KR20100132869A - Therapeutic applications - Google Patents

Abstract

PURPOSE: A method for treating neurodegenerative diseases using Eph A4 antagonist or Eph A4 ligand antagonist is provided to delay clinical progress. CONSTITUTION: A composition for treating neurodegenerative diseases(NDD) contains Eph A4 antagonist or Eph A4 ligand antagonist. The NDD is multiple sclerosis(MS) or modified MS. A biological sample derived from an individual having axonal injury is used as a screening agent. The antagonist preserves function or completeness of nerve tissue or reduces the amount of generation degree of nerve injury. The antagonist is soluble eprine or functional mutant or analogue thereof.

Description

Therapeutic Uses {THERAPEUTIC APPLICATIONS}

The present invention relates to therapeutic neuroprotective molecules and neuroregeneration molecules. In particular, the present invention relates to therapeutic molecules that mitigate the clinical progression of neurodegenerative diseases such as multiple sclerosis (MS) and modified forms of MS.

Specific details of the documents incorporated herein are also described at the end of the present specification.

The nervous system, especially the central nervous system, has limited regenerative capacity, often with shocking consequences after illness or injury. The nervous system is an area of intensive research, with some optimism in the art that some form of acute spinal cord injury can be recovered to some extent through mammalian spinal cord injury models.

Many different molecules, cells and mechanisms are involved in neuronal regeneration. Horner and Gage, Nature. 407 (6807): 963-970, 2000. These are described. In particular, this relates to the Eph and ephrin receptor-ligand teams. In particular, Eph and ephrin, identified in humans, mice and chickens, consist of four phylogenetic groups. The Eph A receptor (currently 10) binds to Group A Ephrin Ligand (6), which is a GPI-linked membrane molecule, and the Eph B Receptor (6) is the Ephrine B ligand (3), a transmembrane molecule To combine. In binding, most of the Eph A receptors are mostly bound to ephrin A ligands, and most of the Eph B receptors are mostly mixed to bind to ephrin B ligands. There are two exceptions to this, Eph A4 which binds to ephrin B3 and EphB2 which binds to ephrin A5.

The intracellular domain of the transmembrane Eph receptor includes a kinase domain that is linked to the membrane by a juxtamembrane domain. The carboxy side of the kinase domain includes a SAM domain and a carboxy-terminal PDZ-binding domain. The extracellular domain consists of two fibronectin type III repeats, a cysteine high content region and an amino-terminal ephrin binding domain. Ephrin B ligands include an extracellular Eph-binding domain, an intracellular portion comprising a short carboxy tail and a carboxy terminal PDZ binding domain. The EphB-ephrin B complex has crystallized and its structure can be used for computer-based structure-based interaction regulator identification. The high affinity linkage between the receptor and the ligand is the G-H loop of the Eph-binding domain, which is introduced into the gap of the Eph receptor, a loop of 15 amino acids in length. The low affinity interaction site appears to particularly promote binding between two Eph-ephrin complexes. Interaction and clustering of receptors induces activation of signal transduction pathways mediated by phosphorylation of residues in the cytoplasmic domain, thereby promoting receptor kinase activity. Bidirectional signal transduction is such that cellular ephrin binding to the Eph receptor bound to the cell can induce signal transduction to either ephrin or the Eph receptor. Antagonists of Eph / ephrine interactions are generally designed to block the activation of receptors or ligands by blocking high or low affinity interactions between members.

Eph was first studied in embryonic development, which was found to be an important axon guide molecule in many nervous systems. In such a system, the complementary expression gradient of the Eph / ephrine molecule bound to a particular cell effectively attacks or inhibits axon growth against its suitable target. Eph / ephrine interactions, including soluble forms of action, are currently thought to participate in a variety of ways in the development, maintenance, and repair of many different systems, including the nervous, vascular and immune systems (Wilkinson, Nat Rev Neurosci). 2 (3): 155-164, 2001, Pasquale, Nat Rev Mol Cell Biol. 6 (6): 462-75, 2005 and Horner and Gage, 2000 (documents described above). In many studies, upregulation of Eph and ephrin and reactive astrocytes in neural tissue have been identified after acute spinal cord injury or hemisection of the spinal cord. Re-expression of these receptors in adults is presumed to be important in regulating neuronal regeneration after acute injury (Goldshmit et al., 2004 (see above), Goldshmit et al., Brain Res Brain Res Rev 52) : 327-45, 2006). Blocking of Eph4 by the use of soluble ligands in astrocytes reduces all direct inhibitory effects of Eph4 on axon regeneration as confirmed in vitro (Goldshmit et al., 2004 (documents described above) being)).

Eph and ephrin polypeptides can be used to activate or inhibit activation of Eph receptors according to their stoichiometry. Eph receptor activation is required for ephrin-Fc ligand clustering or membrane adhesion of ephrin ligands, but blocks Eph activation and subcellular responses when not clustered (Goldshmit et al., 2004 (documents described above); Davis et al., Science 266: 816-9, 1994; Ohta et al., Mech Dev 64: 127-35, 1997; Stein et al., Genes Dev 12: 667-78, 1998).

Soluble Eph-Fc ligands have been found in a number of studies to be effective in vivo, particularly in terms of inhibition of tumor proliferation (Brantley et al., Oncogene 21: 7011-26, 2002; Cheng et al., Neoplasia 5: 445-56, 2003, Dobrzanski et al., Cancer Res 64: 910-9, 2004).

Despite the considerable amount of research achieved on the role of Eph receptors in the nervous system, the control or action of any Eph / ephrine in CNS disease, especially experimental autoimmune encephalomyelitis (EAE), which is a condition of MS and its model It is unknown whether there is.

MS is an inflammatory demyelinating neurodegenerative disease of the CNS. This is the most common cause of neurological disorders in young adults and is a permanently significant neurological disorder over many years.

MS was initially thought to be an inflammatory demyelinating disease characterized by demyelination and relative axonal sparing of axons in local lesions in the CNS for years. Although axon conditions have also been noted (Barnes et al., Brain 114: 1271-80, 1991), it has become apparent in recent years that there is substantial axonal damage and reduced MS lesions (Trapp et al., N Engl J Med 338: 278-85). , 1998; Trapp et al., Curr Opin Neurol 12: 295-302, 1999). Axon reduction is now widely thought to be the cause of irreversible permanent disorders observed in secondary or chronic progressive stages of MS (Bjartmar et al., J Neurol Sci 206: 165-71, 2003). In addition, axon reduction occurs in the earlier relapse-relief phase of the disease, and it has been clarified that it is related to the extent of inflammation within the lesion as shown by histological analysis (Trapp et al., 1998 (see above); Ferguson et al., Brain 120: 393-9, 1997; Kornek et al., Am J Pathol 157: 267-76, 2000) and imaging of human MS patient brains (De Stefano et al., Brain 121 (Pt 8): 1469-77, 1998; Gonen et al., Neurology 54: 15-9, 2000). In addition, axon reduction is not only in the normal appearance of white matter (Bjartmar et al., Neurology 57: 1248-52, 2001), but also in gray matter cortical lesions (Kidd et al., Brain 122 17-26, 1999; Peterson et al., Ann Neurol 50 389-400, 2001). These findings, combined with EAE model analysis that correlate axon reduction with clinical values (Wujek et al., J Neuropathol Exp Neurol 61: 23-32, 2002), suggest that the onset of chronic progressive stages of MS axon It has been hypothesized that it is associated with a threshold reduction in axonal integrity (Bjartmar et al, 2003 (documents described above)). In the relapsing-mitigating phase of MS, remyelination occurs in demyelinated lesions, but there is very little evidence suggesting that damaged axons are regenerated. Since the MOG model of EAE produces substantial axon reduction (> 40% in many areas), this model is a useful tool in this regard (Lo et al., J Neurophysiol 90: 3566-71, 2003; Wu et al. , Neuroimage 37: 1138-47, 2007).

In MS and EAE, one of the pathological hole markers is the development of glial traces in demyelinated plaques, formed by activation of astrocytes (Raine et al., Lab Invest 31: 369-80, 1974; Smith et. al., Brain Res 264: 241-53, 1983). After CNS injury, astrocytes are activated, enlarge and proliferate, and increase the expression of glial fibrillary acidic protein (GFAP). It also produces soluble factor and extracellular matrix molecules. Eph A4, along with Eph A7, A6, A3 and A1, is also expressed in astrocytes of human MS tissue. However, it was not confirmed whether this expression was merely a result of injury or evidence of regeneration, or both (Sobel, Brain Pathol. 15 (1): 35-45, 2005).

Thus, the association of MS and EAE with astrocytic gliosis has been recognized for a long time. However, it is not known what role these gliosis plays in the clinical progression and pathology of MS and EAE. Indeed, some studies have shown that gliosis may have beneficial aspects in these conditions. For example, EAE in GFAP null mice has resulted in more serious clinical outcomes (Liedtke et al., Am J Pathol 152: 251-9, 1998).

Research into the molecular mechanisms and cellular phenomena characteristic of neurodegenerative diseases can lead to increased understanding of complex molecular / cell pathways that are up or down regulated and may be associated with either causing or repairing nerve damage. However, these studies have only reached some of the achievements of interest in terms of treatment. This requires a new treatment for neurodegenerative diseases. Neuroprotective compositions for use in various ranges have been explored.

The present invention relates to therapeutic neuroprotective molecules and neuroregenerating molecules, and in particular, to provide therapeutic molecules that mitigate the clinical progression of neurodegenerative diseases such as multiple sclerosis (MS) and modified forms of MS.

The present invention provides a composition for treating neurodegenerative disease (NDD), comprising an Eph A4 antagonist or an Eph A4 ligand antagonist.

summary

Historically, experimental autoimmune encephalomyelitis (EAE) mouse models have been shown to exhibit histopathological and neuropathologically specific characteristics of multiple sclerosis (MS), providing a useful indicator of the therapeutic effect of putative modulators in MS. Has been proven.

In the present invention, it was surprisingly found that the clinical trend of experimental autoimmune encephalomyelitis (EAE) is less severe in Eph A4 knockout mice, and that axon damage is reduced in Eph A4 knockout mice suffering from EAE compared to EAE controls.

As shown in Figures 1 and 2, Eph A4 deficient mice showed significantly lower limb weakness and axon damage compared to control EAE mice. Thus, clinical trends in neurodegenerative diseases (NDD), such as modified forms of multiple sclerosis and multiple sclerosis (MS), by antagonizing Eph A4 or Eph A4-binding ligands (hereinafter referred to as "Eph A4 ligands") Propose to improve, or weaken.

In this specification, each example may be applied to each other example with the necessary changes except where explicitly stated.

In one embodiment, the invention includes a method of treating a neurodegenerative disease (NDD) in an individual, such as MS, comprising administering to the individual an Eph A4 antagonist or an Eph A4 ligand antagonist. In specific instances, the antagonist weakens the clinical progress of the disease.

In a related embodiment, the present invention includes Eph A4 antagonists or Eph A4 ligand antagonists for use in the treatment of neurodegenerative diseases such as MS in a subject. In specific embodiments, antagonists weaken the clinical progression of the disease.

Similarly, the present invention provides a wide range of uses of Eph A4 antagonists or Eph A4 ligand antagonists in the manufacture of a medicament for the treatment of NDD, such as MS, in a subject. In specific embodiments, antagonists weaken the clinical progression of the disease. "Manufacturing" includes the selection and manufacture of a medicament.

In another similar embodiment, the present invention provides a pharmaceutical composition comprising an Eph A4 antagonist or an Eph A4 ligand antagonist for use in treating NDD, such as MS, in a subject. In specific embodiments, antagonists weaken the clinical progression of the disease. For example, antagonists may reduce the rate, incidence or amount of nerve damage occurring in an individual or preserve the integrity or function of nerve tissue.

In another embodiment, the invention includes a method of treating NDD, such as MS, in a subject, the method comprising the following steps:

i) screening the biological sample obtained from the individual for axonal damage;

ii) administering an Eph A4 antagonist or Eph A4 ligand antagonist under conditions sufficient to reduce the rate, incidence or amount of neuronal damage occurring in the subject or to maintain neural tissue integrity or function.

In this example, the subject may be at risk for NDD or may exhibit clinical symptoms of NDD.

In some examples, NDD is an MS or a modified form of MS. As used herein, "MS" includes MS or a modified form of MS.

In some embodiments, the antagonist is administered at a time and condition sufficient to attenuate clinical progression of the disease. In some embodiments, the subject has or is likely to have mild nerve damage or weak progressive neurological dysfunction. In some embodiments, neuronal dysfunction is limb weakness. In some embodiments, the amount of antagonist is provided in an amount effective to attenuate progressive neuronal dysfunction.

In some embodiments, the antagonist preserves the function or integrity of the nerve tissue and reduces the rate, incidence or amount of nerve damage that occurs in the individual.

The term “injury” includes neuronal (axonic) changes that mean reduced cell integrity or reduced function. The term includes cell death, denaturation and fragmentation. In some embodiments, the term extends to decreased conductivity or reactivity, or demyelination of nerves. Nerve damage can be analyzed by the methods described herein and by any methods that are recognized.

In some embodiments, the antagonist maintains the function or integrity of neural tissues and reduces the rate, incidence or amount of neuronal damage occurring in the subject during the chronic progressive process of NDD, such as MS. In some embodiments, the administration is administered before or at the beginning of the clinical symptoms, or at or before the onset of the recurrence of the clinical symptoms.

In some embodiments, the antagonist is administered to or near the nerve tissue. In some embodiments, administration is topically to the brain, spinal cord or optic nerve.

In some embodiments, the subject is a mammal, including a human.

In some embodiments, the antagonist is administered systemically to be provided at about 1 mg / kg to 100 mg / kg daily, every other day or weekly, or in lower amounts for national delivery.

Eph A4 antagonists or Eph A4 ligand antagonists are described in more detail in the following description and in the detailed description, and in the Examples.

In some embodiments, the antagonist binds to Eph A4 and downregulates its level or activity. In an illustrative example, the antagonist is ephrin, including soluble forms, functional variants or analogs that bind to Eph A4 and downregulate its level or activity. Suitable ephrins are ephrin A, such as ephrin A1, A2, A3, A4, A5, A6, or ephrin B, such as ephrin B2 or B3. In some embodiments, the ephrin is A4, A5, B2 or B3. Ephrin may be a monomer, dimer, tetramer or multimer. In some embodiments, the soluble ephrin is an Fc or His fusion protein. Fc fusions are in particular contemplated.

In another example, the antagonist binds to and inhibits the level or activity of an Eph A4-binding ephrin molecule. In an illustrative example, the antagonist is an Eph A4 receptor, such as a soluble Eph A4 receptor polypeptide or a functional variant or analog thereof. In some embodiments, Eph A4 is a monomer, dimer, tetramer or multimer. In some embodiments, the Eph A4 receptor is Eph A4-Fc or His fusion protein. Fc fusions are in particular contemplated.

In another example, the antagonist is an antisense or inhibitory RNA molecule.

In some embodiments, the antagonist is, but is not limited to, a protein, polypeptide or peptide, such as a stapled peptide or peptide mimetic. In another embodiment, the antagonist is, but is not limited to, organic molecules (typically small or medium sized) such as molecules derived from a library of pharmaceutically acceptable small organic molecules.

In some embodiments, the antagonist is an antibody that binds to Eph A4 or Eph A4 ligand, an antigen-binding fragment or aptamer of the antibody.

The present invention further provides combination therapies, such as targeting Eph A4 signaling to reduce clinical signs of MS or other NDDs such as nerve damage, and also provide immunomodulators, immunosuppressants or anti-inflammatory agents, or disease or immunity or disease. It provides a method of use for improving the inflammatory component of.

Accordingly, the present invention includes a method of treating NDD, such as MS, comprising administering an Eph A4 antagonist or an Eph A4 ligand antagonist with at least one other therapeutic agent or treatment. In a similar example, the present invention provides an Eph A4 antagonist or an Eph A4 ligand antagonist with at least one other therapeutic agent or treatment for use in treating NDD, such as MS. In another related embodiment, the present invention provides an Eph A4 antagonist or an Eph A4 ligand antagonist for treating NDD, such as MS, wherein the Eph A4 antagonist or Eph A4 ligand antagonist must be administered with at least one other therapeutic agent or treatment. do. Likewise, the present invention provides at least one other therapeutic agent or treatment for treating NDD, such as MS, wherein the therapeutic agent or method must be administered with an Eph A4 antagonist or an Eph A4 ligand antagonist. In some embodiments, the antagonist weakens the clinical progress of the disease. "Together" includes sequential or simultaneous administration.

In another aspect, the invention provides an Eph A4 antagonist or Eph A4 in the manufacture of a composition for preserving the function or integrity of neural tissue in an individual or for reducing the rate, incidence or amount of nerve damage occurring in an individual. Provided is the use of a ligand antagonist. In some embodiments, the subject has or is at risk of developing symptoms associated with nerve damage.

In a similar example, the method comprises administering an Eph A4 antagonist or an Eph A4 ligand antagonist to the subject, either preserving the function or integrity of the neural tissue in the subject, or adjusting the rate, incidence, or amount of nerve damage that occurs in the subject. It is provided to reduce. In some embodiments, the subject has or is at risk of developing symptoms associated with nerve damage.

Eph A4 antagonists or Eph A4 ligand antagonists are similarly provided to preserve the function or integrity of nerve tissue, or to reduce the rate, incidence or amount of nerve damage that occurs in an individual. In some embodiments, the subject has or is at risk of developing symptoms associated with nerve damage.

Likewise, the pharmaceutical composition is a composition comprising an Eph A4 antagonist or an Eph A4 ligand antagonist to preserve the function or integrity of nerve tissue or to reduce the rate, incidence or amount of nerve damage occurring in an individual. In some embodiments, the subject has or is at risk of developing symptoms associated with nerve damage.

The pharmaceutical composition is formulated with a pharmaceutically acceptable carrier and / or diluent.

Also provided is a medical kit comprising an Eph A4 antagonist or an Eph A4 ligand antagonist with instructions for use in the treatment of NDD, such as MS.

In addition, the present invention comprises screening a subject-derived biological sample for axon damage, administering an Eph A4 antagonist or Eph A4 ligand antagonist at a time and condition indicated to be sufficient to reduce nerve damage. It further provides a treatment protocol for treating NDD. In some embodiments, the subject is rescreened for axon injury after treatment. In some embodiments, in another example or in addition, a subject-derived sample is examined to determine one or more symptomatic conditions, such as autoimmunity, inflammation or demyelination. In some embodiments, the antagonist is administered prior to nerve damage to prevent nerve damage.

In another embodiment, the present invention provides for the screening of any one or more Eph or ephrin modulators for their ability to attenuate clinical progression of NDD, such as MS. In some embodiments, the agent investigates the ability to maintain the function or integrity of neural tissue or to reduce the rate, incidence or amount of axon damage in a subject as illustrated in the examples.

In another example, screening for identifying agents for treating NDD, such as MS, comprising screening the agent for the ability to modulate the level or activity of a polypeptide having the functional activity of an Eph A4 or Eph A4 ligand. to provide.

The foregoing summary is a comprehensive description of all examples of the invention and is not presented in any way and should not be presented.

Detailed description of the invention

generalization

Abbreviations used herein are shown in Table 1.

Except where expressly stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All materials and methods similar or equivalent to those described herein can be used to practice or test the present invention. Experts will find Sambrook, in particular, for definitions and terms in the art and other methods known to those skilled in the art. et al ., Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, Coliganet al.,Current Protocols In Protein Science, John Wiley & Sons, Inc., 1995-1997, in particular Chapters 1, 5 and 6, and Ausubelet al ., Current Protocols in Molecular Biology , Supplement 47, John Wiley & Sons, New York, 1999.

Throughout the specification, unless otherwise necessary in context, variations such as the terms "comprises" or "comprises" or "comprising" include the constituent elements, integers, or groups of elements or integers, and others. It will be understood that not all other elements, integers or groups of elements or integers are excluded.

"Consisting of" includes, but is not limited to, "before" consisting of. That is, the expression "consisting of" means that the listed elements are necessary or necessary, and other elements may not exist. By "essentially" is meant to include all listed elements preceding the expression and is limited to other elements which do not interfere or contribute to the activity or action specified in the description of the listed elements. Thus, the expression “essentially made” means that the listed elements are necessary or necessary, other elements are not optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

The terms "polypeptide", "protein" and "peptide" are used interchangeably herein.

As used herein, the singular forms "a, an" and "the" also include plural references unless the context clearly dictates otherwise. Thus, for example, "cell" includes not only one cell but also two or more cells; "Formulation" includes one formulation as well as two or more formulations.

The terms “genetic material”, “genetic form”, “nucleic acid”, “nucleotide” and “polynucleotide” include both sense and antisense strands of RNA, cDNA, genomic DNA, synthetic forms and hybrid polymers, and will be apparent to those skilled in the art. As can be, it can include chemically or biochemically modified or non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling substances, methylation, substitution of one or more naturally occurring nucleotides with analogs (eg, morpholine rings), uncharged linkages (eg, methyl phosphonates, phosphoesteres, phosphates) Poamidates, carbamates, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant moieties (e.g., Polypeptides), intercalators (eg, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (eg, Alpha-anomeric nucleic acids, etc.). It also includes synthetic molecules that mimic polynucleotides in terms of their ability to bind to specified sequences, through hydrogen bonding and other chemical interactions. Such molecules are known in the art and, for example, there are peptide linkages that replace phosphate linkages in the backbone of the molecule.

The term "gene" is used in its broadest sense and includes cDNAs corresponding to exons of genes. “Gene” herein also refers to classical genomic genes consisting of transcriptional and / or translational regulatory sequences and / or coding regions and / or untranslated sequences (ie, introns, 5′- and 3′-untranslated sequences); Or mRNA or cDNA corresponding to the coding region (ie exon) of the gene and the 5'- and 3'-untranslated sequences.

By "isolated" it is meant that in its original state substantially or inevitably the components normally accompanying it are present. For example, herein, an “isolated polynucleotide” refers to a DNA fragment in which a polynucleotide, such as a sequence normally adjacent to a fragment, has been removed from a sequence flanked in its naturally occurring state. Or “isolated peptide” or “isolated polypeptide” and the like herein means in vitro isolation and / or purification of the peptide or polypeptide molecule from its original cellular environment and from combination with other components of the cell. do. Without limitation, an isolated composition, complex, polynucleotide, peptide, or polypeptide may refer to a sequence produced by recombinant or synthetic means, or a native sequence separated by purification.

Citations referenced herein should not be construed as an admission that such references are available as "prior art" in this application.

The invention is not limited to specific screening methods for materials. Modifications may be made to the material of a particular formulation and various medical methods. The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting.

"Treat" or "treat" includes prophylactic and protective measures, as well as therapeutic means, which are intended to partially reduce the severity of clinical signs of neurodegenerative diseases or to delay or alleviate the clinical progress of the disease. Without being bound by theory, the experimental results described herein indicate a reduction in the rate, incidence, or amount of axon damage that decreases Eph A4 function maintains the function or integrity of neural tissue or contributes to better clinical outcomes. Means to provide.

Implementation

In one embodiment, the invention includes a method of treating a neurodegenerative disease (NDD), such as MS, comprising administering an Eph A4 antagonist or an Eph A4 ligand antagonist to an individual.

The term “antagonist” in a broad sense means a substance, active substance, modulator, medicament, chemical, pharmaceutically active substance, drug, molecule, and the like, which down-regulates the level or activity of a target molecule. Here, "activity" includes one or more biological functions of the target molecule. In some embodiments, the target is an Eph or ephrin polypeptide molecule. As is known in the art, the activity of these molecules includes, inter alia, intracellular signaling activity, enzymatic activity and binding activity that makes it functional. Signaling activity includes intracellular transfer of signals that act on cellular responses mediated by, for example, expression, phosphorylation or binding of intracellular elements or structural changes; Enzymatic activity includes kinase or other catalytic activity; Binding activity includes low, medium or high affinity binding to complementary Eph, ephrin or other molecules that form a complex or bind to Eph or ephrin. As is known in the art, in some examples, binding is inhibited, while in other examples, binding results in activation and receptor / ligand signaling. These functions are easily evaluated and quantified. The activity of nearby membrane domains, functional domains such as PDZ-domain or SAM-domain, GPI anchors, fibronectin repeats and cysteine high content domains also affects the regulation of functional activity of Eph and ephrin. Methods of monitoring the level or activity of Eph / ephrine or other ligands are known in the art. For example, some antagonists may be used in Eph A4 receptor binding assays such as Davis et. al ., 1994 (documents described above), Murai et al ., Mol Cell Neurosci . 24 (4): 1000-1011, 2004 or PubChem Bioassay AID No. 689 ( Colorimetric assay for HTS discovery of chemical inhibitors of Eph A4 receptor antagonists can be identified. Suitable Eph A4 antagonists and Eph A4 ligand antagonists can inhibit the binding of Eph A4 ligands to Eph A4. Likewise, for example Davis et Other antagonists can be identified using the Eph A4 receptor phosphorylation assay disclosed in al ., 1994 (documents described above). Suitable Eph A4 antagonists and Eph A4 ligand antagonists can inhibit phosphorylation of Eph A4 when exposed to Eph A4 ligand. Functional analysis such as Murai et Some antagonists can be identified using the neutral crest migration assay described in al ., 2004 (documents described above). Suitable Eph A4 antagonists and Eph A4 ligand antagonists can inhibit the function of Eph A4 in vitro or in vivo. Some antagonists may be identified by computer silico method. For example, the crystal structure of the high-affinity ephrin-binding channel of the Eph A4 receptor is described by Qin et. al ., J Biol Chem . 283 (43): 29473-29484, 2008. This structure can be used to logically design suitable Eph A4 antagonists and Eph A4 ligand antagonists.

In some instances, the level or activity of the target polypeptide is currently common in the art, such as by reduction in transcription or translation levels, such as inhibition of activity of a promoter or enhancer, by methylation, or in co-inhibition, antisense or inhibitory RNA strategies. Can be reduced by the use of gene silencing methods. Accordingly, the present invention provides, if necessary, Eph A4 or Eph A4 ligands, for example, by gene therapy, or by inhibiting or promoting the function of a gene, or by the use of gene silencing constructs or antisense or inhibitory RNA oligonucleotides known to those skilled in the art. The use of nucleic acid molecules or vectors, including nucleic acid molecules, to indirectly regulate the level of protein. Methods of regulating and monitoring gene function, including transcription or translation, are known in the art. For example, the method of controlling and monitoring the expression of Eph A4 is described in Fu et. al ., Nature Neuroscience 10 : 67-76 , 2007.

The term “individual” herein refers to a warm blooded animal, in particular a mammal, more preferably a primate, including a lower primate, and even more preferably a human, in which the medical use of the invention may be beneficial. Regardless of human or animal or embryo other than human, an individual can be referred to as an individual, subject, animal or patient. In some instances, the subject is a mammal such as a human.

Modulation or reduction in the level or activity of an individual "Eph", "Eph-binding ligand" or "ephrine" refers to all polypeptide forms or naturally occurring forms of these molecules, such as homologues from other species. It includes. As antagonists these molecules include homologues and naturally occurring analogs of isolated, synthetic or recombinant forms, and portions thereof, such as functional fragments or domains that retain activity, and functional variant forms.

In some embodiments, the subject is at risk of NDD or shows clinical signs of NDD.

Neurodegenerative diseases (NDD) include diseases of the CNS and / or PNS tissues, including peripheral neuropathy, motor neuron disease, amyotrophic lateral sclerosis (Als, Lou Gehrig's disease), Bell's palsy, Alzheimer's disease, Parkinson's disease , Epilepsy, multiple sclerosis, Huntington's chorea, Down's syndrome, hypoxic ischemic encephalopathy, incidental Lewy bodies, amyloid angiopathy, traumatic myelomalacia, Meniere's disease. Peripheral neuropathy is a neurodegenerative disorder that acts on the peripheral nerves, most often occurring as one or a combination of motor, sensory, sensorimotor or autonomic dysfunction. Peripheral neuropathy can be genetically acquired, for example, can result from systemic diseases, or can be caused by toxic substances such as neurotoxic drugs, such as antitumor agents, industrial or environmental pollutants. Peripheral sensory neuropathy is characterized by degeneration of peripheral sensory neurons and can be idiopathic, such as diabetes mellitus (diabetic neuropathy), cytostatic drug therapy in cancer, alcoholism, acquired immunodeficiency syndrome (AIDS), or heredity May occur as a result of genetic predisposition. Genetically acquired peripheral neuropathy includes, for example, Lefsome's disease, Crabe's disease, otitis dystrophic disease, Fabry's disease, Degerin-Sotas syndrome, Riboprotein deficiency, Charco-Marie-Tooth disease (CMT) (also osteomyelitis or Hereditary motor sensory neuropathy (also known as HMSN). Peripheral neuropathy usually acts together on sensory and motor neurons, resulting in hybrid sensory and motor neuropathy, but pure sensory neuropathy and pure motor neuropathy are also known. In some embodiments, the symptoms of NDD progress over months or years, which is referred to as chronic or progressive.

In some embodiments, the neurodegenerative disease is combined with one or more of demyelination, inflammation, and autoimmunity. Examples of diseases associated with demyelination, inflammation, and autoimmunity are multiple sclerosis and its modified diseases, or Schwain Barre syndrome (GBS) and its modified diseases.

In some embodiments, the neurodegenerative disease is multiple sclerosis or MS modified disease. Modified diseases of MS include Marburg's variant, Schilder's disease, Kbal and Devic's concentric sclerosis disease. As an illustrative example, the disease (symptom) is multiple sclerosis.

In some embodiments, the antagonist is administered at a time and condition sufficient to attenuate clinical progression of the disease. In some instances, the individual may or may exhibit mild nerve damage or weak progressive neuronal dysfunction. In some embodiments, neuronal dysfunction is limb weakness. In some instances, the amount of antagonist is provided effectively to attenuate progressive neuronal dysfunction.

In some embodiments, the antagonist maintains the function or integrity of nerve tissue or reduces the rate, incidence or amount of nerve damage that occurs in an individual.

In some embodiments, the antagonist is provided in an “effective amount” sufficient to alleviate or delay the progression of clinical signals of NDD, such as MS.

"Effective amount" means the administration of an active amount effective for treatment, in a single dose or as part of a series, or in a sustained release system, in a subject in the treatment of NDD. The effective amount will vary depending on the individual's health and physical condition, the formulation of the composition used, the assessment of the medical condition and other relevant factors. The amount is expected to be in a relatively wide range that can be determined through routine testing.

Pharmaceutical compositions comprising individual antagonists are contemplated to exhibit therapeutic activity when administered in an amount determined in accordance with a particular case. The variable depends, for example, on human or animal, selected material. Antagonists must be soluble, i.e., agents that are biologically available at sufficiently effective concentrations. A wide variety of dosages can be applied. In consideration of the individual, for example, the preparation may contain about 0.1 mg to 0.9 mg (ie 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg per kg of body weight daily, every other day, weekly or monthly). , 0.8 mg and 0.9 mg), about 15 mg-35 mg, about 1 mg-30 mg or 5-50 mg, or 10 mg-100 mg. Therapeutic antibodies are typically administered at a dosage of about 1-20 mg / kg, but higher or lower dosages are contemplated within the aforementioned ranges. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly, or at appropriate time intervals, or the dose may be proportionally reduced as indicated by the state of emergency.

In some embodiments, the antagonist is administered weekly or daily at about 1 mg-100 mg / kg for systemic delivery or for less than in the case of local delivery.

Administration is generally time and conditions sufficient to alleviate or delay the progression of clinical signs of NDD, such as MS. Clinical signs will vary depending on the neurodegenerative disorder to be treated, but may include symptoms of decreased cognitive performance, sensory performance, or motor performance. Common clinical signs include optic neuritis, transverse spondylitis, brainstem or cerebellum disorders.

The substance may be administered in a conventional manner, such as by oral, intravenous (if water-soluble), intraperitoneal, intramuscular, subcutaneous, intradermal, intrathecal or suppository route or implantation (e.g. use of sustained release material). May be administered. Administration is more systemic, but can be systemic or local. Systemic includes intravenous, intraperitoneal, subcutaneous injection, infusion as well as administration via the oral, rectal and nasal routes, or by convenient inhalation. Other routes of administration that may be considered are by patch, cellular migration, implants, sublingual, intraocular, topical or transdermal. Depending on the severity or grade of the disease and the integrity of the cerebrovascular barrier, suitable antagonists are needed to cross the cerebrovascular barrier. In some embodiments, the substance is administered directly to the PNS or CNS, brain stem or bovine liver.

Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical composition techniques. For example, Remington's Pharmaceutical Sciences , 18th Ed., Mack Publishing, Company, Easton, PA, USA, 1990. The composition may comprise an active substance or a pharmaceutically acceptable salt of the active substance. Such compositions may include, in addition to one of the active substances, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances known in the art. Such substances should be nontoxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation suitable for administration, eg intravenous, oral or parenteral.

A “pharmaceutically acceptable carrier” and / or diluent is a pharmaceutical vehicle composed of a substance that is not inappropriate, ie, must itself cause a substantially deleterious reaction or will not cause a deleterious reaction with the active substance. The carrier may include all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, tonicity modifiers, agents for increasing or decreasing the rate of absorption or scavenging, buffers to maintain pH, chelating agents, membrane or barrier crossing materials. have. Pharmaceutically acceptable salts are salts which are not inappropriate. Compositions or materials containing materials can be administered in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts or metal complexes.

For oral administration, the compounds may be formulated in solid or liquid preparations, such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, in the case of oral liquid preparations (e.g. suspending agents, elixirs and solvents), for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents and the like Conventional pharmaceutical media; Alternatively, in the case of oral solid preparations (eg, powders, capsules, tablets), carriers such as starch, sugar, diluent, granulating agent, lubricant, binder, disintegrating agent and the like can be used. Because of their ease of administration, where solid pharmaceutical carriers are explicitly used, tablets and capsules are the best oral dosage unit form. Tablets may include a binder such as tragacanth, corn starch or gelatin; Disintegrants such as alginic acid; And lubricants such as magnesium stearate. Where appropriate, tablets may be sugar-coded or enteric-coated by standard techniques. The active substance can be encapsulated, making it stable for passage through the gastrointestinal tract. See, eg, international publication WO 96/11698. For parenteral administration, the compounds may be dissolved in pharmaceutical carriers and administered in either solution or suspension. Examples of suitable carriers are water, saline, dextrose solution, fructose solution, ethanol or oils of animal, vegetable or synthetic origin. The carrier may also include other components such as preservatives, suspending agents, solubilizing agents, buffers and the like.

For parenteral administration, the antagonist may be dissolved in a carrier and administered as a solution or suspension. When the substance is administered intradurally, it may be dissolved in cerebrospinal fluid. For transmucosal or transdermal (including patch) delivery, suitable penetrants known in the art are used for the delivery of the antagonist. For inhalation, delivery uses any conventional system, such as dry powder aerosols, liquid delivery systems, air jet nebulizers, propellant systems. For example, the formulation may be administered in the form of aerosol or mist. In addition, the substance can be delivered in the form of long term delivery or long term release. For example, biodegradable microspheres or capsules or other long deliverable polymer constructs may be included in the formulation. Formulations can be modified to modify pharmacokinetics and biodistribution. For a comprehensive description of pharmacokinetics, see for example the Remington literature. In some instances, the formulation may be incorporated into a lipid monolayer or dualism such as liposomes or micelles.

Targeting therapies known in the art can be used to more specifically deliver antagonists to certain types of cells or adjacent tissues such as neurons, oligodendrocytes, astrocytes and the like. Targeting can be helpful if the antagonist is unacceptably toxic, or if the dose is too high or inappropriate for other sites. Or targeting may help to cross the cerebrovascular barrier. Many strategies are known in the art for improving the accessibility of administered antagonists to the nervous system (Misra et. al ., J Pharm Sci 6 : 252-273, 2003).

In some instances, the antagonist is administered to or near the nerve tissue. In some instances, the administration is administered locally to the brain, spinal cord or optic nerve.

The actual amount of active substance administered and the rate and time-path of administration will depend on the nature and severity of the disease. Determination of treatment regimens, such as dosage, timing, etc., is the responsibility of the general practitioner or skilled person, and typically takes into account the condition, delivery site, method of administration, and other factors known to the individual patient. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences (documents described above).

Sustained release preparations that can be prepared are particularly convenient for long term administration of appropriately stable antagonists. An example of a sustained release preparation is a semipermeable matrix of solid hydrophobic polymers containing an antagonist, which matrix is in the form of a tangible object such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactide, air of L-glutamic acid and ethyl-L-glutamate. Copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for 100 days, while certain hydrogels release proteins for shorter periods of time. Small (approximately 200-800 mm 3) unilamellar type liposomes with a lipid content higher than about 30% cholesterol can be used and the selection ratio is adjusted according to the optimal therapy.

Stabilization of the antagonist can be achieved through modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content with titrant additives, and development of specific polymer matrix compositions. The in vivo half-life of an antagonist can be extended using techniques known in the art, such as by the attachment of other elements such as polyethylene glycol (PEG) groups.

The present invention further provides combination therapies, such as Eph A4 signaling targeting, to reduce clinical signs of NDD, such as neuronal damage, such as MS, and can also be used to improve immunomodulatory, immunosuppressive or anti-inflammatory agents or immune or inflammatory components of disease. Provide measures to:

The present invention further provides combination therapies such as Eph A4 signaling targeting to reduce clinical signs of MS or other NDDs, such as nerve damage, and also improves immune or inflammatory components of immunomodulators, immunosuppressants or anti-inflammatory agents or diseases. Provide measures for use. Immunomodulators used in the art include beta-interferon, glatiramer acetate and natalizumab, which are contemplated for combination therapy.

Accordingly, the present invention encompasses methods of treating NDD, such as MS, comprising administering an Eph A4 antagonist or an Eph A4 ligand antagonist with one or more other therapeutic agents or treatments. In a similar example, the present invention provides an Eph A4 antagonist or an Eph A4 ligand antagonist with one or more other therapeutic agents or treatments for use in the treatment of NDD, such as MS. In another related embodiment, the present invention provides an Eph A4 antagonist or an Eph A4 ligand antagonist for use in the treatment of NDD, such as MS, wherein the Eph A4 antagonist or Eph A4 ligand antagonist is administered in combination with one or more other therapeutic agents or treatments. Similarly, the present invention provides one or more therapeutic agents or treatments for use in the treatment of NDD, such as MS, administered with an Eph A4 antagonist or Eph A4 ligand antagonist, which is a therapeutic agent or method. In some embodiments, the antagonist weakens the clinical progress of the disease. "Together" includes sequential or simultaneous administration.

In some instances, the antagonist is administered in combination with current anti-inflammatory agents and other active compounds for therapeutic use in the target disease. The compound is a nonsteroidal anti-inflammatory agent such as corticosteroids such as glatiramer acetate and natalizumab, corticosteroids such as prednisolone, aspirin, ibuprofen and COX-2 inhibitors, methoxtrexate, leflunomide, sulfasalazine disease-modifying anti-rheumatic drugs such as sulfasalazine, azathioprine, cyclosporine, hydroxychloroquine, and D-penicillamine; And biological response modifiers such as cytokines (eg IFN-γ) or cytokine inhibitors.

The term “nerve (nerve) injury” or “axon (neurnal) injury” includes cell death (apoptosis), cell degeneration, cell fragmentation and reduced neuronal conductivity or reactivity. In some embodiments, nerve damage occurs in venus or chronic demyelinated lesions, but damage may also occur in myelinated axons. In certain instances, markers for axon damage allow for combination with clinical progression. In particular, a reduction in the level of axon damage compared to the control means that the subject antagonist can protect the axon from damage sufficient to weaken clinical progression in NDD, such as MS.

Neuronal dysfunction is a CNS or PNS dysfunction. In some embodiments, the neurological dysfunction is a dysfunction of spinal cord function, cognitive motor function, cognitive sensory function or optic nerve function related to clinical progression in NDDs such as MS.

In some embodiments, neuronal dysfunction is limb weakness. In some embodiments, the amount of antagonist is effectively provided for alleviating progressive neuronal dysfunction.

In some embodiments, the antagonist maintains the function or integrity of the nerve tissue or reduces the rate, incidence or amount of nerve damage that occurs in an individual.

The term “injury” includes changes in nerves (axons) which mean loss of cell integrity or loss of function. The term includes cell death, degeneration and fragmentation. In some instances, the term extends to a decrease or demyelination of neuronal conductivity or reactivity.

In some embodiments, the antagonist maintains the function or integrity of the neural tissue and reduces the rate, incidence or amount of axial damage that occurs in the subject during the chronic progression phase of NDD, such as MS. In some embodiments, the administration is prior to or at the onset of the clinical symptom, or prior to or at the onset of relapse of the clinical symptom.

In some embodiments, the present invention includes a method of treating an NDD, such as an MS, comprising the following steps.

i) screening the biological sample obtained from the individual for axonal damage;

ii) administering an Eph A4 antagonist or Eph A4 ligand antagonist under conditions sufficient to reduce the rate, incidence or amount of neuronal damage occurring in the subject or to maintain neural tissue integrity or function.

In some embodiments, the antagonist is administered in proximity to the nerve tissue or nerve tissue. Neural tissue can be, but is not limited to, the brain, CNS, or spinal cord. In some embodiments, topical administration to the brain, spinal cord or optic nerve.

In some embodiments, the subject is a mammal, such as a human.

In some embodiments, the antagonist binds to Eph A4 and down regulates its concentration or activity. As an illustrative example, the antagonist is ephrin, such as soluble forms, functional variants or analogs thereof, that bind to Eph A4 and downregulate its concentration or activity. Soluble ephrins, which are Eph receptor antagonists, are described, for example, in Gale et al ., Neuron . 17 (1): 9-19, 1996 and Davis et as disclosed in al ., 1994 (documents described above), it is well known in the art. These ephrins typically do not contain any membrane anchors and / or transmembrane domains and thus take a soluble form that does not bind to the membrane. Suitable ephrins for use in the present invention are ephrin A such as ephrin A1, A2, A3, A4, A5, A6 or ephrin B such as ephrin B2 or B3. In some embodiments, the ephrin is A4, A5, B2 or B3. Ephrin may be a monomer, dimer, tetramer or multimer. In some embodiments, the soluble ephrin is an Fc or His fusion protein. Fc fusions are in particular contemplated.

In another example, the antagonist binds to and inhibits the level or activity of an Eph A4-binding ephrin molecule. In an illustrative example, the antagonist is an Eph A4 receptor, such as a soluble Eph A4 receptor polypeptide or a functional variant or analog thereof. That is, soluble Eph receptors are well known in the art, as described, for example, in Brantley et al., 2002. (documents described above). These Eph receptors typically do not contain any membrane anchors and / or transmembrane domains, thus taking a soluble form that does not bind to the membrane. Typically, the antagonist is an E4 receptor, such as a soluble E4 receptor polypeptide or a functional variant or analog thereof. In some embodiments, Eph A4 can be a monomer, dimer, tetramer or multimer. In some embodiments, the Eph A4 receptor is Eph A4-Fc or His fusion protein. Fc fusions are in particular contemplated.

In another example, the antagonist is an antisense or inhibitory RNA molecule.

In some embodiments, the antagonist is, but is not limited to, a protein, polypeptide or peptide, such as a spiked peptide or peptide mimetic. Various peptide antagonists are known in the art. These antagonists and other suitable peptide antagonists can be used in the present invention. For example, Murai et antagonists described in al ., 2004 (documents described above). Methods for identifying peptide antagonists are described, for example, in Koolpe et. al ., J Biol Chem ., 280 (17): 17301-17311, 2005. In another embodiment, the antagonist is an organic molecule (typically small or medium sized), such as but not limited to a molecule derived from a pharmaceutically acceptable small organic molecule library. Various organic molecular antagonists are known in the art. Noberini et for which small molecule antagonists are disclosed al ., J Biol Chem ., 283 (43): 29461-29472, 2008. Kolb et also describes methods for screening and purifying small molecule antagonists. al ., Proteins , 73 (1): 11-18, 2008; Chrencik et al ., Structure , 14 (2): 321-330, 2006; And Chrencik et al ., J Biol Chem ., 282 (50): 36505-36513, 2007. These antagonists and other suitable organic molecular antagonists can be used in the present invention. For example, Qin et al . Antagonists described in 2008 (which is the literature described above) can be used.

Isolated recombinant or synthetic polypeptides can be prepared by methods known in the art, such as from genetically engineered host cells comprising an expression system. Experts Sambrook, especially with respect to the other methods known to the definitions and terminology, and those skilled in the art et al ., 1989 (documents described above) and Ausubel et al ., 1999 , supra.

Nucleotide and amino acid sequences for Eph A4 and Eph A4-binding ephrin are disclosed in the appropriate accession numbers in the electronic access database. Insert the appropriate nucleotide sequence into the expression system, Sambrook et al ., 1989 (documents described above) and Examples, which are well understood and can be expressed in suitable host cells by any of a variety of conventional techniques. Polypeptides are recombinant cultures by conventional methods, which generally include chromatography or filtration, and can be recovered and purified from other biological sources.

DNA encoding Eph A4 or ephrin polypeptide can be recombinantly expressed as a fusion protein with the Fc region of a human IgG heavy chain. Soluble forms of ephrin A4, A5 or ephrin B3 are prepared by recombinantly expressing ephrin A5-Fc, ephrin A3-Fc or ephrin B3-Fc fusion proteins. The extracellular domain provides a soluble form of the protein, but any portion of the domain (amino acids 20-546 of human Eph A4) that includes a ligand binding domain (amino acids 20-274) or a functional fragment or variant is contemplated. Signal peptides can be cleaved upon expression and are not shown in FIG. 6. The mouse and human homologues of Eph A4 share 98.2% amino acid sequence identity (536/546). The Fc portion of the fusion protein facilitates protein purification using chromatography and enhances the function, stability, half-life, structure or solubility of the fusion partner. Mouse Fc derived from IgG1 lacks complement and effector function, but human homologues have this function that can interfere with action or activity. Thus, different IgG isotypes can be used that do not modify Fc or interfere with effector cells. As an illustrative example, an IgG4 isotype is used. Eph or ephrine can be used as monomers, dimers, tetramers or multimers as needed.

In some embodiments, the nucleic acid molecule and the virus or other vector comprising the same, if necessary, induce silencing of an Eph A4 or Eph A4 ligand gene, or deliver an antagonist. DNA (gDNA, cDNA), RNA (sense RNA, antisense RNA, mRNAs, tRNA, rRNA, small interfering RNA (SiRNA), double stranded RNA (dsRNA), short hairpin RNA (shRNA), piwi-interacting RNA Nucleic acids such as (PiRNA), microRNA (miRNA), small nuclear RNA (SnoRNA), small nuclear (SnRNA) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes are conveniently used. Methods of making chimeric or fusion constructs capable of producing DNA or RNA in the art are disclosed in the art.

In some embodiments, the antagonist is an antibody that binds to Eph A4 or Eph A4 ligand, an antigen-binding fragment or aptamer of the antibody.

As used herein, an "antibody" refers to a whole antibody, or fragment thereof, such as F (ab ') ₂ , Fab, Fab', Fv, VH or VK fragment, single chain antibody, multimeric single specific antibody or fragment thereof. Or bispecific or multispecific antibodies or antigen-binding fragments thereof that exhibit desirable binding and function blockade. Binding means that the fragment specifically binds with sufficient affinity to the antigen to be therapeutically useful. In addition, the antibody or fragment thereof is selected for its ability to bind and block target activity such as intracellular signaling. Herein, the antibody is a polyclonal antibody or a monoclonal antibody. Antibodies can belong to all immunoglobulin classes and can be, for example, IgGs such as IgG1, IgG2, IgG3, IgG4, IgE, IgM or IgA antibodies. It may be of animal or mammalian origin, for example a murine, rat or human antibody. Alternatively, the antibody may be a chimeric antibody. The term chimeric antibody means herein all antibodies that include moieties derived from various animal species. Particularly non-limiting examples are antibodies in which the variable portion is derived from a mouse and the antibody constant portion is human. Antibodies, wherein one or more CDR sequences are derived from a mouse and the remaining regions of the variable and constant regions are from human immunoglobulins, are generally described as “humanized”. Antigen binding fragments of an antibody include all, some, or portions of the variable enough to exhibit the desired binding to an Eph A4 or Eph A4 ligand.

Methods of making monoclonal and polyclonal antibodies are known in the art, for example Harlow and Lane, Antibodies , A Laboratory Manual , Cold Spring Harbor Laboratory, 1988, section 5 and 6. For example, antibodies according to the invention can be prepared by conventional immunization and recombinant DNA techniques. Thus, for example, polyclonal antibodies can be obtained from animal serum immunized with a target protein or fragment thereof. Any suitable host, eg, BALB / c mice, which is beneficial for obtaining mouse polyclonal antibodies, can be injected with immunogens, serum collected and antibodies recovered therefrom. Monoclonal antibodies can be obtained from hybridomas derived from spleen cells of the immunized animals described above and can be fused to appropriate “infinite proliferating” B-cancer cells. In each case, using standard purification and / or concentration techniques, such as chromatography using Protein A or other affinity chromatography using proteins or fragments thereof, the antibody is either serum or hybridoma. Can be recovered from.

By obtaining a cell line expressing an appropriate antibody, such as a hybridoma, it is possible to identify a variable region gene encoding a desired antibody, including a sequence encoding a CDR. From there, one or more replicable expression vectors are prepared, comprising at least one DNA sequence encoding the variable region of the light or heavy chain of the antibody and optionally another DNA sequence encoding the rest of the heavy and / or light chain, Chimeric antibodies can be prepared by transforming a suitable cell line in which the antibody is produced, such as a non-producing myeloma cell line, such as a mouse NSO line. Certain methods of making antibodies in this manner are generally known and commonly used. Antibody production includes similar methods such as stimulation of an immune response using injection into an animal, as well as production of synthetic antibodies, screening of recombinant immunoglobulin libraries for specific binding molecules, or in vitro stimulation of lymphocyte populations.

Antibodies are particularly useful when the target has a binding site accessible outside the cell. In this respect, the antibody does not have to cross or completely penetrate the cell membrane. Antibodies specific for Eph A4 or ephrin polypeptides can be used, for example as direct antagonists. Antibodies can be generated using methods known in the art and include, for example, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments generated by Fab expression libraries.

In some instances, the antibody of the invention is a CDR-grafted antibody. As used herein, the term "CDR-grafted antibody" means that a heavy and / or light chain is transplanted from a donor antibody (eg, a monoclonal antibody in a mouse) into the heavy and / or light chain variable region structure of a recipient antibody (eg, a human antibody). Mean an antibody molecule comprising one or more CDRs. The structure of CDR-grafted antibodies is fully described in European Patent Application EP-A-0239400, which is incorporated herein by reference. Criteria for selecting structural residues that need to be modified are described in international patent application WO 90/07861, which is incorporated herein by reference.

Substances that may act as Eph A4 signal modulators include organic molecules that can penetrate through (usually small or medium) cell membranes or ion channels or other pores, and cartilage fish-derived antibodies (eg sharks). Antibody; Liu et al ., BMC Biotechnol . 7 : 78, 2007), as well as antigen-binding agents with intracellular delivery ability.

Antigen binding agents or functionally active fragments thereof having intracellular delivery ability include camelid and llama antibodies, scFv antibodies, intracellular antibodies or nanobodies such as scFv intracellular antibodies and V._HH Include antibodies such as intracellular antibodies. Such antigen binding agents are described in Harmsen and De Haard,Appl . Microbiol . Biotechnol. 77(1): 13-22, 2007; Tibaryet al ., Soc . Reprod . Fertil . Suppl. 64: 297-313, 2007; Muyldermans,J. Biotechnol . 74: 277-302, 2001; And the documents cited therein. In some instances, scFv intracellular antibodies that can interfere with protein-protein interactions can be used in the present invention; For example Visintinet al ., J. Biotechnol , 135: 1-15, 2008 and Visintinet al , J. Immunol . Methods , 290(1-2): Refer to the production method disclosed in 135-53, 2008.

Modulators used in the present invention are Constantini et al ., Cancer Biotherm . Radiopharm. 23 (1): 3-24, 2008, such as cell-permeable peptide sequences or nuclear-localizing peptide sequences.

Aptamers are also thought to be antagonists. RNA and DNA aptamers can replace monoclonal antibodies in a variety of applications (Jayasena, Clin . Chem ., 45 (9): 1628-1650, 1999; Morris et. al ., Proc . Natl . Acad . Sci ., USA , 95 (6): 2902-2907, 1998). Aptamers are nucleic acid molecules that have binding affinity specific for nonnucleic acid or nucleic acid molecules through interactions other than the classic Watson-Crick base pairs. Aptamers are described, for example, in US Pat. No. 5,475,096; 5,270,163; 5,589,332; 5,589,332; And 5,741,679. Numerous DNA and RNA aptamers that recognize nonnucleic acid targets have been developed and characterized by SELEX (Gold et. al ., Annu . Rev. Biochaem ., 64 : 763-797.1995; Bacher et al ., Drug Discovery Today , 3 (6): 265-273, 1998).

In some instances, agents that downregulate the formation, expression, or activity of the Eph A4 receptor may be derived from, or are variants or analogs of, Eph A4 polypeptides or coding sequences thereof. Thus, for example, the substance may be a natural protein or hydrocarbon-stapled peptide that is alpha-helix and cell-permeable and may interfere with protein-protein interactions (eg, Wilder et al ., Chem Med Chem . 2 (8): 1149-1151, 2007; And Henchey et al ., Curr Opin Chem Biol . 12 (6): 692-697, 2008).

In some embodiments, the substance is known of Eph A4 or a binding ligand, ephrin A or B ephrin, such as ephrin or another Eph A4 ligand gene such as ephrin A4, A5, B2 or B3, or a modified form thereof or variant thereof. Derived from a nucleic acid molecule having a nucleotide sequence. Variants may be selectively hybridized in medium or high stringent conditions, or include regions of at least about 15 nucleotides, including nucleic acid molecules sufficiently similar to the naturally occurring forms of these molecules or to their complementary forms in whole or in part. In comparison, it has sequence homology of about 60% to 90% or 90 to 98% with a nucleotide sequence defined as a naturally occurring antagonist polypeptide sequence. Preferably the length of the hybridization region is at least about 12 to about 18 nucleobases. Preferably, the% homology between a particular nucleotide sequence and a reference sequence is at least about 80% or at least 85%, more preferably at least about 90%, such as about 95%, 96%, 97%, 98%, 99% or More than that. % Homology between 80% and 100% is included. The length of a nucleotide sequence depends on its function. For example, siRNAs (short interfering RNA) are about 20 to 24 nucleotides in length, while molecules designed to provide dominant negative functions need to be full or substantially full length molecules. As mentioned above, homologues are included. The term "homolog" or "homologs" broadly refers to molecules that are functionally and structurally related, including other types of molecules. Homologs and orthologs are examples of variants.

In some instances, the present invention encompasses the use of full length polypeptides or biologically active sites or peptides, eg, the use of one or more of these molecules as an antagonist of staple peptides. Biologically active sites or peptides include one or more binding domains, which contribute to the target activity of an antagonist, such as a ligand or receptor binding domain. In some instances, peptide antagonists block the G-H loop to limit the high or moderate binding affinity between the Eph and ephrin binding domains. Other binding domains are shown in FIGS. 6, 7 and 8. Biologically active moieties or peptides, such as staple peptides of full length polypeptides, are for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350, 400, 450, 500, 550, 600 to about 640 or about 700, 800, 900, 1000, 1200, or more polypeptides or peptides having amino acid residues. Typically, the length of the ligand / receptor binding site is about 150 to 300 amino acid residues. An example is shown in FIG. 6.

A “variant” antagonist polypeptide deletes (cuts) or adds one or more amino acids at the N- and / or C-terminus of a native protein; Deleting or adding one or more amino acids to one or more sites of a native protein; Substitution of one or more amino acids in one or more sites of a native protein includes proteins derived from native proteins such as soluble Eph A4, Ephrine A or Ephrine B, such as Ephrine A4, A5, B2 or B3. The variant proteins included in the present invention are biologically active, ie they continue to act as inhibitors of Eph A4 or ephrin mediated signaling. Variants include soluble forms lacking membrane anchors and / or transmembrane domains. Antagonist variants are selected based on the action of inhibiting or antagonizing the biological activity of Eph A4 or its cell binding ligand. Such variants can be generated, for example, through genetic polymorphism or human manipulation. Biologically active variants of the native polypeptide are at least 40%, 50%, 60%, 70%, usually at least 40% of the amino acid sequence of the native protein, as determined by a contemporary sequence alignment program using default parameters. At least 75%, 80%, 85%, preferably about 90% to 95% or more, more preferably about 98% or more sequence similarity. Biologically active variants of the antagonist polypeptide are generally 100, 50, or 20 amino acid residues with the polypeptide, or only 1-15, 1-10, such as 6-10, 5, 4, 3, 2, or Even one amino acid residue may be different.

In one example, the functionally active spice variant form of ephrin-A4 is Aasheim et al. al ., Blood , 95 (1): 221-230, 2000. Such spice variants lack nucleotide 146 at the 3 'end of the open reading frame (ORF) of the first site of exon IV. This results in modification of the carboxy terminus and absence of the GPI-signal sequence. COS cells transformed with cDNA produced soluble ephrin-A4.

Eph A4 or Eph A4-binding ephrins that signal antagonist polypeptides or peptides can be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Such methods of operation are known in the art. For example, amino acid sequence variants of ephrin B or ephrin A polypeptides can be prepared by introducing mutations in DNA coding. Mutagenesis and nucleotide sequence modification methods are known in the art. For example, Kunkel, Proc . Natl . Acad . Sci . USA , 82 : 488-492, 1985; Kunkel et al ., Methods in Enzymol ., 154 : 367-382, 1987; US Patent No. 4,873,192; Watson et al ., Molecular Biology of the Gene , Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987; And the documents cited therein. Guidelines for appropriate amino acid substituents that do not affect the biological activity of the protein of interest are described in Dayhoff et. al ., Natl . Biomed . Res . Found in Model 5, 345-358, 1978. Fusion proteins, such as Fc fusions, are particularly proposed. Such fusion proteins usually comprise a linked sequence.

Methods of screening genetic material of combinatorial libraries by point mutations or cleavage and methods of screening cDNA libraries for selectable genetic products are known in the art. Such methods are suitable for rapid screening of gene libraries generated by combinatorial mutations of polypeptides. Recursive ensemble mutagenesis, a technique for increasing the frequency of functional mutations in libraries, can be used to identify useful polypeptide variants in combination with screening assays (Arkin et al. al ., Proc . Natl . Acad . Sci . USA , 89 : 7811-7815, 1992; Delgrave et al ., Protein Engineering , 6 : 327-331, 1993). Conservative substitutions may be desirable, such as exchanging one amino acid for another amino acid with similar properties.

Variant polypeptides may comprise conservative amino acid substitutions at various positions depending on the sequence, as compared to the reference amino acid sequence. "Conservative amino acid substitutions" means that amino acid residues are replaced with other amino acid residues with similar side chains. Family of amino acid residues with similar side chains are known in the art. Amino acid residues may be further subclassified depending on whether the side chain substituents of the residue are cyclic or acyclic, aromatic or nonaromatic, and according to size. If additional polar substituents are present, including carboxyl carbons, if the total carbon number is 4 or less, or if no additional polar substituents are present, the total carbon number is 3 or less, the residues are considered small residues. Small residues are of course always non-aromatic. Depending on their structural properties, amino acid residues can be divided into two or more classes. For naturally occurring protein amino acids, subclasses are shown in Table 2 as described above.

Conservative amino acid substitutions also include groups based on side chains. Whether changes in amino acids will result in functional antagonists can be readily determined by analyzing binding activity to the target and inhibiting Eph A4 mediated signaling, receptor activity, phosphorylation, and the like. Easily evaluable activities are known in the art, for example, nuclear magnetic resonance spectroscopy (NMR), Biacore, kinetic, affinity, and the like, where heteronuclear single quantum coherence (HSQC) spectra are observed. Assays that assess binding or dimerization and oligomerization, detected using pull-down assays. Conservative substitutions are listed in Table 3 as "exemplary substitutions." More preferred substitutions are shown in the list of "preferred substitutions". Amino acid substitutions within the scope of the present invention typically do not significantly alter the effect on the structure of the peptide backbone at the substitution region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains. By selecting a substitution that does not. When a substituent is introduced, the biological activity of the variant is screened.

Antagonists can be modified by those skilled in the art for the purpose of improving activity, stability, or, if necessary, the ability to penetrate the cerebrovascular barrier using endogenous or introduced transport molecules or carriers.

In some instances, analogs of ephrin or Eph polypeptides have improved stability and activity, or reduced undesirable pharmacological properties. They can also be designed to improve the ability to penetrate the cerebrovascular barrier, which is a biofilm, or to work only with certain substrates. Thus, analogs may retain some of the functional properties of the parent molecule, but may have a modified specificity or perform new functions useful in the present invention for the purpose of administering to the subject. Methods of making polypeptide analogs described herein are not limited to modification of the side chains, and include incorporating non-natural amino acids and / or derivatives thereof, using crosslinking agents, or proteinaceous molecules during peptide, polypeptide or protein synthesis. Or using other methods of imposing conformational constraints on analogs thereof.

Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t -Butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and / or the use of D-isomers of amino acids. Examples of non-natural amino acids used herein are shown in Table 4.

Crosslinkers are for example homo- such as bifunctional amido esters, glutaraldehyde, N-hydroxysuccinimide esters having a (CH ₂ ) _n spacer group (where n = 1 to 6) Heterofunctional crosslinking agents, and amino-reactive sites such as N-hydroxysuccinimide and other group specific-reactive sites such as maleimido or dithio moieties (SH) or carbodiimide (COOH) By using bifunctional reagents, they can be used for the purpose of stabilizing 3D conformation. The peptide can also be conformationally restricted, for example, by inserting C _α and N _α -methylamino acids or by introducing a double bond between the C _α and C _β atoms of the amino acid.

In another aspect, the invention provides a method of preparing a composition for preserving the function or integrity of neural tissue in an individual or for reducing the rate, incidence or amount of nerve damage to an individual, Serves the purpose. Neural tissues include CNS and PNS tissues, brain, spinal cord, visual nerves, and the like. In some instances, the subject has or is at risk of suffering from a condition associated with nerve damage.

As a similar example, the present invention comprises administering an Eph A4 antagonist or an Eph A4 ligand antagonist to a subject, thereby preserving the function or integrity of the neural tissue in the subject, or reducing the rate, incidence or amount of nerve damage to the subject. It provides a method to make it. In some instances, the subject has or is at risk of suffering from a condition associated with nerve damage.

Eph A4 antagonists or Eph A4 ligand antagonists are provided for the purpose of preserving the function or integrity of neural tissue in an individual or reducing the rate, incidence or amount of neuronal damage to an individual. In some instances, the subject has or is at risk of suffering from a condition associated with nerve damage.

Similarly, compositions are provided to preserve the function or integrity of neural tissue in an individual, or to reduce the rate, incidence or amount of nerve damage to an individual, including an Eph A4 antagonist or an Eph A4 ligand antagonist. In some instances, the subject has or is at risk of suffering from a condition associated with nerve damage.

Nerve damage can cause a disease or condition that results in axon detachment, neuron degeneration, or demyelination. Examples of conditions that cause nerve damage include traumatic lesions (eg, lesions caused by physical or surgical, and compression injuries); Ischemic lesions (eg, brain or spinal cord infarction and ischemia); Malignant lesions; Infectious lesions (eg, lesions caused by pus or associated with infection by human immunodeficiency virus, Lyme disease, tuberculosis, syphilis or herpes virus); Degenerative lesions such as those referred to herein (eg, lesions associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea or Amyotrophic lateral sclerosis); Lesions associated with nutritional diseases or disorders (e.g. vitamin B12 deficiency, folate deficiency, Wernicke's disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease and alcohol cerebellum) denaturalization); Neurological lesions associated with systemic diseases (eg, lesions associated with diabetes mellitus, systemic lupus erythematosus, carcinoma or sarcoidosis); Lesions caused by toxic substances (eg, alcohol, lead or neurotoxins); And other demyelinating lesions (eg, those associated with human immunodeficiency virus-associated myelopathy, transverse myelopathy of various etiologies, progressive multifocal leukoencepholopathy and central pontine myelinolysis). do.

Also provided is a medical kit comprising the antagonist described herein, with instructions, for use in the treatment of the NDD disease described herein. Also provided is a medical kit comprising an Eph A4 antagonist or an Eph A4 ligand antagonist, with instructions, for use in neuroprotective applications in the treatment of NDD, such as MS.

The present invention further provides a therapeutic protocol for treating NDD, such as MS, in a subject, said protocol being proposed to be sufficient to reduce additional neuronal damage, by screening a subject-derived biological sample for axon damage. Administering an Eph A4 antagonist or an Eph A4 ligand antagonist under conditions and time, and rescreening the subject for axon injury. In some instances, alternatively or additionally, the sample is tested for signs of autoimmunity, inflammation, or demyelination.

The term “sample” means any biological fluid, cell, tissue, organ, or portion thereof, including or potentially including a target nucleic acid molecule or polypeptide. The term includes not only samples present in an individual, but also samples obtained or derived from an individual. For example, the sample may be a tissue section of the sample obtained by biopsy, or a cell present in or suitable for tissue culture. The sample may be a subcellular fraction or extract, or a purified or crude nucleic acid or polypeptide preparation.

The present invention is not limited to any particular method of measuring gene expression, but is not limited to the use of mRNAs, such as RNA, encoding a gene or portions thereof or expression products thereof, such as phosph-NF or phosph-NF proteins. It may be a method of measuring level or activity. Consideration of sample form, availability, and dose may affect the choice of a particular method. For example, if only a small amount of cellular material is available, a method of measuring RNA amount, for example by PCR amplification, may be a suitable option for measuring gene set expression. Alternatively, enzyme immunoassay (ELISA), which measures the amount of polypeptide, may be a suitable means for measuring the expression level of one or more gene set polypeptides.

Various methods for measuring transcriptional expression of genes in tissues are known in the art and the present invention does not rely on any particular method. Gene expression can be measured using hybridization or sequencing, based on methods known in the art. In general, conventional methods include, but are not limited to, northern blotting and in situ hybridization drop tests ( in situ hybridisation dot-blot or other membrane-based techniques, RNAse protection assays, real-time PCR, reverse transcriptase PCR, such as TaqMan RT-PCR, differential display method, MassARRAY-based gene expression Sequenom, bead array, microarray, HiCage (hicoverage expression profiling), immunohistochemistry (IHC), mass spectrometry (e.g., MALDI-TOF analysis), SAGE (serial analysis of gene) expression techniques, and massively parallel signature sequencing (MPSS). Details of the method are described, for example, in Sambrook et. al ., (documents described above), and in Ausubel et al , (documents described above) or online scientific literature. RNA or crude cell lysate preparations isolated on PCR or RT-PCR can be used. As mentioned above, PCR is useful when there are few starting materials. Further information on traditional PCR methods can be found in, for example, Dieffenbach and Dveksler, PCR Primer : A Laboratory Manual , Cold Spring Harbor Press, Plainsview, NY, 1995.

It is known that various forms of affinity binding can be used in the diagnostic methods of the present invention. As an example, specific examples of such affinity binding assays are described below with reference to immunoaffinity binding assays. Affinity binding methods are known as Harlow and Lane, Using Antibodies : A Laboratory General experimental manuals such as Manual , Cold Spring Harbor Laboratory Press, New York, 1999.

In other assays, magnetic antibodies that bind to neurodegenerative markers are used to label the markers, and high T _c superconducting quantum interference devices are used to measure the amount of free and binding antibodies to detect the presence or level of the markers. Liposomal immunomigration, liquid-phase competition strip immunoassay is described, for example, by Glorio-Paulet et. al ., J Agric Food Chem 48 (5): 1678-1682, 2000.

In another embodiment, the present invention provides a method of screening for potency modulators of any one or more Eph or ephrins to reduce clinical progression of NDD, such as MS disease.

In another example, screening methods are provided for identifying therapeutic agents of NDD, such as MS, including screening agents for modulating the level or activity of a polypeptide having a functional activity of an Eph A4 or Eph A4 ligand.

Antagonists can be prepared from natural substances, binding synthetic organic or inorganic compounds, peptides / polypeptides / proteins, nucleic acid molecules. Other display techniques, including libraries or phages, or both, can be used to screen or test suitable materials. Natural materials include those derived from corals, soils, plants or marine or Antarctic environments. Libraries of organic small molecules can be generated and screened using high-throughput techniques known in the art. See, for example, US Pat. No. 5,763,623 and US Application 20060167237. Combination synthesis provides a very useful approach when synthesizing a large number of related compounds that are common to the parent structure or have different substituents of a subset. Such compounds are usually non-oligomers and may be similar in terms of their basic structure and function, for example, changing chain length, ring size or number of substituents. The virtual library consists of compounds prepared from computer virtual experiments in silico ) (see, eg, US Publication No. 20060040322) or in vitro or in vivo assays known in the art. Libraries of small molecules suitable for testing are available in the art (eg, Amezcua et. al ., Structure ( London ) 10 : 1349-1361, 2002). Yeast SPLINT antibody libraries can be used to test intrabodies that may interfere with protein-protein interactions (Visintin et. al , (documents described above). Examples of methods suitable for the synthesis of molecular libraries are known in the art, for example DeWitt et. al ., Proc . Natl . Acad . Sci . USA 90 : 6909, 1993; Erb et al ., Proc . Natl . Acad . Sci . USA 91 : 11422, 1994; Zuckermann et al ., J. Med . Chem . 37 : 2678, 1994; Cho et al ., Science 261 : 1303, 1993; Carrell et al ., Angew . Chem . Int . Ed . Engl . 33 : 2059, 1994; Carell et al., Angew . Chem . Int . Ed . Engl . 33 : 2061, 1994; And Gallop et al ., J. Med. Chem . 37 : 1233, 1994.

Thus, the material may comprise a biological library; Solid or solution phase libraries spatially addressable parallel; Synthetic library methods requiring deconvolution; "1-bead 1-compound" library method; And materials can be obtained using any of a number of approaches from combinatorial library methods known in the art, including synthetic library methods using the choice of affinity chromatography. While biological library approaches are suitable for peptide libraries, the other four approaches can be applied to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, Anticancer). Drug Des . 12 : 145, 1997; US Patent No. 5,738,996; And US Pat. No. 5,807,683). Libraries of the compounds are, for example, solutions (eg Houghten, Bio / Techniques 13 : 412-421, 1992), or beads (Lam, Nature 354 : 82-84, 1991), chips (Fodor, Nature 364 : 555-). 556, 1993), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al ., Proc . Natl . Acad . Sci . USA 89 : 1865-1869, 1992) or phage (Scott and Smith, Science 249 : 386-390, 1990; Devlin, Science 249 : 404-406, 1990; Cwirla et. al ., Proc . Natl . Acad . Sci . USA 87 : 6378-6382, 1990).

As used herein, "hybridization" means that complementary nucleotide sequences are paired to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are sequences associated with the base-pair law. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this respect, the terms “match” and “mismatch” as used herein refer to the possibility of hybridization of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, like the classical A-T and G-C base pairs described above. Mismatched nucleotides are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing is with Watson-Crick, Hoogsteen or reverse Hugstin hydrogen bonds between the complementary nucleosides or nucleotide bases (nucleobases) of the oligomeric compound strands. Like, hydrogen bonds. For example, adenine and thymine are complementary nucleobases that form hydrogen bonds to mate. Hybridization can occur in a variety of conditions known in the art.

Nucleic acid sequences can be identified in the following manner. The subject nucleic acid sequence is used to search a nucleic acid sequence database, such as the GenBank database using the program BLASTM version 2.1 (Altschul et al. al . , Nucleic acids Research 25 : 3389-3402, 1997). This program is used in a ungapped manner. Default filtering is used to remove sequence homology due to low complexity regions.

Amino acid sequences can be identified in the following manner. The subject polypeptide sequence is used to search a polypeptide sequence database, such as the GenBank database (accessible at the website http://www.ncbi.nln.nih.gov/blast/) using the BLASTP program. This program is used in a ungapped manner. Default filtering is used to remove sequence homology due to low complexity regions. The default parameter of BLASTP is used. SEG programs can be used to filter out low complexity sequences.

The term “hybridization under stringent conditions”, and a verbally similar expression, refer to a target nucleic acid molecule (eg, a target nucleic acid molecule immobilized on a DNA or RNA blot, such as a Southern blot or a Northern blot), under defined temperature and salt concentration conditions. Refers to the ability of a nucleic acid molecule to hybridize. For nucleic acid molecules longer than about 100 bases in length, typical stringent hybridization conditions are temperatures of 25 ° C. to 30 ° C. or less (eg 10 ° C.), lower than the melting point (Tm) of native duplex DNA (native duplex). Sambrook et al . , (Documents described above); Ausubel et al . , Current Protocols in Molecular Biology, Greene Publishing, 1987). The Tm of a nucleic acid molecule longer than about 100 bases can be calculated by the formula Tm = 81.5 + 0.41% (G + C-log (Na ⁺ )).

For nucleic acid molecules shorter than 100 bases in length, exemplary stringent conditions are temperature conditions of 5 ° C. to 10 ° C. lower than Tm.

As used herein, the term "oligonucleotide" refers to a nucleic acid molecule that is 100 bases or less in length.

The term "complement", as used in reference to a nucleic acid molecule, refers to a complementary nucleic acid sequence relationship determined by Watson-Crick base pairing. For example, the complementary sequence of nucleic acid sequence 5'CCATG3 'is 5'CATGG3'.

Expressions such as “specifically hybridize to” may, under stringent conditions, bind, duplex or hybridize a molecule only to a specific nucleotide sequence present in a complex mixed (eg, whole cell) DNA or RNA. I mean.

The term "modulation" or "modulator" in the context of a particular target means to directly or indirectly upregulate or downregulate the level, effect or activity of the target.

Short description of the table

Table 1 shows the SEQ ID NOs provided herein.

Table 2 shows amino acid sub-classifications.

Table 3 shows amino acid substitution examples.

Table 4 is a list of non-natural amino acids included in the present invention.

Table 5 is a list of abbreviations.

table One

Sequence identifier summary

SEQ ID NO: Explanation SEQ ID NO: 1 Amino Acid Sequences of MOG 35-55 SEQ ID NO: 2 Mature Amino Acid Sequences of Mouse Eph A4 Human IgG1 Fc SEQ ID NO: 3 Mature amino acid sequence of mouse Eph A4 mouse IgG1 Fc SEQ ID NO: 4 Mature Amino Acid Sequence of Mouse IgG1 Fc Deleting Mouse Eph A4 Ligand Binding Domain SEQ ID NO: 5 Mature amino acid sequence of mouse Eph A4-His ("monomer" Eph A4) SEQ ID NO: 6 Mature amino acid sequence of mouse Eph A4 ligand binding domain deletion-His ("monomer" Eph A4) SEQ ID NO: 7 Mature amino acid sequence of human Eph A4 human IgG4 Fc SEQ ID NO: 8 Mature amino acid sequence of human ephrin A4 human IgG1 Fc SEQ ID NO: 9 Mature amino acid sequence of human ephrin A5 human IgG1 Fc SEQ ID NO: 10 Mature amino acid sequence of human ephrin B3 human IgG1 Fc

TABLE 2

amino acid Subclass

Subclass amino acid acid Aspartic Acid, Glutamic Acid Basic Acyclic: arginine, lysine; Annular: Histidine Majesty Aspartic Acid, Glutamic Acid, Arginine, Lysine, Histidine small Glycine, serine, alanine, threonine, proline Polar / neutral Asparagine, Histidine, Glutamine, Cysteine, Serine, Threonine Polarity / large Asparagine, Glutamine Hydrophobic Tyrosine, Valine, isoleucine, leucine, methionine, phenylalanine, tryptophan Aromatic Tryptophan, Tyrosine, Phenylalanine Residues Affecting Chain Determination Glycine and proline

TABLE 3

Examples of Preferred Amino Acid Substitutions

originally Residue Exemplary Substituents Preferred Substituents Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

table 4

Unusual amino acid code

Table 5

List of abbreviations

Abbreviation EAE Experimental Autoimmune Encephalomyelitis
(experimental autoimmune encephalomyelitis) ELISA Enzyme-linked immunosorbent
(enzyme-linked immunosorbent assay) GFAP Glial fiber protein
(Glial fibrillary acidic protein) NDD Neurodegenerative diseases PBS Phosphate Buffered Saline MOG Myelin oligodendrocyte glycoprotein
(myelin oligodendrocyte glycoprotein) CNS Central nervous system PNS Peripheral nervous system RNA Ribonucleic acid PDZ Postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1) SAM Sterile Alpha Motif
(sterile alpha motif) GPI Glycophosphatidyl Inositol
(glycophosphatidylinositol) MS Multiple sclerosis Eph Erythropoietin-producing hepatocytes
(erythropoietin producing hepatocellular) LIF Leukocyte inhibitor IFNγ Interferon gamma Fc Fragment Crystallization Zone
(fragment crystalisable region) IFNβ Interferon beta DAPI 4 ', 6'-diamino-2-phenylindole

According to the present invention there is provided therapeutic molecules that mitigate clinical progression of neurodegenerative diseases such as therapeutic neuroprotective and neuroregenerating molecules, in particular multiple sclerosis (MS) and modified forms of MS.

The invention will be explained in more detail by the following non-limiting examples.

Example One: Availability Eph  A 4  Generation of receptors

Recombinant mouse Eph A4 mouse Fc (mEph A4mFc) or human Eph A4 human Fc (hEph A4hFc) fusion proteins were prepared by transient transfection in mammalian cells. For cell culture, FreeStyle ™ 293-F cells and mammalian expression vector pcDNA3.1 were obtained from Invitrogen. Cells were cultured in FreeStyle ™ Expression Medium (Invitrogen). All tissue culture medium was supplemented with penicillin / streptomycin / pungezone reagent (Invitrogen) and cells were maintained at 37 ° C. in an incubator under an atmosphere of 8% CO ₂ .

Transient transformation of the expression plasmid encoding mEph A4mFc or hEph A4hFc (see FIG. 6) using FreeStyle ™ 293-F cells was performed using 293fectin transformation reagent (Invitrogen) according to the manufacturer's instructions. Cells (1000 ml) were transformed to a final concentration of 1 × 10 ⁶ viable cells / ml, and on an 2/10 Wave Bioreactor system 2/10 or 20/50 (GE Healthcare) under an atmosphere of 8% CO ₂ 37 Incubated in Cellbag 2L (GE Healthcare) for 5 days at ℃. Incubation conditions are 35 rocks per minute with an angle of 8 °. After transformation, Pluronic F68 (Invitrogen) was added for 4 hours to a final concentration of 0.1% v / v. Twenty four hours after transfection, cell cultures were supplemented with Tryptone N1 (Organotechnie, France) so that the final concentration was 0.5% v / v. Cell culture supernatants were collected by centrifugation at 2500 rpm and then passed through a 0.45 μM filter (Nalgene) prior to purification.

CDNA expression plasmids encoding mouse Eph A4 mouse Fc and human Eph A4 human Fc were generated. Synthetic genes encoding mouse Eph A4 (mEph A4mFc) fused to mouse IgG1 Fc, and human Eph A4 (hEph A4hFc) fused to human IgG4 Fc, respectively, were represented by GenScript Corporation (Piscataway, NJ) and Geneart AG (Regensburg). , Germany). To facilitate translation initiation, a Kozak sequence (GCCACC) was introduced just before the N-terminus of each protein. For the codon bias of the homo sapiens gene, the codon usage of mouse Eph A4 gene, mouse IgG1 Fc gene, human Eph A4 gene and human IgG4 Fc gene was adopted. To link the synthetic cDNA to Nhe I-Xho I digested pcDNA3.1, a Nhe I restriction site was introduced at the 5 'end of the cDNA and an Xho I restriction site was introduced at the 3' end. cDNA was cleaved into Nhe I and Xho I and linked to pcDNA3.1. Mass production of plasmid DNA was done using Qiagen Maxi or Giga Kit according to the manufacturer's instructions. The nucleotide sequence of all plasmid constructs was identified by sequencing the two strands using Big Dye Terminator v3.1 Cycle Sequencing and Applied Biosystems Automated Sequencer. It became.

In order to analyze the protein expression of mEph A4mFc or hEph A4hFc protein, 20 μl of a portion of the culture supernatant was transformed from 4-20% tris-glycine SDS polyacrylic acid from the transformation of the expression construct encoding either mEph A4mFc or hEph A4hFc. Electrophoresis on amide gels and the protein was visualized by staining with Coomassie Blue reagent. Cell culture supernatants containing either mEph A4mFc or hEph A4hFc protein were collected by centrifugation at 2500 rpm and then passed through a 0.45 μM filter (Nalgene) prior to purification.

For purification, the conditioned medium containing Eph A4 Fc was concentrated by target flow filtration. The concentrated medium was purified by affinity chromatography with protein A. Endotoxins were removed by ion exchange chromatography. Aggregate was removed using size exclusion chromatography, ie, separating Fc binding dimer Eph A4 protein from other molecular weight species. At 280, corresponding to 1.3 mg / ml, the protein was quantified based on the absorbance unit 1.0. Purity was determined based on protein visualization by SDS-PAGE and Coomassie Blue reagent.

Example 2: Analysis method

Based on the histological analysis of the degree of inflammatory infiltration, the severity of the disease is described by Butzkueven et. al ., Glia 53 : 696-703, 2006, assess the area of inflammatory infiltration per cross section of the spinal cord. In brief, in some instances, the total inflammatory lesion area of the DAPI stained cross section is calculated as the total percentage area of all inflammatory infiltrates per total area of spinal cord in the same cross section. Calculate for 15-20 sections per mouse. The cross-points are scored on a semi-quantitative scale (Soilu-Hanninen et. al ., J Neurosci Res 59 : 712-721, 2000): 0, no inflammatory cells; 1, almost no inflammatory cells; 2, severe perivascular invasion; 3, rich inflammatory cell infiltration, parenchymal necrosis. The number of lesions per section was quantified.

In the test subjects, immunohistochemical analysis on immune cells was performed. A wide range of T cell (CD4, CD8, T cell receptor), B cell (CD19) and myeloid cell (CD11b, IBA1) markers can be investigated. If any population changes, more specific subpopulation markers are used. These markers are also used in FACS analysis of splenocytes and lymphocytes for the purpose of determining whether there is a significant difference in the immunological status of Eph A4-/-mice after antagonist administration or after EAE induction.

In the lumbar spinal cord dorsal horn white matter and visual nerve sections, the number of mature and immature oligodendrocytes is measured. To compare the density of mature oligodendrocytes, count the mature oligodendrocytes in cross sections immunostained with mouse anti-CC1 antibody (APC Ab-7, Oncogene Research Products, Germany), or PLP-expressing (maturation). ) When oligodendrocytes express the marker dsRed, in the C57BL / 6 background, mature oligodendrocytes are counted from mice hybridized to the transformed PLP-dsRed strain. Precursors of oligodendrocytes are labeled with rabbit anti-NG2 antibody (Chemicon). To analyze oligodendrocyte apoptosis, the cross sections were TUNEL labeled using a TMR In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions (Butzkueven et. al ., 2002 (documents described above). Cross sections were stained with CC-1 antibody. Apoptosis of TUNEL positive cells is confirmed by nuclear fission or condensation using DAPI. The number of apoptotic oligodendrocytes per cross section is measured in the total number of apoptotic cells and in 10 μm cross sections of five 100 μm per spinal cord and visual nerve per animal. Statistical significance is assessed using the Mann Whitney U -test. Butzkueven et , using Luxol fast blue staining of sections al . As described in, 2006 (documents described above), the extent of demyelination and remyelination in the course of disease is measured.

Axon damage can be assessed, for example, by immunostaining against β-amyloid precursor protein (βAPP) and hyperphosphorylation and aggregation of Tau. APP-positive axon end-bulbs and glomeruli, which are indicative of recently acute axons, can be detected in acute MS lesions at the active interface of chronic active lesions and, to a lesser extent, chronic inactive lesions ( Kornek et al ., 2000 (documents described above); Ferguson et al ., 1997 (documents described above), immunostaining for phosphorylated Tau reveals areas of axon damage in MS / EAE plaques (Schneider et. al . J Biol Chem . 279 (53): 55833-95583, 2004). In some methods, methylene blue staining of Image Pro and resin-embedded sections is commonly used, based on automatic quantification of axon numbers and areas at defined spinal cord sites. Dorsal funiculus is used as a comparison area between animals. In some instances, ELISA based analysis of phospho-NF in blood is an acute marker for axon damage. Gresle et al ., J Neurosci Res . 86 (16): 3548-3555, 2008, levels of phospho-NF-H are closely associated with axon damage in neuroinflammatory states in mice. In humans, since CSF pNF-H levels are elevated during relapse in MS subjects, these levels are potentially clinically relevant markers. In addition, high levels of CSF pNF-H are associated with a faster transition to the secondary progression phase over a three-year period, allowing poor clinical outcomes after relapse to be predicted.

Prior to the onset of clinical symptoms, axon loss in the optic nerve was reported in the MOG model of EAE (Hobom et. al ., Brain Pathol 14 : 148-157, 2004), tissues were tested 6 days prior to the onset of clinical symptoms in the EAE model (Butzkueven et. al . , 2002 (documents described above). To brighten the plaque area, cross sections from test subjects were immunostained for phosphorylated Tau and βAPP and counterstained with H & E. The number of βAPPs and bulb / spheres is counted in adjacent appearing white matter (NAWM) and plaques, in the transverse and longitudinal sections of the lumbar spinal cord and optic nerve. In the spinal cord and through the optic nerve, the total density of axons adjacent to or in plaques is measured by automatic counting of methylene blue stained sections of these tissues. At least 5 sections of each tissue per animal, 50 μm apart, were counted and the results expressed as the average density of axons per mm ² . Statistical significance is assessed using the Mann Whitney U -test.

Neuroglial fiber acid protein (GFAP) positive astrocytes in plaques and in adjacent NAWM are immunostained and counted and expressed in astrocytic cells per mm ² at each time point during the test period. In order to measure the number of GFAP stains per field as a percentage of the total number of pixels in the field, image analysis (NIH Image J) is used (see FIG. 3). Digital images of GFAP stained tissue can be obtained at high power (x100) in various (5-10) sites, inside or around the lesion, of at least five cross sections (10 μm), per mouse, per tissue. To test for differences in GFAP expression levels, Western analyzes are performed on spinal cord homogenates. Densitometry is performed by autoradiography with NIH Image J software to determine the relative levels of GFAP bands and normalized to β-actin levels.

Eph A4 expression levels are measured during disease progression in wild type mice. Spinal cord and optic nerve cross sections are immunostained for Eph A4. Levels of Eph A4 expression and phosphorylation are measured in spinal cord tissue. Lumbar spinal cord tissue is homogenized and western analysis is performed on a portion of the lysate. The residue is used for immunoprecipitation by anti-Eph A4 antibody and then probed for phosphotyrosine and Eph A4 expression.

Example  3: Eph  A 4  Deficiency MS  Changing functional outcomes and disease progression in the model

EAE Induction and Clinical Evaluation of

To elucidate the role of Eph A4 in the progression and severity of EAE, and to determine whether blocking Eph A4 is a potential treatment for MS, Eph A4-/-(Dottori et. al ., Proc Natl Acad Sci USA 95 : 13248-53, 1998) and the pathological and histological characteristics as well as the clinical course and severity of EAE in wild-type mice were tested.

Eph A4-/-(Dottori et al . , 1998 (documents described above)) The mice are backcrossed to the C57BL / 6 background so that MOG models of EAE are used with the mice. C57BL / 6 mice use MOG 35-55 (MEVGWRSPFSRVVHLYRNGK (SEQ ID NO: 1)) peptide or (Butzkueven et al ., 2002 (documents described above); Slavin et al ., Autoimmunity 28 : 109-20, 1998; Gold et al) or by manual delivery of MOG-specific T cells are susceptible to the induction of EAE. In the C57BL / 6 background, clinical processes in both MOG models of EAE tend to exhibit a chronic progression pattern, characterized by immune cell infiltration and local demyelination, via the CNS, especially in the spinal cord and optic nerve.

The severity of EAE disease was assessed daily up to 1 month, with additional cohort studies up to 3 months, using the following standard 5-point paraplegic score: 0, no clinical symptoms; 1, fur ruffling or weakening of the tail end; 1.5, tail laxity or weak paralysis of the hind legs; 2, complete paralysis of the tail; 2.5, complete paralysis of the tail and partial paralysis affecting one or two hind legs; 3, complete paralysis of the hind legs; 3.5, unable to turn to the right when lying on his back; 4, complete paralysis of both sides of the hind limb and weakening or dying of the forelimb; And 5, death. Mice with a score of 4 are killed according to the guidelines of the Ethics Committee and take up the CNS organization for histological studies. The onset of clinical symptoms using this model is typically about 10-12 days (Butzkueven et. al ., 2002 (documents described above).

Studies with n = 10 Eph A4-/-mice and n = 10 control wild type litters were performed (see FIG. 1). Clinical symptoms were similarly initiated for the two cohorts, but the extent of the disease was more severe in wild-type mice, with a mean clinical score of 2.3 ± 0.3 (highest score = 4; 60% maximum score in wild-type mice: 2.5 In contrast, Eph A4 null mice had an average score of 1.7 ± 0.2 (highest score = 2.75; maximum score of 60%: 1.5 or less). Thus, the clinical progression of EAE is not as severe as in Eph A4-/-mice.

Example  4: Eph  A 4 -/-And in wild type mice EAE  Immunohistologic Characteristics of Infected Tissue

Analysis of the spinal cord cross-sections derived from mice described in Example 1 showed typical inflammatory infiltration in both wild-type and Eph A4 knockout mice, as assessed by DAPI (unbased) and microglia markers CD11b and IBA1 (unbased). Not only was there no significant difference in the number of lesions per section, but there was no significant difference in the number of DAPI nuclei of the outer white matter.

The levels of phosphorylated-neural microfibers in the blood were evaluated as surrogate markers of axonal damage closely related to the severity of the disease as well as histochemical properties of the axon injury (see Gresle et. al , 2008 (documents described above). Eph A4 knockout mice showed reduced levels of phosphorylated-NF, indicating that axon damage was reduced in these mice (see FIG. 2).

Example 5: Eph  A 4 Is EAE  Expressed on astrocytic cells in lesions

Astrocyte neurogliosis is a hallmark of MS lesions. To assess whether Eph A4 is expressed on astrocytes in EAE lesions, spinal cord sections from C57BL / 6 mice with EAE experimentally induced by administration of MOG35-55 peptide (Butzkueven et al. al ., 2002 (documents described above)) Immunostaining for Eph A4 and Neuroglial Fiber Protein (GFAP) Expression. Eph A4 was positively expressed on all neuroglial fibric acid protein (GFAP) and consequently expressed on active astrocytic cells around the lesion in the mouse spinal cord with 3-point EAE (FIG. 3).

Example 6: CNS  After damage Eph  A 4  Blockers

Because cerebrovascular barriers are destroyed not only after CNS injury, but also during MS and EAE lesions, antagonist access routes are provided only at the site of CNS injury. In order to detect the presence of ephrin A5-Fc, the damaged spinal cord cross section was immunostained using biotylated anti-human IgG, followed by the vector ABC kit and DAB. The PBS treated spinal cord showed no markers, whereas the ephrin A5-Fc treated spinal cord displayed markers at the lesion site, indicating that ephrin A5-Fc can enter the parenchymal organ through the cerebrovascular barrier. (FIG. 4). Since cerebrovascular barriers are disrupted in EAE lesions, treatment of EAE via IP injection of Eph A4 blockers should be a viable way to selectively deliver these substances to the affected CNS region.

Example  7: Noncomplex ( uncomplexed ) Ephrine A5- Fc Add to the astrocytes, IFN γ induction Eph  A 4  Activity is blocked

Wild type astrocytic cells cultured from the neocortex were treated with ephrin A5-Fc, in which some of the ephrin A5-Fc complexed with anti-human IgG Fc to produce an active form of ephrin A5-Fc, The rest was left uncomplexed to block Eph A4 activity. Eph A4 activity was assessed at basal conditions and conditions responsive to cytokines. Under basal conditions, the complex (poly) ephrin A5-Fc increased Eph A4 phosphorylation. Significantly, non-complex (mono) ephrin A5-Fc reduced Eph A4 phosphorylation at both basal and cytokine reaction conditions (FIG. 5). Thus, in addition to regulating neuronal growth cone response, regulation of Eph A4 signaling also directly affects astrocytes.

Example 8: further research

Eph A4 blockers such as soluble Ephrine A5-Fc or Eph A4-Fc are administered to C57BL / 6 mice suffering from MOG induced EAE as described in Example 1. Before, during or after the onset of clinical disease, the assay of Example 2 is used to assess the effect of Eph A4 antagonist, including clinical severity, inflammatory infiltration and oligodendrocyte viability.

In some instances, CHO cell transformants are used that express Eph- and ephrin-human IgG1 Fc recombinant proteins (Coulthard et. al ., Growth Factors 18 : 303-17, 2001). Soluble Ephrine A5-Fc or Eph A4-Fc protein is prepared from these cell lines. In studies with radiolabeled ephrin A5, 65% of IP injected ephrin A5 is rapidly removed into the tissue, with similar absorption rates in all tissues tested. However, blood clearance is relatively low, with 5% of the injected dose circulating for 24 hours. This means that effective dosing inhibitors must be maintained by daily dosing regimens. Fully unclustered ephrin A5-Fc or Eph A4-Fc are tested in EAE diseased animals.

In the spinal cord injury of the cerebrovascular barrier, early studies of EAE used this route of administration before IP-injected ephrin A5-Fc enters the CNS parenchymal organ at the site of CNS injury. Penetration into the CNS parenchyma is assessed by immunostaining for human-IgG. Soluble ephrin is expected to penetrate the cerebrovascular barrier at the site of the EAE lesion, but if such an approach is inadequate, mini-osmotic infusion pumps (Alzet) are inserted into the ventricle 4 and Used together, the pump is implanted under the skin.

In an initial study, IP injection of Ephrine A5-Fc or Eph A4-Fc was used at 500 μg / day / mouse, which was found to be effective for spinal cord studies. Antagonists are administered until disease begins to be induced and tissues are taken for analysis. In some instances, administration is made prior to the onset of clinical symptoms of the disease or prior to the induction of EAE. Subsequent studies involve resetting the start of treatment, for example, by delaying administration until the onset of clinical symptoms or after axon damage has been identified. Dosage is properly optimized.

This specification is intended to describe preferred embodiments of the invention and is not intended to limit the scope of the invention to any one example or specific feature of the invention. Accordingly, those skilled in the art will understand that various modifications and changes can be made to the specific examples illustrated within the scope of the present disclosure without departing from the scope of the present disclosure. All modifications and variations are understood to be included within the scope of the following claims.

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Some drawings include colored portions or all. Color versions of the drawings are available upon request and from the patentee or a suitable patent bureau. Fees may be charged if obtained from the patent office.

1 is a graph showing that Eph A4 knockout mice show a lower severity EAE clinical trend. The onset and severity of the disease is similar between genotypes in the very early stages of the disease, but there are markedly different trends thereafter. Maximum clinical grade reached by Eph A4 knockout mice was lower than control (each n = 10).

FIG. 2 is an ELISA graph of Eph A4 knockout mice suffering from EAE with a trend toward decreased levels of phospho-neufilament-H indicating lower axon damage in EAE than wild-type mice with EAE.

3A to 3D are photographs showing expression of Eph A4 around the EAE lesion. Spinal cords from C57BL / 6 mice, grade 3 MOG-induced EAE, were immunostained for GFAP and Eph A4 expression. a) no obvious background when no primary Eph A4 antibody was added; b) GFAP expressing astrocytes derived from these mice are also expressed; c) Eph A4 expression is observed; d) b & c merge, + DAPI to show expression of Eph A4 on all GFAP positive astrocytes in EAE. Lesions are marked with an asterisk.

Figure 4 is a photograph showing the ephrin A5-Fc enters the CNS at the injury site after IP injection. Mice were treated with (A) PBS or (B) ephrin A5-Fc for 4 days.

5 is a photograph showing that the monomeric ephrin A5-Fc blocks Eph A4 phosphorylation on astrocytes. Astrocytes were treated in vitro with ephrin A5-Fc monomer (blocking) or ephrin A5-Fc polymer (activation) under basal conditions, or with IFNv. Ephrin A5-Fc monomers inhibited phosphorylation of Eph A4 under both these conditions.

6 shows amino acid sequences of Eph A4, Eph A4-binding Ephrine Fc and His fusion proteins.

                         SEQUENCE LISTING <110> University of Melbourne        Howard Florey Institute        TURNLEY, Ann        BUTZKEUVEN, Helmut   <120> Therapeutic applications <130> 30787857 / JEH / DXT <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> mouse <400> 1 Met Glu Val Gly Trp Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr 1 5 10 15 Arg Asn Gly Lys             20 <210> 2 <211> 777 <212> PRT <213> artificial <400> 2 Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp 1 5 10 15 Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu             20 25 30 Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile         35 40 45 Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln Asn Asn Trp     50 55 60 Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile 65 70 75 80 Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met                 85 90 95 Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn             100 105 110 Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys Ile Asp Thr         115 120 125 Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile     130 135 140 Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys 145 150 155 160 Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val                 165 170 175 Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu             180 185 190 Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val         195 200 205 Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro     210 215 220 Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn 225 230 235 240 Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu Cys Gln Ala                 245 250 255 Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys Ala             260 265 270 Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys         275 280 285 Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met     290 295 300 Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val 305 310 315 320 Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly                 325 330 335 Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala             340 345 350 Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr         355 360 365 Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp Leu     370 375 380 Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val 385 390 395 400 Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr                 405 410 415 Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu             420 425 430 Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro         435 440 445 Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln     450 455 460 Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp 465 470 475 480 Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala                 485 490 495 Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr             500 505 510 Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser Gly         515 520 525 Ser Gly Gly Gly Gly Gly Gly Arg Ser Glu Asn Leu Tyr Phe Gln Gly     530 535 540 Thr Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 545 550 555 560 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 565 570 575 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             580 585 590 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr         595 600 605 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     610 615 620 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 625 630 635 640 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 645 650 655 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln             660 665 670 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu         675 680 685 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     690 695 700 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 705 710 715 720 Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu                 725 730 735 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val             740 745 750 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln         755 760 765 Lys Ser Leu Ser Leu Ser Pro Gly Lys     770 775 <210> 3 <211> 750 <212> PRT <213> artificial <400> 3 Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp 1 5 10 15 Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu             20 25 30 Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile         35 40 45 Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln Asn Asn Trp     50 55 60 Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile 65 70 75 80 Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met                 85 90 95 Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn             100 105 110 Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys Ile Asp Thr         115 120 125 Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile     130 135 140 Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys 145 150 155 160 Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val                 165 170 175 Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu             180 185 190 Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val         195 200 205 Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro     210 215 220 Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn 225 230 235 240 Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu Cys Gln Ala                 245 250 255 Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys Ala             260 265 270 Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys         275 280 285 Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met     290 295 300 Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val 305 310 315 320 Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly                 325 330 335 Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala             340 345 350 Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr         355 360 365 Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp Leu     370 375 380 Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val 385 390 395 400 Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr                 405 410 415 Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu             420 425 430 Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro         435 440 445 Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln     450 455 460 Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp 465 470 475 480 Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala                 485 490 495 Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr             500 505 510 Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser Thr         515 520 525 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe     530 535 540 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 545 550 555 560 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val                 565 570 575 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr             580 585 590 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu         595 600 605 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys     610 615 620 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 625 630 635 640 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro                 645 650 655 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile             660 665 670 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly         675 680 685 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp     690 695 700 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 705 710 715 720 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His                 725 730 735 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys             740 745 750 <210> 4 <211> 495 <212> PRT <213> artificial <400> 4 Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys 1 5 10 15 Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser             20 25 30 Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser         35 40 45 Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn     50 55 60 Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr 65 70 75 80 Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly                 85 90 95 Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr             100 105 110 Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp         115 120 125 Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly     130 135 140 Val Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val 145 150 155 160 Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys                 165 170 175 Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg             180 185 190 Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp         195 200 205 Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr     210 215 220 Asp Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg 225 230 235 240 Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val                 245 250 255 Thr Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser             260 265 270 Thr Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val         275 280 285 Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr     290 295 300 Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu 305 310 315 320 Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln                 325 330 335 Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser             340 345 350 Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys         355 360 365 Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile     370 375 380 Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro 385 390 395 400 Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met                 405 410 415 Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn             420 425 430 Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr         435 440 445 Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn     450 455 460 Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu 465 470 475 480 His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys                 485 490 495 <210> 5 <211> 533 <212> PRT <213> artificial <400> 5 Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp 1 5 10 15 Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu             20 25 30 Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile         35 40 45 Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln Asn Asn Trp     50 55 60 Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile 65 70 75 80 Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met                 85 90 95 Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn             100 105 110 Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys Ile Asp Thr         115 120 125 Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile     130 135 140 Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys 145 150 155 160 Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val                 165 170 175 Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu             180 185 190 Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val         195 200 205 Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro     210 215 220 Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn 225 230 235 240 Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu Cys Gln Ala                 245 250 255 Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys Ala             260 265 270 Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys         275 280 285 Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met     290 295 300 Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val 305 310 315 320 Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly                 325 330 335 Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala             340 345 350 Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr         355 360 365 Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp Leu     370 375 380 Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val 385 390 395 400 Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr                 405 410 415 Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu             420 425 430 Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro         435 440 445 Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln     450 455 460 Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp 465 470 475 480 Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala                 485 490 495 Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr             500 505 510 Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser His         515 520 525 His His His His His     530 <210> 6 <211> 278 <212> PRT <213> artificial <400> 6 Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys 1 5 10 15 Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser             20 25 30 Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser         35 40 45 Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn     50 55 60 Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr 65 70 75 80 Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly                 85 90 95 Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr             100 105 110 Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp         115 120 125 Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly     130 135 140 Val Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val 145 150 155 160 Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys                 165 170 175 Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg             180 185 190 Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp         195 200 205 Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr     210 215 220 Asp Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg 225 230 235 240 Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val                 245 250 255 Thr Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser             260 265 270 His His His His His         275 <210> 7 <211> 756 <212> PRT <213> artificial <400> 7 Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp 1 5 10 15 Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu             20 25 30 Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile         35 40 45 Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln Asn Asn Trp     50 55 60 Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile 65 70 75 80 Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met                 85 90 95 Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn             100 105 110 Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys Ile Asp Thr         115 120 125 Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile     130 135 140 Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys 145 150 155 160 Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val                 165 170 175 Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu             180 185 190 Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val         195 200 205 Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro     210 215 220 Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn 225 230 235 240 Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu Cys Gln Ala                 245 250 255 Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Thr Cys Ala             260 265 270 Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys         275 280 285 Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met     290 295 300 Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val 305 310 315 320 Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly                 325 330 335 Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala             340 345 350 Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr         355 360 365 Pro Gln Gln Asn Gly Leu Lys Thr Thr Lys Val Ser Ile Thr Asp Leu     370 375 380 Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val 385 390 395 400 Ser Lys Tyr Asn Pro Asn Pro Asp Gln Ser Val Ser Val Thr Val Thr                 405 410 415 Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu             420 425 430 Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro         435 440 445 Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln     450 455 460 Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp 465 470 475 480 Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala                 485 490 495 Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr             500 505 510 Thr Asn Thr Val Pro Ser Arg Ile Gly Asp Gly Ala Asn Ser Glu         515 520 525 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu     530 535 540 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 545 550 555 560 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser                 565 570 575 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu             580 585 590 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr         595 600 605 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn     610 615 620 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 625 630 635 640 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln                 645 650 655 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val             660 665 670 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val         675 680 685 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro     690 695 700 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 705 710 715 720 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val                 725 730 735 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu             740 745 750 Ser Leu Gly Lys         755 <210> 8 <211> 396 <212> PRT <213> artificial <400> 8 Leu Arg His Val Val Tyr Trp Asn Ser Ser Asn Pro Arg Leu Leu Arg 1 5 10 15 Gly Asp Ala Val Val Glu Leu Gly Leu Asn Asp Tyr Leu Asp Ile Val             20 25 30 Cys Pro His Tyr Glu Gly Pro Gly Pro Pro Glu Gly Pro Glu Thr Phe         35 40 45 Ala Leu Tyr Met Val Asp Trp Pro Gly Tyr Glu Ser Cys Gln Ala Glu     50 55 60 Gly Pro Arg Ala Tyr Lys Arg Trp Val Cys Ser Leu Pro Phe Gly His 65 70 75 80 Val Gln Phe Ser Glu Lys Ile Gln Arg Phe Thr Pro Phe Ser Leu Gly                 85 90 95 Phe Glu Phe Leu Pro Gly Glu Thr Tyr Tyr Tyr Ile Ser Val Pro Thr             100 105 110 Pro Glu Ser Ser Gly Gln Cys Leu Arg Leu Gln Val Ser Val Cys Cys         115 120 125 Lys Glu Arg Lys Ser Glu Ser Ala His Pro Val Gly Ser Arg Gly Ala     130 135 140 Arg Gln Arg Ser Gly Gly Gly Gly Gly Gly Arg Ser Glu Asn Leu Tyr 145 150 155 160 Phe Gln Gly Thr Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro                 165 170 175 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe             180 185 190 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val         195 200 205 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe     210 215 220 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 225 230 235 240 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr                 245 250 255 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val             260 265 270 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala         275 280 285 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg     290 295 300 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 305 310 315 320 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro                 325 330 335 Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser             340 345 350 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln         355 360 365 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His     370 375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 9 <211> 421 <212> PRT <213> artificial <400> 9 Gln Asp Pro Gly Ser Lys Ala Val Ala Asp Arg Tyr Ala Val Tyr Trp 1 5 10 15 Asn Ser Ser Asn Pro Arg Phe Gln Arg Gly Asp Tyr His Ile Asp Val             20 25 30 Cys Ile Asn Asp Tyr Leu Asp Val Phe Cys Pro His Tyr Glu Asp Ser         35 40 45 Val Pro Glu Asp Lys Thr Glu Arg Tyr Val Leu Tyr Met Val Asn Phe     50 55 60 Asp Gly Tyr Ser Ala Cys Asp His Thr Ser Lys Gly Phe Lys Arg Trp 65 70 75 80 Glu Cys Asn Arg Pro His Ser Pro Asn Gly Pro Leu Lys Phe Ser Glu                 85 90 95 Lys Phe Gln Leu Phe Thr Pro Phe Ser Leu Gly Phe Glu Phe Arg Pro             100 105 110 Gly Arg Glu Tyr Phe Tyr Ile Ser Ser Ala Ile Pro Asp Asn Gly Arg         115 120 125 Arg Ser Cys Leu Lys Leu Lys Val Phe Val Arg Pro Thr Asn Ser Cys     130 135 140 Met Lys Thr Ile Gly Val His Asp Arg Val Phe Asp Val Asn Asp Lys 145 150 155 160 Val Glu Asn Ser Leu Glu Pro Ala Asp Asp Thr Val His Glu Ser Ala                 165 170 175 Glu Pro Ser Arg Gly Glu Asn Leu Tyr Phe Gln Gly Thr Glu Pro Lys             180 185 190 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu         195 200 205 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr     210 215 220 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val 225 230 235 240 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val                 245 250 255 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser             260 265 270 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu         275 280 285 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala     290 295 300 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 305 310 315 320 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln                 325 330 335 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala             340 345 350 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr         355 360 365 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu     370 375 380 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 385 390 395 400 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser                 405 410 415 Leu Ser Pro Gly Lys             420 <210> 10 <211> 465 <212> PRT <213> artificial <400> 10 Ala Ser Ile Ser Ala Arg Gln Asp Tyr Lys Asp Asp Asp Asp Lys Thr 1 5 10 15 Arg Arg Leu Ser Leu Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg             20 25 30 Phe Gln Ala Glu Gly Gly Tyr Val Leu Tyr Pro Gln Ile Gly Asp Arg         35 40 45 Leu Asp Leu Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser     50 55 60 Pro Asn Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala Gln Gly 65 70 75 80 Arg Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu Thr Cys Asp                 85 90 95 Arg Pro Asp Leu Asp Leu Arg Phe Thr Ile Lys Phe Gln Glu Tyr Ser             100 105 110 Pro Asn Leu Trp Gly His Glu Phe Arg Ser His His Asp Tyr Tyr Ile         115 120 125 Ile Ala Thr Ser Asp Gly Thr Arg Glu Gly Leu Glu Ser Leu Gln Gly     130 135 140 Gly Val Cys Leu Thr Arg Gly Met Lys Val Leu Leu Arg Val Gly Gln 145 150 155 160 Ser Pro Arg Gly Gly Ala Val Pro Arg Lys Pro Val Ser Glu Met Pro                 165 170 175 Met Glu Arg Asp Arg Gly Ala Ala His Ser Leu Glu Pro Gly Lys Glu             180 185 190 Asn Leu Pro Gly Asp Pro Thr Ser Asn Ala Thr Ser Arg Gly Ala Glu         195 200 205 Gly Pro Leu Pro Pro Pro Gly Ser Gly Gly Gly Gly Gly Gly Arg     210 215 220 Ser Glu Asn Leu Tyr Phe Gln Gly Thr Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 P: \ OPER \ JEH \ Specis \ Convention or Complete \ 30787857-South Korea \ 30787857 Sequence Listing.doc-10 / 06/2009 - One -  

Claims (21)

A composition for the treatment of neurodegenerative diseases (NDD), comprising an Eph A4 antagonist or an Eph A4 ligand antagonist. The method of claim 1, Wherein said antagonist attenuates clinical progression of the disease. The method of claim 1, A composition for treating an individual at risk of or exhibiting clinical symptoms of a neurodegenerative disease (NDD). The method of claim 1, Wherein said neurodegenerative disease is multiple sclerosis (MS) or a modified form of MS. The method of claim 1, A composition, characterized in that it is used as a screening agent for screening a biological sample from an individual for axon damage. The method of claim 1, A composition, characterized in that it is capable of reducing or reducing nerve damage or progressive neuronal dysfunction. The method of claim 6, Said antagonist preserves the function or integrity of nerve tissue or reduces the rate, incidence or amount of nerve damage in a subject. The method of claim 7, wherein The antagonist, in chronically progressive state of neurodegenerative disease (NDD) such as multiple sclerosis (MS), is intended to preserve the function or integrity of nerve tissue or to reduce the rate, incidence or amount of nerve damage occurring in an individual. Characterized in that the composition. The method of claim 6, The neurological dysfunction is a composition characterized in that the extremity weakness. The method of claim 1, A composition characterized in that it is administered before or at the onset of clinical symptoms or at or before the onset of clinical symptoms. The method of claim 1, A composition, which is administered topically to the brain, spinal cord, or optic nerve. The method of claim 1, The antagonist binds to Eph A4, characterized in that to down-regulate the level or activity of Eph A4. The method of claim 12, Wherein said antagonist is a soluble ephrin or a functional variant or analog thereof. The method of claim 13, Wherein said antagonist is soluble ephrin A5, soluble ephrin B2 or soluble ephrin B3. The method of claim 12, The antagonist is a composition characterized in that the antibody or antigen-binding fragment that binds to Eph A4 and down-regulates the level or activity of Eph A4. The method of claim 1, Wherein said antagonist binds to Eph A4-binding ephrin and down regulates the level or activity of Eph A4-binding ephrin. The method of claim 16, Said antagonist is a soluble Eph A4 or a functional variant or analog thereof. The method of claim 17, Wherein said soluble Eph A4 is Eph A4-Fc. The method of claim 16, The antagonist is a composition, characterized in that the antibody or antigen-binding fragment that binds to Eph A4-binding ephrin downregulates the level or activity of Eph A4-binding ephrin. The method according to claim 12 or 16, The antagonist is a nucleic acid, polypeptide, peptide or organic molecule that binds to Eph A4 or Eph A4-binding ephrins or nucleic acids encoding them to downregulate the level or activity of Eph A4 or Eph A4-binding ephrins or nucleic acids. A composition, characterized in that. The method of claim 1, Wherein said antagonist is administered in combination with at least one other therapeutic agent or treatment.

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