WO2020097150A1

WO2020097150A1 - Treatment of traumatic brain injury

Info

Publication number: WO2020097150A1
Application number: PCT/US2019/059981
Authority: WO
Inventors: Kirsty J. DIXON
Original assignee: Virginia Commonwealth University
Current assignee: Virginia Commonwealth University
Priority date: 2018-11-06
Filing date: 2019-11-06
Publication date: 2020-05-14
Anticipated expiration: 2021-05-06

Abstract

A method for treating a subject suffering from traumatic brain injury, includes: administering to the subject a therapeutically effective amount of a selective inhibitor of solTNF-α, whereby the subject is treated. In some embodiments, the selective inhibitor of solTNF-α includes a DN- TNF-α protein and/or a nucleic acid encoding the DN-TNF-α protein. In some embodiments, the DN-TNF-α protein includes XPR01595.

Description

TREATMENT OF TRAUMATIC BRAIN INJURY

TECHNICAL FIELD

10001 j The invention is directed to a method for medical treatment of a subject patient suffering from traumatic brain injur_)' (TBI).

[0002] More particularly, 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.

BACKGROUND ART

[0003] Traumatic brain injury (TBI) 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.

[0004] It is estimated that about 1.7 million incidences of TBI occur in the U.S. ever}' year. Approximately 5.3 million people live with a disability caused by TBI in the U.S. alone. These numbers are greatly expanded when considering the impact of TBI worldwide.

[0005] A computed tomography scan (CT or CAT scan) is the gold standard for the radiological assessment of a TBI patient. A 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. However, most centers have readily available CT scanning, which is a more accurate test and therefore preferable to x-ray.

[0006] Many patients with moderate or severe head injuries are taken directly from the emergency room to the operating room. In many cases, surgery is performed to remove a large hematoma or contusion that is significantly compressing the brain or raising the pressure within the skull .

[0007] Those w'ho survive a TBI may develop conditions that can be debilitating, long- lasting or even permanent. [0008] Currently, there is no medication that can be given to mitigate the more serious damage that often occurs as a result of TBI, for example and without limitation, medication that prevents nerve damage or promotes nerve healing after TBI.

SUMMARY OF INVENTION

Technical Problem.

[0009] Without pharmacotherapy intervention for mitigating the impact of TBI on a subject patient, a serious or potentially fatal outcome can occur.

Solution to Problem

[0010] 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. In other aspects, 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.

[001 1 ] Other features and aspects concerning the invention and/or solutions to the problems will be recognized by one having skill in the art upon a thorough review of the appended details and descriptions, in particular when reviewed in conjunction with the enclosed drawings.

Advantageous Effects of Invention

[0012] It was surprisingly discovered that selective neutralization of soluble tumor necrosis factor -alpha (solTNFa), by systemic administration of a DN-TNF-a protein following TBI in mice, achieved: (i) significantly reduced cortical and hippocampal inflammation, (ii) improved cortical sparing and associated neurological outcome, (iii) mitigation of injury induced by pathophysiological changes in the hippocampus, and (iv) attenuation of cognitive impairment, pain- and depressive-like behaviors. This discover}' provides a reasonable basis to conclude that the solution as described herein, more particularly, 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

TBI.

BRIEF DESCRIPTION OF DRAWINGS

[ 00131 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.

[0014] 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.

[0015 ] FIG.1C shows the amino acid sequence of human TNF-a (SEQ ID O: 3).

[0016] FIG. 2 shows the positions and ammo acid changes in certain TNF-a variants.

[0017] 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.

[0018] Figure 3C shows inflammatory response in the mimed cortex two-weeks following TBI, wherein injur' upregulates GFAP protein (astrogliosis).

[0019] 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.

[0020] Figure 3E shows inflammatory' response in the injured hippocampus two-weeks following TBI, wherein injury upregulates GFAP (astrogliosis).

[0021 ] 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

[0022] 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.

[0023] Figure 31 shows inflammatory' response in the injured cortex two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity). [0024] 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.

[0025] Figure 3K shows inflammatory' response in the injured hippocampus two-weeks following TBI, wherein inj ury upregulates IBA-1 (microglial reactivity).

[0026] 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.

[0027] Figure 3M shows glial reactivity (peri-lesional GFAP) in the ipsi!atera! and contralateral hemisphere following TBI.

[0028] Figure 3N show's glial reactivity (hippocampal GFAP) m the ipsilateral and contralateral hemisphere following TBI.

[0029] Figure 30 shows glial reactivity (peri-lesional IBA-1) in the ipsilateral and contralateral hemisphere following TBI.

[0030] Figure 3P shows glial reactivity (hippocampal IBA-1) in the ipsilateral and contralateral hemisphere following TBI.

[0031 ] Figure 4A show's the amount of cortical tissue loss after TBI.

[0032] Figure 4B shows systemic XPR01595 treatment reduces the amount of cortical tissue loss relative to Vehicle treatment.

[0033] Figure 4C shows a plot summarizing spared cortical tissue according to that of

Figures 4A and 4B.

[0034] 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.

[0035] 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.

[0036] Figure 5 A shows ThyT-YFP mouse hippocampus showing dentate gyrus (DG),

CA1, CA2 and CAS regions.

[0037] Figure 5B show's neurons from CA1 region in naive mice show' extensive dendritic spines, whereas TBI promotes spine regression.

[0038] Figure 5C show's spine regression due to TBI reduced post-synaptic protein expression (PSD-95), which was rescued by XPRO 1595 treatment. [0039] Figure 5D shows XPR01595 rescued LTP in hippocampal slices.

[0040] 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

[0041] Figure 5F show's XPR01595 treatment significantly reduced the amount of hippocampal dendritic beading, compared to vehicle-treated injured mice

[0042] Figure 6A shows a plot indicating cognitive abilities m the Morris w¾ter 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.

[0043] 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.

[0044] 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.

[0045] 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).

[0046] 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.

[0047] Figure 6F shows a plot indicating hindpaw mechanical hypersensitivity

(threshold) in sham- and TBI-, vehicle- and XPR01595-ireated mice post-TBI (baseline injury', and days 3, 7, and 14). XPR01595 treatment prevents the progression of hindpaw hypersensitivity following TBI.

[0048] 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).

[0049] 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

[0050] 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. A method that applies this unexpected finding is disclosed, and 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.

Selective inhibitors of Soluble Tumor Necrosis Factor

[0051] Proteins with TNF-a antagonist activity , and nucleic acids encoding these proteins, 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”.

[0052] Examples of selective inhibitors of solTNF-a are disclosed in US 7,056,695;

US 7,101,974; US 7,144,987; US 7,244,823; US 7,446,174; US 7,662,367; and US 7,687,461; the entire contents of each of winch is hereby incorporated by reference.

[0053] 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. By“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. Thus, a variant of human TNF-a is compared to SEQ ID NO: ! (nucleic acid including codons for 6 histidines), SEQ ID NO:2 (amino acid including 6 N-terminal histidines) or SEQ ID NO:3 (amino acid without 6 N -terminal histidines). 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. As used herein 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 having skill in the art will understand that other variants may be made while retaining the function of inhibiting soluble but not transmembrane TNF-a. [0054] Thus, the proteins useful in various aspects 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.

[0055] In a preferred embodiment 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. By“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. Preferably there is an inhibition in wild-type soluble TNF-a activity in the absence of reduced signaling by transmembrane TNF-a. In a preferred embodiment, the activity⁷ of soluble TNF-a is inhibited while the activity of transmembrane TNF-a is substantially and preferably completely maintained.

[0056] The TNF proteins useful in various embodiments of the invention have modulated activity' as compared to wild type proteins. In a preferred embodiment, 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. By“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. More preferred are 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.

[0057] In one embodiment, 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. Thus, 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. In other words, it is preferable that 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. By“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). In a preferred embodiment, at least a 50% decrease in receptor activation is seen, with greater than 50%, 75%, 80-90% being preferred.

[0058] In some embodiments, the variants of the invention are antagonists of both soluble and transmembrane TNF-a. However, as described herein, 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. Thus, 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. However, 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. In these embodiments, 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, For example, variants that result in at least a 10: 1 selective inhibition of soluble to transmembrane TNF-a activity are preferred, with 50: 1, 100: 1, 200: 1, 500: 1, 1000: 1 or higher find particular use in the invention. Thus, 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. Without being bound by theory, the variants exhibiting such differential inhibition allow- the decrease of inflammation without a corresponding loss in immune response.

[0059] In one embodiment, the affected biological activity of the variants is the activation of receptor signaling by wild type TNF-a proteins. In a preferred embodiment, 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). In a preferred embodiment, the variant TNF-a protein is a variant TNF-a protein that functions as an antagonist of wild type TNF-a. Preferably, 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. By 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. In some embodiments, trimers may be formed comprising only variant TNF-a proteins.

[0060] The 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.

[0061 ] Similarly, variant TNF-a proteins, for example are experimentally tested and validated in in vivo and in in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays. For example, TNF-a activity' assays, such as detecting apoptosis via caspase activity can be used to screen for TNF-a variants that are antagonists of wild type TNF- a. Other assays include using the Sytox green nucleic acid stain to detect TNF-induced cell permeability' in an Actinomycin-D sensitized cell line. As this stain is excluded from live cells, but penetrates dying ceils, this assay also can be used to detect TNF-a variants that are agonists of wild-type TNF-a. By“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. Generally, variant TNF-a proteins that function as agonists of wild type TNF-a are not preferred. However, in some embodiments, variant TNF-a proteins that function as agonists of wild type TNF-a protein are preferred. An example of an NF kappaB assay is presented in Example 7 of U.S. Pat. No. 7,446,174, which is expressly incorporated herein by reference.

[0062] In a preferred embodiment, 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. [0063] In a preferred embodiment, 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. Expressed as a percentage, 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. 1C (SEQ ID NO:3), 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.

[0064] 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). In a similar manner,“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.

[0065] 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. In this embodiment, 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. As used herein, 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.

[0066] In a preferred embodiment, 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 Thus, for example, at position 143, 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, UΊ 15G), Y115E, Y i 151 . Y115H, UP5I, Y115K, Y115L, Y115M, Y115N, Y1 15Q, Y115R, Y115S, Y115T, Y115W, D140K, D140R, D143E, D143K, D143L, D143R, D143N, D143Q, D143R, D143S, F144N, A145D, A145E, A145F, A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, A145Y, EI46K, E146L, E146M, E146N, E146R, E146S and S147R. These may be done either individually or m combination, with any combination being possible. However, as outlined herein, preferred embodiments utilize at least 1 to 8, and preferably more, positions in each variant TNF-a protein.

[0067] In an additional aspect, 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.

[0068] In an additional aspect, 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.

[0069] In an additional aspect, 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.

[0070] In a further aspect, 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. Preferably, 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. In a preferred embodiment, 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. In an additional aspect, 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, K112D, K112E, Y115D, Y115E, Y1 15F, Y115H, Y115I, Y115K, Y115L, YI I5M, Y115N, Y1 15Q, Y115R, Y1 15S, Y1 15T, Y115W, D140K, D140R, D143E, D143K, D143L, D143R, D143N, D143Q, D143R, D143S, F144N, A145D, A145E, A145F, A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, A145Y, E146K, E146L, E146M, E146N, E146R, E146S and S147R.

[0071 ] In another preferred embodiment, 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. In addition, triple, quadruple, quintuple and the like, point variants may be generated.

[0072] In one aspect the invention makes use of TNF-a variants comprising the amino acid substitutions A145R/I97T. In one aspect, the invention provides TNF-a variants comprising the amino acid substitutions VIM, R31C, C69V, Y87H, C101 A, and A145R. In a preferred embodiment, this variant is PEGylated.

[0073] In a preferred embodiment 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”

[0074] For purposes of the present invention, 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. For the Small Domain, the preferred positions to be modified are 75 and/or 97. For the DE Loop, 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. In a preferred embodiment, substitutions at multiple receptor interaction and/or trimerization domains may be combined. Examples include, but are not limited to, simultaneous substitution of amino acids at the large and small domains (e.g. A145R and I97T), large domain and DE loop (A145R and Y87H), and large domain and trimerization domain (A145R and L57F). Additional examples include any and all combinations, e.g., I97T and Y87H (small domain and DE loop). More specifically, theses variants may be in the form of single point variants, for example 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.

[0075] Preferred double point variant positions include 57, 75, 86, 87, 97, 115, 143,

145, and 146; in any combination. In addition, 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. AI45R, 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, 1.571·. I97R, 197T, S86Q, D143N, E146K, A145R and I97T, A145R and either Y87R or Y87H; N34E and V91E; L75E and Y115Q; L75Q and Y115Q; L75E and A145R; and L75Q and A145R.

[0076] Further, 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 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.

[0077] Variant TNF-a proteins may also be identified as being encoded by variant

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. In a preferred embodiment, 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. Thus, having identified a particular amino acid sequence, 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. [0078] In one embodiment, the nucleic acid homology is determined through hybridization studies. Thus, for example, 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. High stringency conditions are known m the art; see for example Maniatis et al., Molecular· Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry' and Molecular Biol ogy— Hybrid! zation with Nucleic Acid Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993), incorporated by reference. Generally, 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. The 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. 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. In another embodiment, 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. In addition, nucleic acid variants encode TNF-a protein variants comprising the am o acid substitutions described herein. In one embodiment, the TNF-a variant encodes a polypeptide variant comprising the amino acid substitutions A145R/197T. In one aspect, 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.

[0079] The variant TNF-a proteins and nucleic acids of the present invention are recombinant. As used herein,“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. As will be appreciated by those in the art, the depiction of a single strand ( Watson”) 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. By the term“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. Thus, 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.

[0080] By“vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0081] It is understood that 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.

[0082] Similarly, 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. For example, 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. For example, 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. Furthermore, 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.

[0083] Also included within the definition of 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.

[0084] 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.

[0085 ] Using the nucleic acids disclosed herein, which encode a variant TNF-a protein, a variety of expression vectors are made. 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.

[0086] Nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, 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.

[0087] In a preferred embodiment, when the endogenous secretory sequence leads to a low level of secretion of the naturally occurring protein or of the variant TNF-a protein, a replacement of the naturally occurring secretory' leader sequence is desired. In this embodiment, an unrelated secretory leader sequence is operably linked to a variant TNF-a encoding nucleic acid leading to increased protein secretion. Thus, 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. In another preferred embodiment, 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

[0088] Generally,“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.

[0089] In general, 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. In a preferred embodiment, 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. In a preferred embodiment, 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.

[0090] In addition, the expression vector may comprise additional elements. For example, 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. Furthermore, for integrating expression vectors, 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

[0091] In addition, in a preferred embodiment, 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. In a preferred embodiment, the expression vector comprises the components described above and a gene encoding a variant TNF-tx protein. As will be appreciated by those in the art, ail combinations are possible and accordingly, as used herein, the combination of components, comprised by one or more vectors, which may be retroviral or not, is referred to herein as a“vector composition’.

[0092] 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. For example, 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. Thus, 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). 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. (See, e.g., U.S. Pat. No. 5,219,740.) 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 w¾ll 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.

[0093] 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.

[0094] 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

-IB- advantages. For a description of adenovirus vectors and their uses see, e.g., Haj -Ahmad and Graham (1986) J. Virol 57:267-274; Beit et al. (1993) J. Virol. 67:5911-5921 ; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L (1988) BioTechmques 6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476.

[0095] In a preferred embodiment, 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. For more on various AAV serotypes, see for example Cearley et al, Molecular Therapy, 16: 1710-1718, 2008, which is expressly incorporated herein in its entirety by reference.

[0096] 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.

[0097] By“adeno-associated virus inverted terminal repeats” or“AAV ITRs” is meant 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.

[0098] The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R.

M. (1994) Human Gene Therapy 5:793-801; Bems, 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. 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. Furthermore, 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.

[0099] 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). The artisan of reasonable skill will be able to determine which factor is appropriate based on the neurological disorder being treated.

[0100] The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally associated with the selected gene. Alternatively, 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. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available.

[0101] Once made, the TNF-a protein may be covalently modified. For instance, 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. These nonproteinaceous polymers may also be used to enhance the variant TNF-a's ability to disrupt receptor binding, and/or in vivo stability. In another preferred embodiment, cysteines are designed into variant or wild type TNF-a in order to incorporate (a) labeling sites for characterization and (b) incorporate PEGylation sites. For example, 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.

[0102] In one preferred embodiment, the optimal chemical modification sites are 21,

23, 31 and 45, taken alone or in any combination. In an even more preferred embodiment, a TNF-a variant of the present invention includes the R31 C mutation.

[0103] In a preferred embodiment, 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.

[0104] In another preferred embodiment, 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.

[0105] Treatment Methods

[0106] The terms ‘treatment”, “treating”, and the like, as used herein 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.

[0107] In one embodiment, 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.

[0108] In one embodiment, 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.

[0109] In some embodiments, the method may comprise subcutaneous injection of the selective inhibitor of solTNF-a for treatment of traumatic brain injury.

[01 10] In another embodiment, the method may comprise intravenous administration of the selective inhibitor of solT F-tx for treatment of traumatic brain injury.

[0111] In an alternative embodiment the method may comprise topical administration of a selective inhibitor of solTNF-a as described herein. In this embodiment the DN-TNF-a may be formulated as a lotion or cream.

[0112] Other methods of administration are further described herein or may be appreciated by one having skill in the art.

Formulations

[01 13] Depending upon the manner of introduction, the pharmaceutical composition may be formulated in a variety of ways. The concentration of the therapeutically active variant TNF-a protein in the formulation may vary from about 0.1 to 100 weight %. In another preferred embodiment, 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.

[01 14] The pharmaceutical compositions for use in embodiments of the present invention comprise a variant TNF-a protein in a form suitable for administration to a patient. In the preferred embodiment, 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-toluenesulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and 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.

[01 15] Pharmaceutical 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! alcohol ; 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 complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). In a preferred embodiment, 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. '‘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.

Controlled Release

[01 16] In addition, 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. Alternatively, 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.

[01 17] 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. Additives are well known in the art, and are used in a variety of formulations. In a further embodiment, the variant TNF-a proteins are added in a micellular formulation; see U.S. Pat. No. 5,833,948, incorporated entirely by reference. Alternatively, liposomes may be employed with the TNF-a proteins to effectively deliver the protein. Combinations of pharmaceutical compositions may be administered. Moreover, the TNF-a compositions of the present invention may be administered in combination with other therapeutics, either substantially simulta eously or co-administered, or serially, as the need may be.

[01 18] 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.

Methods of Administration

[0119] The administration of the selective inhibitor of solTNF-a m accordance with embodiments of the present invention, preferably in the form of a sterile aqueous solution, 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. In some instances, 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).

[0120] Subcutaneous

[0121] 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.

[0122] Intravenous

[0123] As is known in the art, protein therapeutics are often delivered by IV infusion or bolus. The selective inhibitor of solTNF-a may also be delivered using such methods. For example, administration may be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.

[0124] Inhaled

[0125] Pulmonary delivery may be accomplished using an inhaler or nebulizer and 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.

[0126] Oral Delivery_'

[0127] Furthermore, 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.

[0128] Transdermal

[0129] 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.

[0130] Intraocular

[0131] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being suitable for use in embodiments of this invention.

[0132] In a preferred embodiment, the selective inhibitor of solTNF-a is administered as a therapeutic agent, and can be formulated as outlined above. Similarly, 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.

[0133] Dosage

[0134] Dosage may be determined depending on the complication being treated and mechanism of delivery. Typically, 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. Suitably, 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. 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. 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.

[0135] Toxicity

10136] Suitably, 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).

Exemplary Features and Embodiments of the Invention

[0137] Certain preferred embodiments can be summarized as follows:

[0138] A method is disclosed for treating a subject suffering from traumatic brain injury , the method 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”.

[0139] The selective inhibitor of solTNF-a may comprise a DN-TNF-a protein or a nucleic acid encoding the DN-TNF-ot protein.

[0140] The DN-TNF-a protein may comprise XPR01595. Thus, in an embodiment, the method may comprise administering XPR01595 in a dose between 0.1 mg/kg and 10.0 mg/kg.

[0141] 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.

EXAMPLES

Example 1: Neutralization o f so!TNF-a Reduces Cortical and Hippocampal In flammation

Followine TBI in Mice

[0142] Brain inflammation following TBI is known to promote a plethora of undesirable pathologies including neuronal cell death, degeneration and aberrant neural plasticity. The pro-inflammatory cytokine solTNF-a plays a key role in promoting these pathologies by activating TNFR1, which not only enhances the inflammatory_' response, but also directly promotes these events. To determine whether Neutralization of solTNF-a reduces brain inflammation, mice received a TBI (CCI model), followed sixty-minutes later by the administration of XPR01595 (lOmg/kg S.C., twice weekly) or vehicle. After a two-week survival period, we assessed glial reactivity by immunohistochemically labelling cortical and hippocampal tissue with antibodies against GFAP (astrocytes) and IBA-l (microghal/macrophage). Following semi-quantitation of protein immunoreactivity using NIH Image! software, only minimal GFAP and IBA-l reactivity was observed m the uninjured hemisphere, independent of treatment (See for example, Figures 3A, B, G & H: contralateral hippocampus not shown). In comparison, GFAP and IBA-1 are all upregulated in the cortical peri-lesional region and hippocampus in the vehicle-treated injured mice (See for example. Figures 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), indicating that administration of XPR01595 prevents brain inflammation following TBI

[0143] Referring to the drawings, 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.

[0144] Figure 3C show's inflammatory response in the injured cortex two-weeks following TBI, wherein injury upregulates GFAP protein (astrogiiosis).

[0145] 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.

[0146] Figure 3E show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates GFAP protein (astrogiiosis).

[0147] 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

[0148] Figures 3(G-H) show inflammatory response in the uninjured cortex two-weeks following TBI, wherein negligible IBA-1 protein (microglial activity) is expressed.

[0149] Figure 31 shows inflammatory response m the injured cortex two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity).

[0150] 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.

[Of 51] Figure 3K show's inflammatory response in the injured hippocampus two-weeks following TBI, wherein injury upregulates IBA-1 (microglial reactivity).

[0152] 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.

[0153] Figure 3M show's glial reactivity (peri-lesional GFAP) in the ipsilateral and contralateral hemisphere following TBI. [0154] Figure 3N shows glial reactivity (hippocampal GFAP) in the ipsilateral and contralateral hemisphere following TBI.

[0155] Figure 30 show's glial reactivity (peri-lesiona) IBA-l) in the ipsilateral and contralateral hemisphere following TBI.

[0156] 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

Neurological Outcome Following TBI in Mice

[0157] To assess whether neutralization of solTNF-a can prevent neuronal cell death and thus improve tissue viability, 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). Since the injury' site overlies the motor cortex, the animals’ motor function was also assessed using the accelerating rotarod. 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. In comparison, 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.

[0158] To confirm that XPROl 595 is acting directly on the brain to regulate motor function, XPROl 595 or vehicle was infused into the lateral ventricle of the brain immediately following a TBI. Seventeen days following the injury, the motor performance of the mice was assessed on the accelerating rotarod. XPR01595-treated injured mice had significantly improved motor performance, compared to vehicle-treated injured mice (Figure 4E), suggesting that XPROl 595 is having a direct effect on the brain to regulate motor performance.

[0159] With reference to the drawings, 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. [0160] Figure 4A shows the amount of cortical tissue loss after TBI.

[0161 ] Figure 4B shows systemic XPR01595 treatment reduces the amount of cortical tissue loss relative to V ehicle treatment

[0162] Figure 4C shows a plot summarizing spared cortical tissue according to that of

Figures 4A and 4B.

[0163] 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.

[0164] 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.

Example 3: Neutralization of solTNF-a Prevents Hippocampal Dendritic Degeneration and Altered Plasticity

[0165] Since hippocampal inflammation, specifically TNF/TNFR1 activity, promotes pathophysiological changes m the hippocampus, it w-as investigated whether XPROl 595 treatment can prevent injury -induced by these pathophysiological changes. Thy-1 YFP mice display auto-fluorescent labelling of hippocampal neurons (Figure 5.4), which in the CA1 regions of naive mice have extensive dendritic spines (Figure 5B), although the number of spines is drastically reduced in the injured brain three-days after injury. This reduction corresponds with reduced PSD-95 expression seventy-two hours following TBI as identified using Western blotting (Figure 5C), hut can be rescued by systemically treating the mice sixty minutes following injury' with XPR01595, and leads to significant improvements in the long term potentiation of neurons in the CA1 region (Figure 5D: mPSC rise and decay times seventy-two hours after injury; electrophysiology preliminary data n=T animal per group). Within the DG, doublecortin-immunoreactive immature neurons in the naive mouse appear long and curvy without beading (Figure 5E), while vehicle-treated injured mice displayed extensive dendritic degeneration (i.e. beading) two weeks following injury. The number of dendritic beads were markedly reduced by treating mice systemically with XPROl 595 treatment (Figures 5E and 5F). Therefore, the use of XPRO 1595 is shown to be associated with improved hippocampal-related neurological outcomes following TBI.

[0166] With reference to the drawings, Figure 5A shows Thy 1 -YFP mouse hippocampus showing dentate gyrus (DG), CA!, CA2 and CA3 regions. [0167] Figure 5B shows neurons from CA1 region in naive mice show extensive dendritic spines, whereas TBI promotes spine regression.

[0168] Figure 5C shows spine regression due to TBI reduced post-synaptic protein expression (PSD-95), which was rescued by XPR01595 treatment.

[0169] Figure 5D shows XPR01595 rescued LTP in hippocampal slices.

[0170] 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.

[0171 ] 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

[0172] One of the most well-characterized neurological symptoms associated with hippocampal injury is cognitive impairment related to learning and memory . A common behavioral test to quantitate learning and memory' deficits in rodents following TBI is the Morris water maze (MWM). Here the MWM w'as used to assess the level of cognitive impairment seven-days post-TBI. It was observed that systemic XPRQ1595 treatment significantly improved the ability' of injured mice to learn the task (Figures 6(A-B)), and showed attend to memory improvement three-days following completion of the training phase (Figure 6C). This data indicates that XPROl 595 is a significant modulator of cognitive ability, which is likely due to reduced inflammation resulting in less aberrant hippocampal pathophysiology.

[0173] 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. For this the 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. As expected, prior to injury' 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. Conversely, 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.

[0174] Since depression and pain are frequently comorbid following TBI, and chronic pain has links to hippocampal inflammation (TNF/TNFR1 activation), the mechanical von Frey assay was used to assess hindpaw hypersensitivity, as a marker of pain in mice. This is an especially useful test because individuals with injury -induced chronic pain are also at increased risk of developing dermal mechanical and thermal hypersensitivity. Both sham and injured adult mice were systemically treated with either XPR01595 or vehicle twice a week for tw'O- weeks starting sixty-minutes following TBI, and underwent hindpaw von Frey testing prior to injury and on post-injury days 3, 7 and 14 It was observed that all sham injured animals, independent of treatment, experienced a small increase in hypersensitivity three-days following injury' (indicated by reduced mechanical threshold: Figure 6F), but this was transient and quickly returned to baseline levels. In comparison, vehicle-treated inj ured mice were significantly more hypersensitive on day three (almost a 3-fold increase compared to vehicle- treated sham mice), and this remained until the end of testing on day fourteen, suggesting these mice wnre in more pain, which lasted longer than sham-operated mice. Conversely, 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_jury_'.

[0175] Here, it is shown that XPRO 1595 therapy improves hippocampal functions following TBI m mice.

[0176] With reference to the drawings, Figure 6A shows a plot indicating latency

(seconds) to find platform in both Vehicle- and XPROl595-treated mice post-TB! (day 7 thru day 11). XPROl 595 treatment improves learning.

[0177] Figure 6B shows a plot indicating time in platform quadrant (seconds) in both

Vehicle- and XPROl 595-treated mice post-TBI (day 7 thru day 11). XPRO 1595 treatment improves learning.

[0178] 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. [0179] 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).

[0180] 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.

[0181] 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.

Example 5: Neutralization of solTNF-a Modulates the Brain’s Perception of Pain

[0182] Since TNF can regulate peripheral nociceptor activation, as well as central hippocampal control of pain perception, it was investigated whether systemic XPro1595 therapy is primarily acting centrally or peripherally to reduce injury-induced pain. For this, XPR01595 or vehicle was infused directly into the lateral ventricles of the brain following TBI, and neuropathic pain testing was performed similar to that performed following systemic administration. It was observed that vehicle- treated injured mice had significantly greater mechanical hypersensitivity (Figure 7A), compared to baseline testing. Interestingly, infusing XPRO 1595 into the lateral ventricles following an injury prevented this effect, suggesting a significant component of the increased dermal hypersensitivity followin TBI could be due to pain perception within the brain.

[0183] It 'as next investigated whether XPRO 1595 treatment can regulate neuronal activation in the somatosensory cortex, where the perception of pam is processed. In 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. Consistent with this, an extensive number of e- Fos-positive cells were observed in layers II and III of the ipsilateral and contralateral somatosensory cortex of vehicle-treated injured mice (indicated by black arrows m Figure 7B). In contrast, no labelled cells were detected in the somatosensory cortex of XPR01595-treated mice. As a positive control for the immunohistochemistry technique, c-Fos w¾s constitutively expressed in stem cells of the hippocampal DG in all tissue examined (images not shown). [0184] The ability of XP 1101595 infused directly into the brain to improve pain outcomes (Figure 7 A), and the reduction in c-Fos activity m the somatosensory cortex of XProl595-treated injured mice (Figure 7B), suggests XPR01595 may regulate pain perception by preserving hippocampal-cortical connectivity, potentially by preventing both dendritic degeneration and synaptic plasticity.

Conclusion

[0185] Following a short sixty-minute delay after TBI, 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. .

INDU S TRIAL APPLICABILITY

[0186] The invention finds utility in the treatment of traumatic brain injury and is therefore applicable to the medical field.

Claims

1. A method for treating a subject suffering from traumatic bram injury, comprising: administering to the subject a therapeutically effective amount of a selective inhibitor of so!TNF-a, whereby the subject is treated.

2. The method of claim 1, wherein the selective inhibitor of solTNF-a comprises a DN- TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein.

3. The method of claim 2, wherein the DN-TNF-a protein comprises XPROl 595.

4. The method of claim 3, wherein the method comprises administering XPROl 595 in a dose between 0.1 mg/kg and 10.0 mg/kg.

5. The method of claim 2, wherein the DN-TNF-a protein is administered:

intravenously; subcutaneously; orally; via aerosol; via topical application; or via gene therapy.

6. The method of claim 5, wherein the gene therapy comprises mesenchymal stem cells expressing a construct of the DN-TNF-a protein.

7. The method of claim 2, wherein the DN-TNF-a protein is administered via gene modified autologous or allogeneic cellular therapy.

8. A composition comprising selective inhibitor of solTNF-a for use in a method of treating a sub_ject suffering from traumatic brain injury', the method comprising: administering to the subject a therapeutically effective amount of the selective inhibitor of solTNF-a, whereby the subject is treated.

9. The composition of claim 8, wherein the selective inhibitor of so!TNF-a comprises a DN-TNF-a protein and/or a nucleic acid encoding the DN-TNF-a protein.

10 The composition of claim 9, wherein the DN-TNF-a protein comprises XPROl 595.

11. The composition of claim 10, wherein the method comprises administering

XPR01595 in a dose between 0.1 mg/kg and 10.0 mg/kg.

12. The composition of claim 9, wherein the DN-TNF-a protein is administered:

13. The composition of claim 12, wherein the gene therapy comprises mesenchymal stem cells expressing a construct of the DN-TNF-a protein.

14. The composition of claim 12, wherein the DN-TNF-a protein is administered via gene modified autologous or allogeneic cellular therapy.