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Title:  Neurotransmission disorders
United States Patent: 
7,267,820
Issued: 
September 11, 2007

Inventors: 
Vincent; Angela (Oxford, GB), Hoch; Werner (Houston, TX)
Assignee: 
Isis Innovation Limited (Oxford, GB)
Max-Planck Gesellschaft zur Foerderung der Wissenschaften e.V. (Munich, DE)

Appl. No.: 
10/311,575
Filed: 
June 15, 2001
PCT Filed: 
June 15, 2001
PCT No.: 
PCT/GB01/02661
371(c)(1),(2),(4) Date: 
June 06, 2003
PCT Pub. No.: 
WO01/96601
PCT Pub. Date: 
December 20, 2001


 

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Abstract

There is disclosed a method for diagnosing neurotransmission or developmental disorders in a mammal comprising the step of detecting in a bodily fluid of said mammal autoantibodies to an epitope of the muscle specific tyrosine kinase (MuSK). One such method comprises a) contacting said bodily fluid with said MuSK or an antigenic determinant thereof; and b) detecting any antibody-antigen complexes formed between said receptor tyrosine kinase or an antigenic fragment thereof and antibodies present in said bodily fluid, wherein the presence of said complexes is indicative of said mammal suffering from said neurotransmission or developmental disorders. Also disclosed are kits for use in the diagnosis of neurotransmission and subsequent developmental disorders.

Description of the Invention

This application is a national stage filing under 35 U.S.C. .sctn. 371 of PCT International application PCT/GB01/02661, filed Jun. 15, 2001, which was published under PCT Article 21(2) in English.

The present invention is concerned with neurotransmission disorders and, in particular, with a method of diagnosing such disorders in mammals. Also provided by the present invention are kits for use in said diagnosis.

Myasthenia gravis (MG) is a chronic autoimmune disorder of neuromuscular transmission resulting in muscle weakness. The key feature of weakness due to MG is its variability. Patients generally experience a waning of strength throughout the day with a tendency to fatigue later in the day or even towards the end of a particular task. A symptom of MG is often ocular weakness, causing ptosis (drooping eyelids) and/or diplopia (double vision). Other symptoms include leg weakness, dysphagia and slurred or nasal speech. Symptoms of weakness tend to worsen with various stressors, such as, exertion, heat and infection.

In 1960 it was discovered that MG was caused by antibodies against the acetyl choline receptor (AChR) and that it is therefore autoimmune in origin. Today MG is one of the most characterised of neurological disorders which has consequently lead to treatments which vastly improve the length and quality of life of myasthenics. Approximately 10 people in every million of a population contract this disease in one year. There is no racial predominance and 75% of MG patients less than 40 years of age are female and 60% of those older than 40 years are male.

Approximately 80% of patients with MG possess within their plasma autoantibodies that are immunoprecitipatable with radiolabelled AChR. The remaining 20% of MG patients do not, however, exhibit such antibodies in their plasma but do have similar symptoms and respond to the same therapies such as plasma exchange and immunosuppression. Accordingly, it has not been established whether these patients have the same or a distinct and separate MG condition(3,4). Autoantibodies are naturally occurring antibodies directed to an antigen which an individual's immune response recognises as foreign even though that antigen actually originated in the individual. They may be present in the circulatory system as circulating free antibodies or in the form of circulating immune complexes bound to their target depending on the nature of the antigen concerned.

Human plasma from patients who were anti-AChR autoantibodies negative (AAAN or previously known as sero-negative MG), were investigated for alternative autoantibodies and one candidate autoantibody was that one for the MuSK protein.

The present inventors surprisingly found that many of the 20% of MG patients which do not exhibit any autoantibodies to AChR, instead have IgG antibodies directed against the extracellular N-terminal domains of MuSK, a receptor tyrosine kinase located on the cell surface of neuromuscular junctions, indicating that they are afflicted with a form of MG which has a different etiology from MG characterised by circulating autoantibodies to AChR.

The MuSK protein has been sequenced and the protein characterised recently by Valenzuela et al (International patent application number PCT/US96/20696, published as WO97/21811). It is a receptor tyrosine kinase (RTK) located on the cell surface of muscle cells at the neuromuscular junction. Ligands bind to RTKs at the binding site on the extracellular side of the receptor, which induces transmission of a signal cascade to intracellular target proteins. RTKs are classified according to their function and members of these families share high homology in their amino acid sequence as well as functionality.

At the neuromuscular junction (NMJ) where the motor nerve axon dendrites meet the muscle cell basal membrane, important physiological signals are exchanged between these adjacent cells. An example of this is the chemical transmitter acetyl choline which passes through the synaptic cleft from the nerve cell, and is then rapidly and specifically bound by the AChR at the muscle cell wall. This in turn begins a cascade of events which ultimately leads to contraction of the muscle cells.

The post synaptic structure at the muscle cell wall is termed the motor endplate which is densely packed with protein and lipid, thereby giving an electron dense appearance when observed by electron microscopy. The muscle AChRs are present here, and it is believed that signalling gives rise to concentrations of proteins there by two mechanisms; one is altered distribution of pre-existing membrane proteins and the other is by induction of localised transcription of specific genes only by subsynaptic nuclei underlying the NMJ.

Development of the neuromuscular junction is initiated through activation of MuSK. Agrin isoforms, released from the motorneuron, trigger MuSK and muscle acetylcholine receptor (AChR) phosphorylation resulting in clustering of AChRs and other proteins of the postsynaptic apparatus(1). Agrin's ability to cause AChR clustering in cultured myotubes has been shown to be inhibited by anti agrin antibodies. It is currently accepted that agrin does not bind directly to MUSK, but via a hypothetical agrin-binding component termed Myotubule Associated Specificity Component (MASC) (1,11). No disease associated with either MuSK, MASC, or agrins has been reported and their roles in adult muscle have not yet been elucidated.

It has already been shown that anti AChR autoantibody negative MG is caused by humoral IgG antibodies: it can be successfully treated by plasma exchange and other immune therapies(5); transient neonatal MG was reported in the newborn infant of one of the patients with anti-MuSK antibodies(17); and injection of immunoglobulin or IgG preparations into mice caused defects in neuromuscular transmission (5).

The present inventors have therefore now shown that anti-MuSK antibodies have functional effects on agrin-induced AChR clustering in vitro, and direct interference with this agrin/MuSK/AChR pathway may be an important disease mechanism in vivo. MuSK is a relatively new member of the receptor tyrosine kinase (RTK) family. With very few exceptions (for example, see 18), autoantibodies to RTKs have not been implicated in human disorders but the combination of large extracellular domains and functional activities make them attractive potential antigens in other autoimmune conditions. Other members of the RTK family are mutated in inherited diseases, and somatic mutations have been found in various tumors (19). MuSK may prove to be involved in congenital as well as acquired muscle disorders.

Therefore, there is provided by a first aspect of the present invention a method of diagnosing neurotransmission disorders in a mammal comprising the step of detecting in a bodily fluid of said mammal autoantibodies to an epitope of the muscle specific tyrosine kinase, MuSK.

More specifically the neurotransmission disorder will preferably be Myasthenia gravis and more particularly a subclass or subtype of MG which is generally found in patients who do not exhibit the ability to immunprecipitate radiolabelled AChR with their bodily fluids.

This aspect of the invention is particularly advantageous because the identification of this new subclass or subtype of MG patients will allow for more accurate and speedy diagnosis of individuals by medical practitioners. The method according to this aspect of the invention will allow for detection of neurotransmission abnormalities that are either congenital or acquired, for example, postnatally or prenatally from transmission from the mother to the foetus. As set out in more detail in the example provided, some mothers of babies with developmental disorders, such as paralysis and fixed joints were identified as having antibodies to MuSK, which were transferred placentally.

Until now, MuSK has been studied primarily in NMJ development. The presence of antibodies to the extracellular domain of MuSK in an acquired disorder implies that MuSK is functional at the adult NMJ, and implicates MuSK as a novel target for pathogenic autoantibodies causing Myasthenia gravis. The isolation and purification of this anti-MUSK autoantibody will give rise to a useful product which may be exploitable as an indicator of neurotransmission diseases.

Preferably, the method according to the first aspect of the invention, comprises the steps of a) contacting said bodily fluid with said MUSK or an antigenic determinant thereof; and b) detecting any antibody-antigen complexes formed between said MuSK or an antigenic fragment thereof and antibodies present in said bodily fluid, wherein the presence of said complexes is indicative of said mammal suffering from said neurotransmission disorders.

The actual steps of detecting autoantibodies in a sample of bodily fluids may be performed in accordance with immunological assay techniques known per se in the art. Examples of suitable techniques include ELISA, radioimmunoassays and the like. In general terms, such assays use an antigen which may be immobilised on a solid support. A sample to be tested is brought into contact with the antigen and if autoantibodies specific to the protein are present in a sample they will immunologically react with the antigen to form autoantibody-antigen complexes which may then be detected or quantitatively measured. Detection of autoantibody-antigen complexes is preferably carried out using a secondary anti-human immunoglobulin antibody, typically anti-IgG or anti-human IgM, which recognizes general features common to all human IgGs or IgMs, respectively. The secondary antibody is usually conjugated to an enzyme such as, for example, horseradish peroxidase (HRP) so that detecting of autoantibody/antigen/secondary antibody complexes is achieved by addition of an enzyme substrate and subsequent calorimetric, chemiluminescent or fluorescent detection of the enzymatic reaction products.

Thus, in one embodiment the antibody/antigen complex may be detected by a further antibody, such as an anti-IgG antibody. Complexes may alternatively be viewed by microscopy. Other labels or reporter molecules which may be used in a method according to the invention. Preferably, said reporter molecule or label includes any of a heavy metal, a fluorescent or luminescent molecule, radioactive or enzymatic tag. Preferably, the label or reporter molecule is such that the intensity of the signal from the anti-human IgG antibody is indicative of the relative amount of the anti-MuSK autoantibody in the bodily fluid when compared to a positive and negative control reading.

An alternative method of detecting autoantibodies for MuSK or an epitope thereof relies upon the binding of a MuSK or its epitope, together with a revealing label, to the autoantibodies in the serum or bodily fluid. This method comprises contacting MuSK or an epitope or antigenic determinant thereof having a suitable label thereon, with said bodily fluid, immunoprecipitating any antibodies from said bodily fluid and monitoring for said label on any of said antibodies, wherein the presence of said label is indicative of said mammal suffering from said neurotransmission or developmental disorder. Preferably, the label is a radioactive label which may be .sup.125I, or the like. Iodination and immunoprecipitation are standard techniques in the art, the details of which may be found in references (4 and 6).

In a further aspect of the invention, there is provided an assay kit for diagnosing neurotransmission disorders in mammals comprising an epitope of muscle specific tyrosine kinase (MuSK) and means for contacting said MuSK with a bodily fluid from a mammal. Thus advantageously, an assay system for detecting neurotransmission disorders, and particularly Myasthenia gravis in patients who are anti-AChR autoantibody negative (AAAN) is provided. Prior to the present invention there was no basis for providing an immediate clinical diagnosis for such patients.

Also provided by the invention is an isolated or purified autoantibody specific for MuSK. Such an antibody can be detected in bodily fluids of mammals and isolated or purified therefrom using techniques which would be known to the skilled practitioner, such as, immunoabsorption, or immunoaffinity chromatography or high pressure chromatography.

In a further aspect the invention also comprises an isolated or purified antibody specific for an anti-MuSK autoantibody from bodily fluid of a mammal. Such a purified or isolated antibody which is specific for anti-MuSK autoantibody may advantageously be used as a medicament, or in the preparation of a medicament for treating neurotransmission disorders in a mammal, and preferably a human suffering from Myasthenia gravis. Such an antibody may also be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, excipient or diluent therefor. Antibodies, polyclonal or monoclonal may be prepared using techniques which are known in the art. For example, the technique described by Kohler & Milstein (1975, Nature 256:495-497) for developing hybridomas capable of producing monoclonal antibodies may be used. Monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse monoclonal antibodies. Chimeric antibody molecules may be prepared containing a mouse antigen binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6581, Takeda et al., 1985, Nature 314:452). For production of antibody various host animals can be immunized by injection with anti-MuSK autoantibody, or a fragment or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

The present invention includes not only complete antibody molecules but fragments thereof. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques, for example, such fragments include but are not limited to the F(ab').sub.2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragments and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

The antibody which is specific for anti-MuSK autoantibodies may also, advantageously, be used in a diagnostic kit for detecting neurotransmission disorders, such as Myasthenia gravis. As aforementioned any protein which binds to the autoantibody may also be used such as an epitope or fragment of the MuSK protein itself. Such a kit comprises an isolated or purified antibody specific for anti-MuSK autoantibody according to the invention and means for contacting said antibody with a bodily fluid of a said mammal.

In accordance with the present invention a bodily fluid should be taken to mean plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid or nipple aspirate. In general, however, the methods of the invention will be performed on samples of serum or plasma.

In the pharmaceutical composition of the invention, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier can also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, solubility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

The antibody or the MuSK protein or fragment thereof or the pharmaceutical composition of the invention may be administered orally. In this embodiment the antibody, MuSK or its eptopic fragment, or pharmaceutical composition of the invention may be encapsulated and/or combined with suitable carriers in solid dosage forms which would be well known to those of skill in the art.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual.

In a further aspect, the present invention comprises a method of treating a patient suffering from a neurotransmission disorder such as Myasthenia gravis comprising administering to said patient an effective amount of an antibody according to the invention or a MuSK protein or an epitope thereof.

In an even further aspect, the invention comprises a method for making a pharmaceutical formulation for the treatment of neurotransmission disorders, comprising the steps of isolating or purifying an antibody or MuSK protein or fragment thereof according to the invention, manufacturing bulk quantities of said antibody and formulating the antibody in a compound including a pharmaceutically acceptable carrier, diluent or excipient therefor.

In an even further aspect, the invention comprises a method of identifying compounds capable of alleviating or treating neurotransmission disorders, comprising the steps of contacting a candidate compound in the presence of MuSK or an epitope thereof and an antibody capable of binding MuSK, wherein a compound that prevents binding of said antibody to MuSK or an epitope thereof is a candidate for treating neurotransmission disorders. Such compounds may also be used in treating neurotransmission or developmental disorders or in the manufacture of a medicament for treating such disorders. The compounds identified may also, as would be appreciated by those of skill in the art, serve as lead compounds for the development of analogue compounds. The analogues should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to the polypeptides of the invention in substantially the same way as the lead compound. In particular, the analogue compounds have spatial electronic properties which are comparable to the binding region, but can be smaller molecules than the lead compound, frequently having a molecular weight below about 2 kD and preferably below about 1 kD. Identification of analogue compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available; e.g., Rein, Computer-Assisted Modelling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, N.Y., USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art; see also supra. Furthermore, peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used.
 


Claim 1 of 12 Claims

1. A method for diagnosing neurotransmission or developmental disorders related to muscle specific tyrosine kinase (MuSK) in a mammal comprising the step of detecting in a bodily fluid of said mammal autoantibodies to an epitope of muscle specific tyrosine kinase (MuSK).

 

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