CA2452544A1 - Method for treating secondary tissue degeneration associated with central nervous system injury - Google Patents

Abstract

The invention provides a method of reducing the severity of secondary tissue degeneration associated with CNS injury in a subject by administering to a subject having secondary tissue degeneration associated with CNS injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10).

Description

METHOD FOR TREATING SECONDARY TISSUE DEGENERATION
ASSOCIATED WITH CENTRAL NERVOUS SYSTEM INJURY
This invention was made with government support under Contract No. NS37336-O1 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
This invention relates to immunology and, more specifically to treatment of secondary tissue degeneration associated with central nervous system (CNS) injury through neutralization of the chemokine CXCL10.
The CNS consists of the brain and the spinal cord, which form a continuous system containing nerve cells, supporting cells, and nerve fibers. The brain is a cognitive organ that has distinct regions and layers, each associated with the reception and processing of specific stimuli received from sense organs. The human brain is divided into three main areas that each have distinct functions: the forebrain (prosencephalon), mid-brain (mesencephalon) and hindbrain (rhombencephalon). The spinal cord is arranged in segments, with higher segments controlling movement and sensation in upper parts of the body and lower segments controlling the lower parts of the body.
The consequences of CNS injury reflect the organization of its component organs.
The types of disability associated with spinal cord injury vary greatly depending on the type and severity of the injury, the level of the cord at which the injury occurs, and the nerve fiber pathways that are damaged. Severe injury to the spinal cord causes paralysis and complete loss of sensation to the parts of the body controlled by the spinal cord segments below the point of injury. Spinal cord injuries also can lead to many complications, including pressure sores and increased susceptibility to respiratory diseases. Brain injury can lead to a wide spectrum of impairments in different areas, including cognitive functioning, physical abilities, communication, or social/behavioral functioning.
Damage to the central nervous system does not stop immediately after the initial injury, but continues in the hours and days following the initial trauma. These delayed injury processes present windows of opportunity for treatments aimed at reducing the extent of disability resulting from brain and spinal cord injury. In the absence of trauma or disease, most types of immune cells only rarely enter the CNS.
However, when the brain or spinal cord are damaged by trauma or disease, immune cells engulf the area, eliminating debris and releasing a host of powerful regulatory chemicals, both beneficial and harmful.
Influx of immune cells is associated with both acute CNS injury and associated secondary degeneration.
The current standard of care treatment of acute spinal cord injury is high-dose methylprednisolone, a steroid that has to be administered in the first three hours following injury and whose overall efficacy has been questioned in recent years. Symptomatic treatment of spinal cord injury can be achieved with 4-aminopyridine (4-AP), a blocker of potassium channels that prolongs the duration of nerve action potentials and improves conduction in demyelinated axons. However, neither of these agents prevents the complicated pathological cascade associated with CNS trauma.
Thus, there exists a need to have additional methods for treating central nervous system injury, particularly spinal cord injury, and the secondary tissue degeneration that results from trauma to the CNS. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides methods of reducing the severity of secondary tissue degeneration associated with central nervous system (CNS) injury in a subject by administering to a subject having or at risk of developing secondary tissue degeneration associated with CNS injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10). The methods of the invention are useful for the treatment of both spinal cord and brain injuries.
In additional aspects, the present invention provides methods of reducing the severity of secondary tissue degeneration associated with CNS injury in a subject, comprising administering to a subject having secondary tissue degeneration associated with CNS
injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10).
In specific embodiments the subject is a mammal. In other embodiments, the subject of the method is a human. In yet additional embodiments the CNS injury is either a spinal cord injury, a brain injury or both.
In yet further embodiments, the neutralizing agent is an anti-CXCL10 antibody, fragment corresponding to an anti-CXCL10 antibody, small molecule, or polypeptide specific for CXCL10.
An additional aspect of the present invention provides methods for reducing the severity of secondary tissue degeneration associated with CNS injury in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or fragment thereof capable of binding specifically to interferon inducible protein of 10 kDa (CXCL10).
In certain embodiments of the instant invention, the CNS injury is created or induced by a mechanical injury, bruising of the spinal cord, a compression injury of the spinal cord, laceration of the spinal cord, severance of the spinal cord, or a demyelinating condition. In one embodiment, the demyelinating condition is multiple sclerosis.
Also provided by the instant invention are methods of reducing the severity of secondary tissue degeneration associated with pathological CNS condition in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or fragment thereof capable of binding specifically to interferon inducible protein of 10 kDa (CXCL10).
In particular embodiments, the pathological condition is myelin loss. In other embodiments myelin loss is due to acute disseminated encephalomyelitis, post-infectious myelin loss, post-vaccinal myelin loss, acute necrotizing encephalomyelitis, or progressive necrotizing myelopathy.
In further embodiments, the neutralizing agent of the present invention is administered within 5 at least one, two, three, four, five, six, seven, or eight hours of injury. In yet further embodiments, the neutralizing agent is administered within 12 hours, 18 hours or within 24 hours of injury or diagnosis. In other embodiments, the neutralizing agent is an antibody or an antibody fragment. In yet additional embodiments the neutralizing agent is administered alone or in combination with additional agents such as anti-inflammatories and administration occurs at least once and in other embodiments, repeatedly for a number of days. In one embodiment, the number of days is up to 10 days, 15 days, 20 days, 25 days, or at least 30 days.
Also provided by the instant invention are methods of reducing the severity of secondary tissue degeneration associated with CNS injury in a subject, comprising administering to a subject in need thereof an effective amount of a polynucleotide agent capable of reducing the amount of interferon inducible protein of 10 kDa (CXCL10) in a cell.
In various embodiments, the polynucleotide agent may be an antisense oligonucleotide or a ribozyme, wherein said antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.

In yet additional aspects, the instant invention provides methods for reducing the severity of secondary tissue degeneration associated with pathological CNS condition in a subject, comprising administering to a subject in need thereof an effective amount of a polynucleotide agent capable of reducing the amount of interferon inducible protein of 10 kDa (CXCL10) in a cell.
In certain embodiments, the agent may be an antisense oligonucleotide or a ribozyme, wherein said antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.
In still yet additional embodiments, the neutralizing agent of the present invention may be administered in a composition capable of enhancing penetration of the blood-brain barrier, such as liposomes.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows increased CXCL10 mRNA levels after hemisection injury to the adult mouse spinal cord.
Figure 2 shows behavioral deficit following hemisection injury to the adult mouse spinal cord progressively lessened in mice treated with anti-CXCL10 antibody as compared to untreated control mice as measured by the following four kinematic parameters:
2(A) stride length; 2(B) toe spread; 2(C) stride width;
and 2(D) rear paw rotation.

Figure 3 shows 3(A) gross pathology of a spinal cord hemisection lesion from a dorsal view at day 14 post-injury in an untreated mouse; 3(B) a longitudinal section of a spinal cord dorsal hemisection lesion at day 14 post-injury in an untreated mouse; 3(C) gross pathology of a spinal cord hemisection lesion from a dorsal view at day 14 post-injury in an anti-CXCL10 antibody treated mouse; and 3(D) a longitudinal section of a spinal cord dorsal hemisection lesion at day 14 post-injury in an anti-CXCL10 antibody treated mouse. Anti-CXCL10 antibody mediated attenuation of the CD4+ T lymphocyte response to traumatic spinal cord injury is associated with tissue sparing.
Figure 4 shows CD4+ stained light microscopy photographs demonstrating that 4 (A) anti-CXCL10 antibody treatment decreases the robust T-lymphocyte recruitment following hemisection injury in treated mice compared to 4(B) untreated mice.
Figures 5 shows that anti-CXCL10 antibody mediated attenuation of the CD4+ T lymphocyte response to traumatic spinal cord injury is associated with tissue sparing.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a method of treating and/or ameliorating CNS injury, including spinal cord injury and brain injury, by neutralizing interferon inducible protein of 10 kDA (CXCL10) with a specific neutralizing agent. The invention also provides a method of reversing the secondary tissue degeneration associated with CNS injury or trauma as well as a method of facilitating nerve cell regeneration and remyelination by neutralizing interferon inducible protein of 10 kDA (CXCL10) with a specific neutralizing agent.
Neutralization of CXCL10 represents a significant treatment option for CNS injury because it decreases the extent T-cell infiltration that is responsible for the neurological degeneration associated with tissue loss following CNS injury or trauma. Neutralization of CXCL10 targets a cause rather than merely the symptoms of secondary degeneration associated with CNS injury or trauma.
The CNS contains nerve cells, or neurons, that are characterized by long nerve fibers called axons. Axons in the spinal cord carry signals downward from the brain along descending pathways and upward toward the brain along ascending pathways. Many axons in these pathways are covered by sheaths of an insulating substance called myelin, which gives them a whitish appearance; therefore, the region in which they lie is called "white matter." The nerve cells themselves, with their tree-like branches called dendrites that receive signals from other nerve cells, make up "gray matter." Gray matter lies in a butterfly-shaped region in the center of the spinal cord. Both the brain and the spinal cord are enclosed in three membranes (meninges): the pia mater, the innermost layer; the arachnoid, a delicate middle layer; and the dura mater, which is a tougher outer layer.

The spinal cord is organized into segments along its length. Nerves from each segment connect to specific regions of the body. The segments in the neck, or cervical region, referred to as C1 through C8, control signals to the neck, arms, and hands. Those in the thoracic or upper back region, referred to as T1 through T12, relay signals to the torso and some parts of the arms. Those in the upper lumbar or mid-back region just below the ribs, referred to as Ll through L5, control signals to the hips and legs. Finally, the sacral segments, referred to as S1 through S5, lie just below the lumbar segments in the mid-back and control signals to the groin, toes, and some parts of the legs.
The effects of spinal cord injury at different segments reflect this organization.
Several types of cells carry out CNS
functions. Large motor neurons have long axons that control skeletal muscles in the neck, torso, and limbs.
Sensory neurons called dorsal root ganglion cells, whose axons form the nerves that carry information from the body into the spinal cord, are found immediately outside the spinal cord. Spinal interneurons, which lie completely within the spinal cord, help integrate sensory information and generate coordinated signals that control muscles. Glia, or supporting cells, outnumber neurons in the brain and spinal cord and perform a variety of functions. One type of glial cell, the oligodendrocyte, creates the myelin sheaths that insulate axons and improve the speed and reliability of nerve signal transmission. Other glia enclose the spinal cord like the rim and spokes of a wheel, providing compartments for the ascending and descending nerve fiber tracts. Astrocytes, large star-shaped glial cells, regulate the composition of the fluids that surround nerve cells. Some of these cells also form scar tissue after injury. Smaller cells called microglia also become activated in response to injury and help clean up waste products.
5 All of these glial cells produce substances that support neuron survival and influence axon growth.
The brain and spinal cord are confined within bony cavities that protect them, but also render them vulnerable to compression damage caused by swelling or 10 forceful injury. Cells of the CNS have a very high rate of metabolism and rely upon blood glucose for energy. Since the extent to which normal blood flow exceeds the minimum required for healthy functioning, is much smaller in the CNS than in other tissues, CNS
cells are particularly vulnerable to ischemia. Other unique features of the CNS are the "blood-brain-barrier" and the "blood-spinal cord-barrier," which are formed by cells lining blood vessels in the CNS and serve to protect nerve cells by restricting entry of potentially harmful substances and cells of the immune system. Trauma compromises these barriers, contributing to further damage in the brain and spinal cord.
As used herein, the terms "central nervous system injury" and "CNS injury" are intended to refer to an injury or condition of the spinal cord and/or brain that is characterized by an inflammatory response at a site of injury or lesion. Central nervous system injury generally involves a primary mechanical insult, but also encompasses lesions caused by non-traumatic events such as pathological conditions. Injuries of the CNS include contusions caused by bruising of the spinal cord, and compression injuries caused by pressure on the spinal cord. Other types of spinal cord injuries include lacerations, severance and central cord syndrome, which affects the cervical (neck) region of the cord and results from focused damage to a group of nerve fibers called the corticospinal tract that carries signal between the brain and the spinal cord. A CNS injury or lesion can affect one or more of the cell types or tissues of the CNS, including neurons, glial cells and myelin.
Conditions of the CNS include demyelinating conditions including Multiple Sclerosis (MS), which in which demyelination occurs in the white matter of the brain and spinal cord. Demyelination and local inflammation are common to spinal cord injury and demyelinating conditions of the CNS, including Multiple Sclerosis, and influx of T-cells specific for myelin basic protein (MBP) as a result of myelin destruction has been demonstrated in both spinal cord injury and Multiple Sclerosis.
Demyelinating diseases or conditions are an important group of neurological disorders because of the frequency with which they occur and the disability that they cause. Demyelinating diseases have in common a focal or patchy destruction of myelin sheaths that can be accompanied by an inflammatory response. Myelin loss also occurs in other conditions, for example, in genetically determined defects in myelin metabolism, and as a consequence of toxin exposure and infections of oligodendrocytes or Schwann cells. Demyelinating diseases of the CNS include, for example, MS, acute disseminated encephalomyelitis (ADE) including postinfectious and postvaccinal encephalomyelitis, acute necrotizing hemorrhagic encephalomyelitis and progressive (necrotizing) myelopathy.

Injury to the CNS results in an influx of inflammatory cells to the CNS parenchyma as described by Dusart and Schwab, Eur. J. Neurosci., 6(5):712-724 (1994) and Schnell et al., Eur. J. Neurosci., 11(10):3648-3458 (1999). The initial response is dominated by neutrophils, which peak at 1 day post-injury, and accompanies a breakdown of the Blood-brain barrier and upregulation of cell adhesion molecules on the vascular endothelium. Neutrophils display phagocytic and bactericidal properties, and are critical to the removal of microbial intruders and debris (Clark, Dermatol. Clin., 11(4):647-66 (1994)).
Activated neutrophils produce a variety of proinflammatory molecules including proteases, cytokines, chemokines and free radicals as described by Cassatella, Immunol. Today, 16(1):21-26 1994); Kunkel et al., Semin. Cell Biol., 6(6):327-336 (1995); Conner and Grisham, Nutrition 12(4):274-277 (1996); each of which is incorporated herein by reference. The release of these molecules contributes to the recruitment and activation of other inflammatory cells, as well as neuronal injury and glial cell activation as described by Yong, Cytokines, astogliosis and neutrophils following CNS trauma, in Cytokines and the CNS:
Development, Defense and Disease, CRC Press, Boca Baton, Florida (1996). Hematogenous macrophages and activated resident CNS microglia dominate the inflammatory response beginning on the second day post-injury, and are accompanied by infiltrating lymphocytes. Chemoattractants for these cell types are upregulated within hours of spinal cord injury, and adhesion molecules necessary for the adherence of cells to the cerebral endothelium are upregulated after injury Bartholdi and Schwab, Eur. J. Neurosci.
9(7):1422-1438 1997); Hamada et al., J. Neurochem.

66(4):1525-1531 (1996). The expression of CXCL10, a CXC chemokine which attracts T cells, and MIP-la, a CC
chemokine which attracts macrophages, have been shown to be upregulated within hours of spinal cord injury (see Bartholdi, supra, 1997; and McTigue et al., J.
Neurosci. Res. 53(3):368-376 (1998).
The term "secondary tissue degeneration," as used in herein in reference to CNS injury refers to the secondary loss of adjacent cells and tissues that were undamaged or marginally damaged by the initial trauma or lesion to the CNS. The secondary tissue degeneration can affect any of the cell types present at the site of injury or lesion, including neurons and filial cells as well as tissues, including the myelin tissue surrounding the nerve fibers and the vasculature. Secondary tissue degeneration generally is associated with inflammation and CXCL10 mediated recruitment of T cells having specificity for a wide array of cell types and tissues. Secondary tissue degeneration mediated by CXCL10 following CNS injury or trauma also causes blood-brain-barrier disruption, edema, demyelination, axonal damage and neuronal death, which can be preceded by activation of voltage-dependent or agonist-gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases and nucleases, mitochondrial dysfunction and energy depletion, culminating in cell death (Yoles et al., Invest. Ophthalmol. Vis. Sci.
33(13):3586-3591 (1992); Zivin and Choi, Scientific American 265(1):56-63 (1993); Hovda et al., Brain Research 567(1):1-10 (1991); Yoshino et al., Brain Research 561(1):106-119 (1991)), each of which is incorporated herein by reference. CXCL10 mediated neuronal cell death associated with secondary tissue degeneration following CNS injury or trauma can include death by any mechanism, including necrosis, apoptosis, paraptosis or any additional form of cell death, for example, neurodegenerative cell death, such as has been described in a transgenic mouse model of amyotrophic lateral sclerosis (Canto and Gurney, American Journal of Pathololoav 145:1271-1279 (1994), which is incorporated herein by reference).
Inflammation contributes to secondary tissue degeneration associated with a CNS injury (Dusart and Schwab, Eur. J. Neurosci.6(5):712-724 (1994); Blight, Central Nervous System Trauma 2(4):299-315 (1985);
Egerton et al., EMBO J. 11(10):3533-40 (1992); Popovich et al., J Comp,. Neurol., 377(3):443-464 (1997), each of which is incorporated herein by reference). A
successful host response to inflammation generally requires the accumulation of specialized host cells at the site of tissue damage. Inflammation in the injured CNS is characterized by fluid accumulation, and the influx of plasma proteins, neutrophils, T lymphocytes and macrophages. This cellular accumulation is a critical step in the normal inflammatory process and is mediated by a family of secreted chemotactic cytokines that have in common important structural features and a role in pathogenic inflammation. CXCL10 belongs to this protein family of over forty structurally and functionally related proteins known as chemokines.
Chemokines are homologous 8 to 10 kDa heparin binding proteins that possess a conserved structural motif containing two cysteine pairs and are divided into subfamilies based on the relative position of the cysteine residues in the mature protein. There are at least four subfamilies of chemokines, but only two, a-chemokines and (3-chemokines, have been well characterized. In the cx-chemokines, the first two cysteine residues are separated by a single amino acid (CXC), whereas in the ~3-chemokines the first two 5 cysteine residues are adjacent to each other (CC). The C-X-C chemokines include, for example, interleukin-8 (IL-8), human platelet derived factors, CXCL10 and Mig.
Members of the C-C chemokine subfamily include, for example, macrophage chemoattractant and activating 10 factor (MCAF), macrophage inflammatory protein-la (MIP-la), macrophage inflammatory protein-lei (MIP-1(3) and regulated on activation, normal T-cell expressed and secreted (RANTES).
Chemokines selectively attract leukocyte 15 subsets; some chemokines act specifically toward eosinophils, ethers toward monocytes, dendritic cells, or T cells (see Luster, A., New Engl. J. Med. 338: 436-445 (1998), which is incorporated herein by reference).
In general, CC chemokines chemoattract monocytes, eosinophils, basophils, and T cells; and signal through the chemokine receptors CCR1 to CCR9. The CXC
chemokine family can be further divided into two classes based on the presence or absence of an ELR
sequence (Glu-Leu-Arg) near the N terminal preceding the CXC sequence. The ELR-containing CXC chemokines including IL-8 chemoattract neutrophils, while the non-ELR CXC chemokines including CXCL10 and Mig chemoattract lymphocytes.
Chemokines induce cell migration and activation in at least two ways: first, through direct chemoattraction, and, second, by binding to specific G-protein coupled cell-surface receptors on target cells.
More than 10 distinct chemokine receptors, each expressed on different subsets of leukocytes, have been identified. Chemokine receptors are constitutively expressed on some cells, whereas they are inducible on others. CXCR3, the receptor recognized by CXCL10 is expressed on activated T lymphocytes of the T helper type 1 (Th 1) phenotype and Natural Killer (NK) Cells.
Significantly, a central mechanism in the pathology of neuroinflammation associated with secondary tissue degeneration is the migration of activated T cells to the site of injury or lesion.
As used herein, the term "effective amount"
when used in reference to interferon inducible protein of 10 kDa (CXCL10), is intended to mean an amount of a neutralizing agent specific for CXCL10 sufficient to reduce the severity of secondary tissue degeneration associated with CNS injury.
As used herein, "reduction in severity" is intended to refer to an arrest, decrease or reversal in signs and symptoms, physiological indicators, biochemical markers or metabolic indicators of secondary tissue degeneration associated with a CNS
injury. In spinal cord injury, the destruction of nerve fibers that carry motor signals from the brain to the torso and limbs leads to muscle paralysis.
Physiological symptoms of secondary tissue degeneration associated with CNS injury include, for example, neurological impairments and neuroinflammation and clinical symptoms vary greatly depending on the severity of the injury, the segment of the spinal cord at which the injury occurs, and which nerve fibers are damaged. Destruction of sensory nerve fibers can lead to loss of sensations such as touch, pressure, and temperature; it sometimes also causes pain. Other serious consequences can include exaggerated reflexes;
loss of bladder and bowel control; sexual dysfunction;
lost or decreased breathing capacity; impaired cough reflexes; and spasticity. Most people with spinal cord injury regain some functions between a week and six months after injury, but the likelihood of spontaneous recovery diminishes after six months. Spinal cord injuries can lead to many clinical complications, including pressure sores, increased susceptibility to respiratory diseases, and autonomic dysreflexia, a potentially life-threatening increase in blood pressure, sweating, and other autonomic reflexes in reaction to bowel impaction or some other stimulus.
Physiological indicators of secondary tissue degeneration associated with CNS injury include blood-brain-barrier disruption, edema, demyelination, axonal damage and neuronal death and tissue loss. Biochemical markers 4f secondary tissue degeneration associated with CNS injury are, for example, neuronal cell markers, myelin, gamma globulin or the specific molecules that give rise to oligoclonal banding.
Tissue loss in the CNS associated with secondary tissue degeneration can be detected by a variety of clinical methods well known in the art. A neuronal cell marker, for example, NeuN can be utilized to visualize tissue loss in the CNS (Wolf et al., J. Histochem. Cytochem.
44:1167-1171 (1996), which is incorporated herein by reference). In addition, evoked potentials (EP) can be used to measure how quickly nerve impulses travel along the nerve fibers in various parts of the nervous system and computer-assisted tomography (CT) can be used to scan the CNS to detect areas of tissue loss caused by cell death or demyelination of nerve fibers. Magnetic resonance imaging (MRI) also can be used to scan the CNS, but without the use of x-rays. More sensitive than the CT scan, MRI can detect areas of CNS tissue loss that may not be seen by the CT scanner. Moreover, lumbar puncture or spinal tap procedures can be used to draw out cerebrospinal fluid. The fluid can be examined for increased levels of gamma globulin and oligoclonal banding. These and other methods well known in the art can be used to determine the severity of secondary tissue degeneration associated with CNS
injury by measuring tissue loss, including tissue loss caused by neuronal, glial cell death and demyelination of nerve fibers.
As used herein, the term "neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10)" is intended to refer to an agent effecting a decrease in the extent, amount or rate of CXCL10 expression or effecting a decrease in the activity of CXCL10. Neutralizing agents useful for practicing the claimed invention include, for example, binding molecules such as antibodies against CXCL10. A
neutralizing agent can be any molecule that binds CXCL10 with sufficient affinity to decrease CXCL10 activity. Additionally, a neutralizing agent can be any molecule binds to a regulatory molecule or gene region so as to inhibit or promote the function of the regulatory protein or gene region and effect a decrease in the extent or amount or rate of CXCL10 expression or activity. For example, a fragment or peptidomimetic of the CXCR3 receptor that binds CXCL10 with sufficient affinity to decrease CXCL10 activity, is useful for practicing the claimed methods. In addition, examples of neutralizing agents which effect a decrease in CXCL10 expression can include antisense nucleic acids and transcriptional inhibitors.

The invention provides a method of reducing the severity of secondary tissue degeneration associated with CNS injury. The method comprises administering to a subject having secondary tissue degeneration associated with CNS injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10).
As described herein, CXCL10 is involved in mounting a host defense against inflammation of the nervous system. Specifically, CXCL10 coordinates the trafficking of Thl T lymphocytes into the CNS in response to a CNS injury, CNS inflammation, pathological aberration such as an infectious agent or autoagressive immune cells. T lymphocytes can be subdivided into two major categories known as CD4+
cells and CD8+ cells. CD4 and CD8 are surface proteins that facilitate interactions between T cell receptors for antigen and antigen itself which is presented to T
cells by antigen-presenting cells. Antigens recognized by T cells are contained within clefts of major histocompatibility complex (MHC) proteins expressed on the surface of antigen-presenting cells. CD4 cells recognize peptide fragments presented by class II
histocompatibility alleles, CD8 cells recognize fragments presented by class I histocompatibility alleles. The DR2 allele, over-represented in multiple sclerosis, is a MHC class II allele, pointing to a role for CD4+ cells in lesion formation in the CNS.
CD4+ cells can be further subdivided into Thl and Th2 subtypes. Thl cells are responsible for delayed type hypersensitivity responses and secrete numerous cytokines including interleukin-2 (IL-2), a stimulator of T cell proliferation, and interferon, an activator of macrophages, and lymphotoxin, a protein which has the capacity to damage oligodendrocytes, the myelin-forming cells of the CNS. Interferon, in combination with IL-2 activates macrophages which 5 directly strip myelin from nerve fibers as well as secrete tumor necrosis factor-a (TNF-a), a cytokine that damages the myelin-producing oligodendrocytes.
As described herein, neutralization of CXCL10, which is expressed following a CNS injury 10 within and around the injury site or lesion (Figure 1), reduces the secondary tissue degeneration that manifests itself through tissue loss at the injury site and surrounding tissue (Figure 3). As such, neutralizing CXCL10 activity can lead to a reduction in 15 the severity of secondary tissue degeneration associated with CNS injury. A CXCL10 neutralizing agent including an antibody, antisense nucleic acid or other compound identified by the methods described below is useful for treating or reducing the severity 20 of secondary tissue degeneration associated with CNS
ink ury .
Administration of a CXCL10 neutralizing agent targets a variety of distinct CXCL10 mediated destructive events associated with secondary tissue degeneration including, for example, activation of voltage-dependent or agonist-gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases and nucleases, inflammation, mitochondrial dysfunction and energy depletion, culminating in cell death. Administration of a CXCL10-specific neutralizing agent provides an enhanced method of reducing the severity of secondary degeneration that represents a therapeutic improvement by targeting and treating each of these distinct CXCL10 mediated events rather than a single aspect of the secondary tissue degeneration.
In addition, to the unexpected finding that CXCL 10 has such a dramatic effect on secondary tissue degeneration, is the astounding finding that, not only can damage be halted, but CXCL10 neutralizing agents also facilitate myelin regrowth thereby inducing nerve regeneration and remyelination. Therefore, the invention also provides a method of reversing secondary tissue degeneration associated with CNS injury or trauma as well as a method of facilitating nerve cell regeneration and remyelination by neutralizing interferon inducible protein of 10 kDA (CXCL10) with a specific neutralizing agent.
Mechanical injury to the adult mammalian spinal cord results in an increase in vascular permeability, and a widespread activation and recruitment of inflammatory cells (Dusart, I. and M. E.
Schwab (1994), "Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord," Eur J Neurosci 6(5): 712-24) (Schnell, L., S. Fearn, et al. (1999), "Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord," Eur J
Neurosci 11(10): 3648-58). The control and consequences of this robust inflammatory response to spinal cord injury are largely unknown. A greater understanding of neuroimmune interactions can be seen in the field of multiple sclerosis research, where it has recently been demonstrated that attenuation of the T lymphocyte response to demyelinating pathology in the MHV model of multiple sclerosis resulted in diminished histopathogenesis and behavioral impairment (Liu, M.
T., H. S. Keirstead, et al. (2001), "Neutralization of the chemokine cxc110 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis," J Immunol 167(7):4091-7). Given the predominance of T
lymphocytes within sites of spinal cord injury (McTigue, D. M., M. Tani, et al. (1998), "Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury," J Neurosci Res 53(3):368-76), and recent studies suggesting that they play a central role in the anatomical and functional outcome of spinal cord trauma (Hauben, E., O. Butovsky, et al. (2000), "Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion,"
J Neurosci 20(17):6421-30) (Hauben, E., U. Nevo, et al.
(2000), "Autoimmune T cells as potential neuroprotective therapy for spinal cord injury," Lancet 355(9200):286-7), the consequences of inhibiting T
lymphocyte recruitment to sites of spinal cord injury were further investigated. The availability of functionally blocking antibodies to the T lymphocyte chemoattractant CXCL10 (Liu, M. T., H. S. Keirstead, et al. (2001), "Neutralization of the chemokine cxc110 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis," J Immunol 167(7):4091-7) provided an opportunity to test the role of this chemokine in recruiting T lymphocytes to sites of spinal cord trauma, and the contribution of T lymphocytes to posttraumatic histopathogenesis and behavioral impairment. The data herein clearly demonstrate that CXCL10 is upregulated after injury to the adult mammalian spinal cord (Figure 1), and that antibody-mediated neutralization of CXCL10 in injured animals reduced the dramatic T lymphocyte infiltration that normally occurs after spinal cord trauma (Figure 5). These data corroborate the inventors' recent demonstration that administration of anti-CXCL10 antisera to mice with established viral-induced demyelination resulted in a significant reduction in CD4+ T lymphocyte infiltration to the CNS, as well as diminished expression of the TH1-associated proinflammatory cytokine IFN-g (Liu, M. T., H. S.
Keirstead, et al. (2001), "Neutralization of the chemokine cxc110 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis," J Immunol 167(7):4091-7). The attenuation of T lymphocyte infiltration to sites of spinal cord injury following anti-CXCL10 treatment present strong evidence that CXCL10 in particular is a key T lymphocyte chemoattractant during spinal cord injury, consistent with the expression of CXCR3 receptors on the surface of these cells (Rollins, B. J. (1997), "Chemokines,"
Blood 90(3):909-28) (Luster, A. D., J. C. Unkeless, et al. (1985), "Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins," Nature 315(6021):672-6).
Importantly and unexpectedly, the data disclosed herein indicate that anti-CXCL10 treatment alone was sufficient to decrease posttraumatic tissue degeneration and locomotor deficits following injury.
Morphometric analyses indicated that hemisection-injured mice that received anti-CXCL10 treatment had a 68% reduction in tissue loss around the injury site compared to untreated hemisection-injured mice (Figure 3). Neuronal cell counts around the injury site indicated that the reduction in tissue loss in hemisection-injured mice that received anti-CXCL10 treatment was associated with the presence of 438% more neurons around the injury site than untreated hemisection-injured mice. These data indicate that anti-CXCL10 treatment significantly decreased posttraumatic tissue loss following dorsal hemisection inj ury .
Behavioral analyses indicated that hemisection-injured mice that received anti-CXCL10 treatment showed a statistically significant progressive improvement in all kinematic parameters tested during the recovery period (Figure 2). By 14 days post-injury, the behavioral scores for all 4 kinematic parameters were not significantly different from uninjured control mice. This recovery was likely contributed to by reorganization of the sensorimotor pathways caudal to the lesion rather than regeneration of severed fibers. Biochemical and synaptic reorganization has been demonstrated following spinal cord injury, and can contribute to functional improvement (Edgerton, V. R., R. D. Leon, et al. (2001) "Retraining the injured spinal cord," J Physiol 533(Pt 1):15-22). Although little data exist pertaining to synaptogenesis in the injured spinal cord, quantitative electron micrographic studies using the well-documented model of the denervated dentate gyrus indicate that substantial numbers of new synapses are formed on denervated neurons by 10 days post-injury (Steward, O., S. L. Vinsant, et al. (1988), "The process of reinnervation in the dentate gyrus of adult rats: an ultrastructural study of changes in presynaptic terminals as a result of sprouting," J Comp Neurol 267(2):203-10). Although the mechanism of behavioral recovery in the current study is unknown, the data disclosed herein clearly indicate that anti-CXCL10 treatment reduces neurological impairment following spinal cord injury.
A detrimental role for the immune response 5 following extravasation to the injured CNS parenchyma is indicated by the etiology of multiple sclerosis and disease states seen in animal models of multiple sclerosis, in which pro-inflammatory cells are considered central to the development of disease 10 (Olsson, T. (1995), "Cytokine-producing cells in experimental autoimmune encephalomyelitis and multiple sclerosis," Neurology 45(6 Suppl 6):511-5) (Schulze-Koops, H., P. E. Lipsky, et al. (1995), "Elevated Th1- or Th0-like cytokine mRNA in peripheral 15 circulation of patients with rheumatoid arthritis.
Modulation by treatment with anti- ICAM-1 correlates with clinical benefit," J Immunol 155(10):5029-37). A
detrimental role for the immune response in traumatic spinal cord injury is suggested by the temporal 20 association of chemokine expression and immune cell influx with secondary degeneration (Dusart, I. and M.
E. Schwab (1994), "Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord," Eur J Neurosci 6(5):712-24) (Blight, 25 A. R. (1985), "Delayed demyelination and macrophage invasion: a candidate for secondary cell damage in spinal cord injury," Cent New Syst Trauma 2 (4) :299-315) (Popovich, P. G. , P. 4~Iei, et al . (1997) , "Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats," J Comp Neurol 377 (3) :443-64) .

Popovich and colleagues have convincingly demonstrated that clodronate-mediated depletion of hematogenous macrophages leads to a decrease in posttraumatic tissue loss and an improvement in overground locomotion following spinal cord injury.
These findings led the authors to conclude that infiltrating immune cells are effectors of acute secondary degeneration, and suggest that cell-specific immunodepletion may prove therapeutic for spinal cord injury (Popovich, P. G., P. Wei, et al. (1997), "Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats," J Comp Neurol 377(3):443-64). Indeed, activated mononuclear phagocytes release neurotoxins after traumatic CNS
injury (Giulian, D., M. Corpuz, et al. (1993), "Reactive mononuclear phagocytes release neurotoxins after ischemic and traumatic injury to the central nervous system," J Neurosci Res 36(6):681-93), induce NMDA receptor-mediated neurotoxicity (Paini, D., K.
Frei, et al. (1991), "Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate," Neurosci Lett 133:159-162), and activated leukocytes release a wide variety of lytic enzymes as well as reactive oxygen and nitrogen intermediates (Martiney, J. A., C. Cuff, et al. (1998), "Cytokine-induced inflammation in the central nervous system revisited," Neurochem Res 23(3):349-59).
These findings are interesting in light of the present demonstration that anti-CXCL10 treatment leads to a decrease in the infiltration of both T
lymphocytes and hematogenous macrophages to sites of spinal cord injury (Figure 5). As hematogenous macrophages do not express the CXCR3 receptor for CXCL10 it is likely that their depletion is a downstream effect of T lymphocyte depletion; this is supported by the demonstration that activated CD4+ T
lymphocytes secrete RANTES, a chemoattractant for hematogenous macrophages.
Given the complexity of immune cell regulation within sites of CNS disease/trauma and the downstream consequences of immune cell activation within such sites, targeting the recruitment of immune cell populations to sites of injury is a can elucidate the role of particular populations in the secondary degenerative cascade that follows a CNS insult. The results described herein provide new insight into the functional significance of CXCL10 expression during CNS
injury and further provide a basis for methods of treating CNS injuries with a neutralizing agent specific for CXCL10, which is shown herein to be a key chemoattractant during spinal cord injury. Therapies that target CXCL10 as provided by the invention represent a viable treatment strategy for reducing posttraumatic histopathogenesis and behavioral impairment following CNS injury.
A CXCL10 neutralizing agent that binds CXCL10 with sufficient affinity can reduce activity of CXCL10 related to immune cell recruitment, tissue loss or demyelination. A CXCL10-specific neutralizing agent can be a macromolecule, such as polypeptide, nucleic acid, carbohydrate or lipid. A CXCL10-specific neutralizing agent can also be a derivative, analogue or mimetic compound as well as a small organic compound as long as CXCL10 activity is reduced in the presence of the neutralizing agent. The size of a neutralizing agent is not important so long as the molecule exhibits or can be made to exhibit selective neutralizing activity towards CXCL10. For example, a neutralizing agent can be as little as between about one and six, and as large as tens or hundreds of monomer building blocks which constitute a macromolecule or chemical binding molecule. Similarly, an organic compound can be a simple or complex structure so long as it binds CXCL10 with sufficient affinity to reduce activity.
Neutralizing agents specific for CXCL10 can include, for example, antibodies and other receptor or ligand binding polypeptides of the immune system. Such other molecules of the immune system include for example, T cell receptors (TCR) including CD4 cell receptors. A neutralizing agent for CXCL10 also can be a molecule that inhibits the interaction of CXCL10 and a chemokine receptor, for example, the CXCR3 receptor.
Additionally, cell surface receptors such as integrins, growth factor receptors and chemokine receptors, as well as any other receptors or fragments thereof that bind CXCL10, or can be made to bind with sufficient affinity to reduce activity are also neutralizing agents useful for practicing the methods of the invention. Additionally, receptors, growth factors, cytokines or chemokines, for example, which inhibit the expression of CXCL10 or their receptors are also neutralizing agents useful for practicing the methods of the invention. Furthermore, DNA binding polypeptides such as transcription factors and DNA
replication factors are likewise included within the definition of the term binding molecule so long as they have selective binding activity for CXCL10, regulatory molecules that control the expression or activity of CXCL10, or gene regions that control the expression of CXCL10. Finally, polypeptides, nucleic acids and chemical compounds such as those selected from random and combinational libraries are also included within the definition of the term so long as such a molecule binds CXCL10 with sufficient affinity to decrease activity.
Various approaches can be used for identifying neutralizing agents selective for CXLC10.
For example, one approach is to use the information available regarding the structure and function of CXCL10 to generate binding molecule populations from molecules known to function as chemokine binding molecules or known to exhibit or be capable of exhibiting binding affinity specific for CXCL10, such as fragments or mimetics of the CXCR3 receptor found on CD4+ T cells and NK cells. A neutralizing agent specific for CXCL10 can be an antibody and other receptor of the immune repertoire. The normal function of such immune receptors is to bind essentially an infinite number of different antigens and ligands.
Therefore, generating a diverse population of binding molecules from an immune repertoire, for example, can be useful for identifying a neutralizing agent specific for CXCL10.
A neutralizing agent specific for CXCL10 can further be identified from a large population of unknown molecules by methods well known in the art.
Such a population can be a random library of peptides or small molecule compounds. The population can be generated to contain a sufficient diversity of sequence or structure so as to contain a molecule which will bind to the CXCL10 protein or their respective nucleic acids. Those skilled in the art will know what size and diversity is necessary or sufficient for the intended purpose. A population of sufficient size and complexity can be generated so as to have a high probability of containing a CXCL10 neutralizing agent that binds CXCL10 with sufficient affinity to decrease activity. Numerous other types of library molecule 5 populations exist and are described further below.
Any molecule that binds to CXCL10, to a CXCL10 receptor, to a gene region that controls CXCL10 expression, or to a regulatory molecule that modulates CXCL10 activity or expression as well as to any 10 regulatory molecule that modulates CXCL10 receptor expression is a CXCL10-specific neutralizing agent useful for practicing the invention. For example, a CXCL10-specific neutralizing agent can be a regulatory molecule affects CXCL10 expression by reducing or 15 inhibiting the action of a transcription factor that controls or upregulates transcription of CXCL10. In addition, a regulatory molecule that binds with sufficient affinity to a molecule involved in the activation of CXCL10 to reduce CXCL10 activation is a 20 neutralizing agent useful for practicing the methods of the invention.
A neutralizing agent can bind to CXCL10 with sufficient affinity to decrease their activity is useful for practicing the claimed methods of reducing 25 the severity of secondary tissue degeneration associated with CNS injury and in a subject affected with secondary tissue degeneration associated with CNS
injury. In addition, a CXCL10-specific neutralizing agent can decrease CXCL10 activity or expression by 30 binding to a CXCL10 receptor, to a regulatory molecule that modulates the activity or expression of CXCL10, or to a gene region that controls CXCL10 expression. For example, a neutralizing agent useful for practicing the claimed invention can be an antibody against a regulator molecule that modulates CXCL10 expression or activity. Furthermore, as described herein, an anti-CXCL10 antibody is a useful neutralizing agent for practicing the methods of the invention. In addition, since CXCL10 is a secreted protein known to bind to a specific T-cell receptor, a neutralizing agent useful for practicing the invention can be any binding polypeptide, receptor or fragment thereof of the immunoglobulin superfamily of receptors.
Alternatively, it may be desired to use populations of random peptide populations to identify further neutralizing agents specific for CXCL10. Those skilled in the art will know or can determine what type of approach and what type of neutralizing agent is appropriate for practicing the methods of the invention for reducing the severity of secondary tissue degeneration associated with CNS injury in a subject affected with secondary tissue degeneration associated with CNS injury.
A moderate sized population for identification of a CXCL10-specific neutralizing agent can consist of hundreds and thousands of different binding molecules within the population whereas a large sized binding molecule population will consist of tens of thousands and millions of different binding molecule species. More specifically, large and diverse populations of binding molecules for the identification of a neutralizing agent will contain any of about 104, 105, 106, 10', 108, 109, 101°, or more, different binding molecule species. One skilled in the art will know the approximate diversity of the population of binding molecules sufficient to identify a neutralizing agent specific for CXCL10.

Recombinant libraries of binding molecules can be used to identify a neutralizing agent specific for IP-10 since large and diverse populations can be rapidly generated and screened with CXCL10.
Recombinant libraries of expressed polypeptides useful for identifying a neutralizing agent specific for CXCL10 can be engineered in a large number of different ways known in the art. Recombinant library methods similarly allow for the production of a large number of binding molecule populations from naturally occurring repertoires. Whether recombinant or otherwise, essentially any source of binding molecule population can be used so long as the source provides a sufficient size and diversity of different binding molecules to identify a neutralizing agent specific for CXCL10. If desired, a population of binding molecules useful for identifying a neutralizing agent specific for CXCL10 can be a selectively immobilized to a solid support as described by Watkins et al., Anal. Biochem. 256 (92):
169-177 (1998), which is incorporated herein by reference.
A phage expression library in which lysogenic phage cause the release of bacterially expressed binding molecule polypeptides is a specific example of a recombinant library that can be used to identify a neutralizing agent specific for CXCL10. In another type of phage expression library, large numbers of potential binding molecules can be expressed as fusion polypeptides on the periplasmic surface of bacterial cells. Libraries in yeast and higher eukaryotic cells exist as well and are similarly applicable in the methods of the invention. Those skilled in the art will know or can determine what type of library is useful for identifying a neutralizing agent specific for CXCL10.
In addition to the methods described above, which utilize purified polypeptide to screen libraries of compounds for those which specifically bind CXCL10, a neutralizing agent specific for CXCL10 can be identified by using purified polypeptide to produce antibodies. For example, antibodies which are specific for CXCL10 can be used as neutralizing agents of the invention and can be generated using methods that are well known in the art. Neutralizing agents useful for practicing the methods of the invention include both polyclonal and monoclonal antibodies against CXCL10 or any molecule that modulates CXCL10 expression or activity, as well as antigen binding fragments of such antibodies including Fab, F(ab')2, Fd and Fv fragments and the like. In addition, neutralizing agents useful for practicing the methods of the invention encompass non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, complementarity determining region-grafted (CDR-grafted) antibodies and humanized antibodies, as well as antigen-binding fragments thereof .
Methods of preparing and isolating antibodies, including polyclonal and monoclonal antibodies, using peptide immunogens, are well known to those skilled in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), which is incorporated herein by reference. Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Hoogenboom et al., U.S. Patent No. 5,564,332, issued October 15, 1996; Winter and Harris, Immunol. Todav 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).
A CXCL10-specific antibody neutralizing agent can be raised using as an immunogen a substantially purified CXCL10 protein, which can be prepared from natural sources or produced recombinantly, or a peptide portion of a CXCL10 protein including synthetic peptides. A non-immunogenic peptide portion of a CXCL10 protein can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein.
Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see Harlow and Lane, supra, 1988; see, also, Hermanson, Bioconjuaate Techniques, Academic Press, 1996, which is incorporated herein by reference). As described above, an antibody neutralizing agent specific for CXCL10 can also be raised against a regulatory molecule that modulates CXCL10 expression or activity rather than against CXCL10 directly.
A neutralizing agent specific for CXCL10, such as an antibody can be labeled so as to be 5 detectable using methods well known in the art (Hermanson, su ra, 1996; Harlow and Lane, su ra,1988;
chap. 9). For example, a neutralizing agent specific for CXCL10 can be linked to a radioisotope or therapeutic agent by methods well known in the art. A
10 neutralizing agent that directly binds CXCL10 linked to a radioisotope or other moiety capable of visualization can be useful to diagnose or stage the progression of a clinical stage of secondary tissue degeneration associated with CNS injury that is characterized by the 15 organ or tissue-specific presence or absence of CXCL10.
Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (Harlow and Lane, supra, 1988). The production of anti-peptide antibodies 20 commonly involves the use of host animals such as rabbits, mice, guinea pigs, or rats. If a large amount of serum is needed, larger animals such as sheep, goats, horses, pigs, or donkeys can be used. Animals are usually chosen based on the amount of antiserum 25 required and suitable animals include rabbits, mice, rats, guinea pigs, and hamsters. These animals yield a maximum of 25 mL, 100-200 uL and 1-2 mL of serum per single bleed (Harlow and Lane, supra, 1988). Rabbits are very useful for the production of polyclonal 30 antisera, since they can be safely and repeatedly bled and produce high volumes of antiserum. Two injections two to four weeks apart with 15-50~Zg of antigen in a suitable adjuvant such as, for example, Freund's Complete Adjuvant can be followed by blood collection and analysis of the antiserum.
In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988). A peptide portion of a protein such as CXCL10 for use as an immunogen can be determined by methods well known in the art. Spleen cells from a CXCL10 immunized mouse can be fused to an appropriate myeloma cell line to produce hybridoma cells. Cloned hybridoma cell lines can be screened using a labeled CXCL10 protein to identify clones that secrete anti-CXCL10. Hybridomas expressing anti-CXCL10 monoclonal antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibody neutralizing agent.
Neutralizing agents specific for CXCL10 can be used to reduce the severity of secondary tissue degeneration associated with CNS injury in a mammal, including in a human subject. Humanized antibodies can be constructed by conferring essentially any antigen binding specificity onto a human antibody framework.
Methods of constructing humanized antibodies are useful to prepare an antibody neutralizing agent appropriate for practicing the methods of the invention and avoiding host immune responses against the antibody neutralizing agent when used therapeutically.
The antibody neutralizing agents described above can be used to generate therapeutic human neutralizing agents by methods well known in the art such as complementary determining region (CDR)-grafting and optimization of framework and CDR residues. For example, humanization of an antibody neutralizing agent can be accomplished by CDR-grafting as described in Fiorentini at al., Immunotechnoloay 3(1): 45-59 (1997), which is incorporated herein be reference. Briefly, CDR-grafting involves recombinantly splicing CDRs from a nonhuman antibody neutralizing agent into a human framework region to confer binding activity onto the resultant grafted antibody, or variable region binding fragment thereof. Once the CDR-grafted antibody, or variable region binding fragment is made, binding affinity comparable to the nonhuman antibody neutralizing agent can be reacquired by subsequent rounds of affinity maturation strategies known in the art. Humanization of antibody neutralizing agents in the form of rabbit polyclonal antibodies can be accomplished by similar methods as described in Rader et al., J. Biol. Chem. 275(18): 13668-13676 (2000), which is incorporated herein be reference.
Humanization of a nonhuman CXCL10 antibody neutralizing agent can also be achieved by simultaneous optimization of framework and CDR residues, which permits the rapid identification of co-operatively interacting framework and CDR residues, as described in Wu et al., J. Mol. Biol. 294(1): 151-162 (1999), which is incorporated herein by reference. Briefly, a combinatorial library that examines a number of potentially important framework positions is expressed concomitantly with focused CDR libraries consisting of variants containing random single amino acid mutations in the third CDR of the heavy and light chains. By this method, multiple Fab variants containing as few as one nonhuman framework residue and displaying up to approximately 500-fold higher affinity than the initial chimeric Fab can be identified. Screening of combinatorial framework-CDR libraries permits identification of monoclonal antibodies with structures optimized for function, including instances in which the antigen induces conformational changes in the monoclonal antibody. The enhanced humanized variants contain fewer nonhuman framework residues than antibodies humanized by sequential in vitro humanization and affinity maturation strategies known in the art.
As described above, antibody neutralizing agents of the invention include, for example, polyclonal antibodies, monoclonal antibodies as well as recombinant versions and functional fragments thereof.
Recombinant versions of antibody neutralizing agents include a wide variety of constructions ranging from simple expression and co-assembly of encoding heavy and light chain cDNAs to speciality constructs termed designer antibodies. Recombinant methodologies, combined with the extensive characterization of polypeptides within the immunoglobulin superfamily, and particularly antibodies, provides the ability to design and construct a vast number of different types, styles and specificities of binding molecules derived from immunoglobulin variable and constant region binding domains. Specific examples include chimeric antibodies, where the constant region of one antibody is substituted with that of another antibody, and humanized antibodies, described above, where the complementarity determining regions (CDR) from one antibody are substituted with those from another antibody.

Other recombinant versions of antibody neutralizing agents include, for example, functional antibody variants where the variable region binding domain or functional fragments responsible for maintaining antigen binding is fused to an F~ receptor binding domain from the antibody constant region. Such variants are essentially truncated forms of antibodies that remove regions non-essential for antigen and F~
receptor binding. Truncated variants can be have single valency, for example, or alternatively be constructed with multiple valencies depending on the application and need of the user. Additionally, linkers or spacers can be inserted between the antigen and F~ receptor binding domains to optimize binding activity as well as contain additional functional domains fused or attached to effect biological functions other than CXCL10 neutralization. Those skilled in the art will know how to construct recombinant antibody neutralizing agents specific for CXCL10 in light of the art knowledge regarding antibody engineering and given the guidance and. teachings herein. A description of recombinant antibodies, functional fragments and variants and antibody-like molecules can be found, for example, in Antibodv Engineer,ing, 2nd Edition, (Carl A.K. Borrebaeck, Ed.) Oxford University Press, New York,(1995).
Additional functional variants of antibodies that can be used as antibody neutralizing agents include antibody-like molecules other than antigen binding-F~ receptor binding domain fusions. For example, antibodies, functional fragments and fusions thereof containing a F~ receptor binding domain can be produced to be bispecific in that one variable region binding domain exhibits binding activity for one antigen and the other variable region binding domain exhibits binding activity for a second antigen. Such bispecific antibody neutralizing agents can be advantageous in the methods of the invention because a 5 single bispecific antibody will contain two different target antigen binding species. Therefore, a single molecular entity can be administered to achieve neutralization of CXCL10.
An antibody neutralizing agent specific for 10 CXCL10 can also be an immunoadhesion or bispecific immunoadhesion. Immunoadhesions are antibody-like molecules that combine the binding domain of a non-antibody polypeptide with the effector functions of an antibody of an antibody constant domain. The binding 15 domain of the non-antibody polypeptide can be, for example, a ligand or a cell surface receptor having ligand binding activity. Immunoadhesions for use as CXCL10 neutralizing agents can contain at least the F~
receptor binding effector functions of the antibody 20 constant domain. Specific examples of ligands and cell surface receptors that can be used for the antigen binding domain of an immunoadhesion neutralizing agent include, for example, a T cell or NK cell receptor such as the CXCR3 receptor that recognizes CXCL10. Other 25 ligands and ligand receptors known in the art can similarly be used for the antigen binding domain of an immunoadhesion neutralizing agent specific for CXCL10.
In addition, multivalent and multispecific immunoadhesions can be constructed for use as CXCL10 30 neutralizing agents. The construction of bispecific antibodies, immunoadhesions, bispecific immunoadhesions and other heteromultimeric polypeptides which can be used as CXCL10-specific neutralizing agents is the subject matter of, for example, U.S. Patent Numbers 5,807,706 and 5,428,130, which are incorporated herein by reference.
In one embodiment of the invention, the polynucleotides encoding CXCL10, regulatory molecules that modulate the expression or activity of CXCL10, or any fragment thereof, or antisense molecules, can be used as neutralizing agents for therapeutic purposes.
In one aspect, antisense molecules to the CXCL10 encoding nucleic acids can be used to block the transcription or translation of the mRNA.
Specifically, cells can be transformed with sequences complementary to CXCL10 nucleic acids. Such methods are well known in the art, and sense or antisense oligonucleotides or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding CXCL10. Thus, antisense molecules can be used to neutralize CXCL10 activity, or to achieve regulation of gene function.
Expression vectors derived from retroviruses, adenovirus, adeno-associated virus (AAV), herpes or vaccinia viruses, or from various bacterial plasmids can be used for delivery of antisense nucleotide sequences. The viral vector selected should be able to infect the CNS cells and be safe to the host and cause minimal cell transformation. Retroviral vectors and adenoviruses offer an efficient, useful, and presently the best-characterized means of introducing and expressing foreign nucleotide sequences efficiently in mammalian cells. These vectors are well known in the art and have very broad host and cell type ranges, express genes stably and efficiently. Methods well known to those skilled in the art can be used to construct such recombinant vectors and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1999); each of which is incorporated herein by reference. Even in the absence of integration into the DNA, such vectors can continue to transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient expression can last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
Ribozymes, enzymatic RNA molecules, can also be used to catalyze the specific cleavage of CXCL10 mRNA or the mRNA of any regulatory molecule that modulates the expression or activity of CXCL10. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target the mRNA, followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within any potential RNA target are identified by scanning the RNA for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features which can render the oligonucleotide inoperable. The suitability of candidate targets can also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Antisense molecules and ribozymes of the invention can be prepared by any method known in the art for the synthesis of nucleic acid molecules.

The neutralizing agents useful for practicing the methods of the invention can be formulated and administered by those skilled in the art in a manner and in an amount appropriate for the severity of the tissue degeneration to be treated; the rate or amount of tissue loss; the weight, gender, age and health of the subject; the biochemical nature, bioactivity, bioavailability and side effects of the particular compound; and in a manner compatible with concurrent treatment regimens. An appropriate amount and formulation for decreasing the severity of secondary tissue degeneration associated with CNS injury in humans can be extrapolated from credible animal models known in the art of the particular disorder. It is understood, that the dosage of a neutralizing agent specific for CXCL10 has to be adjusted based on the binding affinity of the neutralizing agent for CXCL10, such that a lower dose of a neutralizing agent exhibiting significantly higher binding affinity can be administered compared to the dosage necessary for a neutralizing agent with lower binding affinity. Thus, appropriate dosage will vary with the particular treatment and with the duration of desired treatment;
,however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. A therapeutically effective amount is typically an amount of a neutralizing agent that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 ug/ml to about 100 pg/ml, from about 1.0 pg/ml to about 50 pg/ml, or at least about 2 ~Zg/ml and usually 5 to 10 ug/ml.

It is contemplated that the method of the invention of reducing the severity of secondary tissue degeneration associated with CNS injury in a subject, comprising administering to a subject having secondary tissue degeneration associated with CNS injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10) can further comprise a time-course regimen consisting of repeated treatments starting at the time of injury or trauma. Since secondary tissue degeneration associated with CNS injury is a progressive process that ensues over the days or early weeks post injury, administration of a CXCL10 neutralizing agent can be initiated immediately or as soon as feasible following injury and repeated daily for an appropriate length of time. An appropriate length of time can be determined on a variety of factors known to those skilled in the art and can be, for example, 1, 2, 3, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more days post injury. Treatments also can be administered every second or third day as well as two, three or more times per day of treatment. For example, treatments can be continued for the amount of time that CXCL10 levels are upregulated above their base level following CNS injury or trauma (see Lee et al. Neurochemistry International 36:417-425 (2000), which is incorporated herein by reference). Thus, a time course regimen is useful for practicing the claimed methods because a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10) can be administered starting immediately following the CNS injury, for example, within about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 or more hours and repeated throughout the time period that has been determined to correlate with upregulation of CXCL10 following a CNS injury.
The total amount of neutralizing agent can be 5 administered as a single dose or by infusion over a relatively short period of time, or can be administered in multiple doses administered over a more prolonged period of time. Such considerations will depend on a variety of case-specific factors such as, for example, 10 whether the disease category is characterized by acute episodes or gradual tissue deterioration. For example, for a subject affected with chronic tissue deterioration the neutralizing agent can be administered in a slow-release matrice, which can be 15 implanted for systemic delivery or at the site of the target tissue. Contemplated matrices useful for controlled release of therapeutic compounds are well known in the art, and include materials such as DepoFoamTM, biopolymers, micropumps, and the like.
20 The neutralizing agents of the invention can be administered to the subject by any number of routes known in the art including, for example, systemically, such as intravenously or intraarterially. A CXCL10-specific neutralizing agent can be provided in the form 25 of isolated and substantially purified polypetides and polypeptide fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes, including for 30 example, topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) routes. Intraspinal and intravenous administration of a CXCL10-specific neutralizing agent are particularly suitable routes for practicing the methods of the invention. Intraspinal administration can be performed so as to target the delivery of the neutralizing agent to the site of injury or trauma. In addition, a CXCL10-specific neutralizing agent variant can be incorporated into biodegradable polymers allowing for sustained release of the compound useful for reducing the severity of secondary tissue degeneration associated with CNS injury. Biodegradable polymers and their use are described, for example, in Brem et al., J. Neurosura. 74:441-446 (1991), which is incorporated herein by reference.
A CXCL10-specific neutralizing agent can be administered as a solution or suspension together with a pharmaceutically acceptable medium. Such a pharmaceutically acceptable medium can be, for example, sterile aqueous solvents such as sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution or other physiologically buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable medium can additionally contain physiologically acceptable compounds that act, for example, stabilize the neutralizing agent, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans;
antioxidants such as ascorbic acid or glutathione;
receptor mediated permeabilizers, which can be used to increase permeability of the blood-brain barrier;

chelating agents such as EDTA, which disrupts microbial membranes; divalent metal ions such as calcium or magnesium; low molecular weight proteins; lipids or liposomes; or other stabilizers or excipients. Those skilled in the art understand that the choice of a pharmaceutically acceptable carrier depends on the route of administration of the compound containing the neutralizing agent and on its particular physical and chemical characteristics.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions such as the pharmaceutically acceptable mediums described above. The solutions can additionally contain, for example, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
Other formulations include, for example, aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a lyophilized condition requiring, for example, the addition of the sterile liquid carrier, immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
For applications directed to brain injury that a CXCL10 neutralizing agent can be administered in a formulation that can cross the blood-brain barrier, for example, a formulation that increases the lipophilicity of the CXCL10 neutralizing agent. For example, the neutralizing agent can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols.
I to III, 2nd ed. (CRC Press, Boca Raton FL (1993)).
Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
A neutralizing agent specific for CXCL10 can also be prepared as nanoparticles. Adsorbing peptide compounds onto the surface of nanoparticles has proven effective in delivering peptide drugs to the brain (see Kreuter et al., Brain Res. 674:171-174 (1995)).
Exemplary nanoparticles are colloidal polymer particles of poly-butylcyanoacrylate with a neutralizing agent specific for~CXCL10 adsorbed onto the surface and then coated with polysorbate 80.
Image-guided ultrasound delivery of a CXCL10 neutralizing agent through the blood-brain barrier to selected locations in the brain can be utilized as described in U.S. Patent No. 5,752,515. Briefly, to deliver a CXCL10 neutralizing agent past the blood-brain barrier a selected location in the brain is targeted and ultrasound used to induce a change detectable by imaging in the CNS (CNS) tissues and/or fluids at that location. At least a portion of the brain in the vicinity of the selected location is imaged, for example, via magnetic resonance imaging (MRI), to confirm the location of the change. A
CXCL10-specific neutralizing in the patient's bloodstream can delivered to the confirmed location by applying ultrasound to effect opening of the blood-brain barrier at that location and, thereby, to induce uptake of the neutralizing agent.
In addition, polypeptides called receptor mediated permeabilizers (RMP) can be used to increase the permeability of the blood-brain barrier to molecules such as therapeutic agents or diagnostic agents as described in U.S. Patent Nos. 5,268,164;
5,506,206; and 5,686,416. These receptor mediated permeabilizers can be intravenously co-administered to a host with molecules whose desired destination is the cerebrospinal fluid compartment of the brain. The permeabilizer polypeptides or conformational analogues thereof allow therapeutic agents to penetrate the blood-brain barrier and arrive at their target destination.
In accordance with another embodiment of the present invention, there are provided therapeutic systems, preferably in kit form, containing a CXCL 10 neutralizing agent and instructions for its use in methods of reducing the severity of secondary tissue degeneration associated with central nervous system injury. In one embodiment, for example, the therapeutic agent is an anti-CXCL10 antibody.
Invention kits are useful for reversing the severity of secondary tissue degeneration associated with central nervous system injury.
A suitable kit includes at least one CXCL10 neutralizing agent, as a separately packaged chemical reagents) in an amount sufficient for at least one therapeutic application. Instructions for use of the packaged kit are also typically included. Those of skill in the art can readily incorporate a CXCL10 neutralizing agent into kit form in combination with appropriate buffers and solutions for the practice of 5 the invention methods as described herein.
In current treatment regimes for CNS injury, more than one compound is often administered to an individual for management of the same or different aspects of the disease. Similarly, in the methods of 10 the invention involving decreasing the rate of secondary tissue degeneration, a neutralizing agent specific for CXCL10 can advantageously be formulated with a second therapeutic compound such as an anti-inflammatory compound, immunosuppressive compound or 15 any other compound that manages the same or different aspects of the disease. Such compounds include, for example, methylprednisolone acetate, CM-1 ganglioside and 4-aminopyridine (4-AP). Contemplated methods of reducing the severity of secondary tissue degeneration 20 associated with CNS injury include administering a neutralizing agent specific for CXCL10 alone, in combination with, or in sequence with, such other compounds as well as in combination with other therapies, for example, rehabilitation and neural 25 prostheses. Alternatively, combination therapies can consist of fusion proteins, where the neutralizing agent specific for CXCL10 is linked to a heterologous protein, such as a therapeutic protein.
The following examples are intended to 30 illustrate but not limit the present invention. It is understood that modifications which do not substantially affect the activity of the various ~1 embodiments of this invention are also included within the definition of the invention provided herein.
Accordingly, the following examples are intended to illustrate but not limit the present invention.
Unless described separately below, the experimental procedures corresponding to the Examples set forth below are as follows:
Antibody administration. Age-matched adult female C57/BL6 mice were used for all studies. Mice received intraperitoneal injections of 100mg of monoclonal CXCL 10 antibody (suspended in 200m1 of sterile PBS) 1 day prior to injury, and every other day thereafter until sacrifice. The monoclonal antibodies used in these studies are specific for CXCL 10 and block CXCL 10-induced T cell chemotaxis.
Spinal cord injury. Spinal cord dorsal hemisection lesions were performed under Avertin anesthesia (0.6m1/20g) administered via intraperitoneal injection. A midline incision was made over the spinous processes of T4-L2 and the paravertebral muscles were separated from the vertebrae. A complete dorsal laminectomy of the T9 vertebrae was performed using fine scissors and rongeurs. The column was stabilized with a clamp attached to a micromanipulator.
A microlesion knife was then be used to produce a dorsal hemisection injury. Muscle layers were then sutured and the superficial tissue and skin closed with 4-0 silk. Body temperature was maintained on a heating pad. Postoperative care included bladder voiding and hydration with lactated Ringer's solution.

Kinematic Analysis. All mice were acclimated to a linear running arena for 4 days prior to the onset of functional testing. Kinematic analysis was conducted one day prior to injury, and every day thereafter until sacrifice at approximately midday, and scored independently by two observers blinded to the treatment group. Animals were videotaped using a Hitachi 8mm Video Camcorder VM-E555LA from underneath a plexiglass surface bearing defined lcm grid lines. The videos were then downloaded using an MPEG-2 compression devise, and analyzed using FMV 2.0 software, which allowed the video to be viewed frame by frame for kinematic assessment. Four kinematic parameters were assayed; stride length, stride width, paw rotation and toe spread. Rear paw stride length was defined as the point from which the start of a step with the rear paw through to the end of a step with the same paw (measurements taken on individual sides and for three consecutive steps in mms which were subsequently averaged). Stride width was defined as the width in mms of the hind limb strides (distance from the left outermost lateral hind paw digit to the right outermost contralateral hind paw digit). Toe spread was defined as the width in mms in the hind paw from the most lateral point of the lateral digit to the most medial point of the medial digit (both right and left hind paw respectively). Paw rotation was defined as the angle between the longitudinal axis of the rear paws and the midline axis of the body in degrees. The SPSS 4.0 T-test was used to determine significant differences between treated and untreated groups.
RNase protection assay. Mice were killed at the following post-injury time points by C02 fixation and decapitation: 6hr (n=3), l2hr (n=3), l8hr (n=3), 24hr (n=3), 3days (n=3), 7days (n=3), and l4days (n=2).
Two non-injured mice were used as controls. The spinal cord was removed in two pieces and immediately frozen.
Total RNA was extracted with Trizol reagent (Gibco) and ethanol precipitated. The pellets were dissolved in 50u1 RNase-free water and the RNA concentration was determined. The multiprobe set mCK-5 was used to detect CXCL 10 mRNA transcripts. This included a probe for L32 which was used to as a control for RNA loading.
Analysis was performed on l0ug protein. Fragments were separated by polyacrylamide gel electrophoresis, and visualized by film autoradiography. Autoradiographs were scanned and imaging software was used to quantify the bands.
Mononuclear cell isolation and flow cytometry. A single cell suspension was obtained from spinal cords of mice treated with either anti-CXCL10, or control antibody at days 3 and 14 post-injury. FACS
analysis was performed as previously described (Lane, Liu et al., "A central role for CD4(+) T cells and RANTES in virus-induced central nervous sytem inflammation and demyelination," J Virol, (2000) 74(3):1415-24). Briefly, brains were removed and a single cell suspension was obtained by grinding the tissue. All techniques were performed within sterile tissue culture plates on ice; the plates contained Dulbecco modified Eagle medium supplemented with 10°s fetal bovine serum. Cell suspensions were transferred to 15 ml conical tubes and Percoll (Pharmacia, Uppsala, Sweden) was added for a final concentration of 30%.
One milliliter of 70°s Percoll was underlaid and the cells were spun at 1300 x g for 30 min at 4oC. Cells were removed from the interface and washed twice.
Fluorescein isothiocyanate-conjugated (FITC) rat anti-mouse CD4 and (Phycoerythrin-conjugated PE) rat anti-mouse CD8 (Pharmingen, San piego, CA) were used to detect infiltrating CD4+ and CD8+ T cells respectively. As a control isotype-matched FITC and PE antibody was used. Cells were incubated with antibodies for 1 hour at 40°C, washed, fixed in to paraformaldehyde, and analyzed on a FACStar (Becton Dickinson, Mountain View, Calif.). Percent of cells infiltrating into the CNS were determined on Cell quest flow cytometric software. Positive cells were determined by subtracting experimental sample fluorescence from background and control samples.
Total numbers of CD4 and CD8 T cells were determined by multiplying the percentage of positive cells within the gated population by the number of cells isolated from the spinal cord. Data is presented as the mean + SEM.
H&E Stamina. Longitudinal frozen spinal cord sections were cut and fixed in acetic alcohol.
The sections were stained in Harris' Hematoxylin, washed with tap water, and counterstained in 1°s eosin.
Slides were dehydrated in ascending alcohols and mounted with permount (Fisher Scientific).
Immunohistochemistry. Animals were transcardially perfused with 4°s paraformaldehyde 24 hrs, 3 days, and 14 days post-injury. The spinal cords were removed and sunk in 25% sucrose overnight. The tissue was then embedded in OCT and l2um thick longitudinal cryosections were cut and placed onto slides. Sections were blocked in 10% normal goat serum (NGS; diluted with PBS) for lhr at room temperature.
Primary antisera (rat anti-mouse CD4 monoclonal ab, 1:200 dilution in 10% NGS, PharMingen; rat anti-mouse CDllb, Serotec) were applied to sections overnight at 4°C. Sections were rinsed three times with PBS and goat anti-rat IgG biotinylated antisera (1:200 dilution in 10~ NGS, Vector Laboratories) were applied and 5 slides were incubated lhr at room temperature.
Sections were rinsed three times in PBS and incubated in a methanol: 30o hydrogen peroxide solution (100:1) for l0min. The sections were washed three times in PBS
and incubated in ABC solution (Vector Laboratories) for 10 30min at room temperature. DAB substrate solution (Vector Laboratories) was used to visualize the binding of the antibodies. Sections were dehydrated in ascending alcohols. Permount (Fisher Scientific) was used for mounting. No immunoreactivity was seen when 15 the primary antibody was omitted. The same procedure was used to stain for neurons with the following modifications: The sections were blocked overnight and the primary antisera used were mouse anti-NeuN
monoclonal (Chemicon, 1:100 dilution). The secondary 20 antibody used was biotinylated rat adsorbed horse anti-mouse at (Vector Laboratories, 1:200 dilution).
Neurons and CD4 positive T cells within one millimeter of each side of the injury were counted at a lOX
magnification. Only clearly labeled cells were 25 counted.
EXAMPLE I
Upreaulation of CXCL10 Levels and T Cell Numbers following CNS In'~ury This example describes upregulation of CXCL10 30 levels and T cell numbers following CNS injury.

To determine whether CXCL10 mRNA levels were increased after hemisection injury, hemisection injuries of adult mouse spinal cords were performed on adult C57B16 female mice using a pointed scalpel blade at T9, following removal of the dorsal half of the T9 vertebrae. Total RNA was extracted from hemisectioned spinal cords using TRIzol~ reagent (Life Technologies (GIBCO-BRL) Rockville, MD) at 6 hrs, 12 hrs, 18 hrs, 24 hrs, 3 days, 7 days and 14 days post injury, and the level of IP-10 mRNA transcripts was determined for each time point by ribonuclease protection assay (RPA) using the CK-1 chemokine probe set previously described in Lane et al., J. Virol. 74(3):415-424 (2000), which is incorporated herein by reference. The abundance of mRNA transcripts was determined by scanning autoradiographs to determine the density of individual bands as it related to internal L32 controls using NIH
1.61 image software.
As shown in Figure 1, CXCL10 mRNA levels increased by 6 hours post-injury and then gradually declined, remaining above basal levels even after 14 days post-injury. CXCL10 mRNA was undetectable in uninjured spinal cord tissue.
Spinal cords also were dissected 3 and 14 days post-injury such that the injury site, which was marked at surgery, was centrally located within the dissected tissue. Longitudinal sections in which the central canal was clearly visible were selected and digitalized. For cell counts, the number of CD4 immunopositive cells within the total tissue area extending one millimeter either side of the injury site was determined using stereology. Only immunolabeled cells with a clearly discernable Hoechst stained nucleus were scored.
Quantitative analysis of CD4 immunostained longitudinal tissue sections indicated that CD4+ T
cells were present at an increased density within the region extending 1 mm either side of the injury site at 3 days post-injury in hemisection injured spinal cord, as compared to uninjured spinal cord. The above studies indicate that IP-10 levels and T cell numbers are upregulated after CNS injury.
EXAMPLE II
Neutralization of CXCL10 Levels Diminished T Cell Accumulation within and around the In'Ly Site following CNS Iniury To determine the effect of anti-IP-10 treatment following dorsal hemisection injury, hemisection-injured adult mice received 0.5 ml intraperitoneal injections of anti-IP-10 antibodies (approximately 0.5mg/ml) every other day starting at 1 day prior to injury and continuing until 7 days post-injury. The mice were sacrificed at 3 days or 14 days post-injury and the spinal cords were dissected such that the injury site was centrally located within the dissected tissue. Longitudinal sections in which the central canal was clearly visible were selected and digitalized. For cell counts, the number of CD4 immunopositive cells within the total tissue area extending one millimeter either side of the injury site was determined using stereology. As described in Example I above, only immunolabeled cells with a clearly discernable Hoechst stained nucleus were scored.
Quantitative analysis of CD4 immunostained longitudinal tissue sections indicated that CD4+ T
cells were present at an increased density within the region extending 1 mm either side of the injury site at 3 days post-injury in untreated hemisection injured spinal cord, as compared to anti-CXCL10 treated hemisection injured spinal cord. These studies indicate that neutralization of IP-10 decreased T cell recruitment following CNS injury.
To determine the effect anti-IP 10 treatment on behavioral deficits associated with CNS injury, anti-CXCL10 antibody treated hemisection-injured mice, untreated hemisection-injured mice and uninjured control mice were first acclimated for 4 days prior to hemisection injury and subsequently subjected to daily kinematic analyses from 1 to 13 days following hemisection injury by two observers blinded to the treatment group. Animals were videotaped from underneath a 4'x4' plexiglass surface bearing defined grid lines, and the recording analyzed using Adobe Premiere video editing software (Adobe Systems, Inc., San Jose, CA).
Four kinematic parameters were assessed:
stride length, stride width, paw rotation and toe spread. As shown in Figure 2, all hemisection-injured mice treated with anti-IP-10 antibody had significantly greater stride length, and significantly less stride width, toe spread and paw rotation, then untreated hemisection-injured mice. Progressive behavioral improvement was assessed by comparing, for each kinematic parameter, the data points for all animals over the first three days post-injury with the data points for all animals over the last three days post-injury. Untreated hemisection-injured mice showed no change in kinematic parameters during the recovery period (p>0.05). In contrast, treated hemisection-injured mice showed a statistically significant progressive improvement in all kinematic parameters during the recovery period (p« 0.01).
Spinal cords were dissected 14 days post-injury such that the injury site was centrally located within the dissected tissue. Longitudinal sections in which the central canal was clearly visible were selected and digitalized. The total tissue area extending one millimeter either side of the injury site was measured using an MCID analysis system as described in Zhang et al., J. Comp. Neurol. 371:485-495(1996), which is incorporated herein by reference.
As shown in Figure 3, untreated hemisection-injured mice had on average a 49.4%
reduction in total tissue area extending 1 mm either side of the injury, as compared to uninjured control spinal cords. In contrast, morphometric analyses of anti-IP-10 antibody treated hemisection-injured mice indicated a 20.90 reduction in total tissue area extending lmm on either side of the injury compared to uninjured control spinal cords, which represents a 68°s reduction in tissue loss compared to untreated hemisection-injured mice. The number of NeuN

immunopositive neurons was determined within the total tissue area extending one millimeter either side of the injury site was determined using stereology. The NeuN
monoclonal antibody (MAB377, Chemicon, Temecula, CA), 5 which recognizes neuron-specific nuclear protein and reacts with most neuronal cell types was used according to Manufacturer's instructions. Only immunolabeled cells with a clearly discernable Hoechst-stained nucleus were scored. Quantitative analysis of NeuN
10 immunostained longitudinal tissue sections indicated that NeuN+ neurons were present at an increased density within the region extending 1 mm either side of the injury site in anti-CXCL1010 treated hemisection-injured mice, as compared to untreated 15 hemisection-injured mice.
These data indicate that anti-CXCL10 treatment decreased posttraumatic tissue loss following hemisection injury.
EXAMPLE III
20 Effect of CXCL10 Antibodies on Lymphocyte Infiltration CXCL10 mRNA levels were increased after hemisection injury to the adult mouse spinal cord.
Total RNA was extracted from hemisectioned spinal cords at 6 hrs (n=3), 12 hrs (n=3), 18 hrs (n=3), 24 hrs 25 (n=3), 3 days (n=3), 7 days (n=3) and 14 days (n=3) after injury, and the level of CXCL10 mRNA transcripts was determined for each time point by ribonuclease protection assay (RPA) using the CK-1 chemokine probe set (Lane, Liu et al . , ~~A central role for CD4 (+) T
30 cells and R,ANTES in virus-induced central nervous system inflammation and demyelination," J. Virol., (2000) 74 (3) :1415-24) . The abundance of mRNA
transcripts was determined by scanning autoradiographs to determine the density of individual bands as it related to internal L32 controls. CXCL10 mRNA levels increased by 6 hours post-injury to an average level of 74.8 +/- 21.78 and then gradually declined, remaining above basal levels even after 14 days past-injury with an average level of 41.57 +/- 1.80 (Figure 1). CXCL 10 mRNA was undetectable in uninjured spinal cord tissue.
Treatment with anti-CXCL10 reduced lymphocyte and activated macrophage infiltration into the CNS
following injury. In hemisectioned adult mice, quantitative analyses of CD4 immunostained longitudinal tissue sections indicated that the total number of CD4+
T lymphocytes within the region extending lmm either side of the injury site at 3 days post-injury was 2050 +/- 102 (n=3; Figure 5). CD4+ T lymphocytes were not detected in uninjured spinal cord tissue. These data indicate that CXCL 10 levels and T lymphocyte numbers are upregulated after traumatic injury and support previous studies demonstrating an upregulation of CXCL10 6 hours after contusion injury to adult rats, and a peak of T lymphocyte infiltration within the first week post-injury (McTigue, Tani et al.,"Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury," J. Neurosci. Res., (1998) 53 (3) :368-76) .
In hemisectioned adult mice that received anti-CXCL10 treatment, quantitative analyses of CD4 immunostained longitudinal tissue sections indicated that the total number of CD4+ T lymphocytes within the region extending lmm either side of the injury site at 3 days post-injury was 622 +/- 202 (n=3), representing a 70% decrease relative to untreated hemisection-injured animals (Figure 5).
FACS analysis indicated that the number of CD4+ lymphocytes, CD8+ lymphocytes, and F480/CD45+
macrophages were decreased at both 3 days and 14 days after injury in anti-CXCL10 treated hemisection-injured mice as compared to untreated hemisection-injured mice.
In particular, FACS data summarized in Table 1 below demonstrated a significant reduction in CD4+ T
lymphocytes (66.4°s reduction), CD8+ T lymphocytes (57.4% reduction) and activated macrophage/microglia cells (35.5°s reduction) present within the spinal cords of these animals at day 3 post-injury as compared with T lymphocyte and activated macrophage/microglia levels in untreated hemisection-injured mice. In addition, FACS data demonstrated a significant reduction in CD4+
T lymphocytes (58% reduction), CD8+ T lymphocytes (640 reduction) and activated macrophage/microglia cells (43.2% reduction) present within the spinal cords of treated animals at day 14 post-injury as compared with T lymphocyte and activated macrophage/microglia levels in untreated hemisection-injured mice. (n=4; Table 1).
Table 1. Cellular Infiltration is Reduced in anti-CXCL10 treated Mice.
Number infiltrating cells in of spinalcord Treatment n Days CD4 CD8 F480/CD45 p.i.

Anti-CXCL10 4 3 3.7 103 2.6 103 2.0 105 x x x Anti-CXCL10 4 14 4.2 10' 2.7 103 2.5 105 x x x No treatment 4 3 1.1 10' 6.1 103 3.1 105 x x x No treatment 4 14 1.0 104 7.5 10' 4.4 105 x x x EXAMPLE IV
Effect of CXCL10 Treatment on Tissue Sparing and Function Deficit In order to determine whether an attenuated CD4+ T lymphocyte response to traumatic spinal cord injury was associated with tissue sparing or a decrease in functional deficit, hemisectioned adult mice received intraperitoneal injections of anti-CXCL10 antibodies every other day from 1 day prior to injury to 9 days post-injury and were sacrificed 14 days after injury (n=11). Tissue sparing after hemisection injury was significantly greater in mice treated with anti-CXCL10 antibody as compared to untreated hemisection-injured mice (n=8). Morphometric analyses of longitudinal tissue sections in which the central canal was clearly visible were conducted using the MCID
analysis system, as described in Zhang et al., (1996) (Zhang, Fujiki et al., "Genetic influences on cellular reactions to spinal cord injury: a wound-healing response present in normal mice is impaired in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration," J. Comp. Neurol., (1996) 371(3):485-95). Untreated hemisection-injured mice had on average a 49.4% reduction in total tissue area extending lmm either side of the injury, as compared to uninjured control spinal cords (Figure 3). In contrast, morphometric analyses of anti-CXCL10 antibody treated hemisection-injured mice indicated a 20.9%
reduction in total tissue area extending lmm either side of the injury compared to uninjured control spinal cords, which represents a 68% reduction in tissue loss compared to untreated hemisection-injured mice (Figure 3) .

Consistent with greater tissue sparing, treated hemisection-injured mice contained significantly more neurons around the injury site at 14 days post-injury than untreated hemisection-injured mice. Quantitative analyses of NeuN immunostained longitudinal tissue sections from untreated hemisection-injured mice indicated that the averaged total number of NeuN+ neurons within the region extending lmm either side of the injury site at 14 days post-injury was 267 +/- 72. In contrast, quantitative analysis of NeuN immunostained longitudinal tissue sections from anti-CXCL10 antibody treated hemisection-injured mice indicated that the averaged total number of NeuN+ neurons within the region extending lmm either side of the injury site at 14 days post-injury was 1170 +/- 184, representing 438°s more neurons than the untreated hemisection-injured animals.
These data indicate that anti-CXCL 10 treatment significantly decreased posttraumatic tissue loss following dorsal hemisection injury.
EXAMPLE V
Effect of Anti-CXCL10 Antibodv Treatment on Behavioral Deficit Behavioral deficit following hemisection injury progressively lessened in mice treated with anti-CXCL10 antibody as compared to untreated control mice. Treated and untreated hemisection-injured mice, as well as uninjured control mice (n=8), were subject to daily kinematic analyses from 1-13 days following hemisection injury, by two observers blinded to the treatment group. Animals were videotaped from underneath a 3'x 1' plexiglass surface bearing defined 1cm grid lines, and the recording analyzed using video editing software. Four kinematic parameters were 5 assessed; rear paw stride length, rear paw stride width, rear paw rotation and rear paw toe spread.
These analyses indicated that all hemisection-injured mice treated with anti-CXCL10 antibody had significantly greater rear paw stride length, and 10 significantly less rear paw stride width, rear paw toe spread and rear paw rotation, then untreated hemisection-injured control mice at 14 days post-injury (Figure 2). Progressive behavioral improvement was assessed by comparing the data points for all animals 15 over the first three days post-injury with the data points for all animals over the last three days post-injury, for each kinematic parameter. Untreated hemisection-injured mice showed no change in kinematic parameters during the recovery period (p>0.05). In 20 contrast, treated hemisection-injured mice showed a statistically significant progressive improvement in all kinematic parameters during the recovery period (p<0.01). By 14 days post-injury, the behavioral scores for all 4 kinematic parameters of treated 25 hemisection-injured mice were not significantly different from uninjured control mice.
These data indicate that anti-CXCL10 antibody treatment reduces neurological impairment following spinal cord injury.
30 Throughout this application various publications have been referenced within parentheses.
The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made withQUt departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (28)

We claim:

1. A method of reducing the severity of secondary tissue degeneration associated with central nervous system injury in a subject, comprising administering to a subject having secondary tissue degeneration associated with central nervous system injury an effective amount of a neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10).

2. The method of claim 1 wherein said subject is a mammal.

3. The method of claim 2 wherein said subject is human.

4. The method of claim 1 wherein said central nervous system injury is spinal cord injury.

5. The method of claim 1 wherein said central nervous system injury is brain injury.

6. The method of claim 1 wherein said neutralizing agent specific for interferon inducible protein of 10 kDa (CXCL10) is anti-CXCL10 antibody.

7. A method of reducing the severity of secondary tissue degeneration associated with central nervous system injury in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or fragment thereof capable of binding specifically to interferon inducible protein of kDa (CXCL10).

8. The method of claim 7, wherein said central nervous system injury is selected from the group consisting of a mechanical injury, bruising of the spinal cord, a compression injury of the spinal cord, laceration of the spinal cord, severance of the spinal cord, and a demyelinating condition.

9. The method of claim 8, wherein said demyelinating condition is multiple sclerosis.

10. A method of reducing the severity of secondary tissue degeneration associated with pathological central nervous system condition in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or fragment thereof capable of binding specifically to interferon inducible protein of 10 kDa (CXCL10).

11. The method of claim 10, wherein said pathological condition is myelin loss.

12. The method of claim 11, wherein said myelin loss is selected from the group consisting of acute disseminated encephalomyelitis, post-infectious myelin loss, post-vaccinal myelin loss, acute necrotizing encephalomyelitis, and progressive necrotizing myelopathy.

13. The method of claim 1, wherein said neutralizing agent is administered within one hour of said injury.

14. The method of claim 7, wherein said antibody or fragment thereof is administered within one hour of said injury.

15. The method of claim 10, wherein said antibody or fragment thereof is administered within one day of diagnosis of said condition, and said administration is repeated daily for up to 25 additional days.

16. The method of claim 1, wherein said neutralizing agent is administered daily for at least 25 days after injury.

17. The method of claim 7, wherein said antibody or fragment thereof is administered daily for at least 25 days after injury.

18. A method of reducing the severity of secondary tissue degeneration associated with central nervous system injury in a subject, comprising administering to a subject in need thereof an effective amount of a polynucleotide agent capable of reducing the amount of interferon inducible protein of 10 kDa (CXCL10) in a cell.

19. The method of claim 18, wherein said agent is selected from the group consisting of an antisense oligonucleotide and a ribozyme, wherein said antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.

20. A method of reducing the severity of secondary tissue degeneration associated with pathological central nervous system condition in a subject, comprising administering to a subject in need thereof an effective amount of a polynucleotide agent capable of reducing the amount of interferon inducible protein of 10 kDa (CXCL10) in a cell.

21. The method of claim 20, wherein said agent is selected from the group consisting of an antisense oligonucleotide and a ribozyme, wherein said antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.

22. The method of claim 1, wherein said agent is administered in a composition comprising liposomes capable of enhancing penetration of the blood brain barrier by said agent.

23. The method of claim 7, wherein said antibody or fragment thereof is administered in a composition comprising liposomes capable of enhancing penetration of the blood brain barrier by said antibody or fragment thereof.

24. The method of claim 10, wherein said antibody or fragment thereof is administered in a composition comprising liposomes capable of enhancing penetration of the blood brain barrier by said antibody or fragment thereof.

25. The method of claim 18, wherein said antibody or fragment thereof is administered in a composition comprising liposomes capable of enhancing penetration of the blood brain barrier by said antibody or fragment thereof.

26. The method of claim 20, wherein said antibody or fragment thereof is administered in a composition comprising liposomes capable of enhancing penetration of the blood brain barrier by said antibody or fragment thereof.

27. A composition for use in reducing the severity of secondary tissue degeneration associated with central nervous system injury, comprising a CXCL
neutralizing agent and a physiologically acceptable carrier.

28. A kit comprising a CXCL 10 neutralizing agent and instructions for its use in methods of reducing the severity of secondary tissue degeneration associated with central nervous system injury.