantly reduced in vehicle-treated mice acutely following injury, suggesting the CCI brain injury' model promotes depressive-like symptoms.
  • treating injured mice systemically with XPROl 595 prevented a reduction in their preference for the 2% sucrose solution, suggesting that XPR01595 may affect the onset of depressive-like symptoms, and may be a useful clinical tool.
  • XPRO 1595 -treated inj ured mice experienced less hypersensitivity than vehicle-treated injured mice (1 6-fold increase compared to XPRO l 595-treated sham mice), and this quickly improved such that they were no longer experiencing significantly more hypersensitivity at the end of testing. This indicates that using XPRO 1595 therapy can reduce levels of pain associated with brain in j ury ' .
  • Figure 6A shows a plot indicating latency
  • Figure 6B shows a plot indicating time in platform quadrant (seconds) in both
  • Figure 6C show's a plot indicating cognitive abilities in the Morris w'ater maze test (time in platform quadrant (seconds)) in both Vehicle- and XPROl 595-treated mice post- TBI (day 1 1 and day 14). XPRO 1595 treatment has a tendency to improve learning.
  • Figure 6D shows a plot indicating cognitive abilities in the Morris water maze probe trial (number of quadrants entered (integer)) in both Vehicle- and XPR01595-treated mice post-TBI (day 11 and day 14).
  • Figure 6E shows a plot indicating depressive-like behavior (% sucrose preference) in Vehicle- and XPRO 1595 -treated mice post-TBI (baseline injury, and days 3, 7, and 14).
  • XPR01595 treatment prevent the onset of depressive-like behavior following TBI.
  • Figure 6F show's a plot indicating hindpaw mechanical hypersensitivity threshold) in sham- and TBI-, Vehicle- and XPRQ 1595 -treated mice post-TBI (baseline injur ⁇ ', and days 3, 7, and 14). XPR01595 treatment prevents the progression of hindpaw hypersensitivity following TBI.
  • XPRO 1595 treatment can regulate neuronal activation in the somatosensory cortex, where the perception of pam is processed.
  • mice systemically treated with either XPRO 1595 or vehicle the somatosensory' cortex was immunohistoehemically examined to observe c-Fos expression, as a marker of neural activity in that region.
  • Brain injury' is known to cause a loss of input to the somatosensory cortex, resulting in hyper-excitability of neurons (homeostatic activity' regulation), which increases the number of labelled c-Fos-positive neurons.
  • XProl595 was administered systemically and reduced inflammation (glial reactivity) was observed in cortical and subcortical brain regions, along with improved cortical tissue sparing (equates to improved neuronal survival) and reduced hippocampal dendritic degeneration and plasticity. Tins improved pathophysiology lead to significant improvements in motor function, learning and memory, depressive-like behavior, and pain perception. .
  • the invention finds utility in the treatment of traumatic brain injury and is therefore applicable to the medical field.

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Abstract

L'invention concerne une méthode de traitement d'un sujet souffrant d'une lésion cérébrale traumatique, qui consiste à administrer à celui-ci une quantité thérapeutiquement efficace de l'inhibiteur sélectif de solTNF-α, de manière à traiter ledit sujet. Dans certains modes de réalisation, l'inhibiteur sélectif du solTNF-α, comprend une protéine DN-TNF-α, et/ou un acide nucléique codant pour la protéine DN-TNF-α. Dans certains modes de réalisation, la protéine DN-TNF-V comprend XPR01595.
PCT/US2019/059981 2018-11-06 2019-11-06 Traitement d'une lésion cérébrale traumatique WO2020097150A1 (fr)

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US20150079086A1 (en) * 2010-11-01 2015-03-19 Tact Ip Llc Methods for treatment of brain injury utilizing biologics
US20150239951A1 (en) * 2012-09-10 2015-08-27 Xencor Methods of Treating Neurological Diseases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150079086A1 (en) * 2010-11-01 2015-03-19 Tact Ip Llc Methods for treatment of brain injury utilizing biologics
US20150239951A1 (en) * 2012-09-10 2015-08-27 Xencor Methods of Treating Neurological Diseases

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