JP2005501036A - CXCL10 treatment of secondary tissue degeneration - Google Patents

Abstract

The present invention relates to a subject having secondary tissue degeneration associated with CNS injury by administering a therapeutically effective amount of a neutralizing agent specific for a 10 kDa interferon-inducible protein (CXCL10) in a subject. Methods are provided for reducing severe secondary tissue degeneration associated with central nervous system (CNS) damage. The present invention relates to immunology and more specifically to the treatment of secondary tissue degeneration associated with central nervous system injury by neutralization of the chemokine CXCL10.

Description

（実施例ＩＶ）
（組織スペアリングおよび機能欠失に対するＣＸＣＬ１０処置の効果）
外傷性の脊髄創傷に応答して減弱したＣＤ４＋Ｔリンパ球が、組織スペアリングまたは機能的欠失の減少に関与したか否かを決定するために、ヘミセクションした成体マウスに、抗ＣＸＣＬ１０抗体を、創傷１日前から創傷９日後まで１日おきに腹腔内注射し、そして創傷１４日後に屠殺した（ｎ＝１１）。ヘミセクション創傷後の組織スペアリングは、処置しなかったヘミセクション創傷マウスと比較して、抗ＣＸＣＬ１０抗体で処置したマウスで、有意により大きかった（ｎ＝８）。中心管が明瞭に見える縦方向の組織切片の形態学的分析を、Ｚｈａｎｇら、（１９９６）（Ｚｈａｎｇ，Ｆｕｊｉｋｉら，「Ｇｅｎｅｔｉｃ ｉｎｆｌｕｅｎｃｅ 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）に記載されるようなＭＣＩＤ分析システムを用いて行った。処置していないヘミセクション創傷マウスは、創傷していないコントロール脊髄と比較して、創傷のいずれか一方の側が１ｍｍ拡張している組織領域全体で平均４９．４％の減少を有した（図３）。対照的に、抗ＣＸＣＬ１０抗体処置したヘミセクション創傷マウスの形態計測分析は、創傷していないコントロール脊髄と比較して、創傷のいずれか一方の側が１ｍｍ拡張している組織領域全体で２０．９％の減少を示し、組織の喪失においては、処置していないヘミセクション創傷マウスと比較して、６８％の減少を示す（図３）。
【０１１９】
より大きな組織スペアリングと合わせて、処置したヘミセクション創傷マウスは、創傷１４日後に、処置していないヘミセクション創傷マウスより、創傷部位周囲のより多くの神経細胞を有意に含む。処置していないヘミセクション創傷マウス由来の、縦方向の組織切片を免疫染色したＮｅｕＮの定量分析は、創傷１４日後において、創傷部位のいずれか一方の側が１ｍｍ拡張している領域内のＮｅｕＮ＋ニューロンの平均総数が、２６７＋／−７２であることを示した。対称的に、抗ＣＸＣＬ１０抗体で処置したヘミセクション創傷マウスからの縦方向の組織切片を免疫染色したＮｅｕＮの定量分析は、創傷１４日後において、創傷部位のいずれか一方の側が１ｍｍ拡張している領域内のＮｅｕＮ＋ニューロンの平均総数が、１１７０＋／−１８４であることを示し、処置していないヘミセクション創傷動物よりも４３８％多くのニューロンが存在している。
【０１２０】
これらのデータは、背面のヘミセクション創傷の後の抗ＣＸＣＬ１０処置が、外傷後の組織の消失を有意に減少させたことを示す。
【０１２１】
（実施例Ｖ）
（挙動性欠損に対する抗ＣＸＣＬ１０抗体処置の効果）
抗ＣＸＣＬ１０抗体で処置したマウスにおけるヘミセクション創傷後の挙動欠損は、処置しなかったコントロールと比較して、漸進的に減少した。処置したヘミセクション創傷マウスおよび処置しなかったヘミセクション創傷マウス、ならびに非創傷コントロールマウス（ｎ＝８）を、ヘミセクション創傷１〜１３日後から、処置群について盲目である２人の観察者による毎日の運動学的分析に供した。動物を、規定の１ｃｍグリッド線を有する、３’×１’プレキシガラス表面の低部からビデオ撮影し、ビデオ編集ソフトウェアを用いて記録を分析した。４つの運動学的パラメータを評価した；前後肢のスライド長、前後肢のスライド、前後肢の回転および前後肢のつま先の広がり。これらの分析は、抗ＣＸＣＬ１０抗体で処置した全てのヘミセクション創傷マウスが、創傷１４日後における、処置しなかったヘミセクション創傷コントロールマウスよりも有意により大きな、前後肢のスライド長、有意に少ない前後肢のスライド、前後肢のつま先の広がりおよび前後肢の回転を有することを示した（図２）。進行性の挙動の改善を、創傷後の初めの３日間にわたる全ての動物についてデータ点を、創傷後の最後の３日間にわたる全ての動物についてのデータ点と、各々の運動学的パラメータについて比較することによって評価した。処置しなかったヘミセクション創傷マウスは、回復期間の間、運動学的パラメータにおいて、変化を示さなかった（ｐ＞０．０５）。対称的に、処置したヘミセクション創傷マウスは、回復期間の間、全ての運動学的パラメータにおいて、統計的に有意な進行性の改善を示した（ｐ＜０．０１）。創傷１４日後までに、処置したヘミセクション創傷マウスの４つの全ての挙動学的パラメータについて、挙動スコアは、非創傷コントロールマウスと有意に違わなかった。
【０１２２】
これらのデータは、抗ＣＸＣＬ１０抗体処置が、脊髄創傷後の神経学的障害を低減することを示す。
【０１２３】
本出願を通して、括弧内で、多様な刊行物を参照している。本出願において、本発明が関連する分野の水準、より完全に記載するために、これらの刊行物の開示の全体が、本明細書によって参考として援用される。
【０１２４】
本発明は、開示された実施形態を参照して記載されているが、当業者は、記述された特定の実験が、本発明の単なる例示であることを容易に理解する。本発明の精神を逸脱することなしに、多様な改変がなされ得ることを理解すべきである。従って、本発明は、添付の請求項によってのみ限定される。
【図面の簡単な説明】
【０１２５】
【図１】図１は、成体マウス脊髄に対する半側切断損傷の後の増加したＣＸＣＬ１０ ｍＲＮＡレベルを示す。
【図２−１】図２は、以下の４つの運動学的パラメーターにより測定される未処置コントロールマウスと比較して、抗ＣＸＣＬ１０抗体を用いて処置したマウスにおいて漸減した、成体マウス脊髄に対する半側切断損傷の後の挙動欠損を示す：２（Ａ）またぎ長（ｓｔｒｉｄｅ ｌｅｎｇｔｈ）；２（Ｂ）趾の広がり（ｔｏｅ ｓｐｒｅａｄ）；２（Ｃ）またぎ幅（ｓｔｒｉｄｅ ｗｉｄｔｈ）；および２（Ｄ）後足回転（ｒｅａｒ ｐａｗ ｒｏｔａｔｉｏｎ）。
【図２−２】図２は、以下の４つの運動学的パラメーターにより測定される未処置コントロールマウスと比較して、抗ＣＸＣＬ１０抗体を用いて処置したマウスにおいて漸減した、成体マウス脊髄に対する半側切断損傷の後の挙動欠損を示す：２（Ａ）またぎ長（ｓｔｒｉｄｅ ｌｅｎｇｔｈ）；２（Ｂ）趾の広がり（ｔｏｅ ｓｐｒｅａｄ）；２（Ｃ）またぎ幅（ｓｔｒｉｄｅ ｗｉｄｔｈ）；および２（Ｄ）後足回転（ｒｅａｒ ｐａｗ ｒｏｔａｔｉｏｎ）。
【図３】図３は、以下を示す：３（Ａ）未処置のマウスにおける損傷の１４日後の背面観点からの脊椎半側切断損傷の全体の病理；３（Ｂ）未処置のマウスにおける損傷の１４日後の脊椎の背側の半側切断損傷の長手方向の断面；３（Ｃ）抗ＣＸＣＬ１０抗体処置のマウスにおける損傷の１４日後の背面観点からの脊椎半側切断損傷の全体の病理；および３（Ｄ）抗ＣＸＣＬ１０抗体処置のマウスにおける、損傷の１４日後の脊椎の背側の半側切断損傷の長手方向の断面。外抗ＣＸＣＬ１０抗体に媒介された、傷性脊髄損傷に応答したＣＤ４＋ Ｔリンパ球の弱毒化は、組織節約（ｔｉｓｓｕｅ ｓｐａｒｉｎｇ）に関係する。
【図４】図４は、以下を実証するＣＤ４＋の染色された光学顕微鏡写真を示す：４（Ａ）抗ＣＸＣＬ１０抗体処置は、４（Ｂ）の未処置マウスと比較して、処置したマウスにおける半側切断損傷の後の、強力なＴリンパ球の漸増を低下する。
【図５】図５は、抗ＣＸＣＬ１０抗体により媒介される、外傷性脊椎損傷に対するＣＤ４＋ Ｔリンパ球応答の弱毒化は、組織節約に関係することを示す。【Technical field】
[0001]
This invention was made with government support under Contract No. NS37336-01 awarded by National Institutes of Health. The United States government has certain rights in the invention.
[0002]
The present invention relates to immunology and more specifically to the treatment of secondary tissue degeneration associated with central nervous system (CNS) injury by neutralization of the chemokine CXCL10.
[Background]
[0003]
(Background of the Invention)
The CNS consists of the brain and spinal cord, which form a continuous system containing nerve cells, support cells and nerve fibers. The brain is a cognitive organ with distinct regions and layers, each of which is associated with the reception and processing of specific stimuli received from sensory organs. The human brain is divided into three main areas, each of which has the following distinct functions: forebrain (prosthephalon), mid-brain (mesencephalon) And the hindbrain (rhombenephalon). The spinal cord is lined up with segments that have a higher segment that controls movement and sensation in the upper part of the body and a lower segment that controls the lower part of the body. The result of CNS damage is reflected in the organization of its constituent organs.
[0004]
The type of disorder relates to spinal cord injury that is highly dependent on the type and severity of the injury, the level of the spinal cord that develops the injury, and the pathway of the damaged nerve fiber. Severe damage to the spinal cord causes paralysis of the body part and complete loss of sensation controlled by the spinal cord segment below the point of injury. Spinal cord injury can also induce many complications, including bedsores and high susceptibility to respiratory diseases. Brain damage can induce a wide range of indicators of defects in different areas, including cognitive function, physical ability, communication, or social / behavioral function.
[0005]
Damage to the central nervous system does not stop immediately after the first injury, but lasts hours or days after the first trauma. These delayed damage processes present a good opportunity for treatment aimed at reducing the extent of damage resulting from brain and spinal cord damage. In the absence of trauma or disease, the major types of immune cells rarely enter the CNS. However, when the brain or spinal cord is damaged by trauma or disease, immune cells engulf the area, remove debris, and release powerful regulatory chemicals (both beneficial and harmful) to the host . Immune cell entry is associated with both acute CNS damage and associated secondary degeneration.
[0006]
The current standard healing treatment for acute spinal cord injury is high dose methylprednisolone (steroid), administered the first 3 hours after injury, and overall efficacy has recently been questioned. Symptomatic treatment of spinal cord injury can be achieved with 4-aminopyridine (4-AP), a blocker of potassium channels, extending the duration of neural action potential and improving transmission in demyelinated axons. However, none of these agents protects the complex pathological cascade associated with CNS trauma.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0007]
Thus, there is a need to have additional methods for treating central nervous system injury, particularly secondary tissue degeneration resulting from spinal cord injury and trauma to the CNS. The present invention satisfies this need and provides related advantages as well.
[Means for Solving the Problems]
[0008]
(Summary of the Invention)
The present invention relates to treatment of a neutralizing agent specific for a 10 kDa interferon-inducible protein (CXCL10) in subjects who have or are at risk of developing secondary tissue degeneration associated with CNS injury. A method of reducing severe secondary tissue degeneration associated with central nervous system (CNS) damage in a subject by administering an effective amount is provided. The methods of the present invention are useful for the treatment of both spinal cord injury and brain injury.
[0009]
In a further aspect, the present invention provides a method of reducing severe secondary tissue degeneration associated with CNS injury in a subject, the method comprising secondary tissue degeneration associated with CNS injury. A subject having an effective amount of a neutralizing agent specific for a 10 kDa interferon-inducible protein (CXCL10).
[0010]
In certain embodiments, the subject is a mammal. In other embodiments, the subject of the method is a human. In still further embodiments, the CNS injury is either spinal cord injury, brain injury, or both.
[0011]
In still further embodiments, the neutralizing agent is an anti-CXCL10 antibody, a fragment corresponding to an anti-CXCL10 antibody, a small molecule or polypeptide specific for CXCL10.
[0012]
A further aspect of the invention provides a method of reducing severe secondary tissue degeneration associated with CNS injury in a subject, which method is specific for a 10 kDa interferon-inducible protein (CXCL10). Administering an effective amount of an antibody or fragment thereof capable of binding to a subject in need of reduced severe secondary tissue degeneration.
[0013]
In certain embodiments of the invention, the CNS injury is caused or induced by mechanical injury, spinal cord contusion, spinal cord compression injury, spinal cord laceration, spinal cord disruption, or demyelinating condition. In one embodiment, the demyelinating condition is multiple sclerosis.
[0014]
Also provided by the present invention is a method of reducing severe secondary tissue degeneration associated with a pathological CNS condition in a subject, which method is specific for a 10 kDa interferon-inducible protein (CXCL10). Administering an effective amount of an antibody or fragment thereof capable of binding to a subject in need of reduced severe secondary tissue degeneration.
[0015]
In certain embodiments, the pathological condition is myelin loss. In other embodiments, the myelin loss is due to acute disseminated encephalomyelitis, post-infection myelin loss, post-vaccine myelin loss, acute necrotizing encephalomyelitis, or progressive necrotic myelopathy.
[0016]
In further embodiments, the neutralizing agent of the invention is administered within at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours of injury. In still further embodiments, the neutralizing agent is administered within 12 hours, 18 hours, or 24 hours of injury or diagnosis. In other embodiments, the neutralizing agent is an antibody or antibody fragment. In still further embodiments, the neutralizing agent is administered alone or in combination with an additional agent, such as an anti-inflammatory agent, and administration occurs repeatedly at least once, and in other embodiments, several days. In one embodiment, the number of days is up to 10 days, up to 15 days, up to 20 days, up to 25 days, or up to at least 30 days.
[0017]
Provided by the present invention is also a method of reducing the severity of secondary tissue degeneration associated with CNS injury in a subject, which method allows a subject in need thereof to be subcellular. Administering an effective amount of a polynucleotide agent that can reduce the amount of the 10 kDa interferon-inducing protein (CXCL10).
[0018]
In various embodiments, the polynucleotide agent can be an antisense oligonucleotide or ribozyme, wherein the antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.
[0019]
In yet a further aspect, the present invention provides a method of reducing the severity of secondary tissue degeneration associated with a pathological CNS condition in a subject, the method comprising a subject in need thereof And administering an effective amount of a polynucleotide agent capable of reducing the amount of 10 kDa interferon-inducing protein (CXCL10) in the cell.
[0020]
In certain embodiments, the agent can be an antisense oligonucleotide or ribozyme, wherein the antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10.
[0021]
In still further embodiments, the neutralizing agents of the invention can be administered in a composition (eg, a liposome) that can increase blood brain barrier penetration.
[0022]
(Detailed description of the invention)
The present invention relates to a method of treating and / or reducing CNS injury, including spinal cord injury and brain injury, by neutralizing 10 kDa interferon-inducible protein (CXCL10) with a specific neutralizing agent. The present invention also provides a method for reversing secondary tissue degeneration associated with CNS injury or trauma, as well as neutralizing 10 kDa interferon-inducible protein (CXCL10) with a specific neutralizing agent. Methods for promoting regeneration and remyelination are provided.
[0023]
Neutralization of CXCL10 presents a significant treatment option for CNS injury. This is because it reduces the extent of T cell infiltration, which is responsible for neurological degeneration associated with tissue loss following CNS injury or trauma. CXCL10 neutralization targets the cause rather than just the symptoms of secondary degeneration associated with CNS injury or trauma.
[0024]
The CNS includes nerve cells, or neurons, that are characterized by long nerve fibers called axons. Axons in the spinal cord carry signals along the descending pathway, downward from the brain, and upward along the ascending pathway. Many axons in these pathways are covered by a sheath of insulating material called myelin, which gives a whitish appearance; therefore, the areas where they are are called “white matter”. Nerve cells themselves have tree-like branches called dendrites that receive signals from other neurons, and constitute “grey matter”. The gray matter is in the butterfly area in the center of the spinal cord. Both the brain and spinal cord are surrounded by three membranes (meninges): the buffy coat, the innermost layer; the arachnoid membrane, the delicate intermediate layer; and the dura mater (which is the strong outer layer).
[0025]
The spinal cord is organized into segments along its length. The nerves from each segment connect to specific areas of the body. The neck region, referred to as C1-C8, controls the signals to the neck, arms and hands. Segments in the chest or upper back region, referred to as T1-T12, relay signals to several parts of the torso and arms. Segments in the upper waist region or midback region just below the ribs, referred to as L1-L5, control signals to the hips and limbs. Finally, the sacral segment, referred to as S1-S5, is directly below the waist segment in the mid-back and controls signals to the groin, toes, and some parts of the limb. The impact of spinal cord injury in different segments reflects this configuration.
[0026]
Several types of cells perform CNS functions. Large motor nerves have long axons that control skeletal muscles in the neck, torso, and limbs. Sensory nerves called spinal ganglion cells, whose axons form nerves that carry information from the body to the spinal cord, are found just outside the spinal cord. Interneurons of the spinal cord, completely within the spinal cord, help to integrate sensory information and produce coordinated signals that control the muscles. Glia, or supporting cells, are more numerous and perform various functions than neurons in the brain and spinal cord. One type of glial cell, the oligodendrocyte, creates a myelin sheath that insulates axons and increases the speed and reliability of nerve signaling. Other glia enclose wheel rims and spoke-like spinal cords and provide compartments for ascending and descending nerve fiber tubes. Astrocytes (large stellate glial cells) regulate the composition of the fluid surrounding nerve cells. Some of these cells also form scar tissue after injury. Smaller cells, called microglia, are also activated in response to injury and help clean up waste products. All of these glial cells produce substances that support neuronal survival and affect axonal growth.
[0027]
The brain and spinal cord are confined within the bone cavity that protects them, but they also make them vulnerable to compression damage caused by swelling or severe damage. CNS cells have a very high metabolic rate and depend on blood sugar for energy. CNS cells are particularly vulnerable to ischemia because the extent to which healthy blood flow divides the minimum required for normal function is much smaller in the CNS than in other tissues. Other inherent features of the CNS are the “blood brain barrier” and “blood spinal barrier”, which are formed by cells lining blood vessels in the CNS, and potentially harmful substances and cells of the immune system It acts to protect nerve cells by restricting their entry. Trauma damages these barriers and causes further damage to the brain and spinal cord.
[0028]
As used herein, the terms “central nervous system injury” and “CNS injury” refer to spinal and / or brain injury or condition characterized by an inflammatory response at the site of injury or lesion. Is intended. Central nervous system damage generally involves lesions that are primarily accompanied by mechanical damage but are caused by atraumatic events such as pathological conditions. CNS injuries include bruises caused by spinal cord contusions and compression injuries caused by pressure on the spinal cord. Other types of spinal cord injury include rupture, separation, and central spinal cord syndrome, which affect the cervical (neck) region of the cord and is called the corticospinal tract that carries signals between the brain and spinal cord Result from intensive damage to a group of nerve fibers. CNS damage or lesions can affect one or more cell types or tissues of the CNS, including neurons, glial cells and myelin. CNS conditions include demyelinating conditions, including multiple sclerosis (MS), where demyelination occurs in the white matter of the brain and spinal cord. Demyelination and local inflammation are common to spinal cord injury and demyelinating states of the CNS, including multiple sclerosis, and the influx of T cells specific for myelin basic protein (MBP) as a result of myelin destruction It has been demonstrated in both spinal cord injury and multiple sclerosis.
[0029]
Demyelinating diseases or demyelinating conditions are an important group of neurological disorders due to the frequency with which they occur and the disorders they cause. Demyelinating diseases have in common the localized or patchy destruction of the myelin sheath that can be accompanied by an inflammatory response. Myelin loss also occurs in other conditions, such as genetically determined defects in myelin metabolism, and as a result of oligodendrocyte or Schwann cell toxin exposure and infection. Demyelinating diseases of CNS include, for example, MS, acute disseminated encephalomyelitis (ADE), including post-infectious encephalomyelitis and post-vaccine encephalomyelitis, acute necrotizing hemorrhagic encephalomyelitis, and progressive ( (Necrotic) myelopathy.
[0030]
Damage to the CNS is described by Dusart and Schwab, Eur. J. et al. Neurosci. , 6 (5): 712-724 (1994) and Schnell et al., Eur. J. et al. Neurosci. , 11 (10): 3648-3458 (1999), resulting in the influx of inflammatory cells to the CNS parenchyma. The initial response is dominated by neutrophils, which is maximal one day after injury and involves the disruption of the blood brain barrier and the upregulation of cell adhesion molecules to the vascular endothelium. Neutrophils exhibit cellular and bactericidal properties and are important for removal of microbial invaders and microbial debris (Clark, Dermatol. Clin., 11 (4): 647-66 (1994)). Activated neutrophils produce a variety of pro-inflammatory 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): 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 to neuronal damage and glial activation, as described by: Yong, Cytokines, astogliosis and neutrophils following CNS trauma, Cytokines and the CNS: Development, Defence and Disease, CRC Press, Boca Raton, Florida (1996). Blood-derived macrophages and activated resident CNS microglia contribute to the inflammatory response beginning 2 days after injury and involve infiltrating lymphocytes. The chemoattractants for these cell types are up-regulated within hours of spinal cord injury, and the adhesion molecules required for cell adhesion to the cerebral endothelium are up-regulated after injury: Bartholdi and Schwab, Eur. J. et al. Neurosci. 9 (7): 1422-1438 1997; Hamada et al., J. Biol. Neurochem. 66 (4): 1525-1531 (1996). Expression of CXCL10 (CXC chemokine that induces T cells) and MIP-1a (CC chemokine that induces macrophages) has been shown to be upregulated within hours of spinal cord injury (Bartholdi, supra, 1997). And McTigue et al., J. Neurosci. Res. 53 (3): 368-376 (1998)).
[0031]
As used herein with respect to CNS injury, the term “secondary tissue degeneration” refers to adjacent cells that have not been damaged or have been slightly damaged by an initial trauma or lesion to the CNS. And secondary loss of the organization. This secondary tissue degeneration can affect any cell type present at the site of injury or lesion, including neurons and glial cells, and tissues, including myelin tissue surrounding nerve fibers and vasculature . Secondary tissue degeneration is generally associated with CXCL10-mediated T cell recruitment of T cells with specificity for inflammation and a broad array of cell types and tissues. Secondary tissue degeneration mediated by CXCL10 after CNS injury or trauma also results in blood-brain barrier disruption, edema, demyelination, axon damage and nerve death (these are activity of voltage-dependent or antagonist-gated channels) ), Ion leakage, activation of calcium-dependent enzymes (eg, proteases, lipases and nucleases), mitochondrial dysfunction and energy depletion, 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): 1-1 0 (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 after CNS injury or trauma includes necrosis, apoptosis, paraptosis, or any additional form of cell death (eg, amyotrophic lateral sclerosis) Neurodegenerative cells, as described in a transgenic mouse model of disease (Canto and Gurney, American Journal of Pathology 145: 1271-1279 (1994), which is incorporated herein by reference) Death by any mechanism can be mentioned, including death).
[0032]
Inflammation contributes to secondary tissue degeneration associated with CNS injury (Dusart and Schwab, Eur. J. Neurosci. 6 (5): 712-724 (1994); Bright, 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), these Each of which is incorporated herein by reference). A successful host response to inflammation generally requires the accumulation of differentiated host cells at the site of tissue damage. Inflammation in the injured CNS is characterized by fluid accumulation and influx of plasma proteins, neutrophils, T lymphocytes, and macrophages. This cellular accumulation is an important step in normal inflammatory processes and is mediated by a family of secreted chemotactic cytokines with common important structural properties and a role in pathogenic inflammation. CXCL10 belongs to this protein family of over 40 structurally and functionally related proteins known as chemokines.
[0033]
Chemokines are similar to 8-10 kDa heparin-binding proteins with a conserved structural motif that includes two cysteine pairs, and are divided into subfamilies based on the relative position of cysteine residues in the mature protein. There are at least four subfamilies of chemokines, but only two (α-chemokines and β-chemokines) are well characterized. In α-chemokines, the first two cysteine residues are separated by a single amino acid (CXC), whereas in β-chemokines, the first two cysteine residues are adjacent to each other (CC ). Examples of C—X—C chemokines include interleukin-8 (IL-8), human platelet-derived factor, CXCL10, and Mig. Members of the CC chemokine subfamily include, for example, macrophage chemoattractant and activator (MCAF), macrophage inflammatory protein-1α (MIP-α), macrophage inflammatory protein-1β (MIP-β) and regulated on activation. , Normal T-cell expressed and secreted (RANTES).
[0034]
Chemokines selectively attract a subset of leukocytes; some chemokines act specifically on eosinophils, others are specific for 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 their chemokine receptors CCR1-CCR9. This 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-terminus in front of the CXC sequence. Its ELR-containing CXC chemokines (including IL-8) chemoattract neutrophils, while non-ELR CXC chemokines (including CXCL10 and Mig) chemoattract lymphocytes.
[0035]
Chemokines are capable of cell migration and binding by binding to specific G protein-coupled cell surface receptors on target cells, at least in two ways: first, through direct chemoattraction, and secondly. Induces cell activation. Over 10 distinct chemokine receptors, each expressed on a different subset of leukocytes, have been identified. Chemokine receptors are constitutively expressed on some cells, while those chemokine receptors are inducible on other cells. CXCR3 (a receptor recognized by CXCL10) is expressed on activated T lymphocytes and natural killer (NK) cells of the T helper type 1 (Th 1) phenotype. Importantly, a major 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.
[0036]
As used herein, the term “effective amount” when used in reference to the 10 kDa interferon-inducible protein (CXCL10) is the severity of secondary tissue degeneration associated with CNS injury. It is intended to mean the amount of CXCL10 specific neutralizing agent sufficient to reduce
[0037]
As used herein, “reducing severity” refers to signs or symptoms, physiological indicators, biochemical markers or metabolic indicators of secondary tissue degeneration associated with CNS injury. It is intended to refer to stopping, decreasing or reversing. In spinal cord injury, the destruction of nerve fibers that carry movement signals from the brain to the torso and limbs leads to muscle paralysis. Physiological symptoms of secondary tissue damage associated with CNS injury include, for example, neurological deficits and neurogenic inflammation, and clinical symptoms include the severity of the damage, the segment of the spinal cord where the damage exists And changes dramatically depending on the segment of the spinal cord where nerve fibers are damaged. The destruction of sensory nerve fibers can lead to sensory (eg tactile, pressure and temperature) loss; it also sometimes causes pain. Other serious consequences include excessive reflexes; loss of bladder and bowel control; sexual dysfunction; loss or decline in respiratory capacity; deficiencies in cough reflexes; and spasticity. Most people with spinal cord injury recover some function between 1 week and 6 months after injury, but the chances of spontaneous recovery are almost gone beyond 6 months. Spinal cord injury can result in many clinical complications (including bed sores), respiratory diseases and autonomic dysflexia, a potentially fatal disease that increases blood pressure and sweats, and intestinal impact or May lead to increased sensitivity to other autonomic reflexes in response to some other stimulus.
[0038]
Physiological indicators of secondary tissue degeneration associated with CNS injury include blood brain barrier disruption, edema, demyelination, axonal injury and nerve death, and tissue loss. Secondary tissue degeneration biochemical markers associated with CNS damage are, for example, neuronal markers, myelin, gamma globulin or specific molecules that produce oligoclonal banding. CNS tissue loss associated with secondary tissue degeneration can be detected by various clinical methods known in the art. Neuronal markers, such as NeuN, can be used to visualize tissue loss in the CNS (Wolf et al., J. Histochem. Cytochem. 44: 1167-1171 (1996), which is described herein. In addition, the evoked potential (EP) can be used to measure how quickly nerve impulses are transmitted along nerve fibers in various parts of the nervous system, and Computed tomography (CT) can be used to scan the CNS to detect areas of tissue loss caused by cell death or demyelination of nerve fibers, as well as nuclear magnetic resonance imaging (MRI). Can be used to scan the CNS, but there is no use of X-rays, MRI is more sensitive than CT scans A region of CNS tissue loss that cannot be observed by a CT scanning instrument can be detected, and a lumbar puncture or spinal puncture procedure can be used to extract cerebrospinal fluid. Can be examined for elevated levels of oligoclonal banding These or other methods well known in the art measure tissue loss (including tissue loss caused by nerve glial cell death and nerve fiber demyelination) And can be used to determine the severity of secondary tissue degeneration associated with CNS injury.
[0039]
As used herein, the term “neutralizing agent specific for the 10 kDa interferon-inducible protein (CXCL10)” provides a decrease in the extent, amount or rate of CXCL10 expression or reduces the activity of CXCL10. It is intended to refer to the factors given. Useful neutralizing agents for practicing the claimed invention include, for example, binding molecules such as antibodies to CXCL10. The neutralizing agent can be any molecule that binds to CXCL10 with sufficient affinity to reduce CXCL10 activity. Furthermore, the neutralizing agent inhibits or promotes the function of the regulatory protein or gene region, and any molecule that binds to the regulatory molecule or gene region to give a reduced degree, amount or rate of CXCL10 expression or its activity. It can be. For example, CXCR3 receptor fragments or peptidomimetics that bind CXCL10 with sufficient affinity to reduce CXCL10 activity are useful in practicing the claimed methods. In addition, examples of neutralizing agents that confer CXCL10 expression may include antisense nucleic acids and transcription inhibitors.
[0040]
The present invention provides a method for reducing the severity of secondary tissue damage associated with CNS injury. The method includes administering an effective amount of a 10 kDa neutralizing agent specific for an interferon-inducible protein (CXCL10) to a subject having secondary tissue degeneration associated with CNS injury.
[0041]
As described herein, CXCL10 is involved in the process of providing host defense against nervous system inflammation. Specifically, CXCL10 regulates Th1 T lymphocyte transport to the CNS in response to CNS injury, CNS inflammation, physiological abnormalities (eg, infectious agents or autoimmune cells). T lymphocytes are CD4 ⁺ Cells and CD8 ⁺ It can be subdivided into two main categories known as cells. CD4 and CD8 are surface proteins that promote the interaction between the T cell receptor for the antigen and the antigen itself that is presented to the T cell for presentation by the antigen presenting cell. Antigens recognized by T cells are contained in the cleft of a major histocompatibility complex (MHC) protein expressed on the surface of antigen presenting cells. CD4 cells recognize peptide fragments presented by class II histocompatibility alleles, and CD8 cells recognize fragments presented by class I histocompatibility alleles. The DR2 allele is an MHC class II allele that is over-presented in multiple sclerosis, which means that CD4 in lesion formation in the CNS ⁺ Shows a role for cells.
[0042]
CD4 ⁺ The cells can be further subdivided into Th1 and Th2 subtypes. Thl cells are responsible for the delayed hypersensitivity response and many cytokines (interleukin 2 (IL-2), a stimulator of T cell proliferation, and interferon, an activator of macrophages, and oligodendrocytes) Secretes lymphotoxin, a protein that has the ability to damage (myelinating cells of the CNS) Interferon, in combination with IL-2, activates macrophages that strip myelin directly from nerve fibers and myelination Secretes tumor necrosis factor α (TNF-α), a cytokine that damages sex oligodendrocytes.
[0043]
As described herein, neutralization of CXCL10 expressed after CNS injury in or around the injury site or lesion (injury) (FIG. 1) results in tissue loss at the injury site and surrounding tissues. Reduces the secondary tissue degeneration that manifests itself through (FIG. 3). Consequently, neutralizing CXCL10 activity can lead to a reduction in the severity of secondary tissue degeneration associated with CNS injury. CXCL10 neutralizing agents (including antibodies, antisense nucleic acids or other compounds identified by the methods described below) are useful for treating or reducing the severity of secondary tissue damage associated with CNS damage. is there.
[0044]
Administration of a CXCL10 neutralizing agent can result in a variety of distinct CXCL10-mediated destruction events associated with secondary tissue degeneration (eg, activation of voltage-gated or agonist-gated channels, ion leakage, calcium-dependent enzymes (eg, , Proteases, lipases, and nucleases), inflammation, mitochondrial dysfunction and energy depletion, cell death (including culminating). Administration of a CXCL10-specific neutralizing agent presents a therapeutic improvement by targeting and treating each of these distinct CXCL10-mediated events rather than a single aspect of secondary tissue degeneration. An enhanced method for reducing the severity of secondary modification is provided.
[0045]
Moreover, in addition to the unexpected finding that CXCL10 has such a dramatic effect on secondary tissue degeneration, the surprising finding is that not only can the damage be stopped, but CXCL10 neutralizing agent can also be Is to induce regrowth and remyelination. Accordingly, the present invention also provides a method of recovering secondary tissue degeneration or trauma associated with CNS injury by neutralizing a 10 kDa interferon-inducible protein (CXCL10) using a specific neutralizing agent and Methods of promoting neuronal cell remodeling and remyelination are provided.
[0046]
Mechanical damage to the spinal cord of adult mammals results in increased vascular permeability and extensive activation and recruitment of inflammatory cells (Dusart, I and ME Schwab (1994), “Secondary cell death and the inframmatory. reaction after harmalization of the rat spinal cord ”, Eur J Neurosci 6 (5): 712-24) (Schnell, L., S. Fernand et al., National Institute of Medicine, 1999),“ Acture inflames ”. : Differences between brain and spinal cord ", E ur J Neurosci 11 (10): 3648-58). The control and consequences of this strong inflammatory response to spinal cord injury are not well known. A full understanding of neuroimmune interactions can be found in the field of multiple sclerosis research, where this is an abnormal attenuation of T lymphocyte responses to demyelinating pathology in the MHV model of multiple sclerosis In recent years, it has been demonstrated to reduce tissue development and behavioral defects (Liu, MT, HS Keirstead, et al. (2001), “Neutralization of the chemokine cxc110 reduces indegradation cell invasion in the evolution of the cell. function in a virtual model of multiple sclerosis ", J Immunol 167 (7): 4091-7). T lymphocytes predominate at the site of spinal cord injury (McTigue, DM, M. Tani, et al. (1998), “Selective chemokin mRNA accumulation in the spinal cord after injuries 53”). (3): 368-76), and recent studies suggest that they play a central role in the structural and functional consequences of spinal cord trauma (Hauben, E., O.). Butovsky et al. (2000), "Passive or active immunization with myelin basic protein promoters recovery from spin cor. conjution ", J Neurosci 20 (17): 6421-30) (Hauben, E., U. Nevo et al. (2000)," Autoimmune T cells as potentior therapeutic for 9 " 7) The results of inhibiting the recruitment of T lymphocytes to the site of spinal cord injury were further investigated. The ability to functionally block antibodies to the T lymphocyte chemoattractant CXCL10 (Liu, MT, HS Keirstead et al. (2001), “Neutralization of the chemikinetic and vascularization immunity in vivo immunity in vivo immunization in vivo "function in a viral model of multiple sclerosis", J Immunol 167 (7): 4091-7) is responsible for the role of this chemokine in the recruitment of T lymphocytes to sites of spinal cord trauma, and abnormal tissue development and behavioral defects after trauma It provided an opportunity to test the contribution of T lymphocytes. The data herein show that CXCL10 was up-regulated after spinal cord injury in adult mammals (FIG. 1) and that antibody-mediated neutralization of CXCL10 in injured animals is a dramatic occurrence that normally occurs after spinal cord injury. It clearly demonstrated that reduced T lymphocyte infiltration was reduced (FIG. 5). These data show that administration of anti-CXCL10 antiserum to CNS to mice with established virus-induced demyelination is CD4 ⁺ Significantly reduces T lymphocyte infiltration and decreases the expression of the TH1-related proinflammatory cytokine IFN-g (Liu, MT, HS Keirstead et al. (2001), “Neutralization of the the Chemoline cxc110 reduces information cell inversion and demyelination and improves neurological function in a virulence in dev. Attenuation of T lymphocyte infiltration into the site of spinal cord injury after anti-CXCL10 treatment indicates that CXCL10 is a particularly important T lymphocyte chemoattractant during spinal cord injury and expression of the CXCR3 receptor (Rollins, BJ (1997). ), “Chemokines”, Blood 90 (3): 909-28) (Luster, AD, JC Unkeless et al. (1985), “Gamma-interferon transcriptional regulations”). an early-response gene con- taining homology to platelet proteins ", Nature 315 (6021): 672-6).
[0047]
Important and unpredictable, the data disclosed herein was that anti-CXCL10 treatment alone was sufficient to reduce post-traumatic tissue degeneration and post-injury motor deficits It is shown that. Morphogenesis analysis revealed that hemi-resected injured mice that received anti-CXCL10 treatment had a 68% reduction in tissue damage around the injured site compared to untreated hemi-resected injured mice (FIG. 3) showed that. The number of neuronal cells around the injury site is related to the presence of more than 438% of neurons around the injury site in the reduction of tissue loss in semi-excision injury mice treated with anti-CXCL10 treatment than in untreated half-excision injury mice did. These data indicate that anti-CXCL10 treatment significantly reduces post-traumatic tissue loss after dorsal hemirectomy.
[0048]
Behavioral analysis showed that semi-resected injured mice that received anti-CXCL10 treatment showed a statistically significant continuous improvement in kinematic parameters tested during the recovery phase (FIG. 2). By 14 days after injury, the behavioral scores for all four kinematic parameters were not significantly different from undamaged control mice. This recovery was more likely to be contributed by reconstruction of the caudal sensorimotor pathway of the lesion (injury) than the severe fiber reconstruction. Biochemical and synaptic rearrangements have been demonstrated after spinal cord injury and can contribute to functional improvement (Edgerton, VR, RD Leon et al. (2001) “Retraining the injured”. spinal cord ", J Physiol 533 (Pt 1): 15-22). Although there was little data related to synaptogenesis in the injured spinal cord, there was substantially more by quantitative electron micrograph studies using a well-documented model of denervated dentate gyrus. Numbers of new synapses are formed in denervated neurons by 10 days after injury (Steward, O., SL Vinsant et al. (1988), "The process of reinnervation in the dentate of adult rats" : An ultrastructural study of changes in presynthetic terminals as a result of sprouting ", J Comp Neurol 267 (2): 203-1. 0). Although the mechanism of behavioral recovery in the current study is unknown, the data disclosed herein clearly show that anti-CXCL10 treatment reduces neurological deficits after spinal cord injury.
[0049]
A detrimental role for the immune response after overflowing to damaged CNS parenchyma is indicated by the etiology of multiple sclerosis and the disease state seen in animal models of multiple sclerosis, where pro-inflammatory cells Is considered the center of disease development (Olsson, T. (1995), “Cytokine-producing cells in experimental autoimmune essence of globulitis and multiple sclerosis” Neurology 45 (6: Sol. , PE Lipsky et al. (1995), “Elevated Th1-or Th0-like cytokine mRNA in peripheral circulation of p. tients 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 a transient association of chemokine expression and influx of immune cells with secondary degeneration (Dusart, I. and M.E. Schwab ( 1994) “Secondary cell death and the infralamablative reaction after dorsal healdion of the rat spin cord” Eur J Neuroci 6 (5): 712-24) (Bliht. for secondary cell damage in spinal cord injury "Ce nt Nerv Syst Trauma 2 (4): 299-315) (Popovich, P.G., P.Wei et al. (1997) "Cellular inflammatory response after spine brew squeeze in Drawe-Durage in Sprague-Durage". ): 443-64)).
[0050]
Popovich and co-workers are satisfied that clodronate-mediated hematopoietic macrophage removal leads to reduced traumatic tissue loss and improved overground locomotion following spinal cord injury Have demonstrated so. These findings conclude to the authors that infiltrating immune cells are a serious secondary degeneration effector and can prove that cell-specific immune loss is a treatment for spinal cord injury. Led to suggest. (Popovich, PG, P. Wei et al. (1997), “Cellular information response after spinal cord in-jury in Sprague-Dawley and Lewis rats” J Comp. In fact, activated monocyte phagocytes release neurotoxins after traumatic CNS injury (Guilian, D., M. Corpuz et al. (1993), “Reactive mononuclear phagocyte esthetic tomato ischemic chemistry” central nervous system "J Neurosci Res 36 (6): 681-93), which induces NMDA receptor-mediated neurotoxicity (Paini, D., K. Frei, et al. (1991)" Murine brain macromolecules induce in nepti vitro by secreting gluta ate "Neurosci Lett 133: 159-162), and activated leukocytes release a wide variety of lytic enzymes and reactive oxygen and reactive nitrogen intermediates (Martiney, JA, C. Cuff). (1998), “Cytokine-induced information in the central nervous system revised” Neurochem Res 23 (3): 349-59).
[0051]
These findings are interesting in view of the current confirmation that anti-CXCL10 treatment leads to a decrease in infiltration of both T lymphocytes and hematopoietic macrophages at the site of spinal cord injury (FIG. 5). Since hematopoietic macrophages do not express the CXCR3 receptor for CXCL10, the loss of these macrophages may be a downstream effect of T lymphocyte loss; this indicates that activated CD4 + T lymphocytes are RANTES (for hematopoietic macrophages). Of chemoattractants) by demonstrating their secretion.
[0052]
If the regulation of immune cells within a site of CNS disease / trauma is complex and immune cell activation within such a site is a downstream effect, recruitment of the immune cell population to the site of injury Targeting) may elucidate the role of specific populations in secondary degeneration cascades that are subject to CNS insults. The results described herein provide new insight into the functional importance of CXCL10 expression during CNS injury and further treat CNS with neutralizing agents specific for CXCL10 Provides a basis for how to do. CXCL10 has been shown herein to be an important chemoattractant during spinal cord injury. Therapies targeting CXCL10 as provided by the present invention represent a viable treatment strategy for reducing post-traumatic abnormal tissue development and behavioral disorders after CNS injury.
[0053]
A CXCL10 neutralizing agent that binds CXCL10 with sufficient affinity may reduce the activity of CXCL10 with respect to immune cell recruitment, tissue loss or demyelination. The CXCL10 specific neutralizing agent can be a macromolecule (eg, a polypeptide, nucleic acid, carbohydrate or lipid). CXCL10-specific neutralizing agents can also be derived compounds, analogs or mimetic compounds, as well as small organic compounds, so long as CXCL10 activity is reduced in the presence of the neutralizing agent. The size of the neutralizing agent is not critical as long as the molecule exhibits selective neutralization activity or can be made to exhibit selective neutralization for CXCL10. For example, the neutralizing agent can be between about 1 and 6 monomer building blocks, and tens or hundreds of monomer building blocks, the monomer building blocks being macromolecules or chemically linked molecules. Configure. Similarly, an organic compound can be a single structure or a complex structure as long as it binds to CXCL10 with sufficient affinity to reduce activity.
[0054]
Neutralizing agents specific for CXCL10 can include, for example, antibodies and other receptors or ligands that bind polypeptides of the immune system. Such other molecules of the immune system include, for example, T cell receptors (TCRs) including the CD4 cell receptor. A neutralizing agent for CXCL10 can also be a molecule that inhibits the interaction of CXCL10 with a chemokine receptor (eg, CXCR3 receptor). In addition, cell surface receptors such as integrins, growth factor receptors and chemokine receptors, and any other receptor that can be made to bind with sufficient affinity to bind or reduce activity. Fragments are also useful neutralizing agents for carrying out the method of the invention. In addition, for example, receptors, growth factors, cytokines or chemokines that inhibit the expression of CXCL10 or its receptors are also useful neutralizing agents for carrying out the methods of the invention. In addition, DNA binding polypeptides such as transcription factors and DNA replication factors have selective binding activity to CXCL10, regulatory molecules that control CXCL10 expression or activity, or gene regions that control CXCL10 expression. As long as it appears to be included within the definition of the term binding molecule. Finally, polypeptides, nucleic acids and compounds, such as selected from random and combinatorial libraries, are also of the term as long as they bind CXCL10 with sufficient affinity to reduce their activity. Included in the rules.
[0055]
Various approaches can be used for the identification of selective neutralizing agents for CXCL10. For example, one approach is to use information available regarding the structure and function of CXCL10 to generate a binding molecule population from molecules known to function as chemokine binding molecules, or to bind specifically to CXCL10. A population of binding molecules is made from molecules known to present or capable of presenting affinity (eg, CXCR3 receptor fragments or mimetics found on CD4 + T cells and NK cells). Neutralizing agents specific for CXCL10 can be antibodies and other molecules of the immune repertoire. The normal function of such immunoreceptors is to bind an essentially infinite number of different antibodies and ligands. Thus, generating a diverse population of binding molecules from the immune repertoire can be useful, for example, to identify neutralizing agents specific for CXCL10.
[0056]
Neutralizing agents specific for CXCL10 can be further 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. This population can be generated to include sufficiently diverse sequences or structures to contain molecules that bind to the CXCL10 proteins or their respective nucleic acids. Those skilled in the art will recognize which sizes and diversity are necessary or sufficient for the intended purpose. A population of sufficient size and complexity can be generated to likely contain a CXCL10 neutralizing agent that binds CXCL10 with sufficient affinity to reduce activity. Many other types of library molecular populations exist and are described further below.
[0057]
Any molecule that binds to CXCL10, to CXCL10 receptor, to a gene region that regulates CXCL10 expression, or to a regulatory molecule that regulates CXCL10 activity or expression, and any regulation that regulates expression of CXCL10 receptor The molecule is a CXCL10 specific neutralizing agent useful for practicing the present invention. For example, a CXCL10-specific neutralizing agent can be a regulatory molecule that affects CXCL10 expression by reducing or inhibiting the activity of a transcription factor that regulates or upregulates the transcription of CXCL10. Furthermore, regulatory molecules that bind z to molecules involved in CXCL10 activation with sufficient affinity to reduce CXCL10 activation are useful neutralizing agents for carrying out the methods of the invention.
[0058]
A neutralizing agent that can bind to CXCL10 with sufficient affinity to reduce the activity of CXCL10 reduces the severity of secondary tissue degeneration associated with CNS injury, and is associated with CNS injury. It is useful to perform on a subject suffering from secondary tissue degeneration. Furthermore, CXCL10-specific neutralizing agents reduce CXCL10 activity or expression by binding to the CXCL10 receptor, to regulatory molecules that regulate CXCL10 activity or expression, or to gene regions that control CXCL10 expression. obtain. For example, a neutralizing agent useful for practicing the invention can be an antibody against a regulatory molecule that modulates CXCL10 expression or activity. Furthermore, as described herein, anti-CXCL10 antibodies are useful neutralizing agents for practicing the methods of the invention. Further, since CXCL10 is a secreted protein known to bind to specific T cell receptors, neutralizing agents useful in the practice of the present invention are binding properties of any of the immunoglobulin superfamily of receptors. It can be a polypeptide, a receptor or a fragment thereof. Alternatively, it may be desirable to use a population of random peptide populations to identify additional neutralizing agents specific for CXCL10. Those skilled in the art will recognize which type of approach and which type of neutralizing agent is the severity of secondary tissue degeneration associated with CNS injury in a subject suffering from secondary tissue degeneration associated with CNS injury. Can be recognized or determined to be suitable for performing the method of the present invention.
[0059]
A moderately sized population for identification of CXCL10 specific neutralizing agents can consist of hundreds and thousands of different binding molecules in the population, while a large sized binding molecule population is Tens of millions and millions of different binding molecular species. More specifically, a large and diverse population of binding molecules for the identification of neutralizing agents is any about 10 ⁴ , About 10 ⁵ , About 10 ⁶ , About 10 ⁷ , About 10 ⁸ , About 10 ⁹ , About 10 ¹⁰ Any of the above may include different binding molecular species. One skilled in the art will recognize the appropriate diversity of populations of binding molecules sufficient to identify neutralizing agents specific for CXCL10.
[0060]
Recombinant libraries of binding molecules can be used to identify neutralizing agents specific for IP-10. This is because large and diverse populations can be rapidly generated and screened using CXCL10. Recombinant libraries of expressed polypeptides useful for identifying neutralizing agents specific for CXCL10 can be generated in a number of different ways known in the art. Recombinant library methods also allow for the generation of very large populations of binding molecules from naturally occurring repertoires. Whether recombinant or otherwise, essentially any source of the binding molecule population, with a sufficient size and size to identify a neutralizing agent specific for CXCL10. As long as it provides binding molecules of different diversity, they can be used. If desired, populations of binding molecules useful for identifying neutralizing agents specific for CXCL10 are described in Watkins et al., Anal. Biochem. 256 (92): 169-177 (1998), which is incorporated herein by reference, can be selectively immobilized on a solid support.
[0061]
A phage expression library in which a lytic phage results in the release of a binding molecule polypeptide expressed by a bacterium is one example of a recombinant library that can be used to identify neutralizing agents specific for CXCL10. . In another type of phage expression library, a large number of potential binding molecules can be expressed as fusion polypeptides on the periplasmic surface of bacterial cells. Libraries in yeast and higher eukaryotic cells also exist and are equally applicable to the methods of the invention. One skilled in the art can recognize or determine which type of library is useful for identifying neutralizing agents specific for CXCL10.
[0062]
For compounds that specifically bind CXCL10, in addition to the methods described above that utilize purified polypeptides to screen a library of compounds, a neutralizing agent specific for CXCL10 can be used to produce purified poly Can be identified by using peptides. For example, antibodies that are specific for CXCL10 can be used as neutralizing agents of the present invention and can be generated using methods well known in the art. Neutralizing agents useful for carrying out the methods of the invention include both monoclonal and polyclonal antibodies, as well as Fab, F (ab ′) 2, against any molecule that modulates CXCL10 or CXCL10 activity or expression, Examples include antigen-binding fragments of such antibodies that contain Fd and Fv fragments and the like. Further, neutralizing agents useful for carrying out the methods of the invention include, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, complementarity determining region grafted (CDR grafted) antibodies and humanized antibodies, and Includes non-naturally occurring antibodies including their antigen-binding fragments.
[0063]
Methods for preparing and isolating antibodies, including polyclonal and monoclonal antibodies, using peptide immunogens are well known to those of skill 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 produced, for example, by Huse et al., Science 246: 1275-1281 (1989) (herein As described in U.S. Pat. No. 5,036,097) by screening a combinatorial library of variable heavy and variable light chains. These and other methods of making, for example, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single chain antibodies and bifunctional antibodies are well known to those skilled in the art (issued by Hoogenboom et al., Oct. 15, 1996). US Pat. No. 5,564,332; Winter and Harris, Immunol. Today 14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989); Harlow and Lane, supra, 1988). Hillyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering Press, 2nd edition (Oxford University Press 1995); ; Each of which is incorporated by reference herein).
[0064]
A substantially purified CXCL10 protein, which can be prepared from a natural source or recombinantly produced, or a peptide portion of a CXCL10 protein, including synthetic peptides, can be used as an immunogen. Can be triggered. The non-immunogenic peptide portion of the CXCL10 protein can be obtained by binding the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) or as a fusion protein By expressing the part, it can be made immunogenic. Various other carrier molecules and methods for attaching haptens to carrier molecules are well known in the art (see Harlow and Lane, supra, 1988; also Hermanson, Bioconjugate Technologies, Academic Press 1996 ( This is incorporated herein by reference). As noted above, CXCL10 specific antibody neutralizing agents can also be raised against regulatory molecules that modulate CXCL10 expression or activity, rather than directly to CXCL10.
[0065]
Neutralizing agents (eg, antibodies) specific for CXCL10 can be detectably labeled using methods well known in the art (Hermanson, supra, 1996; Harlow and Lane, supra, 1988). ; Chapter 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 neutralizing agent that binds directly to CXCL10, linked to a visible radioisotope or other moiety, is capable of treating CNS damage characterized by the presence or absence of organ-specific or tissue-specific CXCL10. It may be useful for diagnosing or staging the clinical stage progression of associated secondary tissue degeneration.
[0066]
Methods for raising polyclonal antibodies in, for example, rabbits, goats, mice or other mammals are well known in the art (Harlow and Lane, supra, 1988). Production of anti-peptide antibodies usually involves the use of a host animal such as a rabbit, mouse, guinea pig or rat. If large amounts of serum are needed, larger animals such as sheep, goats, horses, pigs or donkeys can be used. Animals are usually selected based on the amount of antiserum required. Suitable animals include rabbits, mice, rats, guinea pigs, and hamsters. These animals produce up to 25 mL, 100-200 μL, and 1-2 mL of serum per blood draw (Harlow and Lane, supra, 1988). Rabbits are very useful for the production of polyclonal antisera. This is because rabbits can be drawn safely and repeatedly and can produce high volumes of antisera. For example, blood can be collected and analyzed for antisera after two injections at 2-4 week intervals with 15-50 μg of antigen in a suitable adjuvant such as Freund's complete adjuvant Can be made.
[0067]
In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988). The peptide portion of a protein (eg, CXCL10) for use as an immunogen can be determined by methods well known in the art. Spleen cells from mice immunized with CXCL10 can be fused with an appropriate myeloma cell line to produce hybridoma cells. Cloned hybridoma cell lines can be screened with labeled CXCL10 protein to identify clones that secrete anti-CXCL10. Hybridomas expressing anti-CXCL10 monoclonal antibodies with the desired specificity and affinity can be isolated and utilized as a sustained source of antibody neutralizing agents.
[0068]
A neutralizing agent specific for CXCL10 can be used to reduce the severity of secondary tissue degeneration associated with CNS injury in mammals, including human subjects. Humanized antibodies can be constructed by conferring essentially any antigen binding specificity to the human antibody framework. Methods for constructing humanized antibodies prepare appropriate antibody neutralizing agents to perform the methods of the invention and to avoid host immune responses to antibody neutralizing agents when used therapeutically. Useful for.
[0069]
Using the antibody neutralizing agents described above, therapeutic humans by methods well known in the art (eg, complementarity determining region (CDR) grafting, and optimization of framework and CDR residues). A neutralizing agent can be produced. For example, humanization of antibody neutralizing agents can be achieved by CDR grafting as described in Fiorentini et al., Immunotechnology 3 (1): 45-59 (1997), which is incorporated herein by reference. Can be achieved. Briefly, CDR grafting recombines CDRs from non-human antibody neutralizing agents into a human framework region in order to confer binding activity to the resulting grafted antibody or variable region binding fragment thereof. Splicing. Once a CDR-grafted antibody or variable region binding fragment is made, binding affinity comparable to a non-human antibody neutralizing agent can be regained by subsequent rounds of affinity mutation strategies known in the art. Humanization of antibody neutralizing agents in the form of rabbit polyclonal antibodies is described by Rader et al. Biol. Chem. 275 (18): 13668-13676 (2000), which can be accomplished by methods similar to those described in this specification.
[0070]
Humanization of non-human CXCL10 antibody neutralizing agents is also described in Wu et al. Mol. Biol. 294 (1): 151-162 (1999), which is incorporated herein by reference, as well as co-optimization of framework and CDR residues (which includes framework and Enabling rapid identification of cooperative interactions with CDR residues). Briefly, a combinatorial library that tests a large number of potentially important framework positions is a focused consisting of variants containing single amino acid mutations randomly in the third CDR of the heavy and light chains. ) It is expressed simultaneously with the CDR library. By this method, multiple Fab variants can be identified that contain as few as a few non-human framework residues and show up to about 500-fold higher affinity than the original chimeric Fab. Screening a combinatorial library of framework-CDRs allows the identification of monoclonal antibodies with structures optimized for function (including when the antigen induces conformational changes in the monoclonal antibody). Variants with enhanced humanization contain fewer non-human framework residues than antibodies humanized by sequential in vitro humanization and affinity maturation known in the art.
[0071]
As described above, antibody neutralizing agents of the present invention include, for example, polyclonal antibodies, monoclonal antibodies, and recombinant versions and functional fragments thereof. Recombinant versions of antibody neutralizers include a wide variety of constructs, ranging from simple expression and coassembly of cDNAs encoding heavy and light chains to specialized constructs called designer antibodies. Recombinant methodologies combined with extensive characterization of polypeptides in the immunoglobulin superfamily (especially antibodies), resulting in a vast number of different types, styles and specificities derived from immunoglobulin variable and constant region binding domains The ability to design and construct sex binding molecules is provided. As specific examples, chimeric antibodies (wherein one antibody constant region is replaced by another antibody constant region) and the above humanized antibody (here, complementarity determination from one antibody) Region (CDR) is replaced with a complementarity-determining region from another antibody).
[0072]
Other recombinant versions of antibody neutralizing agents include, for example, functional antibody variants, where the variable region binding domain or functional fragment responsible for maintaining antigen binding is derived from the antibody constant region. F _c Fused to the receptor binding domain. Such variants basically consist of antigen binding and F _c It is a shortened form of an antibody in which a region not essential for receptor binding is removed. A shortened variant can have a single valency, or can be constructed with multiple valencies, eg, depending on the application and user requirements. In addition, the linker or spacer may be combined with an antigen and F to optimize binding activity and include additional functional domains fused or added to provide biological functions other than neutralization of CXCL10. _c It can be inserted between the receptor binding domain. The skilled artisan knows how to construct a recombinant antibody neutralizing agent specific for CXCL10 in the light of insights in the art regarding antibody manipulation and the guidance and teachings provided herein. A description of recombinant antibodies, functional fragments and variants, and antibody-like molecules can be found, for example, in Antibody Engineering, 2nd edition (Carl, AK Borrebaeck), Oxford University Press, New York (1995). Can be issued.
[0073]
Additional functional variants of antibodies that can be used as antibody neutralizing agents include antigen binding domain-F. _c Antibody-like molecules other than fusions of receptor binding domains are included. For example, F _c An antibody comprising a receptor binding domain, functional fragments and fusions thereof, wherein 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. Can be generated to be bispecific in terms. Such bispecific antibody neutralizing agents may be beneficial in the methods of the invention. This is because a single bispecific antibody contains two different target antigen binding species. Thus, a single molecular entity can be administered to achieve neutralization of CXCL10.
[0074]
The antibody neutralizing agent specific for CXCL10 can also be an immunoadhesion or a bispecific immunoadhesive. An immunoadhesive is an antibody-like molecule that combines the binding domain of a non-antibody polypeptide with the antibody effector functions of an antibody constant domain. The binding domain of the non-antibody polypeptide can be, for example, a ligand or a cell surface receptor having ligand binding activity. The immunoadhesive for use as a CXCL10 neutralizing agent is at least F of the antibody constant domain _c Receptor binding effector functions may be included. Specific examples of ligands and cell surface receptors that can be used in the antigen binding domain of an immunoadhesive neutralizer include, for example, T cell receptors or NK cell receptors, such as the CXCR3 receptor that recognizes CXCL10. Other ligands and ligand receptors known in the art can also be used for the antigen binding domain of immunoadhesive neutralizers specific for CXCL10. In addition, multivalent and multispecific immunoadhesives can be constructed for use as CXCL10 neutralizing agents. The construction of bispecific antibodies, immunoadhesives, bispecific immunoadhesives and other heteromultimeric polypeptides that can be used as CXCL10 specific neutralizing agents is described, for example, in US Pat. No. 5,807,706. Nos. And 5,428,130, which are incorporated herein by reference.
[0075]
In one embodiment of the invention, a polynucleotide encoding CXCL10, a regulatory molecule that modulates CXCL10 expression or activity, or any fragment thereof, or an antisense molecule is used as a neutralizing agent for therapeutic purposes. Can be done. In one aspect, antisense molecules against the CXCL10 encoding nucleic acid can be used to block transcription or translation of the mRNA. Specifically, cells can be transformed with sequences complementary to the nucleic acid of CXCL10. 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 region of the sequence encoding CXCL10. Thus, antisense molecules can be used to neutralize CXCL10 activity or to achieve regulation of gene function.
[0076]
Expression vectors from retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses or vaccinia viruses, or expression vectors from various bacterial plasmids can be used for delivery of antisense nucleotide sequences. The chosen viral vector should be able to infect CNS cells and be safe for the host and cause minimal cell transformation. Retroviral vectors and adenoviruses provide an efficient, useful, and currently best-characterized means for efficiently introducing and expressing foreign nucleotide sequences into mammalian cells. These vectors are well known in the art and have a very broad host range and cell type range to allow stable and efficient gene expression. Such recombinant vectors can be constructed using methods well known to those skilled in the art. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, New York (1989) and Ausubel et al., Current Protocol, Current Protocol. (Each of which is incorporated herein by reference). Even in the absence of integration into DNA, such vectors can continue to transcribe RNA molecules until they are stalled by endogenous nucleases. Transient expression can last for more than a month with a non-replicating vector and can last even longer if the appropriate replication elements are part of the vector system.
[0077]
Ribozymes (enzymatic RNA molecules) can also be used to catalyze specific cleavage of CXCL10 mRNA or any regulatory molecule mRNA that regulates CXCL10 expression or activity. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to its mRNA complementary target, followed by internal nucleotide strand scission cleavage. Specific ribozyme cleavage sites within any potential RNA target are identified by scanning the RNA for ribozyme cleavage sites including the following sequences: GUA, GUU, and GUC. Once identified, a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural properties that can render the oligonucleotide incapacitated. The suitability of candidate targets can also be assessed by testing the availability for hybridization with complementary oligonucleotides using a ribonuclease protection assay. The antisense molecules and ribozymes of the present invention can be prepared by any method known in the art for the synthesis of nucleic acid molecules.
[0078]
Useful neutralizing agents for practicing the methods of the invention include the severity of the tissue degeneration being treated; the rate or amount of tissue damage; the body weight, sex, age and health status of the subject; Chemical properties, biological activity, bioavailability and side effects; and can be formulated and administered by those skilled in the art in an appropriate manner and amount in a manner compatible with the combined treatment regimen. Appropriate amounts and formulations to reduce the severity of secondary tissue degeneration associated with CNS damage in humans can be estimated from reliable animal models for specific disorders known in the art. A medium specific to CXCL10 so that a lower dose of a neutralizing agent exhibiting significantly higher binding affinity can be administered compared to the dosage required for a neutralizing agent having a lower binding affinity. It will be appreciated that the dosage of the sumpant must be adjusted based on the binding affinity of the neutralizing agent for CXCL10. Thus, the appropriate dosage will vary depending on the particular treatment and depending on the duration of treatment desired; however, dosages of between about 10 μg and about 1 mg per kg body weight per day are therapeutic. It is expected to be used for physical treatment. A therapeutically effective amount is typically about 0.1 μg / ml to about 100 μg / ml, about 1.0 μg / ml to about 50 μg / ml when administered in a physiologically acceptable composition. ml, or at least about 2 μg / ml, and usually the amount of neutralizing agent that is sufficient to achieve a plasma concentration of 5-10 μg / ml.
[0079]
A method of the present invention for reducing the severity of secondary tissue degeneration associated with CNS injury in a subject can provide a 10 kDa interferon-inducing protein (10 kDa) to a subject with secondary tissue degeneration associated with CNS injury ( Administering an effective amount of a neutralizing agent specific for CXCL10), the method may further comprise a time course regimen consisting of repeated treatment starting at the time of injury or trauma Is contemplated. Since secondary tissue damage associated with CNS injury is a progressive process that occurs several days or early weeks after injury, administration of CXCL10 neutralizing agent can be initiated immediately or as soon as possible after injury, And can be repeated daily for an appropriate length of time. Appropriate length of time can be determined for various factors known to those skilled in the art and include, for example, 1 day, 2 days, 3 days, 5 days, 6 days, 6 days, 7 days, 8 days after injury. It can be days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or longer. Treatment can also be administered every 2 days or every 3 days, and can be administered two, three or more times on the day of treatment. For example, treatment is continued for an amount of time that CXCL10 levels are upregulated above its base level after injury or trauma of CNS (see Lee et al., Neurochemistry International 36: 417-425 (2000)). (This is incorporated herein by reference). Thus, a time course regimen is useful for performing the present method. Because the neutralizing agent specific for 10 kDa interferon-inducing protein (CXCL10) is immediately after CNS injury (for example, within about 10 minutes, within about 20 minutes, within about 30 minutes, within about 40 minutes, within about 50 minutes) Or 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 hours Within, or longer), and can be repeated for a time determined to correlate with the upregulation of CXCL10 after CNS injury.
[0080]
The total amount of neutralizing agent can be administered as a single dose, can be administered by infusion over a relatively short period of time, or can be administered in multiple doses administered over a longer period of time. Such considerations depend on a variety of case-specific factors (eg, whether the disease category is characterized by acute episodes or gradual tissue degradation). For example, for a subject suffering from chronic tissue degradation, the neutralizing agent can be administered in a sustained release matrix, which can be implanted for systemic administration or implanted at a target tissue site. Can be done. Contemplated matrices useful for the controlled release of therapeutic compounds are well known in the art. Such matrices include materials (eg, DepoFoam ™, biopolymers, micropumps, etc.).
[0081]
The neutralizing agent of the present invention can be administered to a subject by a number of routes known in the art (eg, systemic administration (eg, intravenous administration or intraarterial administration)). The CXCL10 specific neutralizing agent is in the form of isolated and substantially purified polypeptides and polypeptide fragments in pharmaceutically acceptable formulations using formulation methods known to those skilled in the art. Can be provided. These formulations can be administered using standard routes (local, transdermal, intraperitoneal, intracranial, intraventricular, intracerebral, intravaginal, intrauterine, oral, rectal, or parenteral. Routes, including intravenous, intraspinal, subcutaneous, or intramuscular routes). Intraspinal and intravenous administration of CXCL10 specific neutralizing agents are particularly suitable routes for carrying out the methods of the present invention. Intraspinal administration can be performed to target delivery of the neutralizing agent to the site of injury or trauma. In addition, CXCL10-specific neutralizing agent variants can be incorporated into the biodegradable polymer, thereby slowing down the useful compounds to reduce the severity of secondary tissue degeneration associated with CNS injury. It can be released. Biodegradable polymers and their use are described, for example, in Brem et al. Neurosurg. 74: 441-446 (1991), which is incorporated herein by reference.
[0082]
The CXCL10 specific neutralizing agent can be administered as a solution or suspension together with a pharmaceutically acceptable vehicle. Such pharmaceutically acceptable vehicles include, for example, sterile aqueous solvents (eg, sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution, or other physiological buffered physiology). Saline) or other solvents or vehicles such as glycols, glycerol, oils (eg olive oil) or injectable organic esters. The pharmaceutically acceptable medium further includes a physiologically acceptable compound that acts (eg, a compound that stabilizes the neutralizing agent, a compound that increases its solubility, or a compound that increases its absorption). obtain. Such physiologically acceptable compounds include, for example, carbohydrates (eg, glucose, sucrose, or dextran); antioxidants (eg, ascorbic acid or glutathione); receptor-mediated penetrants (which are blood -Can be used to increase the permeability of the brain barrier); chelating agents (eg EDTA) (which disrupt microbial membranes); divalent metal ions (eg calcium or magnesium); low molecular weight proteins; Lipids or liposomes; or other stabilizers or excipients. Those skilled in the art will appreciate that the selection of a pharmaceutically acceptable carrier will depend on the route of administration of the compound, including the neutralizing agent, as well as its specific physical and chemical characteristics.
[0083]
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions (eg, the pharmaceutically acceptable vehicles described above). The solution may further include, for example, a buffer, a bacteriostatic agent, and a solute that renders the formulation isotonic with the blood of the intended recipient. Other formulations include, for example, aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The formulation can be presented in unit dose containers or multi-dose containers (eg, sealed ampoules and sealed vials) and stored in a lyophilized condition requiring the addition of a sterile liquid carrier, for example, just prior to use. Extemporaneous injection solutions and immediate injection suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
[0084]
For applications directed to brain injury, where the CXCL10 neutralizing agent can be administered in a formulation that can cross the blood-brain barrier, for example, administered in a formulation that increases the lipophilicity of the CXCL10 neutralizing agent. obtain. For example, the neutralizing agent can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I-III, 2nd edition (CRC Press, Boca Raton FL (1993)) from phospholipids or other lipids. The resulting liposome is a non-toxic, physiologically acceptable and metabolizable carrier that is relatively simple to make and administer.
[0085]
Neutralizing agents specific for CXCL10 can also be prepared as nanoparticles. Adsorption of peptide compounds on the surface of nanoparticles has been shown to be effective in delivering peptide drugs to the brain (see Kreuter et al., Brain Res. 674: 171-174 (1995). about). An exemplary nanoparticle is a colloidal polymer particle of polybutyl cyanoacrylate with a neutralizing agent specific for CXCL10 adsorbed on the surface and coated with polysorbate 80.
[0086]
Image-guided ultrasound delivery of CXCL10 neutralizing agent through the brain-blood barrier at selected locations in the brain can be utilized as described in US Pat. No. 5,752,515. Briefly, to deliver a CXCL10 neutralizing agent through the blood-brain barrier, a selected location in the brain is targeted and the tissue and / or fluid in the CNS at that location (CNS) Ultrasound is used to induce a detectable change by imaging. At least a portion of the brain in the vicinity of the selected location is imaged to confirm the location of the change, eg, via magnetic resonance imaging (MRI). CXCL10-specific neutralizing agent in the patient's bloodstream is confirmed by applying ultrasound to bring the brain-blood barrier opening to that location, thereby inducing neutralizer uptake. Can be delivered to.
[0087]
In addition, polypeptides called receptor-mediated penetrants (RMPs) are described, for example, in US Pat. Nos. 5,268,164; 5,506,206; and 5,686,416. As such, it can be used to increase the permeability of the blood barrier to a molecule (eg, a therapeutic or diagnostic agent). These receptor-mediated permeabilizers can be co-administered intravenously to the host along with a molecule whose desired destination is cerebrospinal fluid. The penetrant polypeptide or conformational analog thereof allows the therapeutic agent to penetrate the blood-brain barrier to reach its target destination.
[0088]
In accordance with another embodiment of the present invention, a therapeutic system comprising a CXCL10 neutralizing agent and instructions for use in a method for reducing the severity of secondary tissue degeneration associated with the central nervous system Is preferably present in kit form. In one embodiment, for example, the therapeutic agent is an anti-CXCL10 antibody. The kit of the present invention is useful for reversing the severity of secondary tissue degeneration associated with central nervous system injury.
[0089]
A suitable kit includes at least one CXCL10 neutralizing agent as a separate packaged chemical reagent in an amount sufficient for at least one therapeutic application. Instructions for use of the packaged kit are also typically included. One skilled in the art can readily incorporate the CXCL10 neutralizing agent in the form of a kit in combination with suitable buffers and solutions for carrying out the methods of the invention as described herein.
[0090]
In current treatment regimens 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 the invention comprising reducing the rate of secondary tissue degeneration, a neutralizing agent specific for CXCL10 is a second therapeutic compound (eg, an anti-inflammatory compound, an immunosuppressive compound, Or any other compound that manages the same or different aspects of the disease). Such compounds include, for example, methylprednisolone acetate, GM-1 ganglioside and 4-aminopyridine (4-AP). Contemplated methods for reducing the severity of secondary tissue degeneration associated with CNS injury include neutralizing agents specific for CXCL10, either alone or in combination with such other compounds. Or sequentially and in combination with other treatments (eg, rehabilitation and neuroprosthesis). Alternatively, the combination therapy consists of a fusion protein in which a neutralizing agent specific for CXCL10 is bound to a heterologous protein (eg, a therapeutic protein).
[0091]
The following examples are intended to illustrate the invention but are not intended to limit the invention. It is understood that modifications that do not substantially affect the activity of the various embodiments of the invention are also encompassed within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate the invention but are not intended to limit the invention.
[0092]
Unless otherwise indicated below, the experimental procedures corresponding to the following examples are as follows.
[0093]
Antibody administration Age-matched adult female C57 / BL6 mice were used for all studies. Mice were given an intraperitoneal injection of 100 mg monoclonal CXCL10 antibody (suspended in 200 ml sterile PBS) one day prior to injury and every day thereafter until sacrifice. The monoclonal antibodies used in these studies are specific for CXCL10 and block CXCL10-induced T cell chemotaxis.
[0094]
Spinal cord injury Spinal dorsal hemisection injury was performed under Avertin anesthetic (0.6 ml / 20 mg) administered via intraperitoneal injection. A midline incision was made over the spinous process of T4-L2, and the paravertebral muscle was separated from the vertebrae. A complete posterior arch resection of the T9 vertebra was performed using fine scissors and bone forceps. The column was stabilized using a clamp attached to a fine manipulator. Subsequently, a dorsal hemisection lesion was created using a microdissection knife. The muscle layer was then sutured and superficial tissue and skin were closed with 4-0 silk. Body temperature was maintained on a heating pad. Post-surgical care included bladder urination and hydration with lactated Ringer's solution.
[0095]
Kinematic analysis All mice were acclimated to a linear motor field for 4 days before the start of the functional test. Kinematic analysis was performed one day prior to injury, and approximately every day until subsequent sacrifice, and scored separately by two observers who were unaware of the treatment group. The animals were videotaped using a Hitachi 8mm Video Cameror VM-E555LA from below the plexiglass surface carrying a defined 1 cm grid line. The video is then downloaded using an MPEG-2 compression device and using FMV 2.0 software (which allows the video to be viewed frame by frame for kinematic evaluation). And analyzed. Four kinematic parameters (stride length; stride width; paw rotation and toe spread) were assayed. The hind paw stride length was defined as the point from the start of the step using the hind paw to the end of the step using the same paw (measurements were taken on each side for 3 consecutive steps (mm) and then averaged ). The stride width was defined as the width of the hind limb study (mm) (distance from the outermost left hind toe to the outermost right contralateral hind toe). The toe width was defined as the width (mm) in the hind foot from the rearmost point of the rear finger to the middlemost point of the middle finger (both left hind foot and right hind finger, respectively). Paw rotation was defined as the angle (in degrees) between the hindfoot longitudinal axis and the body midline. The SPSS 4.0 T test was used to determine significant differences between the treated and untreated groups.
[0096]
RNASE protection assay Mice were treated at the following time points following injury due to CO2 fixation and decapitation (6 hours (n = 3), 12 hours (n = 3), 18 hours (n = 3), 24 hours (n = 3 3) (n = 3), 7 days (n = 3), and 14 days (n = 2)). Two uninjured mice were used as controls. The spinal cord was removed as two pieces and immediately frozen. Total RNA was extracted using Trizol reagent (Gibco) reagent and ethanol precipitated. The pellet was dissolved in 50 μl of RNASE free water and the RNA concentration was determined. Multi-probe set mCK-5 was used to detect CXCL10 mRNA transcripts. This multi-probe set included a probe for L32 for use as a control for RNA loading. Analysis was performed on 10 μg protein. Fragments were separated by polyacrylamide gel electrophoresis and visualized by film autoradiography. Autoradiographs were scanned and the bands were quantified using imaging software.
[0097]
Mononuclear Cell Isolation and Flow Cytometry Single cell suspensions were obtained from the spinal cord of mice treated with either anti-CXCL10 antibody or control antibody on days 3 and 14 after injury. FACS analysis was performed as previously described (Lane, Liu et al., “A central role for CD4 (+) T cells and RANTES in virus-inventive central system inflation 2000”). (3): 1415-24). Briefly, a single cell suspension was obtained by removing the brain and disrupting the tissue. All techniques were performed on sterile tissue culture plates on ice. The plate contained Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. The cell suspension was transferred to a 15 ml triangular tube and Percoll (Pharmacia, Uppsala, Sweden) was added at a final concentration of 30%. 1 ml of 70% Percoll was placed on the bottom layer and the cells were centrifuged at 1300 × g for 30 minutes at 4 ° C. 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 Diego, Calif.) Were used, respectively, for invasive CD4 + T cells and invasive CD8 + T Cells were detected. As controls, FITC antibody and PE antibody having the same isotype were used. Cells were incubated with antibody for 1 hour at 40 ° C., washed, fixed in 1% paraformaldehyde, and analyzed on a FACStar (Becton Dickinson, Mountain View, Calif.). The percentage of cells that infiltrated into the CNS was determined with Cell quest flow cytometry software. Positive cells were determined by subtracting the fluorescence of the experimental sample from the fluorescence of the background and control samples. The total number of CD4 T cells and CD8 T cells was determined by multiplying the percentage of positive cells in the population through the gate by the number of cells isolated from the spinal cord. Data are presented as mean + standard error mean.
[0098]
H & E staining Longitudinal frozen spinal cord sections were cut and fixed in acetic alcohol. The sections were stained in Harris hematrixin, washed with tap water, and counterstained in 1% eosin. Slides were dehydrated in increasing alcohol and encapsulated in paramount (Fisher Scientific).
[0099]
Immunohistochemistry Animals were transcardia perfused with 4% paraformaldehyde for 24 hours, 3 days, and 14 days after injury. The spinal cord was removed and submerged in 25% sucrose overnight. The tissue was then embedded in OCT, 12 μm thick longitudinal axial cryosections were cut and placed on a slide. Sections were blocked in 10% normal goat serum (NGS: diluted with PBS) for 1 hour at room temperature. Primary antiserum (rat anti-mouse CD4 monoclonal antibody, diluted 1: 200 in 10% NGS, PharMingen; rat anti-mouse CD11b, Serotec) was applied to sections overnight at 4 ° C. Sections were rinsed 3 times with PBS and goat anti-rat IgG biotinylated antiserum (1: 200 dilution in 10% NGS, Vector Laboratories) was applied and the slides were incubated for 1 hour at room temperature. Sections were rinsed 3 times in PBS and incubated in methanol: 30% hydrogen peroxide solution (100: 1) for 10 minutes. The sections were rinsed three times in PBS and incubated for 30 minutes at room temperature in ABC solution (Vector Laboratories). DAB substrate solution (Vector Laboratories) was used to visualize antibody binding. Sections were dehydrated in increasing alcohol. Permount (Fisher Scientific) was used for encapsulation. No immune response was observed when the primary antibody was removed. Neurons were stained using the same procedure, with the following modifications: sections were blocked overnight, and the primary antiserum used was mouse anti-NeuN monoclonal (Chemicon, 1: 100 dilution). The secondary antibody used was a biotinylated rat-adsorbed horse anti-mouse antibody (Vector Laboratories, diluted 1: 200). Neurons and CD4 positive T cells in 1 ml on each side of the injury were counted at 10 × magnification. Only clearly labeled cells were counted.
【Example】
[0100]
Example I
(Upregulation of CXCL10 level and T cell count after CNS injury)
This example describes the upregulation of CXCL10 levels and T cell numbers after CNS injury.
[0101]
To determine if CXCL10 mRNA levels were increased after hemisection injury, hemisection injury of the adult mouse spinal cord was performed on adult C57B16 female mice at T9 using a sharp blade. Thereafter, the dorsal half of the T9 vertebra was removed. Total RNA was obtained from hemisectioned spinal cord using TRIzol® reagent (Life Technologies (GIBCO-BRL) Rockville, MD) at 6, 12, 18, 24 hours after injury. Extracted on days 3, 7 and 14, and the level of IP-10 mRNA transcripts was determined by Lane et al. Virol. 74 (3): 415-424 (2000), which is incorporated herein by reference, each by a ribonuclease protection assay (RPA) using the CK-1 chemokine probe set previously described. Determined at time. The amount of mRNA transcript was determined by scanning autoradiography, and the intensity of individual bands was determined for the internal L32 control using NIH 1.61 imaging software.
[0102]
As shown in FIG. 1, CXCL10 mRNA levels increased by 6 hours after injury, then gradually declined and remained above basal levels even 14 days after injury. CXCL10 mRNA was not detectable in intact spinal cord tissue.
[0103]
The spinal cord was also dissected at 3 and 14 days after injury so that the injury site marked at the time of surgery was located centrally within the dissected tissue. A longitudinal section where the central tube is clearly visible was selected and digitized. For cell counts, the number of CD4 immunopositive cells within the total tissue area extending 1 millimeter on either side of the injury site was determined using steric analysis. Only immunolabeled cells with clearly discernable Hoechst stained nuclei were scored.
[0104]
Quantitative analysis of longitudinal tissue sections immunostained with CD4 showed that CD4 + T cells were 1 mm on either side of the injury site in the hemisection damaged spinal cord 3 days after injury compared to the uninjured spinal cord. It was shown to exist at an increased density in the extended region. The above studies show that IP-10 levels and T cell numbers are upregulated after CNS injury.
[0105]
Example II
(Neutralization of T cell accumulation with reduced CXCL10 levels in and around the injury site after CNS injury)
To determine the effect of anti-IP-10 treatment after dorsal hemisection injury, hemisection-injured adult mice are started 1 day prior to injury and continued every other day until 7 days after injury. An intraperitoneal injection of 0.5 ml of anti-IP-10 antibody (approximately 0.5 mg / ml) was performed. The mice were sacrificed 3 or 14 days after injury and the spinal cord was dissected so that the injury site was centered within the dissected tissue. A longitudinal section where the central tube is clearly visible was selected and digitized. For cell counts, the number of CD4 immunopositive cells within the total tissue area extending 1 millimeter on either side of the injury site was determined using steric analysis. Only immunolabeled cells with clearly discernable Hoechst stained nuclei were scored as described in Example I above.
[0106]
Quantitative analysis of longitudinal tissue sections immunostained with CD4 showed that CD4 + T cells were damaged at 3 days after injury in untreated hemisection damaged spinal cord compared to anti-CXCL10 treated hemisection damaged spinal cord. It was shown to be present at an increased density in the region extending 1 mm on either side. These studies show that neutralization of IP-10 reduced T cell recruitment after CNS injury.
[0107]
To determine the effect of anti-IP10 treatment on behavioral deficits associated with CNS injury, anti-CXCL10 antibody-treated hemisection-damaged mice, untreated hemisection-damaged mice and undamaged control mice were Were subjected to kinematic analysis daily from 1 to 13 days after hemisection injury by two observers who were first adapted for 4 days and then blinded for the treatment group. Animals were videotaped from below a 4'x4 'plexiglass surface with defined grid lines and the recordings were analyzed using Adobe Premiere video editing software (Adobe Systems, Inc., San Jose, CA). .
[0108]
Four kinematic parameters were evaluated: stride length, stride width, limb rotation and toe spread. As shown in FIG. 2, all hemisection damaged mice treated with anti-IP-10 antibody have a significantly larger stride length and significantly smaller than untreated hemisection damaged mice It had stride width, toe spread and limb rotation. Progressive behavioral improvements are compared for each kinematic parameter with data points for all animals over the first 3 days after injury to data points for all animals over the last 3 days after injury. Was evaluated by Untreated hemisection-damaged mice showed no change in kinematic parameters during the recovery period (p> 0.05). In contrast, treated hemisection-damaged mice showed a statistically significant progressive improvement for all kinematic parameters during the recovery period (p << 0.01).
[0109]
The spinal cord was dissected 14 days after injury so that the site of injury was centered within the dissected tissue. The longitudinal section where the central tube is clearly visible was selected and digitized. The total tissue area extending 1 mm on either side of the injury site was determined according to Zhang et al. Comp. Neurol. 371: 485-495 (1996), which is incorporated by reference herein, was measured using an MCID analysis system.
[0110]
As shown in FIG. 3, untreated hemisection-injured mice have an average 49.4% reduction in total tissue area that extends 1 mm on either side of the injury compared to an uninjured control spinal cord. did. In contrast, morphological analysis of anti-IP-10 antibody treated hemisection damaged mice showed 20.9% in total tissue area extending 1 mm on either side of the injury compared to the intact control spinal cord. This represents a 68% reduction in tissue loss compared to untreated hemisection damaged mice. The number of NeuN immunopositive neurons determined within a total tissue area extending 1 mm on either side of the injury site was determined using stereoanalysis. NeuN monoclonal antibody (MAB377, Chemicon, Temecula, CA) that recognizes neuron-specific nuclear proteins and reacts with most neuronal cell types was used according to the manufacturer's instructions. Only immunolabeled cells with clearly discernable Hoechst stained nuclei were scored. Quantitative analysis of longitudinal tissue sections immunostained with NeuN showed that NeuN + neurons extended 1 mm to either side of the injury site in anti-CXCL1010 treated hemisection damaged mice compared to untreated hemisection damaged mice It was shown to exist at an increased density within the region.
[0111]
These data indicate that anti-CXCL10 treatment reduced post-traumatic tissue loss after hemisection injury.
[0112]
Example III
(Effect of CXCL10 antibody on lymphocyte infiltration)
CXCL10 mRNA levels increased after hemisection injury to the adult mouse spinal cord. Total RNA was analyzed at 6 hours after injury (n = 3), 12 hours (n = 3), 18 hours (n = 3), 24 hours (n = 3), 3 days (n = 3) At 7 days (n = 3) and 14 days (n = 3), ribonuclease protection assay using CK-1 chemokine probe set, extracted from hemisection damaged spinal cord and CXCL10 mRNA transcript levels (RPA) (Lane, Liu et al., “A central role for CD4 (+) T cells and RANTES in virus-induced central system information and delineation”, J. Virol. ) For each time point. The amount of mRNA transcript was determined by scanning autoradiography and compared to the internal L32 control to determine the intensity of individual bands. CXCL10 mRNA levels rose to an average level of 74.8 ± 21.78 by 6 hours after injury and then gradually declined and remained above basal levels even 14 days after injury, 41.57 The average level is ± 1.80 (FIG. 1). CXCL10 mRNA was not detectable in intact spinal cord tissue.
[0113]
Treatment with anti-CXCL10 reduced lymphocytes and activated macrophage infiltration into the CNS after wounding. In a hemisectioned adult mouse, quantitative analysis of CD4 immunostained longitudinal tissue sections showed that the total number of CD4 + T lymphocytes in the area where either side of the wound area was expanded 1 mm after 3 days of wounding. , 2050 +/− 102 (n = 3, FIG. 5). CD4 + T lymphocytes were not detected in unwounded spinal cord tissue. These data indicate that CXCL 10 levels and T lymphocyte counts are upregulated after trauma, up-regulation of CXCL10 6 hours after contusion wounds in adult rats, and within the first week after wounding Support previous studies demonstrating the peak of T lymphocyte infiltration (McTigue, Tani et al., “Selective chemokin mRNA accumulation in the spinal cord after conjugation injury,” J. Neurosci. : 368-76).
[0114]
In adult mice that received hemisectioned anti-CXCL10 treatment, quantitative analysis of CD4 immunostained longitudinal tissue sections showed that within 3 days after wounding, either side of the wound area expanded 1 mm. The total number of CD4 + T lymphocytes was 622 +/− 202 (n = 3), showing a 70% reduction compared to untreated hemisection wound animals (FIG. 5).
[0115]
FACS analysis showed that CD4 + lymphocytes, CD8 + lymphocytes, and F480 / CD45 + in anti-CXCL10 treated hemisection wound mice compared to untreated hemisection wound mice at both 3 and 14 days after wounding. It showed that the number of macrophages decreased.
[0116]
In particular, the FACS data summarized in Table 1 below is present in the spinal cord of these animals 3 days after wounding compared to T lymphocytes and activated macrophage / glia levels in untreated hemisection wound mice Demonstrated a significant decrease in CD4 + T lymphocytes (66.4% decrease), CD8 + T lymphocytes (57.4% decrease) and activated macrophages / microglia (35.5% decrease). Furthermore, FACS data showed that CD4 + T lymphocytes present in the spinal cord of treated animals 14 days after wounding (58%) compared to T lymphocytes and activated macrophage / glia levels in untreated hemisection wound mice. Decrease), CD8 + T lymphocytes (64% decrease) and activated macrophages / microglia (43.2% decrease) demonstrated a significant decrease (n = 4; Table 1).
[0117]
(Table 1. Cellular infiltration is reduced in anti-CXCL10 treated mice)
[0118]
[Table 1]

Example IV
(Effect of CXCL10 treatment on tissue sparing and loss of function)
To determine whether CD4 + T lymphocytes attenuated in response to traumatic spinal cord wounds were involved in tissue sparing or decreased functional loss, hemisectioned adult mice were treated with anti-CXCL10 antibody. Intraperitoneal injections were made every other day from 1 day before wounding to 9 days after wounding and sacrificed 14 days after wounding (n = 11). Tissue sparing after hemisection wounds was significantly greater in mice treated with anti-CXCL10 antibody compared to untreated hemisection wound mice (n = 8). Morphological analysis of longitudinal tissue sections in which the central tube is clearly visible is described in Zhang et al., (1996) (Zhang, Fujiki et al. using a MCID analysis system such as described in is implied in mice carrying a mutation (Wlds) that causes delayed Walledian generation, "J. Comp. Neurol., (1996) 371 (3): 485-95). . Untreated hemisection wound mice had an average of 49.4% reduction across the entire tissue area expanding 1 mm on either side of the wound compared to the unwounded control spinal cord (FIG. 3). ). In contrast, morphometric analysis of anti-CXCL10 antibody treated hemisection wound mice showed 20.9% over the entire tissue area that was expanded 1 mm on either side of the wound compared to the unwounded control spinal cord. And a 68% reduction in tissue loss compared to untreated hemisection wound mice (FIG. 3).
[0119]
Combined with greater tissue sparing, treated hemisection wound mice contain significantly more neurons around the wound site than untreated hemisection wound mice after 14 days of wounding. Quantitative analysis of NeuN immunostained longitudinal tissue sections from untreated hemisection wound mice showed that NeuN + neurons in a region that expanded 1 mm on either side of the wound site 14 days after wounding. The average total number was 267 +/− 72. In contrast, the quantitative analysis of NeuN immunostained longitudinal tissue sections from hemisection wound mice treated with anti-CXCL10 antibody shows that after 14 days of wound, either side of the wound site is expanded by 1 mm. The average total number of NeuN + neurons in the body is 1170 +/− 184, with 438% more neurons than untreated hemisection wound animals.
[0120]
These data indicate that anti-CXCL10 treatment after dorsal hemisection wound significantly reduced tissue loss after trauma.
[0121]
(Example V)
(Effect of anti-CXCL10 antibody treatment on behavioral deficits)
The behavioral deficits after hemisection wounds in mice treated with anti-CXCL10 antibody were progressively reduced compared to untreated controls. Treated and untreated hemisection wound mice as well as non-wound control mice (n = 8) daily from 1-13 days after hemisection wound wound by two observers who are blind to the treatment group. Were subjected to kinematic analysis. The animals were videotaped from the bottom of a 3 ′ × 1 ′ plexiglass surface with a defined 1 cm grid line and the recordings analyzed using video editing software. Four kinematic parameters were evaluated; slide length of the front and rear limbs, slide of the front and rear limbs, rotation of the front and rear limbs and toe spread of the front and rear limbs. These analyzes showed that all hemisection wound mice treated with anti-CXCL10 antibody had significantly larger front and rear limb slide lengths, significantly fewer front and rear limbs than untreated hemisection wound control mice after 14 days of wounding. It has been shown that it has a toe spread of the front and rear limbs and a rotation of the front and rear limbs (FIG. 2). Progressive behavioral improvements are compared for each kinematic parameter with data points for all animals over the first 3 days after wounding and data points for all animals over the last 3 days after wounding Was evaluated by Untreated hemisection wounded mice showed no change in kinematic parameters during the recovery period (p> 0.05). In contrast, treated hemisection wound mice showed a statistically significant progressive improvement in all kinematic parameters during the recovery period (p <0.01). By 14 days after wounding, the behavioral scores for all four behavioral parameters of treated hemisection wound mice were not significantly different from non-wound control mice.
[0122]
These data indicate that anti-CXCL10 antibody treatment reduces neurological impairment after spinal cord wounding.
[0123]
Throughout this application, various publications are referenced within parentheses. In this application, the entire disclosures of these publications are hereby incorporated by reference in order to more fully describe the level of the field to which the present invention pertains.
[0124]
Although the present invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments described are merely illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the accompanying claims.
[Brief description of the drawings]
[0125]
FIG. 1 shows increased CXCL10 mRNA levels after hemisection injury to adult mouse spinal cord.
FIG. 2 shows a hemilateral to adult mouse spinal cord tapering in mice treated with anti-CXCL10 antibody compared to untreated control mice as measured by the following four kinematic parameters: Shows behavioral deficits after cut injury: 2 (A) stride length; 2 (B) toe spread; 2 (C) stride width; and after 2 (D) Leg paw rotation.
FIG. 2 shows the hemilateral to adult mouse spinal cord tapering in mice treated with anti-CXCL10 antibody compared to untreated control mice as measured by the following four kinematic parameters: Shows behavioral deficits after cut injury: 2 (A) stride length; 2 (B) toe spread; 2 (C) stride width; and after 2 (D) Leg paw rotation.
FIG. 3 shows the following: 3 (A) Overall pathology of spinal hemisection injury from dorsal view 14 days after injury in untreated mice; 3 (B) Injury in untreated mice Longitudinal section of dorsal hemisection injury 14 days after spine; 3 (C) Overall pathology of spinal hemisection injury from dorsal view 14 days after injury in anti-CXCL10 antibody treated mice; and 3 (D) Longitudinal section of dorsal hemisection injury 14 days after injury in anti-CXCL10 antibody treated mice. Attenuation of CD4 + T lymphocytes in response to traumatic spinal cord injury mediated by external anti-CXCL10 antibody is associated with tissue sparing.
FIG. 4 shows CD4 + stained light micrographs demonstrating the following: 4 (A) anti-CXCL10 antibody treatment in treated mice compared to 4 (B) untreated mice Reduces recruitment of strong T lymphocytes after hemisection injury.
FIG. 5 shows that attenuation of the CD4 + T lymphocyte response to traumatic spinal injury mediated by anti-CXCL10 antibody is associated with tissue saving.

Claims (28)

A method of reducing severe secondary tissue degeneration associated with central nervous system injury in a subject, the method comprising: Administering an effective amount of a neutralizing agent specific for a 10 kDa interferon-inducible protein (CXCL10). The method of claim 1, wherein the subject is a mammal. The method of claim 2, wherein the subject is a human. The method of claim 1, wherein the central nervous system injury is spinal cord injury. The method of claim 1, wherein the central nervous system injury is brain injury. The method according to claim 1, wherein the neutralizing agent specific for the 10 kDa interferon-inducible protein (CXCL10) is an anti-CXCL10 antibody. A method of reducing severe secondary tissue degeneration associated with central nervous system injury in a subject, comprising: an antibody capable of specifically binding to a 10 kDa interferon-inducible protein (CXCL10) or Administering an effective amount of the fragment to a subject in need thereof. 8. The method of claim 7, wherein the central nervous system injury is selected from the group consisting of mechanical injury, spinal cord contusion, spinal cord compression injury, spinal cord laceration, spinal cord disruption, and demyelinating condition. 9. The method of claim 8, wherein the demyelinating condition is multiple sclerosis. A method of reducing severe secondary tissue degeneration associated with a pathological central nervous system condition in a subject, wherein the method specifically binds to a 10 kDa interferon-inducible protein (CXCL10). Administering an effective amount of the resulting antibody or fragment thereof to a subject in need thereof. 11. The method of claim 10, wherein the pathological condition is myelin loss. 12. The method of claim 11, wherein the myelin loss is selected from the group consisting of acute disseminated encephalomyelitis, post-infection myelin loss, post-vaccine myelin loss, acute necrotizing encephalomyelitis, and progressive necrotizing myelopathy. . The method of claim 1, wherein the neutralizing agent is administered within 1 hour of the injury. 8. The method of claim 7, wherein the antibody or fragment thereof is administered within 1 hour of the injury. 11. The method of claim 10, wherein the antibody or fragment thereof is administered within 1 day of diagnosis of the condition, and the administration is repeated daily for an additional 25 days. 2. The method of claim 1, wherein the neutralizing agent is administered daily until at least 25 days after injury. 8. The method of claim 7, wherein the antibody or fragment thereof is administered daily until at least 25 days after injury. A method of reducing severe secondary tissue degeneration associated with central nervous system injury in a subject, which can reduce the amount of intracellular 10 kDa interferon-inducible protein (CXCL10). Administering an effective amount of the polynucleotide agents to a subject in need thereof. 19. The method of claim 18, wherein the agent is selected from the group consisting of an antisense oligonucleotide and a ribozyme, wherein the antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10. A method of reducing severe secondary tissue degeneration associated with a pathological central nervous system condition in a subject, the method comprising measuring the amount of intracellular 10 kDa interferon-inducible protein (CXCL10). Administering an effective amount of a polynucleotide agent that can be reduced to a subject in need thereof. 21. The method of claim 20, wherein the agent is selected from the group consisting of an antisense oligonucleotide and a ribozyme, wherein the antisense oligonucleotide or ribozyme specifically binds to a polynucleotide encoding CXCL10. The method of claim 1, wherein the agent is administered in a composition containing liposomes that can enhance penetration of the blood brain barrier by the agent. 8. The method of claim 7, wherein the antibody or fragment thereof is administered in a composition comprising a liposome capable of enhancing blood brain barrier penetration by the antibody or fragment thereof. 11. The method of claim 10, wherein the antibody or fragment thereof is administered in a composition comprising a liposome capable of enhancing blood brain barrier penetration by the antibody or fragment thereof. 19. The method of claim 18, wherein the antibody or fragment thereof is administered in a composition comprising a liposome capable of enhancing blood brain barrier penetration by the antibody or fragment thereof. 21. The method of claim 20, wherein the antibody or fragment thereof is administered in a composition comprising a liposome capable of enhancing blood brain barrier penetration by the antibody or fragment thereof. A composition for use in reducing severe secondary tissue degeneration associated with central nervous system injury, comprising: a CXCL10 neutralizing agent and a pharmaceutically acceptable carrier. A composition comprising. A kit comprising a CXCL10 neutralizing agent and instructions for use in a method of reducing severe secondary tissue degeneration associated with central nervous system injury.

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