WO2017089606A1 - Therapy to increase remyelination - Google Patents

Abstract

The present invention relates to a therapy for the treatment of demyelination, including multiple sclerosis, based upon suppressing the action of Ephrin-B1, Ephrin-B2 and Ephrin-B3. The demyelination may also be associated with other diseases and/or stroke, radiation injury, heavy metal poisoning, compressive injury of the spinal cord or brain, traumatic injury to the brain or spine or chemical injury.

Description

Therapy to increase remyelination

The present invention relates to a therapy for the treatment of demyelination, including multiple sclerosis, based upon suppressing the action of Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

Background to the Invention

Myelin has a vital role in the function of the nervous system by insulating neurons. Myelin sheaths are layered, lipid-rich structures that wrap around axons to mediate salutatory signal conduction along them and provide them with trophic support. The speed of signal conduction is greater in myelinated neurons because the action potential travels differently through myelinated cells.

Sections of the axon are coated with myelin, and the breaks between each section are called the nodes of anvier. The insulating effect of the myelin means that depolarisation only occurs at the nodes of Ranvier, causing the action potential to jump from one node to the next.

Demyelination is the damage to the myelin sheath around nerves. This can be caused by a specific demyelinating disease (as discussed in more detail below) or by traumatic injuries of the brain and spinal cord, stroke and radiation injuries, for example. Loss of myelin may be caused by loss of oxygen or physical compression.

Myelin is a target of demyelinating diseases, which have diverse symptoms but are grouped together because they all cause damage to the myelin sheaths that surrounds neurons; this is demyelination. One of the most common demyelinating diseases is multiple sclerosis (MS), although many others exist. There are many causes of such diseases, including genetic factors, due to exposure to environmental toxins or infection, caused by an immune response, or, as yet, of unknown cause. The chronic demyelination that follows is associated with axonal loss of function, so providing a possible mechanism for progressive disability in patients suffering from diseases in which chronic demyelination occurs.

Myelin is present in the central nervous system (CNS) and peripheral nervous system (PNS); however it is thought that only the central nervous system is affected in several demyelinating diseases such as MS. MS is thought to involve an immune-mediated process in which an abnormal response of the body's immune system is directed against the CNS, in particular the myelin that is wrapped around the axons. In response to myelin degeneration multipotent parenchymal progenitor cells called oligodendrocyte progenitor cells (OPCs) are activated and recruited to the damaged areas. These can regenerate myelin to some extent, but the process is often incomplete, leaving axons permanently demyelinated and vulnerable to degeneration. These unrepaired areas causes the symptoms seen in patients with demyelination of neurons, including blurred double vision, clumsiness, hemiparesis, paresthesias, impaired muscle coordination, loss of sensation, unsteady gait, and hearing and speech problems. emyelination of lesions often remains incomplete despite the presence of OPCs, particularly in MS. The reasons for remyelination failure in MS are still not fully understood, and are expected to be complex. However, it has been demonstrated that OPCs that fail to differentiate into remyelinating oligodendrocytes are present in a proportion of demyelinated lesions. Failure of OPC differentiation is a major cause of remyelination failure.

Differentiation of OPCs is inhibited, inter alia, by myelin debris, and thus the phagocytic clearance of myelin debris is an important step in remyelination. This essential role is performed by

macrophages, who also contribute to remyelination by secreting cytokines. However, it has been demonstrated that acute MS lesions have accumulated myelin proteins.

The present inventors have been examining myelin proteins to determine whether there are molecular substrates in myelin that are responsible for inhibiting OPC differentiation (Kotter et al , J. Neurosci 26 (2006) : 328-332) in the work leading up to the present invention. A number of potential inhibitors have been proposed including axonal PSA-NCAM in chronically demyelinated lesions, inhibitors that form part of the glial scar such as astrocytic hyaluronan and notch-jagged signalling. It has previously been postulated that a transmembrane signalling protein, Ephrin-B3 is an inhibitor of remyelination by inhibiting OPC differentiation and activation. The present inventors have confirmed this hypothesis, but have noted that neutralising the effect of Ephrin-B3 alone is not sufficient to allow OPC differentiation to normal levels in vivo. Thus, work has been conducted on determining further proteins in myelin debris that may be inhibiting OPC determination in order to provide a more effective treatment.

Axon remyelination is distinct from axon regeneration in general. Axon regeneration is the process of promoting the reformation of destroyed or severed neuronal axons. Axons are slender projections of a nerve cell or neuron that conduct electrical impulses which are transmitted from the neuron's cell body to recipient cells via synapses. Axons or nerve fibres themselves are microscopic in diameter but may be significantly longer extending to a meter or even longer. Axons of many neurons are sheathed in myelin, which is a lipid rich membrane structure performing a protection and signal accelerating function. Myelin is made up primarily of a glycolipid called

galactocerebroside. The intertwining of the hydrocarbon chains of sphingomyelin serve to strengthen the myelin sheath.

Demyelination is the act of loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative, in particular autoimmune diseases. Contrary thereto, axonal loss is the destruction of the neuronal axon. There are important phenotypic, cellular, and molecular differences between demyelination and axonal loss. Similarly, the processes of myelin and axon regeneration, i.e. remyelination and axon regeneration, are characterised by distinct phenotypic, cellular and molecular processes. Axon regeneration primarily constitutes the regrowth of the axon from neuronal cells and reformation of new synapses, whereas remyelination is the process of developing a new myelin sheath on existing but demyelinated axons, which is mediated by differentiation of OPCs in the vicinity of the neural cells, or OPCs transplanted into the diseased CNS or cerebrospinal fluid (CSF) cavities, into new myelinating oligodendrocytes.

Axonal integrity is tightly coupled with functional myelin, as subtle changes in the molecular composition of myelin can result in mid- to long term axonal degeneration under otherwise physiological conditions. The trophic function of myelin sheaths is also very likely to be of significance for maintaining axonal integrity in both acute and chronic CNS pathology, a situation in which denuded axons become more prone to injury or degeneration than axons bearing functionally intact myelin sheaths. Thus, the present invention also aims at methods and means by which remyelination is enhanced therapeutically and by which axons consequently are protected by formation of myelin sheaths.

Ephrins are also known as Ephrin ligands or Eph family receptor interacting proteins, and are a family of proteins that serve as the ligands of the Ephrin receptor (Ephs), which are receptor protein- tyrosine kinases ( TKs). Ephrins are membrane-bound proteins, as are their receptors. Thus, interaction of ligand and receptor involve cellxell interactions. These binding partners have a wide range of biological functions that influence cell behaviour during both embryogenesis and adult life. Ephrin ligands are divided into two subclasses of Ephrin-A and Ephrin-B based on their structure and linkage to the cell membrane. The present invention is concerned with Ephrin-B ligands. These are attached to the membrane by a single transmembrane domain that contains a short cytoplasmic PDZ-binding motif. Only three Ephrin-B ligands have thus far been identified in humans, Ephrin-Bl, B2 and B3. Ephrins and Ephs are thought to play important roles during embryonic development, including during the development of the central nervous system. Furthermore, the Ephrin-B2/EphB4 and Ephrin-B3/EphBl receptor pairs contribute to vasculogenesis in normal and pathological conditions. Additionally, these cell signalling molecules have increased expression in some types of cancer cells. Ephrin-B3 had previously been linked by one of the present inventors as a possible inhibitor of remyelination, but no connection with other Ephrin-B molecules has previously been observed.

Ephrin receptors are also split into two sub-classes, based upon the similarity of their extracellular domains and their affinities for binding Ephrin-A or Ephrin-B ligands. EphBl, EphB2, EphB3 are known to be a receptor for all Ephrin-B ligands. Ephrin-B2 is a preferred ligand for EphB4, which may also act as a receptor for the other Ephrin-Bs. However, EphA4 is a receptor for Ephrin-B2 and Ephrin-B3 in addition to Ephrin-A ligands.

The present inventors have found that it is these Ephrin-B molecules, such as those in the myelin debris around the demyelinated lesions, that have the effect of inhibiting OPC differentiation. The present invention seeks to compensate for the adverse effects of these inhibitors, which facilitates OPC differentiation, leading to remyelination and thus treatment of demyelination.

Summary of the Invention

The present inventors have found that it is necessary to suppress the action of at least two of three inhibitors of OPC differentiation in order to promote remyelination. These three inhibitors are Ephrin-Bl, Ephrin-B2 and Ephrin-B3. Previous work by one of the inventors indicated that Ephrin-B3 was an inhibitor of differentiation of OPCs, and by inhibiting differentiation, remyelination was also prevented. Work conducted and presented here confirms that Ephrin-B3 is indeed an inhibitor of OPC differentiation and thus remyelination. However, when the inventors sought to investigate further, they found that myelin derived from an Ephrin-B3 knock-out model (see Example 6) did not lead to the level of OPC differentiation expected, thus indicating that there are other remyelination inhibitors present in myelin debris. It is known that in the Ephrin-B3 knock-out mice higher levels of Ephrin-Bl and Ephrin-B2 are expressed, which appeared to be compensating for the lack of Ephrin- B3. Furthermore, the inventors found that in addition to Ephrin-B3, Ephrin-Bl and B2 also display inhibitory activity,and are present in myelin debris (Figure 1). Finally, enrichment for inhibitory activity by column chromatography was also associated with an increased presence of Ephrin-Bl and B2 in the fractions (Figure 15a) and also Ephrin-B3 (Figure 15b). Thus, for effective OPC

differentiation, the inventors have found that the action of Ephrin-B3 together with Ephrin-Bl and/or Ephrin-B2 must be suppressed. It is preferred that all of Ephrin-Bl, Ephrin-B2 and Ephrin-B3 are suppressed.

Thus, according to an aspect, the present invention relates to a composition comprising a suppressant agent or suppressant agents against Ephrin-B3 and at least one of Ephrin-Bl and Ephrin- B2 for use in treating demyelination. Preferably, the composition comprises a suppressant agent or agents against Ephrin-B3 and Ephrin-B2, at least.

For example, the composition may comprise a suppressant agent or suppressant agents against Ephrin-B3 and Ephrin-Bl or Ephrin-B3 and Ephrin-B2.

Preferably, the composition comprises a suppressant agent or suppressant agents against Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

Furthermore, the present invention relates to a method of treating demyelination, said method comprising administering to the subject a therapeutically effective amount of a composition comprising a suppressant agent or suppressant agents against Ephrin-B3 and at least one of Ephrin- Bl and Ephrin-B2.

Preferably, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a suppressant agent or suppressant agents against Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

According to the present invention, three inhibitors of OPC differentiation, and thus inhibitors of remyelination have been identified. Suppression of at least two of these three inhibitors is desirable in order to prevent inhibition of OPC differentiation and encourage remyelination. The at least two Ephrin-B ligands preferably includes Ephrin-B3. In an alternative embodiment, suppression of all three inhibitors is desirable. Suppression of any combination of these identified inhibitors can be simultaneous, sequential or separate. A combined preparation may be provided for such use. Suppression of these inhibitors can involve one agent capable of suppressing Ephrin-B3 and at least one of Ephrin-Bl and Ephrin-B2, or may involve two or more (or a plurality) of agents, each of which is capable of suppressing one or more of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3. In order to promote remyelination it is preferred that Ephrin-B3 and at least one of Ephrin-Bl and Ephrin-B2 should be suppressed. Preferably, all three inhibitors should be suppressed.

In one aspect, a single suppressant agent is used, which is active against Ephrin-B3 and one of Ephrin-Bl and Ephrin-B2. Thus, the single suppressant agent may be active against Ephrin-B3 and Ephrin-Bl or Ephrin-B3 and Ephrin-B2.

In another aspect, a single suppressant agent is used, which is active against Ephrin-Bl, Ephrin-B2 and Ephrin-B3. Thus, this agent is capable of suppressing all three Ephrin-B molecules.

If two suppressant agents are provided, then a suppressant agent active against Ephrin-B3 must be provided. A different suppressant agent active against either Ephrin-Bl or Ephrin-B2 is also provided in the composition.

In an alternative aspect, a plurality of suppressant agents are provided. As a composition, these suppressant agents are active against Ephrin-Bl, Ephrin-B2 and Ephrin-B3. Each individual suppressant agent may be active against one or more of:

a) Ephrin-Bl,

b) Ephrin-B2 and/or

c) Ephrin-B3

Thus, the composition may comprise a suppressant agent that is active against Ephrin-Bl, a suppressant agent that is active against Ephrin-B2 and a suppressant agent that is active against Ephrin-B3. Alternatively, one suppressant agent may be active against Ephrin-Bl and Ephrin-B2, and a second suppressant agent may be active against Ephrin-B3. Otherwise, one suppressant agent may be active against Ephrin-Bl and Ephrin-B3, and a second suppressant agent may be active against Ephrin-B2. Further, one suppressant agent may be active against Ephrin-B2 and Ephrin-B3, and a second suppressant agent may be active against Ephrin-Bl. Thus, in the composition, there may be any suitable combination of agents capable of suppressing Ephrin-Bl, Ephrin-B2 and Ephrin- B3. The suppressant agents against Ephrin-B3, Ephrin-Bl and/or Ephrin-B2 will preferably act by suppressing the inhibitory effect of Ephrin-B3, Ephrin-Bl and/or Ephrin-B2 on the differentiation of oligodendrocyte progenitor cells (OPCs). It is this inhibition of differentiation that is thought to contribute to prevention of remyelination. The Ephrin-Bl, Ephrin-B2 and Ephrin-B3 which are thought to contribute to this effect are postulated to be found in the myelin debris around lesions in the myelin sheath.

The suppressant agent may be any agent that prevents the interaction of Ephrin-Bl, Ephrin-B2 or Ephrin-B3, as appropriate, with its receptor. Thus, the suppressant agent or agents may suppress the action of Ephrin-Bl, B2 or B3 by acting directly against these ligands themselves. Alternatively, the suppressant agent or suppressant agent may be one that is active against the receptors for Ephrin-Bl, B2 or B3, as appropriate. For all of the Ephrin-Bs, the receptors are EphBl, EphB2, EphB3 and EphB4. Ephrin-B2 and Ephrin-B3 are also capable of interacting with EphA4 as a receptor.

Preferably, said receptors are one or more, two or more, three or more, or all of EphBl, EphB2, EphB3 and/or EphA4.

Since there are multiple Eph receptors that are capable of interacting with the individual Ephrin-B ligands, it may be necessary to include a suppressant agent or agents which are active against one or more receptor for Ephrin-B3 and one or more of Ephrin-Bl and/or Ephrin-B2. It is the action of the Ephrin-B3 and one or more of Ephrin-Bl and/or B2 that is suppressed at the level of their receptors. Since they these ligands mainly share one receptor subclass, with the exception of Ephrin-B2 and Ephrin-B3 which also interact with EphA4, suppressing the action of one receptor will have an impact for ability of all three Ephrin-Bs to inhibit OPC differentiation. Thus, the composition may comprise a suppressant agent or suppressant agents that are active against any one or more of EphBl, EphB2, EphB3, EphB4 and/or EphA4. Preferably, the composition comprises a suppressant agent or suppressant agents that are active against any one, two, three or all of EphBl, EphB2, EphB3 and/or EphA4.

Alternatively, the suppressant agent may be an agent that directly binds toEphrin-B3, Ephrin-Bl and/or Ephrin-B2.

Alternatively, the suppressant agent may also target or silence the gene or gene expression of Ephrin-B3, Ephrin-Bl and/or Ephrin-B2. Alternatively, the suppressant agent may also target or silence the gene or gene expression of EphBl, EphB2, EphB3, EphB4 and/or EphA4, preferably EphBl, EphB2, EphB3, and/or EphA4.

The suppressant agent or agents may be biological agents. Such biological agents include antibodies and derivatives thereof, Ribonucleic acid (RNA), Deoxyribonucleic acid (DNA), peptides and proteins. In a particular embodiment, the RNA or DNA forms an aptamer. In a different embodiment, the nucleic acid is an antisense nucleic acid, small interfering RNA (siRNA), microRNA (miRNA), CRISPRs (clusters of regularly interspaced short palindromic repeats) or ribozyme.

The suppressant agent or agents may be an antibody or binding fragment thereof. The antibody is preferably a monoclonal antibody or a binding fragment thereof.

The suppressant agent or agents may be a chemical agent, such as a small chemical entity or inhibitor.

The chemical entity may alternatively inhibit the expression of any one or more of Ephrin-Bl, Ephrin- B2 and/or Ephrin-B3 genes.

Any suitable combination of suppressant agent types can be used in the composition of the present invention.

In an alternative aspect, the present invention relates to a composition comprising a suppressant agent or suppressant agents against one or more of EphBl, EphB2, EphB3, EphB4 or EphA4, for use in treating demyelination. Preferably, the composition is active against two or more of, three or more of, four or more of or all of EphBl, EphB2, EphB3, EphB4 or EphA4. In a preferred

embodiment, the receptors are selected from the list comprising: EphBl, EphB2, EphB3 or EphA4.

In a further alternative aspect, the present invention relates to a composition comprising a suppressant agent or suppressant agents against two or more of

a) Ephrin-B3,

b) Ephrin-Bl,

c) Ephrin-B2,

d) EphBl,

e) EphB2, f) EphB3,

g) EphB4 an/or

h) EphA4

for use in treating demyelination.

It is preferred that two or more targets from the above list are selected, or three, four, five, six, seven or all of the targets are selected, and thus a suppressant agent or agents for these targets are supplied. Preferably, the second or further target is selected from any one of (a-f) or (h). Even more preferably, the composition comprises a suppressant agent against Ephrin-B3 which may also be active against any one of (b) to (h). Alternatively, the composition may comprise suppressant agents which are active against Ephrin-B3 and any one or more of (b) to (h).

The composition is for use in treating demyelination. Demyelination is the presence of neurons with lesions in the myelin. Demyelination may be caused by many factors, including demyelinating disease, traumatic injury to the brain, spine and nerves, radiation injury, stroke and other forms of physical damage, including compression from degenerative changes of surrounding structures such as in cervical spondylotic myelopathy. The composition is preferably not used for promoting axonal regrowth or regeneration.

The treatment may be for one demyelinating disease or demyelinating diseases. Demyelinating disease involves damage to the myelin sheath surrounding an axon, by whatever means.

Demyelinating diseases that can be treated using the composition of the present invention include, but are not limited to: neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, post-infectious encephalitis, and demyelinating injuries.

Exemplary demyelinating diseases include, but are not limited to: Multiple Sclerosis, Acute

Disseminated Encephalomyelitis, Transverse Myelitis, Schilder's Disease, Balo's Disease, Clinically Isolated Syndrome, Alexander's Disease, Canavan Disease, Cockayne's Syndrome, Pelizaeus- Merzbacher's Disease, Optic Neuritis, Neuromyelitis Optica, HTLV-I Associated Myelopathy, Hereditary Leukencephalopathy, Guillain-Barre Syndrome, Central Pontine Myelinosis, Deep White Matter Ischemia, Progressive Multifocal Leukoencephalopathy, Demyelinating HIV Encephalitis, Demyelinating Radiation Injury, Acquired Toxic-metabolic Disorders, Posterior Reversible Encephalopathy Syndrome, Central Pontine Myelinolysis, leukodystrophies, Adrenoleukodystroph, Krabbe's globoid cell and/or metachromatic leukodystrophy.

Other disease in which demyelination occurs include cervical spondylotic myelopathy resulting from cervical stenosis, traumatic injury to the brain or spinal cord, hypoxic injury to the central nervous system including stroke and neonatal hypoxic injury.

The features of any aspect of the invention may be combined with other aspects of the invention. Further features are described below and in the claims.

Description of Figures

Figure 1 shows Ephrin-Bl, Ephrin-B2, and Ephrin-B3 inhibiting oligodendrocyte progenitor cell (OPC) differentiation. There is provided a bar graph showing an inhibition in the expression of 04+ and Mbp+ by OPCs cultured in differentiation medium in the presence of recombinant human Ephrin-Bl, B2, B3 (2d differentiation, n = 3; ANOVA: 04 ***P < 0.0001, Mbp ***P < 0.0001; Dunnett's post-hoc test: PLL vs. Ephrin-Bl or Ephrin-B2 or Ephrin-B3 at 10 μg/cm²: P < 0.0001). Error bars indicate ±SEM.

Figure 2 shows that increasing doses of Ephrin-B3 result in increasing levels of inhibition of oligodendrocyte progenitor cell (OPC) differentiation. There is provided a bar graph showing an inhibition in the expression of 04+ and Mbp+ by OPCs cultured in differentiation medium in the presence of recombinant human Ephrin-B3. Ephrin-B3 inhibited OPC differentiation in a dose- dependent manner (2d differentiation, n = 3; ANOVA: 04 ***P < 0.0001, Mbp ***P < 0.0001;

Dunnett's post-hoc test: PLL vs. MPE or vs Ephrin-B3 at 2.5, 5, 10, 20 or 40 μg/cm²: P < 0.0001. (n) RT-qPCR confirmed reduced expression of Mbp mRNA in the presence of Ephrin-B3 (n = 3; t-test; **P < 0.001). Error bars indicate ±SEM.

Figure 3 (a and b) provides data showing a) Ephrin-B3 inhibiting myelin basic protein (Mbp) expression in OPCs on a transcriptional level. RT-qPCR confirmed reduced expression of Mbp mRNA in the presence of Ephrin-B3 (n = 3; t-test; **P < 0.001). There is shown a bar chart of qPCR for MBP versus treatment with PLL or Ephrin-B3. b) Presence of Ephrin-B3 also down regulates Mbp expression in late stage progenitors and mature oligodendrocytes. (2d differentiation; n = 3; Student T test: ***p < 0.001; Dunnett's post-hoc test: PLL (72hr) vs. PLL(48h) EphrinB3-Fc (24h)). There is shown a bar chart of % positive cells versus treatment with PLL, PLL (48h) and Ephrin-B3-Fc (24h), and Ephrin-B3-Fc alone. Figure 4 illustrates an increased Ephrin-B3 inhibitory activity when Ephrin-B3-Fc was pre-clustered with anti-Fc antibodies. When OPCs were exposed to recombinant Ephrin-B3-Fc pre-clustered with anti-Fc antibodies mimicking surface bound Ephrin-B3 the inhibitory activity was increased (2d differentiation; n = 3; ANOVA: Mbp ***P < 0.0001; Dunnett's post-hoc test: Ephrin-B3 vs. Ephrin-B3 + IgG-Fc at 1, 5 or 10 μg/ml): P < 0.0001. There is shown a line graph of % of M bp-positive cells versus concentration of test agent (PLL, Ephrin-B3, Fc-lgG or Ephrin-B3-Fc). Error bars indicate ±SEM.

Figure 5 illustrates Ephrin-B3 impairing the OPC process formation via a bar chart of the results (% Phalloidin-positive cells versus test agent (PLL, MPE, EPhrin-B3 or Ephrin-B3-Fc)). Ephrin-B3 also induced a reduction in the complexity of OPC processes and a corresponding increase of earlier morphological stages (2d differentiation; I, mono/bipolar; II, primary; III, secondary; IV, membranous branches), (n = 3; ANOVA: P < 0.0001). Error bars indicate ± SEM.

Figure 6 shows the plotted results of Ephrin-B3 infusion induced an impairment of CNS

remyelination (Rank value versus treatment). It demonstrates remyelination after 28 days. The extent of remyelination was assessed on methylene blue and Azur-ll-stained semithin sections. Rank analysis of remyelination demonstrated that Ephrin-B3 infusion induced a significant impairment of CNS remyelination as compared to controls (IgG-Fc control n = 6; Ephrin-B3 n = 5; Mann-Whitney U test; **P < 0.001).

Figure 7 provides data showing the quantification of demyelination axons in the experimental demyelinated lesions in presence and absence of Ephrin-B3 and is shown on a plot of % of total axons versus treatment. Quantification of demyelinated axons confirms that the presence of Ephrin- B3 impairs CNS remyelination (PBS control n-5, IgG-Fc control n= 6; Ephrin-B3 -IgG-Fc n= 5; t test; ***P <0.0001). Error bars indicate ±SEM.

Referring to Figure 8 (a and b), a) EM analysis demonstrated successful formation of compact myelin sheath in control lesion infused with Ig-Fc, b) whereas in Ephrin-B3-infused lesions axons remained demyelinated. These are cell photographs via an electron microscope.

Figure 9 (a to c), a) 30 days post-partum significantly more axons in the corpus callosum of Ephrin- B3^{_ "} mice were myelinated as compared to WT littermates. This was accompanied by a corresponding decrease in the number of nude axons, b) The relative thickness of the myelin sheaths (G-ratios) remained unchanged. (WT: n = 9; Ephrin-B3^_/": n = 9; ANOVA: ***P < 0.0001). c) After three months the number of the myelinated axons in the corpus callosum in wild type and Ephrin-B3^{_ ~} mice were comparable. (WT: n = 5; Ephrin-B3^{_ ~}: n = 5; ANOVA: P > 0.1). Shown are bar charts of % of axons versus treatment group (Figures 9a and 9c) or G-ratio against treatment groups (9b). Error bars indicate ± SEM.

Figure 10 shows that myelin protein extract (M PE) from Ephrin-B2/Ephrin-B3 double knock-out mice and Ephrin-Bl/Ephrin-B3 double knock-out mice is less inhibitory to OPC differentiation as compared to MPE from Ephrin-B3 alone. (2d differentiation, n = 3; ANOVA: 04 * * * P < 0.0001;

Dunnett's post-hoc test: PLL vs. MPE, Eb3-/-, EB3/1-/- or EB2/3- P < 0.0001). The level of OPC differentiation was quantified based on expression of 04 . Bar graph (% positive cells versus treatment type) demonstrates a significant increase of 04/Mbp-positive oligodendrocyte cells when they were differentiated in presence of MPE from Ephrin-B3/Ephrin-Bl and Ephrin-B3/Ephrin-B2 knock out mice in comparison to M PE from Ephrin-B3 knock-out mice.

Figure ll(a to e) provides data illustrating antibody-mediated masking of Ephrin-B. a) Neutralizing Ephrin-B3 epitopes using Ephrin-B3-specific antibodies (Abl, Ab2) restored the ability of OPCs to differentiate in the presence of inhibitory MPE substrates whereas the use of unspecific antibodies (IgG) had no effect (n = 3; ANOVA: 04 * * * P < 0.0001, Mbp * * * P < 0.0001; Dunnett's post-hoc test; MPE vs. MPE + Abl & Ab2: P < 0.0001; MPE vs. MPE + IgG; P > 0.1). The bar chart shows % of positive cells versus treatment type. ( b) Quantitative analysis of Olig2-immunohistochemical stainings cells showed comparable levels of total oligodendrocyte lineage cells in control and EphrinB3 antibodies infused lesions, (c) In contrast, EphrinB3-antibody treated animals displayed increased OPC differentiation as the number of Olig2+/CCl+ mature oligodendrocytes were increased. (Control-lgG: n = 6, EphrinB3-Ab(l+2): n = 6, Student t test 0.05; Olig2-CCL). (d) Infusion of EphrinB3-specific antibodies (Abl+2) into CCP lesions also accelerated CNS remyelination as evident from the rank analysis 14 dpi (Control-lgG: n = 8, EphrinB3-Ab(l+2): n = 5, Mann-Whitney U test * * P < 0.01). (e) Manual quantification of remyelinated and demyelinated axons within lesions confirmed accelerated remyelination in Anti-EphrinB3 treated animals (Control-lgG: n = 4, EphrinB3- Ab(l+2): n = 4, t test * * P < 0.01). Error bars indicate ±SEM. The data demonstrates that the administration of EphrinB3 antibodies did not alter the number of OPCs but instead enabled efficient differentiation. Figures 11a to lie are plots of the data for the data obtained for each treatment.

Figure 12 shows the expression levels of Ephrin-B3 in chromic active MS lesions. Ephrin-B3 was detected in human white matter (WM1,2) and chronic active MS lesion extracts (MS1-3) by immunoprecipitation and Western blotting. Figure 13 shows the neutralisation of Ephrin-B3 in acute MS lesion. Neutralisation of Ephrin-B3 in acute MS lesion extracts (AL1-3) using antibodies raised against two distinct Ephrin-B3 epitopes (Abl, Ab2) induced OPCs differentiation into 04+ and Mbp+ Oligodendrocytes as compared to treatment with unspecific antibodies (IgG); (n = 3; ANOVA: 04 ***P < 0.0001, Mbp ***P < 0.0001; Dunnett's post-hoc test; MPE vs. MPE + Abl & Ab2: P < 0.0001; MPE vs. MPE + IgG; P > 0.1). Error bars indicate ±SEM. The bar chart shows % positive cells versus treatment.

Figure 14 shows the neutralisation of Ephrin-B3 in chronic-active MS lesion. Treatment of chronic active MS lesion extracts (MS1-3) with Ephrin-B3-specific antibodies (Abl, Ab2) significantly increased in the percentage of 04+ and Mbp+ OPCs as compared to IgG-controls. (n = 3; ANOVA: 04 ***P < 0.0001, Mbp ***P < 0.0001; Dunnett's post-hoc test; MPE vs. MPE + Abl & Ab2: P < 0.0001; MPE vs. MPE + IgG; P > 0.1). Error bars indicate ± SEM. The bar chart shows % positive cells versus treatment.

Figure 15 (a and b) shows the enrichment of EphrinBs inhibitory fractions following chromatographic fractination of myelin protein extracts. Figure 15a shows the enrichment of Ephrin-Bl which is in parallel with increased inhibition of OPC differentiation. It also confirms the presence of Ephrin-B2. Figure 15b shows that the presence of Ephrin-B3 also increases when inhibitory activity is enriched. Both are photographs of Western blots.

Figure 16 illustrates the expression level of Ephrin-Bl-B3 and its receptors in the brain and

Oligodendrocytes following RT-PCR. The data demonstrates that OPCs expresses EphBl-3 and EphA4 receptors at both the transcriptional and the protein levels. The latter receptors all bind to various Ephrin-B ligands. This is a photograph of an immunoblot.

Figure 17 (a to d) illustrates EphrinB3 induced phosphorylation of EphA4 and EphB2 RTKs in OPCs (immunoprecipitation followed by WB; pTyr phosphorylated Eph-RTK, t-Eph total Eph-RTK;

quantification of phosphorylated Eph-RTK relative to total Eph-RTK (ROD), n = 3: t test; EphA4 ***P < 0.05; EphBl P > 0.1; EphB2 **P < 0.01; EphB3 P > 0.1). This revealed a strong activation of EphA4 and EphB2 receptors in response to Ephrin-B3-Fc. Error bars indicate ±SEM. Figures 17a-d show a bar chart showing ROD when PLL or Ephrin-B3-lgG-Fc are added to the relevant receptor (Figure 17a - EphA4, Figure 17b - EphBl, Figure 17c - EphB2 and Figure 17d). Also shown on these figures is the Western blot photographs of the immunoprecipitation of OPC protein extracts with antibodies against individual Eph receptors (Figure 17a - EphA4, Figure 17b - EphBl, Figure 17c - EphB2 and Figure 17d), the blot being conducted with anti-phosphotyrosine antibodies. Detailed Description

The present invention relates to a composition comprising a suppressant agent or suppressant agents against Ephrin-B3 and at least one of Ephrin-Bl and Ephrin-B2 for use in treating

demyelination.

One or more suppressant agents are present in the composition.

In one aspect, the suppressant agent is active against Ephrin-B3 and either Ephrin-Bl or Ephrin-B2. Alternatively, two or more suppressant agents may be present, one active against Ephrin-B3 and another active against either Ephrin-Bl or Ephrin-B2.

In an alternative aspect, should one suppressant agent be present, this may be active against all of Ephrin-Bl, Ephrin-B2 and Ephrin-B3. Should two or more suppressant agents be present in the composition, each may have a different target, such that the combined action of the composition is to suppress the action of Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

Should the composition comprise a plurality of (two or more) suppressant agents, each individual suppressant agent may be active against one or more of: a) Ephrin-Bl,

b) Ephrin-B2 and/or

c) Ephrin-B3

Thus, the composition may comprise a suppressant agent that is active against Ephrin-Bl, a suppressant agent that is active against Ephrin-B2 and a suppressant agent that is active against Ephrin-B3. Alternatively, one suppressant agent may be active against Ephrin-Bl and Ephrin-B2, and a second suppressant agent may be active against Ephrin-B3. Otherwise, one suppressant agent may be active against Ephrin-Bl and Ephrin-B3, and a second suppressant agent may be active against Ephrin-B2. Further, one suppressant agent may be active against Ephrin-B2 and Ephrin-B3, and a second suppressant agent may be active against Ephrin-Bl. Thus, in the composition, there may be any suitable combination of agents capable of suppressing Ephrin-Bl, Ephrin-B2 and Ephrin- B3. Any suitable combination of suppressant agents may be used in the composition of the present invention.

The suppressant agent or suppressant agents are agents that are capable of suppressing the action of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3. In particular, these agents are capable of suppressing the inhibitory action of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 on the differentiation of OPCs.

Alternatively put, these agents are capable of inhibiting, silencing, neutralizing, masking, sequestering, removing or repressing the action of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 on the differentiation of OPCs. As used herein, suppressant agent and agent capable of suppressing are used interchangeably.

The composition may be provided in an amount sufficient to suppress the inhibitory action on the differentiation of the OPCs. The sufficient amount of said composition may be predetermined, for example in a batch assay, or supplied in a trial in continually escalating doses, and the suppressive effect is monitored. The composition is thus provided in a therapeutically effective amount.

It will be understood by those skilled in the art that the suppression can be carried out by any suitable agent. Thus, the suppression can take place by an agent physically binding to the Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 and preventing it from interacting with receptors on the OPC. Such suppression can also take place by the suppressant agent or agents being active against one or more of EphBl, EphB2, EphB3, EphB4 or EphA4, preferably EphBl, EphB2, EphB3, or EphA4. Alternatively, the suppression can take place by an agent that prevents Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 expression. Such an agent may work at any level of the expression of the gene, such as at transcription (m NA production) or translation (peptide synthesis) of the relevant gene or genes.

Since the Ephrin-B3 and at least one of Ephrin-Bl and Ephrin-B2 suppressed according to the invention may form part of the myelin debris, all of the molecule may be available for binding. Therefore, suppressant agents that target or bind to the intracellular domains of Ephrin-Bl, Ephrin- B2 and/or Ephrin-B3 may be particularly preferred, since this would target only those molecules present in the debris, and not Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 present on cell surfaces.

Ephrin-B molecules contain an intracellular tail with highly conserved tyrosine residues and a PDZ- binding motif at the C-terminus. This tail functions as a mechanism for reverse signalling, where signalling occurs into the ligand-containing cell, as opposed to the cell with the receptor. The suppressant agent or suppressant agents may be a biological molecule. This biological molecule has the ability to bind specifically to Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 as required.

Alternatively, the biological molecule may bind to a receptor for Ephrin-Bl, Ephrin-B2 and/or Ephrin- B3 as required. Such receptors may include one or more of EphBl, EphB2, EphB3, EphB4 or EphA4, preferably one or more of EphBl, EphB2, EphB3, or EphA4. Binding specifically refers to the ability of the agent to bind to its specified target in preference to other components present in a cell, under physiological conditions. The biological agent may have high affinity for Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3, or receptors thereof. In general, high-affinity binding results from greater intermolecular force between the agent and its target, while low-affinity ligand binding involves less intermolecular force between the agent and its target. In general, high-affinity binding involves a longer residence time for the agent with its target than is the case for low-affinity binding. Affinity is determined by strength of binding between the two entities, which occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. In relation to the present invention, the affinity of binding is under physiological conditions.

Biological molecules capable of suppressing the activity of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 include antibodies and derivatives thereof, peptides, proteins, DNA or NA. Single-stranded DNA or RNA (ssDNA or ssRNA) molecules may form aptamers that can bind Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3, or receptors thereof, with high affinity and specificity.

It is preferred that the biological agents, including antibodies and aptamers, bind to Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3.

In one aspect, the suppressant agent or suppressant agents of the composition may comprise an antibody or a binding fragment thereof. The antibody or fragment may be derivatised or modified as required. Antibody as used herein covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments or synthetic polypeptides carrying one or more antigen-binding sequences, insofar as they exhibit the desired biological ability. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD or IgA), or subclass (e.g., IgGi, lgG2, lgG2a, lgG3, lgG4, IgAl or lgA2). Native or wildtype antibodies are composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. Thus, an antibody binding fragment can be chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and bispecific antibodies. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Also included are domain antibodies (dAbs), single-domain antibodies (sdAbs or nanobodies), diabodies, camelid antibodies and engineered camelid antibodies. Furthermore, for administration to humans, the antibodies and fragments thereof may be humanised antibodies. Humanised forms of non-human (e.g., ovine) antibodies are chimeric immunoglobulins,

immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or ScFv) which contain sequences derived from non-human immunoglobulin. The humanised antibody may also comprise at least a portion of a human immunoglobulin constant region (Fc) or other antibody component.

Monoclonal antibodies (mAb or moAb) are identical monospecific antibodies that are made by identical immune cells cloned from a unique parent cell.

An antibody or fragment thereof that binds to one or more of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 means an antibody or polypeptide derived therefrom (a derivative) which binds specifically to Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3, including, but not limited to, molecules which inhibit or substantially reduce the binding of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 to their receptors or inhibit Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 activity. Monoclonal antibodies are highly specific, being directed against a single target site, epitope or determinant.

An antibody or fragment thereof that binds to one or more of EphBl, EphB2, EphB3, EPhB4 or EphA4 means an antibody or polypeptide derived therefrom (a derivative) which binds specifically to EphBl, EphB2, EphB3, EphB4 or EphA4, including, but not limited to, molecules which inhibit or substantially reduce the binding of EphBl, EphB2, EphB3, EphB4 or EphA4 to their ligands (Ephrin- Bl, Ephrin-B2 and/or Ephrin-B3) and prevent the activity of these ligands. Monoclonal antibodies are highly specific, being directed against a single target site, epitope or determinant. Preferred is an antibody or fragment thereof that binds to any one or more, two or more, three or more or all of EphBl, EphB2, EphB3, or EphA4.

According to one aspect of the invention, a suppressant agent may be used against Ephrin-Bl, Ephrin-B2 and Ephrin-B3. It is preferred that this suppressant agent is an antibody or binding fragment thereof. Thus, an antibody may be provided that binds to all Ephrin-Bs in order to suppress their activity. Such antibodies are commercially available, from sources including Monoclonal Ephrin-B protein Antibody from Genxbio Health Sciences Pvt. Ltd (India), Phopspho- Ephrin B (Tyr324/329) antibody from Cell Signalling Technology, (Massachusetts, US), Anti-Ephrin B1/B2 and B3 (phosphor Y324) antibody from Abeam (Cambridge, UK).

Dual-specificity antibodies are also commercially available, such as Ephrin B1/B2 antibody from Acris Antibodies, Inc. (San Diego, US), for use with additional suppressant agents in order to suppress all Ephrin-B molecules. Additionally, dual-specificity antibodies may be used as the sole suppressant agent in the composition, for example an Ephrin-B3/Ephrin-Bl antibody or Ephrin-B3/Ephrin-B2 antibody.

Antibodies are commercially available for the Ephrin receptors, some of which may bind to all receptors in a class. Thus an anti-EphB antibody is available from antibodies-online (Germany) , which is capable of binding to all of the EphBs.

The suppressant agent or suppressant agents may be nucleic acid aptamers. Aptamers are short single-stranded nucleic acid oligomers (DNA or NA) with sequence-dependent structures, characterized by stems, loops, bulges, hairpins, pseudoknots, triplexes, or quadruplexes. These simple secondary structures can fold further to give complex tertiary structures, allowing aptamers to form complementary shapes with the whole or part of their target. Aptamers can be designed to most peptide targets by a suitably skilled person.

Aptamers may have very high binding affinities, ranging from low nanomolar (nM) to picomolar (pM) and are highly specific to their given target. With aptamers it has been possible to discriminate between very closely related targets, and thus if aptamers are used, it is likely that they will be specific for Ephrin-Bl, B2 or B3. However, it may be possible to develop a single aptamer that can recognise a conserved target sequence across two or three of the Ephrin-Bs Aptamers may also be developed to receptors for Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 as required. Thus, the aptamers may target any one or more of EphBl, EphB2, EphB3, EphB4 or EphA4, preferably any one or more of EphBl, EphB2, EphB3 or EphA4.

The suppressant agent or suppressant agents may be a peptide or protein with specificity for Ephrin- Bl, Ephrin-B2 and/or Ephrin-B3. This peptide or protein could prevent the Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 from interacting with the relevant target on the OPCs, or mark the Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 for clearance by the immune system, such as by a macrophage. The peptide or protein could similarly have specificity for one or more receptor for Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 as required, such as any one or more of EphBl, EphB2, EphB3, EphB4 or EphA4. This would prevent the interaction of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 with their receptors.

Alternatively, the suppressant agent or suppressant agents may be a chemical, such as a small molecule inhibitor of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3, or a small chemical entity that binds to Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 and prevents it interacting with the relevant target on the OPCs. Furthermore, the chemical could be a small chemical entity that binds to the Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 in and marks it for clearance by the immune system, such as by macrophages. In a further alternative, the chemical agent could have specificity for any one or more of EphBl, EphB2, EphB3, EphB4 or EphA4, and prevent Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 from interacting with the receptors, and thus suppress the action of these ligands.

In an alternative embodiment, it is the gene for Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 that is suppressed by the suppressant agent. Alternatively, the gene for the receptors of Ephrin-Bl, Ephrin- B2 and/or Ephrin-B3 may be repressed. This includes the genes for EphBl, EphB2, EphB3, EphB4 or EphA4, preferably EphBl, EphB2, EphB3 or EphA4. However, it is preferred that the gene for Ephrin- Bl, Ephrin-B2 and/or Ephrin-B3 is suppressed.

In such a gene-suppression scenario, the suppressant agent may be a nucleic acid, such an antisense nucleic acid, small interfering NA (siRNA), microRNA (miRNA), CRISPRs (clusters of regularly interspaced short palindromic repeats) or ribozyme; a chemical suppressor of expression, a regulator of expression or any other agent which acts to suppress the expression of any one or more of these genes. siRNA and miRNA act via RNA interference to silence gene expression via the destruction of messenger RNA (mRNA).

Thus, the suppressant agent or suppressant agents may work in any suitable way to suppress the action of Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 on the differentiation of OPCs, and may be any combination of suitable agents. The suppressant agent or agents may act directly against Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3 . This is preferred. Alternatively, the suppressant agent or agents may act indirectly against Ephrin-Bl, Ephrin-B2 and/or Ephrin-B3, via action on the receptors for these ligands. Such receptors include EphBl, EphB2, EphB3, EphB4 or EphA4. Where more than one suppressant agent is used in the composition, it will be understood that each agent need not be the same type. Thus, the composition may comprise an antibody and aptamer and a small molecule inhibitor, or an si NA, an antibody and a ribozyme, for example. Thus, the composition may be any suitable combination of suppressant agents. There may further be a mixture of agent(s) that are active against the Ephrin-B ligands and agent(s) that are active against the relevant Eph receptors.

In situations where more than one suppressant agent is present in the composition, the composition may be provided as a combined preparation for simultaneous, sequential or separate use. Thus, all of the suppressant agents are not required to be physically combined in a sole preparation.

The suppressant agent or suppressant agents of the invention may be administered by any available route, such as via the oral, inhaled, intranasal, sublingual, intravenous, subcutaneous, epidurally (injection or infusion into the epidural space), intracerebral (into the cerebrum),

intracerebroventricular (into the cerebral ventricles) and dermal routes, and each agent may require a different route. The route of administration will be dictated by the nature of the suppressant agent. Antibodies, for example, are generally administered intravenously or subcutaneously. Small chemical entities, for example, may be administered orally or intranasally. The suppressant agent or suppressant agents may be administered systemically or locally. It may be preferable to administer the suppressant agents locally at the site of demyelination. The suppressant agent or suppressant agents may be formulation as immediate release or modified, sustained or controlled release preparations. Any pharmaceutically appropriate excipients may be included. A preferred composition may include a CNS penetrating or CNS targeted suppressant agent or agents.

The composition of the present invention is for treating demyelination , including demyelinating disease, and demyelination associated with stroke, radiation injury, traumatic brain and spinal injury and damage by chemicals.

Demyelination is the degradation of myelin sheaths around the axons of neurons. Such neurons may be otherwise healthy. The causes of demyelination are many, but the effect is the same, the myelin is stripped back, and not replenished, leaving the axon without sufficient insulation. It is thought by the inventors that the degraded myelin remains around the sites of demyelination, and prevents remyelination by inhibiting the differentiation of OPCs. Thus, by suppressing the inhibitory nature of the components of the degraded myelin on the OPCs, the OPCs can differentiate and promote remyelination.

Demyelination can be caused by heavy metal poisoning, in particular mercury poisoning.

Alternatively, myelin damage may be caused by radiation injury, traumatic injury, oxygen deprivation and physical compression.

With the inventive treatment (reversible) axon demyelination can be reversed by remyelination. Contrary thereto, axon destruction is irreversible and requires a complete regrowth of a new axon which in turn has to form new neuronal connections and synapses. According to the treatment by the composition of the invention synapses and neural connections are maintained. The inventive treatment is directed specifically at myelin damage, i.e. the treatment of myelin damage but not of axon destruction. Thus, the present invention does not extend to methods of regenerating or growing new axons. The cellular target of the present invention are oligodendrocyte precursor cells (OPCs) (i.e. adult, neural, tissue stem cells) and pre-oligodendrocytes. But also in general any neuronal precursor cells that can be differentiated into active oligodendrocytes are targeted. The first step in remyelination is the population of an area of demyelination with sufficient OPCs. These might come from cells that are already present in the demyelinated area, or they may be recruited from surrounding intact white matter or may be artificially brought to the affected area by transplantation. This initial recruitment phase may involve OPC proliferation and is also likely to involve their migration. The contribution made by OPC migration to remyelination is likely to be determined by the extent to which OPCs survive in areas of demyelination. Once recruited, the OPCs must differentiate into myelin-sheath-forming oligodendrocytes for remyelination to be completed. During this differentiation phase, the OPCs differentiate into pre-myelinating oligodendrocytes that engage the demyelinated axon, before finally becoming mature

oligodendrocytes as their sheet-like processes form spiral wraps around axons and the cytoplasmic contents of the wraps are extruded to form compacted myelin sheaths (Franklin, Nature Reviews Neuroscience 3 (2002) : 705-714).

Demyelinated axons to be treated according to the present invention are preferably found or associated with diseases or conditions selected from Multiple Sclerosis, stroke, nerve injury, in particular spinal cord injury. Nerve injury may be of central nervous system axons, of corticospinal tract axons, of dorsal column axons, or of optic nerve axons. Furthermore, the denuded axons to be treated according to the present intervention include known subtypes of Multiple Sclerosis, in particular relapse-remitting MS, secondary progressive MS, and primary progressive MS as well as fulminant forms of MS. Furthermore, MS types to be treated include MS classified as Type 1, as MS Type 2, as MS Type 3, and as MS Type 4 according to their pathogenesis (Lassmann et al . , TRENDS in Molecular Medicine 7 ; 3 (2001) : 115-121).

The damaged myelin sheaths can be of central nerve system (CNS) axons, in particular of corticospinal tract axons, of dorsal column axons, optic nerve axons, optic tract axons or of axons in the penumbra after a stroke or after spinal cord injury. Another characteristic of the damage of the myelin sheath according to the invention can be a chronic damage, or damage as a result of a chronic disease. Preferably in the chronic damage or chronic disease a glial scar is formed. In further preferred embodiments the damage of the myelin sheath can be an acute or sub-acute damage, preferably resulting in the presence of myelin associated inhibitors, in particular Ephrin-Bl, Ephrin- B2 and Ephrin-B3. In particular the myelin sheath damage is treated after an acute surge during the chronic disease or is treated continuously.

The importance of myelin sheaths and their restoration to spinal cord injury (SCI) oligodendrocyte death is a prominent feature of SCI. Furthermore, following SCI chronic progressive demyelination may occur. Whereas preventing oligodendrocyte cell death has beneficial effects on the recovery following spinal cord injury, the transplantation of OPCs improves white matter sparing as well as it promotes functional recovery. In the light of these findings, enhancing remyelination has been identified as important strategy to ease the devastating clinical manifestations of SCI.

The present invention may treat any demyelinating disease. The demyelinating disease is preferably characterised by damaged myelin sheaths.

The demyelinating disease may be selected from the list comprising: neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, postinfectious encephalitis, and demyelinating injuries.

More particularly, the demyelinating disease may be selected from the list comprising Multiple Sclerosis, Acute Disseminated Encephalomyelitis, Transverse Myelitis, Schilder's Disease, Balo's Disease, Clinically Isolated Syndrome, Alexander's Disease, Canavan Disease, Cockayne's Syndrome, Pelizaeus- Merzbacher's Disease, Optic Neuritis, Neuromyelitis Optica, HTLV-I Associated

Myelopathy, Hereditary Leukencephalopathy, Guillain-Barre Syndrome, Central Pontine Myelinosis, Deep White Matter Ischemia, Progressive Multifocal Leukoencephalopathy, Demyelinating HIV Encephalitis, Demyelinating Radiation Injury, Acquired Toxic-metabolic Disorders, Posterior Reversible Encephalopathy Syndrome, Central Pontine Myelinolysis, leukodystrophies, Adrenoleukodystroph, Krabbe's globoid cell and/or metachromatic leukodystrophy.

In one embodiment, the demyelinating disease is multiple sclerosis (MS). MS is characterized by loss of myelin with sparing of axon cylinders in MS plaques. The plaques usually occur in a perivenular distribution and are associated with a neuroglial reaction and infiltration of mononuclear cells and lymphocytes. Active demyelination is accompanied by transient breakdown of the blood-brain barrier. Chronic lesions show predominantly gliosis in the form of glial scar tissue. MS plaques are distributed throughout the white matter of the optic nerves, chiasm and tracts, the cerebrum, the brain stem, the cerebellum, and the spinal cord.

In preferred embodiments of the present invention the agent is for the administration after complete demyelination of at least a portion of the neuronal axons of a subject, which can e.g. be in respects undamaged. This portion of neuronal axons is preferably a portion of central nerve system axons, of corticospinal tract axons, of dorsal column axons, optic nerve axons, optic tract axons or of axons in the penumbra after a stroke or after spinal cord injury, and especially preferred an optic nerve axon.

The subject for the inventive treatment is preferably a mammal, in particular a human.

The protein sequence for human Ephrin-Bl may be found in GenBank: Accession number

AAH52979.1 The protein sequence for human Ephrin-B2 may be found in GenBank: Accession number AAH69342.1. The protein sequence for human Ephrin-B3 may be found in GenBank:

Accession number AAH42944.1. The protein sequence for EphBl may be found in NCBI Reference Sequence: NP_004432.1. The protein sequence for EphB2 may be found in GenBank: AAH67861.1. The protein sequence for EphB3 may be found in NCBI Reference Sequence: NP_004434.2. The protein sequence for EphA4 may be found in GenBank: AAI05003.1. These database entries are incorporated by reference.

The invention will now be further described with reference to the following non-limiting examples.

Examples Example 1: Materials and Methods

Characterization of chronic active MS lesions

Post mortem MS lesion tissue was provided by the UK MS tissue bank. Chronic active lesions were identified by H&E staining and defined by the presence of immune cells (HLA+ T-lymphocytes) and the absence of foamy macrophages. Active lesions contained foamy macrophages.

Immunohistochemistry of MS lesions

Immunohistochemistry on MS lesions was performed as described in Waldvogel et al, 2006 (Nat Proc 1:2719-2732).

Sections were stained with antibodies to Nkx2.2 (Developmental Studies Hybridoma Bank ; 1:150) and 3tubulin (Miliipore; 1:500), HLA (Millipore; 1:150), MBP (Millipore; 1:300), GFAP (Dako, Abeam 1:500) degenerated MBP (Millipore, 1:500). Appropriate Alexa 488, 555 or 594-conjugated secondary antibodies (Invitrogen) were used. Cell nuclei were visualized with DAPI (Sigma-Aldrich). A list with details of the antibodies used is included in Table 1.

Antibody Source Application Dilution

04 Sigma Immunocytochemistry 1:500

Immunocytochemistry,

Mbp Millipore 1:300

Immunohistochemistry

Western Blot

Pip Millipore Western Blot 1:5000

A2B5 Millipore Immunocytochemistry 1: 300 Immunocytochemistry

1:1000 Immunoprecipitation

EphrinB3 Abeam (Ab2)

Neutralization assay

(2.5ug/ml) In vivo experiment

Immunoprecipitation Neutralization assay

EphrinB3 R&D (Abl) 2.5 ug/ml

In vivo experiment Immunohistochemistry

Immunocytochemistry

1:500

EphA4 Abgent Immunoprecipitation

1:2500 Immunohistochemsitry

Immunocytochemistry 1:500

EphBl Abgent

Immunoprecipitation 1:2500

Immunocytochemistry 1:500

EphB2 Abgent

Immunoprecipitation 1:2500

Immunocytochemistry 1:500

EphB3 Abgent

Immunoprecipitation 1:2500

Marcks Sigma Immunocytochemistry 1:1000

Rho A Millipore Rho A assay 1:2500 p-FAK Cell Signaling Immunoprecipitation 1:3000 t-FAK Cell Signaling Immunoprecipitation 1:3000

Immunocytochemistry

Developmental Studies

Nkx2.2 1:300

Hybridoma Bank

Immunohistochemsitry

MOG Millipore Immunohistochemistry 1:500 dMBP Millipore Immunohistochemistry 1:500

GFAP Abeam Immunohistochemistry 1:500

Preparation of myelin protein extracts (MPE)

Myelin (rat and human) was purified from mechanically dissociated brains by two rounds of discontinuous density gradient centrifugation and osmotic disintegration. Protein extracts were prepared by dissolving myelin pellets with 1% N-octyl^-d-glucopyranoside (Sigma) , 0.2 M sodium phosphate pH 6.8, 0.1 M Na₂S0₄(Sigma), and 1 mM ethylenediaminetetraacetic acid(Sigma).

Enrichment of inhibitory activity in MPE by column chromatography

MPE (50mg) was filtered, desalted and concentrated (Amicon ultrafiltration cell, Millipore) with 50 mM sodium acetate (pH 4). Column chromatography was performed by FPLC (Pharmacia Fine Chemicals, GE Healthcare). First, MPE was separated by cationic chromatography (Econo-Pac CM cartridges, 1 ml, BioRad). To test the resulting fractions for inhibitory activity, OPCs were exposed to substrates that were prepared using equal volumes (ΙΟΟμΙ) of eluates, and the number of 04+ OPCs was determined. Further separation of inhibitory fractions was achieved using anionic chromatography (EconoPac High Q cartridge, BioRad) and gel exclusion chromatography (Sephacryl S100 column, GE Healthcare). Protease inhibitors (Thermo Scientific) were used during all stages.

Proteomic analysis of MS lesions and MPE fractions

Protein extracts were precipitated with ammonium acetate in methanol, separated by ID SDS-PAGE, and stained with colloidal Coomassie blue. Each track was cut into 1 mm slices, (16 per track). Mass spectrometry experiments were performed using an LTQ linear ion trap instrument fitted with a nanospray ion source (Thermo, San Jose, CA). The LTQ was operated in a data-dependent manner, and ions with a charge state of 2+ or 3+ (indicative of a tryptic peptide) were automatically isolated, fragmented by CID and an MS/MS spectrum generated. The separation of peptides was performed by reverse-phase chromatography using an Agilent 1200 (Agilent Technologies, Santa Clara, CA), a HPLC pump at a flow rate of 300 nL/min and a LC-Packings (Dionex, Sunnyvale, CA) PepMap 100 column (C18, 75 uM i.d. x 150 mm, 3 uM particle size). Peptides were loaded onto a LC-Packings pre- column (Acclaim PepMap 100 C18, 5 uM particle size, 100 A, 300uM i.d x 5mm) in 0.1% formic acid for 5 min at a flow rate of 20 uL/min to desalt samples. A gradient employed of 5-55 % B in 60 min was used to elute peptides (Solvent A = 0.1 % formic acid in water and solvent B = 5% acetonitrile with 0.1% formic acid in water). Data were processed using Bioworks Browser (version 3.3.1 SPl, ThermoFisher), and searched using MASCOT (Matrix Science Ltd) using a fixed modification of carbamidomethyl and a variable modification of oxidation (M). The databases used were NCBInr 060629 Rattus or Homo sapiens with a Peptide Mass Tolerance of ± 1 Da and a Fragment Mass Tolerance of ± 0.8 Da.

Preparation of primary OPC cultures

Primary OPC cultures were isolated from neonatal (p0-2) Sprague Dawley rat forebrains as in Baer et al., 2009 (Brain 132:465-481) and Syed et al., 2013 (EMBO Mol Med 5: 1918-1934). Differentiation was induced with Sato's medium containing 0.5% FCS. Only cultures > 94% A2B5+ cells were used. OPCs were plated at a density of 2xl0⁴ cells (8-well chamber slides) or 3x10^s cells (6-well plates).

Immunocytochemistry

Immunocytochemistry on OPCs was conducted as in. 04, Nkx2.2, Mbp-positive cells were quantified relative to DAPI-stained nuclei in >20 randomly selected eye fields on an Olympus X80 microscope. The morphology of OPCs stained with phalloidin was categorized as follows: I: mono/bipolar; II: multipolar, primary branches; III: multipolar, secondary branches; IV: membranous processes.

Preparation of MS lesion, MPE, and Ephrin-B3 substrates

MS lesion extract (see below), MPE and Ephrin-B3-Fc (R&D) substrates were prepared by overnight incubation on PLL (Sigma Aldrich) coated dishes as in Syed et al., 2008 (NeuroSurg focus 24;E5) and Syed et al., 2011 (J. Neurosci 31: 3719-3728) .

Pre-clustering of recombinant Ephrin-B3-Fc Ephrin-B3-Fc fragments (R&D) were mixed with anti-human Fc-lgG (Millipore) (ratio=l:5) and incubated for 2h at room temperature prior to addition to the tissue culture medium.

Effects ofEphrin-B3 on late stage OPCs

OPCs were differentiated in Sato's differentiation medium for 48h, and subsequently exposed to pre-clustered Ephrin-B3 suspended in Sato's medium for another 24h. Cells were then fixed with 4% PFA and assessed for 04 and Mbp expression.

Neutralization of Ephrin-B3 epitopes in MPE

Ephrin-B3 epitopes in MPE substrates and MS lesion extracts were neutralized by incubation with anti-Ephrin-B3 antibodies (Abeam and R&D; ratio: 1:1) in Sato's differentiation medium for 2h at room temperature prior to cell seeding.

TUNEL assay

To detect cell death, TUNEL assays (Promega) were conducted and the percentage of apoptotic nuclei determined Baer et al., 2009.

Proliferation assay

The OPC were cultured in Sato's differentiation medium for 24h. Subsequently, cells were fixed and stained with Olig2 (1:1000) and PCNA (1:500). Number of cells were counted in random field with > 200 cells per experiment. A minimum of three biological replicates were conducted.

Reverse transcriptase-PCR and q-RT PCR

RNA was extracted using RNeasy Mini Kit (Qiagen). Reverse transcription (first strand cDNA synthesis kit for RT-PCR, Roche Applied Science) and second-round PCR was performed using GoTaq DNA polymerase (Promega). Primers used are summarized in Supplementary Table 7. q-RT-PCR was conducted on an Applied Biosystems 7500HT Fast Real-time PCR system Baer et al., 2009 and Syed et al., 2011. Triplicate measurements were made on a minimum of three biological replicates. siRNA mediated gene-silencing

siRNA transfections of purified OPCs were conducted with lipofectamine RNAiMAX transfection reagent (Invitrogen) in OPTI-MEM as in Baer et al., 2009. The knockdown efficiency was established by qPCR. Animal experiments

Immunogold electron microscopy

Perfusion-fixed (4% formaldehyde, 0.2% glutaraldehyde) corpus callosum white matter was cryoprotected (2.3M sucrose), mounted onto aluminum pins, and frozen in liquid nitrogen. Ultrathin cryosections (Leica UC6 cryo-ultramicrotome) were collected (2% methylcellulose:2.3M sucrose=l:l), blocked (1% BSA), and incubated with antibodies to Ephrin-B3 (1:200; R&D). Gold conjugated secondary antibodies (Aurion) were used to visualize Ephrin-B3 epitopes on a LEO EM912 Omega transmission electron microscope (Zeiss) fitted with an on-axis 2048 ^χ 2048 CCD camera (Proscan).

Ephrin-B3 knockout and double-knockout mice

Ephrin-B3and double- knockout mice were kindly provided by Amparo Acker-Palmer and genotyped as previously in Senturk et ai., 2011(Nature 472:356-360).

Induction of focal demyelination

Female Sprague-Dawley rats were anaesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and positioned in a stereotactic frame. Demyelination was induced bilaterally by stereotactic injection of ethidium bromide (0.01%, 4 μΙ) into the CCP- young animals (10.4 mm caudal, 2.6 mm lateral and 7.07 mm ventral to bregma) and older animals (11.1 mm caudal, 2.8 mm lateral and 7.8 mm ventral to bregma)

Infusion of Ephrin-B3-Fc-lgG and anti-Ephrin-B3 antibodies into demyelinated lesions

Ephrin-B3 and anti-Ephrin-B3 antibodies were administered via osmotic pumps (ALZA Corporation) at 10 and 3 dpi, respectively (200 μg/ml in PBS). Ephrin-B3-Fc (R&D Systems) was pre-clustered with anti-human-Fc (Chemicon) for 2 h at room temperature (Ephrin-B3-Fc:anti-Fc=10:l) prior to pump filling. Control animals received anti-Fc IgG only. Anti-Ephrin-B3 antibodies (R&D:Abcam ratio=l:l). Controls received Human IgG. Animals were perfused at 28 and 14 dpi, respectively.

Immunohistochemistry of CCP lesions Rats were anaesthetized and transcardially perfused with 4% PFA. The brains post-fixed, cryoprotected (30% sucrose) and snap frozen. 15μιτι cryostat sections were stained with antibodies to EphA4 (1:500), Nkx2.2 (1:150), 3tubulin (1:500).

In situ hybridization

In situ hybridization was conducted on cryostat sections using digoxigenin-labeled complementary RNA probes for Msrb, and Pdgfr- as in Han et al., 2008 (Nature 451;1076-1081) and Syed et al., 2011 . Lesions were identified on digital images of solochrome-cyanine-stained sections, and the lesion area was determined using Image J 1.43b (http://rsb.info.nih.gov/ij/). ISH-stained cells in the lesions were counted on digitized adjacent sections. To rule out effects attributable to the size of the lesion, only lesions < 0.4 mm² were included for analysis, Syed et al., 2011.

Histological analysis of remyelination

Remyelination was assessed on tissue fixed with 4% glutaraldehyde, osmicated and processed into resin (TAAB Laboratories) as in Kotter et al., 2006 (J Neurosci 26:328-332) . Sections (1 μιτι) were stained with methylene blue and Azur-ll. Rank analysis was conducted as in Syed et al., 2011 . Lesions with the greatest extent of remyelination were assigned the highest rank value. In addition, remyelinated and demyelinated axons were manually counted on a minimum of 3 digitised lOOx images of resin embedded lesions.

Electron microscopy

Ultrathin sections (50nm, Leica Ultracut S ultramicrotome) were stained with aqueous 4% uranylacetate and lead citrate. Electron micrographs were obtained as outlined above (Kotter et al., 2006; Syed et al., 2011).

Statistical analysis / Experimental design

All analyses were conducted with researcher blind to treatment group. Experimental animals were randomized to treatment group. Data were analyzed using GraphPad software (Prism). Multiple group comparisons were conducted using one-way ANOVA followed by Dunnett's post test. Two- tailed Student's t-test was used to test receptor activation, qPCR data. Cellular responses in vivo were analyzed using Student's t-test. For rank analysis a two-tailed Mann-Whitney U test was used.

Example 2, Identification of Ephrin-B3 as a myelin-associated inhibitor of OPC differentiation. To identify the protein responsible for the inhibition of OPC differentiation, an assay was developed for biochemical separation of protein fractions and enrichment of inhibitory activity. This fractionation may include three steps: 1) enriching inhibitory activity by carboxymethyl (CM), 2) High Q column ion exchange chromatography, and 3) Sephadex S100 mediated size exclusion. For the purpose of estimating the size of the inhibitory protein, fractions may be obtained following CM and High Q separation, which were spiked with two proteins of known molecular weight (immunoglobulin G (IgG), 150 kDa, and lysozyme, 14 kDa) before further separation on S100 Sephadex size-exclusion columns. The addition IgG and lysozyme to protein fractions did not change the distribution of the inhibitory activity. Based on the localization of the inhibitory activity relative to the reference proteins, the size of the predicted protein was calculated to be approximately 51 kDa. Taking into account that post-translational modifications may alter the size of the protein core, a search on PubMed Protein Database was conducted to identify signalling proteins expressed in the CNS that fall within a size range of 30-55 kDa. (Search terms: "030000:055000 [Molecular Weight! and membrane and signalling and ligand and brain"). From these searches, Ephrin-Bl-B3, a group of transmembrane proteins forming part of the Ephrin family, emerged as possible candidate proteins. The effects of Ephrin-Bl-B3 on OPC differentiation can be tested by exposing cells to substrates prepared from Ephrin-Bl-3. Amongst these, Ephrin-B3 had the most potent inhibitory effect on differentiation of oligodendrocyte linage cells (Figure 1) Immunoprecipitation of Ephrin-B3 in CNS, myelin extracts and column chromatography fractions demonstrated enrichment of Ephrin-B3 (and Bl) in parallel with enrichment for OPC-differentiation-inhibiting activity (Figure 15a and b). The enrichment and the potent inhibitory effects observed implies an important role for Ephrin-B3. This is supported by the data presented in Figures 17a to 17d. The B class of transmembrane Ephrins preferentially bind to EphB receptors. In addition, the EphA4 receptor can also recognize Ephrin-B3. We found that OPCs express EphBl-3 and EphA4 receptors at both the transcriptional and the protein levels (Fig. 17a-17d). To investigate which of these receptors respond to the addition of recombinant EphrinB3-Fc to Sato's differentiation medium, we conducted immunoprecipitation of OPC protein extracts with antibodies against individual Eph receptors followed by Western blotting with anti-phosphotyrosine antibodies. This revealed a strong activation of EphA4 and EphB2 receptors in response to EphrinB3-lgG -Fc (Fig. 17a-d).

Example 3, Ephrin-B3 inhibits OPC differentiation

OPCs may be exposed to substrates containing recombinant Ephrin-B3-Fc fragments, in which the transmembrane segment was replaced with an Fc tag to make it soluble, a concentration-dependent impairment of 04 and MBP expression was detected (Figure 2) In addition, Ephrin-B3 may inhibit myelin protein extracts (Mbp) expression, as confirmed by RT-qPCR assay, which showed reduced expression of Mbp mRNA in the presence of Ephrin-B3 (Figure 3a). In addition, the presence of Ephrin-B3-Fc has been shown to downregulate Mbp expression in Oligodendrocytes (Figure 3b). Furthermore, Ephrin-B3-Fc may also negatively affected process formation and resulted in the majority of OPCs being arrested at an immature monopolar/bipolar stage (Figure 5).

Example 4, Clustering Ephrin epitopes increases the inhibition of OPC differentiation

Ephrin-B and its receptors (EphR) signalling occurs by direct cell-cell interactions and results in the oligomerization of receptor-ligand complexes. One possible feature of Ephrin signalling is that clustering of Ephrin-Eph receptors can modulate the signalling process. In most cases ligand- receptor clustering leads to enhanced signalling. In one possible example, OPCs may be exposed to aggregated Ephrin-B3-Fc using anti-Fc-lgG in order to mimic interactions with membrane bound Ephrin-B3. Referring to Figure 4, there is provided an increase in Ephrin-B3 inhibitory activity when Ephrin-B3-Fc was pre-clustered with anti-Fc antibodies. Conversely, fewer cells differentiated when exposed to pre-clustered Ephrin-B3 than non-clustered Ephrin-B3 (Figure 4).

Example 5, Ephrin-B3 inhibits CNS remyelination

Having established an important regulatory function of Ephrin-B3 on OPC differentiation in vitro, the effects of Ephrin-B3 on myelin regeneration were assessed. For this purpose, a well characterized model of remyelination, in which a focal demyelination lesion is induced by stereotactic injection of ethidium bromide into the rat caudal cerebellar peduncle (CCP). This can result in the rapid demyelination and recruitment of new OPCs. Around day 10 post lesion induction, the recruited OPCs start to differentiate into myelin-forming oligodendrocytes. 28 days post lesion induction the lesions are fully remyelinated.

The extent of remyelination at 28 dpi was assessed on semithin resin sections (Figure 6). As expected at this time point, remyelination in control animals was almost complete with the majority of axons bearing thin myelin sheaths that are characteristic of remyelination. In contrast, in lesions infused with Ephrin-B3-Fc the majority of axons remained demyelinated. A significant difference was found between the groups by investigator-blinded rank analysis (Figure 6) and quantification of remyelinated and demyelinated axons (Figure 7). In a further embodiment, Figures 8a and b provide Electron Microscopy (EM) analysis of the Myelin sheath formation. Figures 8a and b demonstrated successful formation of compact myelin sheaths in the control (Figure 8a) whereas, Ephrin-B3- infused lesions axons remained demyelinated (Figure 8b). In another embodiment, Stereotactic administration of pre-clustered Ephrin-B3-Fc into intact CCP white matter did not affect the integrity of mature myelin sheaths, as there were no detection of any changes in naive (non-lesioned) CCPs following infusion.

Example 6, Loss of Ephrin-B3 and antibody-mediated masking of Ephrin-B3 epitopes neutralizes the inhibitory effects of myelin extracts on OPC differentiation and promotes CNS remyelination

In this example, the inventors next analyzed developmental myelination in Ephrin-B3 deficient mice and found more myelinated axons in the corpus callosum at 1 month in Ephrin-B3 deficient mice than wild-type mice (Figure 9a).The relative thickness of myelin sheaths as measured by G-ratios was the same in wild-type and knockout mice (Figure 9b). At 3 months, the number of myelinated axons was comparable amongst the groups (Figure 9c), indicating that loss or Ephrin-B3 results in accelerated myelination without affecting myelin thickness or the final extent of myelination.

To investigate the role of Ephrin-B3, in conjunction with Ephrin-Bl or Ephrin-B2, with respect to the OPC-differentiation-inhibiting effects of myelin, OPCs were exposed to myelin protein extracts from Ephrin-B3 Ko mice or double knock-out mice (Ephrin-B3 and Ephrin-Bl or Ephrin-B3 and Ephrin- B2)(Figure 10). Myelin protein extract (MPE) from Ephrin-B3 and Ephrin-B-1 or Ephrin-B2-deficient mice was significantly less inhibitory than MPE. Notably, MPE from double knock-out mice were less inhibitory than the MPE from the single knock-out of Ephrin-B3, supporting the need to suppress Ephrin B3 and one or more of Ephrin-Bl or B2. The MPE from the double knock-out of Ephrin-B2 and Ephrin-B3 had the least inhibitory effect of the MPEs tested.

In another example, to determine whether antibody-mediated masking of Ephrin-B3 epitopes could represent a viable therapeutic approach, myelin protein extracts were treated with two different commercially available antibodies directed against two distinct peptide sequences of Ephrin-B3. Both antibodies induced 04 and MBP expression of OPCs that were plated on the antibody-bound substrates (Figure 11a). A combination of the two antibodies resulted in neutralization of the inhibitory effects of myelin, restoring OPC differentiation to levels observed in cells plated on PLL (Poly-L-Lysine) control substrate (Figure 11a). Focal areas of demyelination were induced using ethidium bromide in the CCPs of 9-12-month-old female Sprague Dawley rats. Owing to their greater age, remyelination was expected to be less efficient and delayed. Aged animals serve as a clinically relevant model for remyelination in young to middle-aged adults. Stereotactic infusion of anti-Ephrin-B3 antibodies was initiated at 3 dpi. Remyelination was assessed on semithin resin-embedded sections at 14 dpi. As expected, remyelination in control animals that received infusions of control IgG, or PBS alone was limited. In contrast, widespread remyelination was detected in animals that received anti-Ephrin-B3 antibodies. The increase in remyelination was confirmed by both, histological rank analysis and manual counts of axons within the lesions (Figures lib and c). Assessment of the lesions by electron microscopy confirmed the presence of thin myelin sheaths typical for remyelination throughout the lesions in anti-Ephrin-B3 treated animals. Axons in control lesions remained largely demyelinated but the relative density of axons between the groups was comparable. The data demonstrate that the administration of Ephrin-B3 antibodies did not alter the number of OPCs but instead enabled efficient differentiation (Figures lid and e).

Example 7, Ephrin is present in MS lesion and its lesions and its neutralization promotes OPC differentiation.

Because Ephrin-B3 in the adult CNS is predominantly expressed by oligodendrocytes, the main source of Ephrin-B3 present in MS white matter lesions is likely to be myelin debris that accumulates as a consequence of demyelination. Experimental findings demonstrated that the clearance of myelin debris is mediated by macrophages (Kotter et al., 2006). In MS, the presence of foamy macrophages defines the acute lesion stage. As time passes, macrophages disappear from the lesions whilst other inflammatory cells continue to persist. This demarcates the subacute, or chronic active lesion stage. Chronic (silent) lesions are defined by the complete absence of immune cells (Kotter et al., 2011). Investigating the presence of myelin proteins including Ephrin-B3 in subacute (chronic active) lesions therefore would be useful to determine 1) the efficiency of macrophage- mediated clearance of myelin debris, and 2) whether myelin associated inhibitors are present for prolonged time periods at biologically relevant levels.

Immunoblots with antibodies specific to Ephrin-B3 confirmed the presence of Ephrin-B3 MS lesions (Figure 12). To confirm that the axons within the lesions were indeed demyelinated, electron micrographs may be obtained from sections of the same lesions. In the absence of intact myelin sheaths and phagocytic cells, the myelin proteins detected were most likely to be associated with interstitial debris. Therefore, demyelination in MS may result in accumulation of extracellular myelin debris. Moreover, the fact that Ephrin-B3 (and other myelin molecules) were found in subacute lesions indicated that the phagocytic clearance of myelin debris in MS lesions may remain incomplete.

In a further example, the effects of antibody-mediated masking of Ephrin-B3 epitopes in MS lesions were investigated. OPCs were cultured on lesion extracts from acute and subacute (chronic active) lesions that were incubated with antibodies binding Ephrin-B3 or unspecific IgG. The treatment of anti Ephrin-B3-antibodies efficiently reduced the inhibitory effects of acute MS lesion extracts (Figure 13). Consistent with the presence of Ephrin-B3 in chronic active lesions, the treatment with anti Ephrin-B3-antibodies was also able to induce OPC differentiation on extracts from later lesion stages (Figure 14). However, the inhibitory activity in chronic active lesions was less pronounced. An alternative hypothesis is that the lesion composition, and specifically the presence of OPC differentiation inhibitors in MS lesions changes over time. In conclusion, the data suggest that inhibitory effects of myelin debris accumulating in acute MS lesions can be neutralised by antibody- mediated masking of Ephrin-B3 epitopes.

Claims

Claims

1. A composition comprising a suppressant agent or suppressant agents against Ephrin-B3 and at least one of Ephrin-Bl and Ephrin-B2 for use in treating demyelination.

2. The composition of claim 1 comprising a suppressant agent or suppressant agents against Ephrin-Bl, Ephrin-B2 and Ephrin-B3 for use in treating demyelination.

3. The composition of claim 1 wherein said suppressant agent is active against Ephrin-B3 and any one of Ephrin-Bl or Ephrin-B2.

4. The composition of claim 2 wherein said suppressant agent is active against Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

5. The composition of claim 1 wherein said suppressant agents comprise two or more

agents, each of which are active against any one or more of the following:

a) Ephrin-Bl

b) Ephrin-B2 and/or

c) Ephrin-B3.

6. The composition of claim 5 wherein the composition is a combined preparation for

simultaneous, separate or sequential use.

7. The composition of claim 1, 2 or 5 wherein said agents comprise three agents, each of which are active against one of Ephrin-Bl, Ephrin-B2 or Ephrin-B3.

8. The composition of any preceding claim wherein said suppressant agent or suppressant agents suppress the inhibitory action of Ephrin-B3, Ephrin-Bl and/or Ephrin-B2 on differentiation of oligodendrocyte progenitor cells.

9. The composition of any preceding claim wherein the suppressant agent or suppressant agents suppress Ephrin-B3, Ephrin-Bl and/or Ephrin-B2 by acting against one or more of EphBl, EphB2, EphB3, EphB4 or EphA4.

10. The composition of claim 9, wherein the suppressant agent or suppressant agents

suppress Ephrin-B3, Ephrin-Bl and/or Ephrin-B2 by acting against one or more of EphBl, EphB2, EphB3 or EphA4.

11. The composition of any one of claims 1 to 8 wherein said suppressant agent suppresses gene expression of Ephrin-B3, Ephrin-Bl and/or Ephrin-B2

12. The composition of any one of claims 1 to 10 wherein said agent suppressed gene

expression of any one or more of EphBl, EphB2, EphB3, EphB4 or EphA4.

13. The composition of any one of claims 1 to 10 wherein said suppressant agent or at least one of said suppressant agents is an antibody or binding fragment thereof.

14. The composition of claim 13 wherein said antibody is a monoclonal antibody or a binding fragment thereof.

15. The composition of claim 4 wherein said suppressant agent is a monoclonal antibody or binding fragment thereof specific for Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

16. The composition of claim 15 wherein said antibody or binding fragment thereof binds to a conserved epitope on Ephrin-Bl, Ephrin-B2 and Ephrin-B3.

17. The composition of any one of claims 1 to 10 wherein said suppressant agent or at least one of said suppressant agents is selected from the list comprising: a chemical, small molecule inhibitor, polynucleotide, polypeptide, peptide, protein, NA, DNA or aptamer.

18. The composition of claim 11 or claim 12 wherein said suppressant agent is selected from an antisense nucleic acid, siRNA, miRNA, CRISPR or a ribozyme.

19. The composition of any one of claims 1 to 10 wherein each suppressant agent is

individually selected from the list comprising: an antibody or binding fragment thereof, a chemical, a small molecule inhibitor, a polynucleotide, a polypeptide, a peptide, a protein, an RNA molecule, a DNA molecule, an aptamer, an antisense nucleic acid, siRNA, miRNA, CRISPR or ribozyme.

20. The composition of any preceding claim wherein said demyelination is associated with stroke, radiation injury, heavy metal poisoning, traumatic injury to the brain or spine or chemical injury.

21. The composition of any one of claims 1 to 19 wherein said demyelination is caused by a demyelinating disease.

22. The composition of claim 21 wherein said demyelinating disease is characterised by damaged myelin sheaths.

23. The composition of claim 21 or 22 wherein said demyelinating disease is selected from the list comprising: neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, post-infectious encephalitis, and demyelinating injuries.

24. The composition of any one of claims 21 to 22 wherein said demyelinating disease is selected from the list comprising Multiple Sclerosis, Acute Disseminated

Encephalomyelitis, Transverse Myelitis, Schilder's Disease, Balo's Disease, Clinically Isolated Syndrome, Alexander's Disease, Canavan Disease, Cockayne's Syndrome, Pelizaeus- Merzbacher's Disease, Optic Neuritis, Neuromyelitis Optica, HTLV-I Associated Myelopathy, Hereditary Leukencephalopathy, Guillain-Barre Syndrome, Central Pontine Myelinosis, Deep White Matter Ischemia, Progressive Multifocal Leukoencephalopathy, Demyelinating HIV Encephalitis, Demyelinating Radiation Injury, Acquired Toxic-metabolic Disorders, Posterior Reversible Encephalopathy Syndrome, Central Pontine Myelinolysis, leukodystrophies, Adrenoleukodystroph, Krabbe's globoid cell and/or metachromatic leukodystrophy.

25. The composition of any of claims 21 to 24, wherein said demyelinating disease is multiple sclerosis (MS).

26. A method of treating demyelination , said method comprising administering to the subject a therapeutically effective amount of a composition comprising a suppressant agent or suppressant agents against Ephrin-B3, Ephrin-Bl and/or Ephrin-B2.

27. The method of claim 25 wherein said composition is as described in any one of claims 1 to 19.

28. The method of claim 26 wherein said demyelination is associated with stroke, radiation injury, heavy metal poisoning, compressive injury of the spinal cord or brain, traumatic injury to the brain or spine or chemical injury.

29. The method of claim 26 wherein said demyelination is associated with a demyelinating disease.

30. The method of claim 29 wherein said demyelinating disease is demyelinating disease is selected from the list comprising: neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, post-infectious encephalitis, and demyelinating injuries.

31. The method of claim 29 or 30 wherein said demyelinating disease is selected from the list comprising: Multiple Sclerosis, Acute Disseminated Encephalomyelitis, Transverse Myelitis, Schilder's Disease, Balo's Disease, Clinically Isolated Syndrome, Alexander's Disease, Canavan Disease, Cockayne's Syndrome, Pelizaeus- Merzbacher's Disease, Optic Neuritis, Neuromyelitis Optica, HTLV-I Associated Myelopathy, Hereditary

Leukencephalopathy, Guillain-Barre Syndrome, Central Pontine Myelinosis, Deep White Matter Ischemia, Progressive Multifocal Leukoencephalopathy, Demyelinating HIV Encephalitis, Demyelinating Radiation Injury, Acquired Toxic-metabolic Disorders, Posterior Reversible Encephalopathy Syndrome, Central Pontine Myelinolysis, leukodystrophies, Adrenoleukodystroph, Krabbe's globoid cell and/or metachromatic leukodystrophy.

32. The method of any one of claims 29 to 31 wherein said demyelinating disease is multiple sclerosis (MS).