CN110824156B - Diagnosis of neuroautoimmune disease - Google Patents

Abstract

The present invention relates to a method for diagnosing a disease comprising the step of detecting autoantibodies bound to DNM1 in a sample comprising antibodies of a patient, to a polypeptide comprising DNM1 or variants thereof, to the use of said polypeptide for diagnosing a disease, to autoantibodies bound to DNM1 for diagnosing a disease, to a method for isolating autoantibodies bound to DNM1, to a pharmaceutical composition or medical device comprising said polypeptide according to the invention, to a kit for diagnosing a disease comprising said polypeptide or said medical device, and to the use of said polypeptide or autoantibody for preparing a kit or medical device.

Description

Diagnosis of neuroautoimmune disease

Technical Field

The present invention relates to a method for diagnosing a disease comprising the step of detecting autoantibodies bound to DNM1 in a sample comprising antibodies of a patient, to a polypeptide comprising DNM1 or variants thereof, to the use of said polypeptide for diagnosing a disease, to autoantibodies bound to DNM1, to the use of autoantibodies for diagnosing a disease, to a method for isolating autoantibodies bound to DNM1, to a pharmaceutical composition or medical device comprising said polypeptide according to the invention, to a kit for diagnosing a disease comprising said polypeptide or said medical device, and to the use of said polypeptide or autoantibody for preparing a kit or medical device.

Background

The development of diagnostic systems for neurological diseases is a continuing challenge in biomedical science, in large part because many of the symptoms encountered can be explained by a wide variety of causes, including genetic diseases, drug abuse, malnutrition, infection, injury, psychotic diseases, immunodeficiency and cancer.

Since neurological diseases are rarely associated with unique characteristic patterns of clinical symptoms, it is often difficult to provide reliable diagnosis based solely on observations and examinations of affected patients or their medical history.

The importance of early diagnosis is not emphasized. Many neurological disorders, most notably alzheimer's disease and parkinson's disease and multiple sclerosis, cannot be cured, but drugs are available that can be used to slow their progression. In addition, some rare types of cancer are associated with neurological symptoms. The earlier the diagnosis, the better the opportunity to utilize the available therapeutic spectrum for the full benefit of the patient.

This is especially the case in the case of neurological diseases associated with autoantibodies. In some cases, the association between the specific detectable autoantibody and the condition is strong enough to allow immediate diagnosis.

However, even if this is not the case, detection of autoantibodies may indicate to the attending physician the means of treatment that may be used to ameliorate the condition of the patient. There are a wide variety of widely used immunosuppressants that can be used regardless of the nature of the autoantibody target. Alternatively, blood component separation may be used to remove autoantibodies from the patient's blood. In many cases, patients continue to live normally after early diagnosis and treatment of autoimmune diseases of the nervous system.

Diagnostic assays based on autoantibody detection can also confirm diagnosis of diseases other than those associated with autoantibodies. If the result is that the blood sample is free of specific autoantibodies, this may help the attending physician to rule out a range of possibilities and thus narrow down the range of conditions that appear to be possible.

Examples of neurological conditions consistent with the appearance of autoantibodies include: neuromyelitis optica (a disease characterized by loss of visual perception and spinal cord function), and anti-NMDA receptor encephalitis (which is associated with autonomic dysfunction), hypopnea, cerebellar ataxia, hemiparalysis, loss of consciousness, or stress. While the involvement of autoantibodies and the nature of these conditions themselves have been poorly understood previously, many of these diseases can now be diagnosed and effectively treated due to the availability of assays based on autoantibody detection.

Therefore, it is of paramount importance to develop new methods to distinguish between autoantibody-related and non-autoantibody-related neurological diseases.

WO1997/021729 and US 5693476 disclose that NSF, synaptic fusion proteins and VAMP proteins are used as part of an artificially formed complex to identify substances that modulate synaptic transmission, but they do not disclose the presence of autoantibodies that bind to DNM1, let alone the diagnostic usefulness of said autoantibodies.

Nicot et al report in murine experimental autoimmune encephalitis that STX1B transcription and protein levels decreased (Nicot A, ratnakar PV, ron Y, chen CC, elkabes S.Regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal dysfunctions.brain.2003 Feb;126 (Pt 2): 398-412), but do not disclose the presence of autoantibodies that bind DNM1, let alone the diagnostic usefulness of such autoantibodies.

Hirai et al report autoantibodies against VAMP2 in 21% of patients with type 1diabetes by an in vitro transcription/translation immunoprecipitation protocol against secreted vesicle-associated proteins (Hirai H, miura J, hu Y, larsson H, larsson K, lernmark A, ivarsson SA, wu T, kingman A, tzisource AG, notkins AL.selective screening of secretory vesicle-associated proteins for autoantigens in type 1diabetes:VAMP2and NPY are new minor autoantigens.Clin Immunol.2008Jun;127 (3): 366-74). In the same report, the authors disclose that they did not detect any antibodies to STX1A, STX1A being a protein with 97.6% homology to STX 1B. They did not disclose the presence of autoantibodies that bind to DNM1, let alone the diagnostic usefulness of said autoantibodies.

Disclosure of Invention

The problem underlying the present invention is to provide new reagents, devices and methods which can be used to support the diagnosis and treatment of autoimmune diseases, preferably of the nervous system or associated with a nervous system disease or a symptom of the nervous system, more preferably selected from the group comprising stiff human syndrome and encephalitis, preferably encephalitis.

Another problem underlying the present invention is to provide new agents, devices and methods which can be used to distinguish autoimmune diseases (in particular of the nervous system, more preferably selected from the group comprising stiff human syndrome and encephalitis, preferably encephalitis) from other diseases besides autoimmune diseases, to a large extent in order to determine the most promising therapeutic regimen, more particularly to determine whether immunosuppressive therapy is moderate.

The problem underlying the present invention is solved by the subject matter of the appended independent and dependent claims.

In a first aspect, the invention relates to the use of an agent for detecting an autoantibody against DNM1 in the preparation of a formulation for diagnosing an autoimmune disease in a subject.

In a preferred embodiment, the formulation is for contact with a sample from the subject.

In a preferred embodiment, the agent is selected from a DNM1 polypeptide or a homolog or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide, and the formulation is used to contact the sample from the subject with a DNM1 polypeptide or a homolog or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide, and to detect binding of an antibody to the polypeptide or homolog.

In preferred embodiments, the DNM1 polypeptide or homologue comprises a sequence according to SEQ ID NO. 1 or a sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity thereto.

In a preferred embodiment, the sample is a body fluid comprising antibodies selected from the group consisting of whole blood, serum, cerebrospinal fluid and saliva.

In a preferred embodiment, the DNM1 polypeptide or homolog or polypeptide comprising one or more epitopes derived from a DNM1 polypeptide is derived from a cerebellum lysate.

In a preferred embodiment, a sample from a subject is contacted with a fragment of a polypeptide or homolog according to the invention or a homolog of said fragment, said fragment having at least 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acids of the sequence according to SEQ ID NO:1, the homolog of said fragment having at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity thereto, and binding of an antibody from the sample to the fragment of the polypeptide or homolog is detected.

In preferred embodiments, the polypeptide or homolog comprises other amino acids that are N-terminal or C-terminal attached and facilitate purification of the polypeptide or homolog.

In a preferred embodiment, the polypeptide or homolog or fragment thereof is linked to a reporter molecule or solid phase.

In preferred embodiments, the subject has or the autoimmune disease is associated with one or more symptoms selected from the group consisting of: the trunk muscles (including parathoracic and lumbar muscles and abdominal muscles, abdominal wall muscles and proximal leg muscles) are progressively stiff, tonic gait, excessive lumbar lordosis, chronic pain, proximal limb and axial muscle spasms, sensitivity to touch and sound, excessive fright, myoclonus, depression, anxiety, phobia, fever, headache, confusion, dysarthria, dysphagia, ocular vibration, vibration hallucination, dizziness, nausea, ataxia, dizziness, seizures, epilepsy and tremors.

In a preferred embodiment, the disease is an autoimmune disease of the nervous system selected from the group consisting of stiff human syndrome, paraneoplastic stiff human syndrome, progressive Encephalomyelitis (PERM) with tonic and myoclonus and encephalitis, and cerebellar encephalitis.

In a second aspect, the present invention relates to the use of a DNM1 polypeptide or homologue according to the invention or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide or a fragment thereof in the manufacture of a medicament for the diagnosis of an autoimmune disease, characterized in that a sample from a subject is contacted with a polypeptide or homologue or fragment according to the invention, and the binding of antibodies from the sample to the polypeptide or homologue or fragment is detected.

In a preferred embodiment, binding of antibodies from the sample to the polypeptide or homolog or fragment is detected using immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays, preferably those from radiolabeled immunoassays, enzyme immunoassays, more preferably ELISA, chemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence, protein arrays, luminescent assays and spot assays, preferably western or dot blot.

In a preferred embodiment, the disease is an autoimmune disease of the nervous system selected from the group consisting of stiff human syndrome, paraneoplastic stiff human syndrome, progressive Encephalomyelitis (PERM) with tonic and myoclonus and encephalitis, and cerebellar encephalitis.

In a third aspect, the present invention relates to a test kit for detecting antibodies, characterized in that it comprises a DNM1 polypeptide or a homologue according to the invention or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide or a fragment thereof.

In a fourth aspect, the present invention relates to a pharmaceutical composition, characterized in that it comprises a DNM1 polypeptide or a homologue according to the invention or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide or a fragment thereof.

In a fifth aspect, the invention relates to a medical device coated with a DNM1 polypeptide or a homologue according to the invention or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide or a fragment thereof or coated with a pharmaceutical composition according to the invention.

In a preferred embodiment, the medical device is selected from the group consisting of slides, preferably for microscopy, biochips, microtiter plates, test strips, membranes, preferably linear dot blots (line blot), chromatographic columns and beads, preferably magnetic beads.

In a sixth aspect, the present invention relates to the use of a DNM1 polypeptide or homologue according to the present invention or a polypeptide comprising one or more epitopes derived from a DNM1 polypeptide or a fragment thereof, for the manufacture of a pharmaceutical composition or medical device for the treatment of an autoimmune disease in a subject, wherein the treatment comprises the steps of:

a. Blood or plasma is taken from a subject,

b. contacting blood or plasma with a pharmaceutical composition according to the invention or a medical device according to the invention to remove disease-related antibodies, an

c. Blood or plasma is reapplied to the subject.

In a preferred embodiment, the disease is an autoimmune disease of the nervous system selected from the group consisting of stiff human syndrome, paraneoplastic stiff human syndrome, progressive Encephalomyelitis (PERM) with tonic and myoclonus and encephalitis, and cerebellar encephalitis.

In a further aspect, the problem is solved by a method for diagnosing a disease comprising the step of detecting an autoantibody binding to DNM1 in a sample comprising antibodies of a patient.

In a further aspect, the problem is solved by a polypeptide comprising a polypeptide selected from the group consisting of DNM1 comprising polypeptides or variants thereof, preferably immobilized, more preferably immobilized on a solid support.

In a further aspect, the problem is solved by the use of a polypeptide according to the invention for the diagnosis of a disease, preferably comprising the step of detecting an autoantibody binding to DNM1 in a sample.

In a further aspect, the problem is solved by a polypeptide according to the invention for use in the treatment of a disease.

In a further aspect, this problem is solved by an autoantibody, preferably an isolated autoantibody which binds to DNM1, wherein the antibody is preferably complexed with a polypeptide according to the invention.

In a further aspect, the problem is solved by the use of an autoantibody according to the invention for diagnosing a disease.

In a further aspect, the problem is solved by a method for isolating an autoantibody that binds to DNM1, comprising the steps of:

a) Contacting a sample comprising the autoantibody with a polypeptide according to the invention under conditions compatible with complex formation, wherein the autoantibody binds to the polypeptide,

b) Isolating the complex formed in step a),

c) Dissociating the complex separated in step b), and

d) Separating the autoantibody from the polypeptide.

In a further aspect, the problem is solved by a pharmaceutical composition, or a medical device, preferably a diagnostic device, comprising a polypeptide according to the invention.

In a further aspect, the problem is solved by a kit for diagnosing a disease comprising a polypeptide according to the invention or a medical device according to the invention, wherein preferably the kit further comprises means for detecting a complex comprising a polypeptide according to the invention and/or an antibody that binds to DNM 1.

In another aspect, the method according to the invention is a method for calibrating a diagnostic test system or for determining the reliability and/or the sufficient capacity (capacity) of such a test system or treatment system for removing autoantibodies from the blood of a patient. In the case of diagnostic test systems, no autoantibodies are detected in the sample of the patient to be diagnosed, but in an artificial solution of known composition, in particular comprising a defined concentration of autoantibodies or a defined concentration of recombinant antibodies bound to autoantigens. The artificial solution can be used as a positive control. As used herein, the term "calibration" may be understood as using an antibody that binds to DNM1 or a variant thereof on a diagnostic test system to obtain qualitative, semi-quantitative or quantitative data for the antibody bound to the corresponding antigen. Preferably, the antigen may be DNM1 or a variant expressed in cells of a tissue section or cells transfected with a nucleic acid molecule comprising genetic information to express the antigen of interest. The diagnostic system may be any system allowing detection of autoantibodies in a sample, e.g. a medical device or a diagnostic device according to the invention.

In the case of a therapeutic system, such as a blood component separation (apharesis) device, the method may be used to develop such a system and test its reliability and/or efficiency and/or adequate capacity. For example, after a blood component separation run or before starting a blood component separation run, a solution comprising a defined concentration of antibodies that bind to a polypeptide of the invention may be contacted with the system, and the method according to the invention may be used to confirm that the system is capable of depleting the antibody solution.

In a preferred embodiment, the patient has one or more, preferably two or more symptoms selected from the group comprising: the trunk muscles (including parathoracic and lumbar muscles and abdominal muscles, abdominal wall muscles and proximal leg muscles) are progressively stiff, tonic gait, excessive lumbar lordosis, chronic pain, proximal limb and axial muscle spasms, sensitivity to touch and sound, excessive fright, myoclonus, depression, anxiety, phobia, fever, headache, confusion, dysarthria, dysphagia, ocular vibration, vibration hallucination, dizziness, nausea, ataxia, paresthesia, muscular atrophy, dizziness, seizures and tremors.

In a further aspect, the problem is solved by the use of a polypeptide according to the invention or an autoantibody according to the invention or an antibody against DNM1 or a medical device according to the invention for the preparation of a kit, a medical device, preferably a diagnostic device, preferably for the diagnosis of a disease.

In a preferred embodiment, the disease is a neurological disease, preferably an autoimmune disease of the nervous system, more preferably selected from the group comprising stiff person syndrome and encephalitis, preferably encephalitis. In a preferred embodiment, the method or use according to the invention is intended to determine whether a disease, preferably a neurological disease, has an autoimmune component, preferably a component suitable for immunosuppressive therapy.

In a preferred embodiment, the sample is a body fluid comprising antibodies, preferably selected from the group comprising whole blood, serum, cerebrospinal fluid and saliva.

In a preferred embodiment, the autoantibodies or complexes are detected using a method selected from the group comprising: immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays such as those from the group comprising radiolabeled immunoassays, enzyme immunoassays, more preferably ELISA, chemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence.

In a preferred embodiment, the medical device is selected from the group comprising: slides, preferably for microscopy, biochips, microtiter plates, test strips, membranes, preferably linear dot blots (line blot), chromatographic columns and beads, preferably magnetic beads.

In a preferred embodiment, the autoantibodies or complexes are detected using a method selected from the group consisting of: immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays such as those from the group comprising radiolabeled immunoassays, enzyme immunoassays, more preferably ELISA, chemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence.

The present invention is based on the surprising finding of the inventors that autoantibodies to DNM1 are present and can be detected in samples from many patients suffering from neurological symptoms, but not in samples obtained from healthy subjects.

Furthermore, the present invention is based on the surprising finding of the inventors that novel neurological diseases can be diagnosed by detecting autoantibodies against a polypeptide of DNM 1.

Without wishing to be bound by any theory, the presence of such autoantibodies suggests that the activity and function of DNM1 and/or downstream effectors is impaired in patients with such autoantibodies, thereby causing neurological symptoms.

Dynamin 1 (DNM 1) is a extracellular peripheral membrane protein that is highly expressed in the brain, particularly in the Cell bodies and axons of neurons, compared to the SNARE protein (Hong W, lev S Tethering the assembly of SNARE complexes. Trends Cell biol. 2014:35-43).

DNM1 is associated with SNARE (soluble NSF attachment protein receptor) complexes involved in the docking and/or fusion of synaptic vesicles in neurons with presynaptic membranes. DNM1 thus regulates neurotransmitter release, including release of gamma-aminobutyric acid (GABA) and glycine (S.ddhof TC, rizo J.synthetic vesicle exochytosis.cold Spring Harb Perspect biol.201CDED 1;3 (12). Pi: a 005637) from inhibitory neurons.

DNM1 is a 97kDa polypeptide containing 864 amino acids. The proteins have mechanochemical properties for tubing and severing the membrane, and are involved in clathrin-mediated endocytosis and other vesicle transport processes. Actin and other cytoskeletal proteins act as binding partners for DNM1, which can also self-assemble resulting in stimulation of gtpase activity.

The present application relates to polypeptides comprising mammalian, preferably human DNM1, or antigenic variants reactive with autoantibodies binding DNM1. Mammalian DNM1 includes those from humans, monkeys, mice, rats, rabbits, guinea pigs, or pigs, with human DNM1 being preferred.

In a more preferred embodiment DNM1 is a polypeptide encoded by database code Q05193, preferably Q05193-1 (UniProt). The database code of the corresponding cDNA is NM-004408 (NCBI).

The teachings of the present application can be implemented not only by using polypeptides, in particular polypeptides comprising the natural sequence of DNM1, or nucleic acids having the exact sequence explicitly (e.g. by function, name, sequence or accession number) or implicitly mentioned in the present application, but also by using variants of such polypeptides or nucleic acids.

In a preferred embodiment, the term "variant" as used herein may refer to at least a fragment of the full-length sequence referred to, more specifically one or more amino acid or nucleic acid sequences truncated at one or both ends by one or more amino acids relative to the full-length sequence. Such fragments comprise or encode a peptide of at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 consecutive amino acids having the original sequence or variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

In another preferred embodiment, the term "variant" refers not only to at least one fragment, but also to a polypeptide or fragment thereof comprising at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95% of the reference amino acid sequence mentioned or fragment thereofAn amino acid sequence that is 96%, 97%, 98%, or 99% identical, wherein other amino acids than those essential for biological activity (e.g., the ability of an antigen-binding (self) antibody) or folding or structure of a polypeptide are deleted or substituted, and/or one or more such essential amino acids are substituted in a conservative manner, and/or amino acids are added, such that the biological activity of the polypeptide is preserved. The prior art includes a variety of different methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity, see, e.g., arthur Lesk (2008), introduction to bioinformatics, oxford University Press,2008,3 ^rd edition. In a preferred embodiment, clustalW software (Larkin, m.a., blackshields, g., brown, n.p., chenna, r., mcGettigan, P.A., mcWilliam, h., valentin, f., wallace, i.m., wilm, a., lopez, r., thompson, j.d., gibson, t.j., higgins, d.g. (2007) Clustal W and Clustal X version.0.bioenformats, 23, 2947-2948) is used, wherein default settings are employed.

In a preferred embodiment, the variant is a linear, non-folding polypeptide, which is optionally denatured.

In preferred embodiments, the polypeptides and variants thereof may also comprise chemical modifications, such as isotopic labeling, or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation, and the like. The person skilled in the art is familiar with methods for modifying polypeptides. Any modification is designed so that it does not eliminate the biological activity of the variant.

In addition, variants may also be produced by fusion with other known polypeptides or variants thereof and comprise an active portion or domain, preferably having at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when aligned with the active portion of a reference sequence, wherein the term "active portion" as used herein refers to an amino acid sequence that is shorter than the full-length amino acid sequence, or in the case of a nucleic acid sequence encodes an amino acid sequence that is shorter than the full-length amino acid sequence, and/or is a variant of the native sequence, but retains at least some biological activity.

In a preferred embodiment, the term "variant" of a nucleic acid includes a nucleic acid whose complementary strand hybridizes to a reference nucleic acid or a wild-type nucleic acid, preferably under stringent conditions. The stringency of hybridization reactions is readily determinable by one of ordinary skill in the art and is generally an empirical calculation depending on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization is generally dependent on the ability of denatured DNA to re-anneal to complementary strands present in the environment at a temperature below their melting temperature: the higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures will tend to make the reaction conditions tighter, while lower reaction temperatures will be less stringent. For additional details and explanation of the stringency of hybridization reactions, see Ausubel, f.m. (1995), current Protocols in Molecular biology, john Wiley & Sons, inc. Furthermore, the person skilled in the art can follow the guidance given in handbook Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, boehringer Mannheim GmbH, mannheim, germany and in Liebl, w., ehrmann, m., ludwig, w., and Schleifer, k.h. (1991) International Journal of Systematic Bacteriology 41:41:255-260 on how to identify DNA sequences by means of hybridization. In a preferred embodiment, stringent conditions are applied to any hybridization, i.e., hybridization occurs only if the probe has 70% or more identity to the target sequence. Probes having a lower degree of identity with respect to the target sequence can hybridize, but such hybrids are unstable and will be removed in a wash step under stringent conditions, e.g., reducing the salt concentration to 2 XSSC or, optionally and subsequently, to 0.5 XSSC, with temperatures (in increasing order of preference) of about 50℃to 68 ℃, about 52℃to 68 ℃, about 54℃to 68 ℃, about 56℃to 68 ℃, about 58℃to 68 ℃, about 60℃to 68 ℃, about 62℃to 68 ℃, about 64℃to 68 ℃, about 66℃to 68 ℃. In particularly preferred embodiments, the temperature is from about 64 ℃ to 68 ℃ or from about 66 ℃ to 68 ℃. It is possible to adjust the salt concentration to 0.2 XSSC, or even 0.1 XSSC. Nucleic acid sequences having a degree of identity of at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to a reference sequence or wild-type sequence can be isolated. In a preferred embodiment, the term variant of a nucleic acid sequence as used herein refers to any nucleic acid sequence which, according to the degeneracy of the genetic code, encodes the same amino acid sequence as the reference nucleic acid sequence, as well as variants thereof.

Variants of the polypeptide have biological activity. In a preferred embodiment, such biological activity is the ability to bind to an autoantibody that binds to DNM1 as found in patients suffering from an autoimmune disease associated with such an autoantibody, preferably associated with a neurological disease such as stiff syndrome, paraneoplastic stiff syndrome, progressive encephalomyelitis with tonic and myoclonus and encephalitis, preferably stiff syndrome associated with such an autoantibody. For example, it may be checked whether variants of DNM1 have such biological activity by determining whether the variant of interest binds to an autoantibody from a patient sample, which autoantibody binds to wild type DNM1, preferably by using an indirect immunofluorescence assay of mammalian cells expressing DNM1 as described in the experimental part of the application.

When used in the practice of the teachings of the present application, polypeptides comprising DNM1 or variants thereof according to the present application (including autoantibodies according to the present application) may be provided in any form and to any degree of purification, from liquid samples, tissues or cells comprising the polypeptides in endogenous form, more preferably cells overexpressing the polypeptides, crude or enriched lysates of such cells, up to purified and/or isolated polypeptides, which are optionally substantially pure. In a preferred embodiment, the polypeptide is a native polypeptide, wherein the term "native polypeptide" as used herein refers to a folded polypeptide, more preferably to a folded polypeptide purified from a tissue or cell, more preferably from a mammalian cell or tissue, optionally from a non-recombinant tissue or cell. In another preferred embodiment, the polypeptide is a recombinant protein, wherein the term "recombinant" as used herein refers to a polypeptide produced at any stage of the production process by using genetic engineering methods, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for over-expression in a cell or tissue or by engineering the sequence of the polypeptide itself. Those skilled in the art are familiar with methods for engineering nucleic acids and encoded polypeptides (e.g., described in Sambrook, j., fritsch, e.f. and manitis, t. (1989), molecular Cloning, CSH or in Brown t.a. (1986), gene Cloning-an interaction, chapman & Hall) and methods for producing and purifying native or recombinant polypeptides (e.g., handbooks "Strategies for Protein Purification", "Antibody Purification", "Purifying Challenging Proteins" (2009/2010), published by GE Healthcare Life Sciences, in Burgess, r.r., deutscher, m.p. (2009), guide to Protein Purification). In a preferred embodiment, a polypeptide is pure if at least 60%, 70%, 80%, 90%, 95% or 99% of the polypeptide in the respective sample consists of the polypeptide as determined by SDS polyacrylamide gel electrophoresis followed by coomassie blue staining and a visualization assay.

If the polypeptide of the invention is provided in the form of a tissue, it is preferred that the tissue is mammalian tissue, e.g. human, rat, primate, donkey, mouse, goat, horse, sheep, pig or cow tissue, more preferably brain tissue, most preferably cerebellum. If a cell lysate is used, it is preferred that the cell lysate comprises a membrane associated with the cell surface or is in fact an enriched fraction of the membrane. If the polypeptide is provided in the form of a recombinant cell, it is preferred that the recombinant cell is a eukaryotic cell, such as a yeast cell, more preferably a cell from a multicellular eukaryotic organism, such as a plant, mammal, frog or insect, most preferably from a mammal, such as a rat, human, primate, donkey, mouse, goat, horse, sheep, pig or cow.

The polypeptides (including any variants) used in the practice of the teachings of the present invention are preferably designed such that they comprise at least one epitope that is recognized by an autoantibody that binds to DNM1 and/or specifically binds to an autoantibody that binds to DNM 1. Any epitope is more preferably an epitope recognized by only such an autoantibody than an antibody other than an autoantibody against DNM 1. In one embodiment, such an epitope comprises 6, 7, 8,9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9 but not more than 16 consecutive amino acids from DNM 1. Those skilled in the art are familiar with guidelines for designing peptides with sufficient immunogenicity, such as those described in Jackson, d.c., fitzmaurice, c.j., brown, l.e., zeng, w. (1999), preparation and properties of totally synthetic immunogenes, vaccine, volume 18, 3-4, month 9 1999, pages 355-361; and Black, m., trent, a., tirrell, m., and Olive, c. (2010), advances in the design and delivery of peptide subunit vaccines with a focus on Toll-like receptor agonists, expert Rev Vaccines, month 2 2010, 9 (2): 157-173. In short, it is desirable that the peptides meet as much as possible the following requirements: (a) it has a high degree of hydrophilicity; (b) It comprises one or more residues selected from the group comprising aspartic acid, proline, tyrosine and phenylalanine; (c) For higher specificity, there is no homology or very low homology with other known peptides or polypeptides; (d) it needs to be sufficiently soluble; and (e) it does not contain glycosylation or phosphorylation sites unless needed for a particular reason. Alternatively, bioinformatics methods may be followed, such as those described by Moreau, V., fleury, C., piquer, D., nguyen, C., novali, N., villard, S., laune, D., granier, C., and Molina, F. (2008), PEPEPOP: computational design of immunogenic peptides, BMC Bioinformatics 2008, 9:71.

When used according to the invention, the polypeptides of the invention comprising DNM1 or variants thereof may be provided in any kind of conformation. For example, the polypeptide may be a substantially unfolded, partially folded, or fully folded polypeptide. In a preferred embodiment, the polypeptide is folded in the sense that the epitope necessary for binding to the autoantibodies of the invention or the protein or variant thereof takes in its entirety the fold taken by the native protein in its natural environment. Those skilled in the art are familiar with methods suitable for determining whether a polypeptide is folded, and if so which structure it has, for example limited proteolysis, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy or X-ray crystallography (see, e.g., banasazak l.j. (2008), foundations of Structural Biology, academic Press; or Teng q. (2013), structural Biology: practical Applications, springer), preferably using circular dichroism.

The polypeptides of the invention may be fusion proteins comprising other amino acid sequences than those taken from DNM1, in particular C-terminal or N-terminal tags, preferably C-terminal tags, which in a preferred embodiment are used herein as additional sequence motifs or polypeptides with functions, which have some biological or physical function and which may for example be used for purification, immobilization, precipitation or identification of the polypeptides of the invention. In a more preferred embodiment, the tag is a sequence or domain capable of binding specifically to a ligand, e.g. selected from the group comprising: his tag, thioredoxin, maltose binding protein, tag of glutathione S-transferase, fluorescent tag, e.g. selected from the group comprising green fluorescent proteins.

The polypeptide of the invention may be an immobilized polypeptide. In a preferred embodiment, the term "immobilized" as used herein refers to molecules bound to a solid support that is insoluble in aqueous solution, more preferably bound by covalent bonds, electrostatic interactions, encapsulation or entrapment (e.g. by denaturing the globular polypeptides in a gel), or by hydrophobic interactions, most preferably by one or more covalent bonds. A variety of different suitable supports, such as paper, polystyrene, metal, silicon or glass surfaces, microfluidic channels, membranes, beads, such as magnetic beads, column chromatography media, biochips, polyacrylamide gels, etc., have been described in the literature, for example in Kim, d. And Herr, a.e. (2013), protein immobilization techniques for microfluidic assays, biomicrofluidics 7 (4), 041501. In this way, the immobilized molecules can be separated from the aqueous solution together with the insoluble carrier in a straightforward manner, for example by filtration, centrifugation or decantation. The immobilized molecules may be immobilized in a reversible or irreversible manner. Immobilization is reversible, for example, if the molecule interacts with the carrier through ionic interactions that can be masked by the addition of high concentrations of salts, or if the molecule binds through cleavable covalent bonds such as disulfide bridges, which can be cleaved by the addition of thiol-containing reagents. Conversely, immobilization is irreversible if the molecule is tethered to the support by a covalent bond that cannot be cleaved in aqueous solution (e.g., a bond formed by the reaction of epoxide groups and amine groups that are often used to couple lysine side chains to the affinity column). The protein may be immobilized indirectly, for example by immobilizing an antibody or other entity having affinity for the molecule, followed by the formation of a complex such that the molecule-antibody complex is immobilized. Various means for immobilizing molecules are described in the literature, for example in Kim, d., herr and a.e. (2013), protein immobilizsation techniques for microfluidic assays, biocofluidics 7 (4), 041501. In addition, various reagents and kits for immobilization reactions are commercially available, for example from Pierce Biotechnology.

It is essential that the sample for diagnosis according to the detection of autoantibodies according to the application comprises antibodies, also called immunoglobulins. Typically, a body fluid sample comprises immunoglobulins from a representative whole set of subjects. However, once provided, the sample may be subjected to further processing, which may include fractionation, centrifugation, enrichment, or isolation of the subject's whole immunoglobulin or any immunoglobulin class, which may affect the relative distribution of the various classes of immunoglobulins.

The reagents, devices, methods and uses described throughout the present application may be used for disease diagnosis. In a preferred embodiment, the disease is a neurological disease. In a more preferred embodiment, the term "neurological disease" as used herein refers to any disease associated with defects in the nervous system, and in another preferred embodiment, the term "PNS" (abbreviation for paraneoplastic neurological syndrome) refers to a systemic disorder caused indirectly by the presence of a tumor, for example, by the release of a substance such as a hormone or cytokine that is not normally produced by the production of cells of tumor origin or by the production or production and release of bioactive cells at increased concentrations. The tumor may be too small to detect.

In a preferred embodiment, the term "diagnostic" as used herein refers to any type of procedure intended for the following purposes: obtaining information helpful in assessing whether a patient is in the past, at the time of diagnosis, or in the future, suffering from or likely to suffer from a disease or disorder than an average or comparison subject (which preferably has similar symptoms); finding out how the disease is progressing or may progress in the future; or evaluating the responsiveness of a patient to a treatment (e.g., administration of an immunosuppressive drug). In other words, the term "diagnosis" includes not only diagnosis, but also the process of predicting and/or monitoring a disease or disorder.

In many cases, a simple test (in other words, determining whether a detectable level of antibody is present in a sample) is sufficient for diagnosis. If autoantibodies can be detected, this would be helpful information to the clinician's diagnosis and would indicate an increased likelihood of the patient suffering from the disease. In a preferred embodiment, an autoantibody is considered detectable if it can be detected using one or more methods selected from the group comprising immunoprecipitation, indirect immunofluorescence, ELISA or linear dot blotting, preferably immunoprecipitation. Experimental details are described in the experimental section of the present application or as in textbooks or practice manuals available at the priority date of the present application. In a preferred embodiment, the relative concentration of antibodies in serum can be determined as compared to the levels that can be found in average healthy subjects. Although in many cases it may be sufficient to determine whether an autoantibody is present or detectable in a sample, a method performed to obtain information helpful for diagnosis may comprise determining whether the concentration is at least 0.1-fold, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000-fold higher than found in average healthy subjects. In a preferred embodiment, the relative concentration of autoantibodies is determined using one or more methods selected from the group comprising semi-quantitative immunoprecipitation, semi-quantitative indirect immunofluorescence, ELISA or semi-quantitative linear dot blotting, preferably ELISA. Experimental details are described in the experimental section of the present application or as in textbooks or practice manuals available at the priority date of the present application.

Those skilled in the art will appreciate that a clinician typically does not merely conclude whether a patient is or is likely to be suffering from a disease, condition, or disorder based on a single diagnostic parameter, but rather needs to consider other aspects, such as the presence of other autoantibodies, markers, blood parameters, clinical assessment of patient symptoms, or the results of medical imaging or other non-invasive methods (e.g., polysomnography) in order to arrive at a conclusive diagnosis. See Baenkler h.w. (2012), general aspects of autoimmune diagnostics, renz, h., autoimmune diagnostics,2012, de Gruyter, page 3. Diagnostic reagents or methods may also be of value in that one disease may be excluded, thus allowing indirect diagnosis of another disease. In a preferred embodiment, the meaning of any symptoms or diseases mentioned throughout the present application is consistent with the understanding of those skilled in the art at the date of application or preferably the earliest priority date of the present application, as evidenced by textbooks and scientific publications.

Thus, the term "diagnosis" preferably does not imply that the diagnostic method or agent according to the application will be definitive and sufficient for performing a diagnosis based on a single test (let alone a parameter), but may refer to contributions to a so-called "differential diagnosis" (i.e. a systematic diagnostic procedure that considers the likelihood of a range of possible conditions based on a range of diagnostic parameters). Thus, the method, polypeptide or use of the application (which is optionally used to determine whether a patient is suffering from a disease) may comprise: obtaining a sample from a patient (preferably a human patient); determining the presence or absence of an autoantibody which binds to DNM1 in said sample, wherein said determination is performed by contacting the sample with a polypeptide of the application and detecting the presence or absence of binding between said polypeptide and said autoantibody, preferably by using a labelled secondary antibody, wherein said autoantibody, if present in the sample, binds to said polypeptide; and diagnosing the patient as suffering from or more likely to suffer from the neurological disorder if autoantibodies are determined to be present in the sample.

In a preferred embodiment, the method according to the invention comprises detecting more than one autoantibody from the group comprising autoantibodies to each of the polypeptides NSF, STX1B, VAMP2, GAD65, GAD67, IA-2, ZNT8 and dual carrier in addition to the autoantibody to DNM 1. In a more preferred embodiment, the further detection step may involve: a) Detecting autoantibodies from a group comprising autoantibodies against each of the polypeptides GAD65, GAD67, IA-2, ZNT8 and bian, preferably GAD65 and GAD67, and b) detecting autoantibodies from a group comprising autoantibodies against each of the polypeptides NSF, STX1B, VAMP2, preferably NSF.

The term "diagnosis" may also refer to a method or agent for distinguishing between two or more conditions associated with similar or identical symptoms.

The term "diagnosis" may also refer to a method or agent for selecting the most promising therapeutic regimen for a patient. In other words, the method or agent may involve selecting a treatment regimen for the subject. For example, detection of an autoantibody may indicate that immunosuppressive therapy will be selected, which may include administration of one or more immunosuppressive drugs to a patient.

The present invention relates to complexes comprising antibodies, preferably autoantibodies, which bind to polypeptides of the invention. Such complexes may be used or detected as part of a method for diagnosing a disease. If autoantibodies against DNM1 are to be detected, a liquid sample comprising antibodies from the subject can be used to carry out the method. Such a liquid sample may be any body fluid from a subject comprising a representative set of antibodies, preferably a sample from a subject comprising antibodies of the IgG immunoglobulin class. For example, the sample may be cerebrospinal fluid (CSF), blood or serum, lymph, interstitial fluid, preferably serum or CSF, more preferably serum.

The step of contacting a liquid sample comprising an antibody with a polypeptide of the invention may be performed by: incubating the immobilized form of the polypeptide in the presence of a sample comprising the antibody under conditions compatible with the formation of a complex comprising the respective polypeptide and an antibody, preferably an autoantibody, binding to the polypeptide of the invention. Subsequently, the liquid sample, which at that time is depleted of antibodies that bind to the polypeptides of the invention, may be removed, followed by one or more washing steps. Finally, complexes comprising the antibody and the polypeptide may be detected. In a preferred embodiment, the term "conditions compatible with complex formation" is conditions that allow specific antigen-antibody interactions to establish a complex comprising the polypeptide and the antibody. In a preferred embodiment, such conditions may include incubating the polypeptide in a 1:100 diluted sample in PBS buffer for 30 minutes at 25 ℃. In a preferred embodiment, the term "autoantibody" as used herein refers to an antibody that specifically binds to an endogenous molecule of an animal (preferably a mammal) that produces said autoantibody, wherein the level of such antibody is more preferably increased compared to a normal healthy human or a human not suffering from the disease, preferably a healthy human. In a most preferred embodiment, the autoantibody is an autoantibody that binds to DNM 1.

The method according to the invention is preferably an in vitro method.

In a preferred embodiment, the detection of a complex for use in a prognosis, diagnosis, method or test kit according to the invention comprises using a method selected from the group comprising: immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeling immunoassays, such as those selected from the group comprising radiolabeled immunoassays, enzyme immunoassays, preferably ELISA, chemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence techniques. Those skilled in the art are familiar with these methods, which are also described in the prior art, for example in Zane, h.d. (2001), immunology-Theoretical & Practical Concepts in Laboratory Medicine, w.b. Saundrs Company, in particular chapter 14.

Alternatively, a sample comprising a tissue comprising a polypeptide of the invention other than a liquid sample may be used. The tissue sample is preferably derived from a tissue expressing endogenous DNM1, preferably expressing the polypeptide at an increased level compared to average tissue in a corresponding organism, preferably a human. Such a sample, which may be in the form of a tissue section immobilized on a carrier such as a slide for microscopic analysis, may then be contacted with an antibody of the invention, preferably an autoantibody, which binds to a polypeptide of the invention. Preferably, the antibodies are labeled to allow differentiation from endogenous antibodies that bind to the polypeptides of the invention, thereby allowing detection, and optionally quantification, of the newly formed complexes. If the amount of complex formed is less than that found in a sample taken from a healthy subject, the subject from which the sample is taken under examination may suffer from the disease.

Any data that demonstrates the presence or absence of a complex comprising an antibody and a polypeptide of the invention can be correlated with reference data. For example, detection of the complex indicates that the patient providing the sample being analyzed has suffered from, is suffering from, or is likely to suffer from a disease in the future. If the patient has been previously diagnosed and the method for obtaining diagnostically relevant information is run again, the amount of complex detected in these two runs may be correlated to learn information about disease progression and/or treatment success. For example, if an increase in the amount of complex is found, this implies that the condition is progressing and may manifest symptoms in the future, and/or that any treatment attempted is unsuccessful.

In a preferred embodiment, the diagnostic method according to the invention is carried out using a blot such as a microtiter plate, a membrane, a dot blot or a linear dot blot. Those skilled in The art are familiar with experimental setup, which is described in The prior art (Raoult, D. And Dasch, G.A. (1989), the line blot: an immunoassay for monoclonal and other anti-bodies.Its application to The serotyping of gram-negative bacteria.J. immunol. Methods,125 (1-2), 57-65; WO 201304540).

In another preferred embodiment, the prognosis, diagnosis, method or test kit according to the teachings of the present invention contemplates the use of indirect immunofluorescence. Those skilled in the art are familiar with such techniques and the preparation of suitable samples, which are described in the prior art (US 4647543; voigt, j., krause, c.,e, saschenbrecker, S., hahn, M, danckwards, M, feirer, C, ens, K, fechner, K, barth, E, martinetz, T.and>W. (2012), automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells, "Clinical and Developmental Immunology, vol.2012, doi:10.1155/2012/65105; bonilla, E., francis, L., allam, F.et al, immuno-fluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients, clinical Immunology, vol.124, no.1, pp.18-21,2007). Suitable reagents, devices and software packages are commercially available, for example from EUROIMMUN, lubeck, germany.

The sample may be tested to determine only the presence of autoantibodies that bind to DNM1, but diagnostic methods, tests, devices, etc. are preferred to determine the presence of autoantibodies to one or more other polypeptides, preferably associated with a neurological autoimmune disease, preferably selected from the group comprising: hu, yo, ri, CV2, PNMA1, PNMA2, DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, neurocartilaginous protein, CARPVIII, zic4, sox1, ma, MAG, MP0, MBP, GAD65, amphiregulin, restorer protein, GABA A receptor (EP 13189172.3), GABA B receptor (EP 2483417), glycine receptor, desmin (gephyrin), igLON5 (2016/0349275), DPPX (US 2015/0247847), aquaporin-4, MOG, NMDA receptor, AMPA receptor, GRM1, GRM5, LGI1, VGCC and mGluR1 and CASPR2, the antigen being preferably immobilized, for example on a medical device such as a linear dot blot. In a more preferred embodiment, autoantibodies to DNM1 and autoantibodies to GAD65 are detected. Autoantibodies directed against one or more of the diagnostic markers neurocartilaginous protein (EP 15001186), ITPR1 (EP 14003703.7), NBC1 (EP 14003958.7), ATP1A3 (also known as the α3 subunit of the human neuronal Na (+)/K (+) atpase (EP 14171561.5)), flotillin1/2 (EP 3101424) and RGS8 (EP 17000666.2) have been described in the prior art in addition.

According to the teachings of the present invention, antibodies, preferably autoantibodies, are provided that bind to the polypeptides of the present invention for use in the diagnosis of disease. Those skilled in the art are familiar with methods for purifying antibodies, such as those described in Hermanson, G.T., mallia, A.K., and Smith, P.K. (1992), immobilized Affinity Ligand Techniques, san Diego: academic Press. Briefly, an antigen that specifically binds to an antibody of interest (an antigen thereof is a polypeptide of the invention) is immobilized and used to purify the antibody of interest from an appropriate source via affinity chromatography. A liquid sample comprising antibodies from a patient suffering from a disease may be used as a source.

According to the present invention, antibodies, such as autoantibodies, are provided which are capable of specifically binding to the polypeptides of the invention. In a preferred embodiment, the term "antibody" as used herein refers to any immunoglobulin-based binding moiety, more preferably comprising at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, including but not limited to monoclonal and polyclonal antibodies and variants of antibodies, particularly fragments, which are capable of binding to the respective antigen, more preferably specifically binding thereto. In a preferred embodiment, the term "specifically binds" as used herein means that the binding ratio is 1X 10 ^-5 M, more preferably 1X 10 ^-7 M, more preferably 1X 10 ^-8 M, more preferably 1X 10 ^-9 M, more preferably 1X 10 ^-10 M, more preferably 1X 10 ^-11 M, more preferably 1X 10 ^-12 The binding reaction is stronger, characterized by the dissociation constant of M, by using BiacThe ore apparatus was determined by surface plasmon resonance in PBS buffer at pH 7 at 25 ℃. The antibody may be part of an autoantibody preparation which is heterologous or may be a homologous autoantibody, wherein the heterologous preparation comprises a number of different autoantibody species, as obtainable by preparation from human donor serum, for example by affinity chromatography using an immobilised antigen to purify any autoantibody capable of binding to the antigen. The antibody may be glycosylated or non-glycosylated. Those skilled in the art are familiar with methods that can be used to identify, generate and purify antibodies and variants thereof, such as those described in EP 2423226A2 and references therein. The antibodies may be used as diagnostic reagents, alone or in combination, for example in combination with a polypeptide of the invention.

The present invention provides a method for isolating an antibody, preferably an autoantibody, which binds to a polypeptide of the invention comprising the steps of: a) contacting a sample comprising the antibody with a polypeptide of the invention, thereby forming a complex, b) separating the complex formed in step a), c) dissociating the complex separated in step b), and d) separating the antibody from the polypeptide of the invention. Samples from patients suffering from the novel neurological disorders identified by the inventors can be used as a source of antibodies. Suitable methods are described in the prior art, for example in handbooks "Affinity chromatography", "Strategies for Protein Purification" and "Antibody Purification" (2009/2010) published by GE Healthcare Life Sciences, and in Philips, terry, m., analytical techniques in immunochemistry,1992,Marcel Dekker,Inc.

The present invention provides pharmaceutical compositions comprising a polypeptide of the invention, preferably suitable for administration to a subject, preferably a mammalian subject, more preferably a human. Such pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered, for example, by oral route, by parenteral route, by inhalation spray, by topical route, by eye drops, by rectal route, by nasal route, by buccal route, by vaginal route or via an implanted reservoir, wherein the term "parenteral route" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions may be provided in suitable dosage forms, such as capsules, tablets, and aqueous suspensions and solutions, preferably in sterile form. It may be used in a method of treating a disease comprising administering to a subject an effective amount of a polypeptide of the invention. In a preferred embodiment, the invention provides a vaccine comprising a polypeptide of the invention, optionally comprising an auxiliary agent such as an adjuvant or buffer; and the use of the polypeptides of the invention for the preparation of a vaccine.

Within the scope of the present invention, a medical or diagnostic device is provided comprising a reagent preferably coated with a reagent for detecting an (auto) antibody of the invention and/or a polypeptide of the invention. Preferably, such medical or diagnostic devices comprise a polypeptide of the invention in a form that allows it to be contacted with an aqueous solution, more preferably a liquid human sample, in a direct manner. In particular, the comprised polypeptides of the invention may be immobilized on the surface of a carrier, preferably selected from the group comprising: glass plates or slides, biochips, microtiter plates, beads, e.g., magnetic beads, blood component separation (apharesis) devices, chromatographic columns, membranes, and the like. Exemplary medical devices include linear dot blots, microtiter plates, microscope slides, beads, preferably magnetic beads, and biochips. In addition to the polypeptides of the invention, the medical or diagnostic device may comprise additional polypeptides, such as positive or negative controls, e.g. samples with or without antibodies binding to the polypeptide of interest, or known other antigens binding to diagnostically valuable autoantibodies, in particular those involved in other diseases associated with one or more of the same or similar symptoms.

The teachings of the present invention provide kits that are preferably used for diagnosing diseases. Such a kit may comprise instructions detailing how to use the kit, and means for contacting a polypeptide of the invention with a sample of body fluid from a subject, preferably a human subject, such as a linear dot blot, wherein the polypeptide of the invention is immobilized on the linear dot blot. Further, the kit may comprise a positive control, such as a collection of autoantibodies or recombinant antibodies known to bind to a polypeptide of the invention; and negative controls, such as proteins having no detectable affinity with the polypeptides of the invention, e.g., bovine serum albumin. Finally, such kits may comprise standard solutions of antibodies or antigens for preparing a calibration curve.

In a preferred embodiment, the kit comprises means for detecting autoantibodies bound to a polypeptide of the invention, preferably by detecting a complex comprising a polypeptide of the invention and an antibody bound to a polypeptide of the invention. Such means are preferably reagents which bind to the complex and modify the complex or carry a label so that the complex is detectable. For example, the means may be a labelled antibody which binds to the polypeptide or to the constant region of the primary antibody at a binding site other than that recognized by the primary antibody. Alternatively, the means may be a secondary antibody which binds to the constant region of the autoantibody, preferably a secondary antibody specific for a mammalian IgG class antibody. Many methods and tools for detecting such complexes have been described in the prior art, for example in Philips, terry, m., analytical techniques in immunochemistry,1992,Marcel Dekker,Inc.

The polypeptide of the invention comprising a polypeptide of DNM1 or a variant thereof may be produced or provided in the form of a cell comprising and/or expressing a nucleic acid encoding the polypeptide. If a nucleic acid comprising a sequence encoding a polypeptide of the invention or a variant thereof is used, such a nucleic acid may be an unmodified nucleic acid. In a preferred embodiment, the nucleic acid is a nucleic acid which does not itself occur in nature and comprises at least one modification, such as isotopic content or chemical modification, such as methylation, sequence modification, labeling, etc., as compared to the natural nucleic acid, which is indicative of synthetic origin. In a preferred embodiment, the nucleic acid is a recombinant nucleic acid, and in a more preferred embodiment, is part of a vector in which it may be functionally linked to a promoter that allows expression (preferably overexpression) of the nucleic acid. Those skilled in the art are familiar with a wide variety of suitable carriers, which are commercially available, for example from origin. For example, vectors encoding fusion constructs with C-terminal GFP may be used. The cells may be eukaryotic or prokaryotic cells, preferably eukaryotic cells, such as yeast cells, and more preferably mammalian cells, more preferably human cells such as HEK293 cells. Examples of mammalian cells include HEK293, CHO or COS-7 cells. The cell comprising a nucleic acid encoding a polypeptide of the invention may be a recombinant cell or an isolated cell, wherein the term "isolated" means that the cell is enriched such that there are few or virtually no other differentiated or typed cells compared to the wild-type environment of the cell.

The teachings of the present invention may be used not only for diagnosis, but also for the prevention or treatment of a disease, more particularly a method for the prevention or treatment of a disease, comprising the steps of: a) Lowering the concentration of autoantibodies bound to a polypeptide of the invention in the blood of a subject, and/or b) administering one or more immunosuppressive pharmaceutical substances, preferably selected from the group comprising: rituximab, prednisone, methoprene, cyclophosphamide, mycophenolate mofetil, intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate, azathioprine, and/or pharmaceutical compositions.

In a preferred embodiment, the invention provides the use of a means for detecting autoantibodies against DNM1 or detecting a nucleic acid or variant encoding DNM1 or a vector or cell comprising said nucleic acid in the preparation of a kit for diagnosing a disease such as stiff human syndrome. In another preferred embodiment, the invention provides the use of a reagent for detecting autoantibodies against DNM1, or detecting a nucleic acid encoding DNM1 or variant thereof, or a vector or cell comprising said nucleic acid, for the preparation of a kit for diagnosing a disease such as stiff human syndrome.

In a preferred embodiment, any method or use according to the invention may be used for non-diagnostic use, i.e. for determining the presence of autoantibodies against DNM1 in addition to diagnosing a patient. For example, the method or use may be used to test the efficiency of a medical device designed to remove autoantibodies from a patient's blood in vitro, wherein the test is performed on a liquid other than the patient's blood. In a preferred embodiment, any method or use according to the invention may be used for generating an autoantibody profile, preferably for detecting a disease in a mammal, preferably a human. In a preferred embodiment, any method or use may be used to detect disease-related markers in a sample from a patient with a neurological disease.

Drawings

FIG. 1 shows an image of a blue silver stained gel after immunoprecipitation of total lysate to demonstrate the pull down of DNM1 by patient serum (pull-down).

Fig. 2 shows an image of a blue silver stained gel showing the pull down of DNM1 after frozen immunoprecipitation.

Figure 3 shows that the patient's serum shows reactivity towards the enrichment of DNM1 from the cerebellum.

Figure 4 shows that the patient cohort depicts a significantly higher prevalence of AAb for DNM1 (prevvalance) compared to the control.

The invention includes a series of sequences, more particularly

SEQ ID NO:19(DNM1)

MGNRGMEDLIPLVNRLQDAFSAIGQNADLDLPQIAVVGGQSAGKSSVLENFVGRDFLPRGSGIVTRRPLVLQLVNATTEYAEFLHCKGKKFTDFEEVRLEIEAETDRVTGTNKGISPVPINLRVYSPHVLNLTLVDLPGMTKVPVGDQPPDIEFQIRDMLMQFVTKENCLILAVSPANSDLANSDALKVAKEVDPQGQRTIGVITKLDLMDEGTDARDVLENKLLPLRRGYIGVVNRSQKDIDGKKDITAALAAERKFFLSHPSYRHLADRMGTPYLQKVLNQQLTNHIRDTLPGLRNKLQSQLLSIEKEVEEYKNFRPDDPARKTKALLQMVQQFAVDFEKRIEGSGDQIDTYELSGGARINRIFHERFPFELVKMEFDEKELRREISYAIKNIHGIRTGLFTPDMAFETIVKKQVKKIREPCLKCVDMVISELISTVRQCTKKLQQYPRLREEMERIVTTHIREREGRTKEQVMLLIDIELAYMNTNHEDFIGFANAQQRSNQMNKKKTSGNQDEILVIRKGWLTINNIGIMKGGSKEYWFVLTAENLSWYKDDEEKEKKYMLSVDNLKLRDVEKGFMSSKHIFALFNTEQRNVYKDYRQLELACETQEEVDSWKASFLRAGVYPERVGDKEKASETEENGSDSFMHSMDPQLERQVETIRNLVDSYMAIVNKTVRDLMPKTIMHLMINNTKEFIFSELLANLYSCGDQNTLMEESAEQAQRRDEMLRMYHALKEALSIIGDINTTTVSTPMPPPVDDSWLQVQSVPAGRRSPTSSPTPQRRAPAVPPARPGSRGPAPGPPPAGSALGGAPPVPSRPGASPDPFGPPPQVPSRPNRAPPGVPSRSGQASPSRPESPRPPFDL

SEQ ID NO:20

gggcgggggccccgcggcgcaggcagtctgggcgcgcggctgcagcggcggagccggagtcggagccgggagcgctagcggcagccggatcgcagcctgcggggcccgccgcagccatgggcaaccgcggcatggaagatctcatcccgctggtcaaccggctgcaagacgccttctctgccatcggccagaacgcggacctcgacctgccgcagatcgctgtggtgggcggccagagcgccggcaagagctcggtgctcgagaatttcgtaggcagggacttcttgcctcgaggatctggcattgtcacccgacgtcccctggtcttgcagctggtcaatgcaaccacagaatatgccgagttcctgcactgcaagggaaagaaattcaccgacttcgaggaggtgcgccttgagatcgaggccgagaccgacagggtcaccggcaccaacaagggcatctcgccggtgcctatcaacctccgcgtctactcgccgcacgtgctgaacctgaccctggtggacctgcccggaatgaccaaggtcccggtgggggaccaacctcccgacatcgagttccagatccgagacatgcttatgcagtttgtcaccaaggagaactgcctcatcctggccgtgtcccccgccaactctgacctggccaattctgacgccctcaaggtcgccaaggaggtggacccccagggccagcgcaccatcggggtcatcaccaagctggacctgatggacgagggcacagatgcccgtgatgtgctggagaacaagctgctccccctgcgcagaggctacattggagtggtgaaccggagccagaaggacattgatggcaagaaggacattaccgccgccttggctgctgaacgaaagttcttcctctcccatccatcttatcgccacttggctgaccgtatgggcacgccctacctgcagaaggtcctcaatcagcaactgacgaaccacatccgggacacactgccggggctgcggaacaagctgcagagccagctactgtccattgagaaggaggtggaggaatacaagaacttccgccctgatgacccagctcgcaagaccaaggccctgctgcagatggtccagcagttcgccgtagactttgagaagcgcattgagggctcaggagatcagatcgacacctacgaactgtcagggggagcccgcattaaccgaatcttccacgagcgcttccctttcgagctggtcaagatggagtttgatgagaaggaactccgaagggagatcagctatgctatcaagaatatccatggcattagaacggggctgtttaccccagacatggcctttgagaccattgtgaaaaagcaggtgaagaagatccgagaaccgtgtctcaagtgtgtggacatggttatctcggagctaatcagcaccgttagacagtgcaccaagaagctccagcagtacccgcggctacgggaggagatggagcgcatcgtgaccacccacatccgggagcgcgagggccgcactaaggagcaggtcatgcttctcatcgatatcgagctggcttacatgaacaccaaccatgaggacttcataggctttgccaatgctcagcagaggagcaaccagatgaacaagaagaagacttcagggaaccaggatgagattctggtcatccgcaagggctggctgactatcaataatattggcatcatgaaagggggctccaaggagtactggtttgtgctgactgctgagaatctgtcctggtacaaggatgatgaggagaaagagaagaaatacatgctgtctgtggacaacctcaagctgcgggacgtggagaagggctttatgtcgagcaagcatatctttgccctctttaacacggagcagaggaatgtctacaaggattatcggcagctggagctagcctgtgagacacaggaggaggtggacagctggaaggcctccttcctgagggctggcgtgtaccctgagcgtgttggggacaaagagaaagccagcgagaccgaggagaatggctccgacagcttcatgcattccatggacccacagctggaacggcaagtggagaccatccggaatcttgtggactcatacatggccattgtcaacaagaccgtgagggacctcatgcccaagaccatcatgcacctcatgattaacaataccaaggagttcatcttctcggagctgctggccaacctgtactcgtgtggggaccagaacacgctgatggaggagtcggcggagcaggcacagcggcgcgacgagatgctgcgcatgtaccacgcactgaaggaggcgctcagcatcatcggcgacatcaacacgaccaccgtcagcacgcccatgcccccgcccgtggacgactcctggctgcaggtgcagagcgtaccggccggacgcaggtcgcccacgtccagccccacgccgcagcgccgagcccccgccgtgcccccagcccggcccgggtcgcggggccctgctcctgggcctccgcctgctgggtccgccctggggggggcgccccccgtgccctccaggccgggggcttcccctgaccctttcggccctccccctcaggtgccctcgcgccccaaccgcgccccgcccggggtccccagccgatcgggtcaggcaagtccatcccgtcctgagagccccaggccccccttcgacctctaaacagatccctcctcttctcggagacctccctttccaagcctgcctggacggctgttctgtgacttgacagtggctcccccagccccaaagccagcccccttcatctgtgacttaatctgttgtagtggtgagctgatacattcaggtgtgaccgttggtgaaaacttgtgccccttctgtggtatgcccttgccctgttctataaatatctataaatactcatatatatacacacctacacatggccaaccgcctcgcctctagcgctgggaatcagtcactgtgctatccttgtggagtcttgtggcccaactaccagagaacgctgtcccccgacatcccactccaaagtgtgccacctccagtgagcctccttgtcatgcccggcctgtggacagccagcccccgccatccctcccaccccctaccaagcatgggggtgctgtgcaggcagccgtgtggcctgacagtttctaccagtcctgctgtccctcggctgagaataaaacccatttctggatgatggggaatgtcaaaaaaaaaaaaaaa

The invention is further illustrated by the following non-limiting examples from which other features, embodiments, aspects and advantages of the invention may be derived.

Detailed Description

Examples

Summary of the inventionsummary

Method: serological investigation was performed on two patients (P1-P2) with idiopathic encephalitis and with autoimmune background. For this purpose, serum from two patients and matched cerebrospinal fluid (CSF) from P2 was obtained by indirect immunofluorescenceAssay (IFA) and immunoblotting comprehensive autoantibody screening was performed. Immunoprecipitation with cerebellum lysate followed by Mass Spectrometry (MS) was used to identify autoantigens by Western Blotting (WB) with monospecific animal antibodies against the respective target antigens, as well as by recombinant expression in HEK293 cells and use of recombinant proteins in immunoassays. In addition, patient sera with neurological symptoms and defined anti-neural autoantibodies, sera with staining patterns similar to those of patients 1 and 2 without known autoantibody reactivity, and negative control sera were screened against autoantibodies. All sera were analyzed by IFA or western blot with other recombinant SNARE complex proteins as substrates.

Patient(s)

The control group included 45 healthy donors, 33 patients with neurological symptoms and with defined anti-neuroautoantibodies (3 x anti-CASPR 2, 3x anti-NMDAR, 3x anti-LGI 1, 3x anti-Hu, 3x anti-Ri, 2x anti-Yo/anti-Ri, 3x anti-Yo, 3x anti-AQP 4, 10x anti-GAD 65) and 10 sera with similar staining patterns as P1 and P2 without known autoantibody reactivity.

Indirect immunofluorescence assay (IFA)

IFA was performed using slides with biochip arrays of frozen sections of brain tissue (hippocampus of rat, cerebellum of rat and monkey) in combination with recombinant HEK293 cells expressing 30 different brain antigens, respectively Hu, yo, ri, CV2, PNMA2, ITPR1, homer 3, CARP VIII, ARHGAP26, ZIC4, dnar/Tr, GAD65, GAD67, amphiregulin, restorer protein, GABA _B Receptors, glycine receptor, DPPX, igLON5, glutamate receptor (types NMDA, AMPA, mGluR, mGluR5, GLURD 2), LGI1, CASPR2, AQP4 (M1 and M23), MOG, ATP1A3, NCDN (EUROIMMUN, FA 111a-1003-51, FA 1112-1003-50, FA-1128-1003-50, FA112d-1003-1, FA 112M-1003-50, FA 1151-1003-50,Miske R,Hahn S,Rosenkranz T,M uller M, dettmann IM, mindorf S, denno Y, brakopp S, scharf M, teegen B, probst C, melzer N, meinck HM, terberg C, W,Komorowski L.,2016,Autoantibodies against glutamate receptorδ2 after allogenic stem cell transplantation.Neurol Neuroimmunol Neuroinflamm.,3(4):e255；Scharf M,Miske R,Heidenreich F,Giess R,Landwehr P,IM,Begemann N,Denno Y,Tiede S,/>C,Schlumberger W,Unger M,Teegen B,/>W,Probst C,Komorowski L,2015,Neuronal Na+/K+ATPase is an autoantibody target in paraneoplastic neurologic syndrome,Neurology；84(16):1673-9；Miske R,Gross CC,Scharf M,Golombeck KS,Hartwig M,Bhatia U,Schulte-Mecklenbeck A,/>K,Strippel C,/>L,Synofzik M,Lohmann H,Dettmann IM,Deppe M,Mindorf S,Warnecke T,Denno Y,Teegen B,Probst C,Brakopp S,Wandinger KP,Wiendl H,/>W, meuth SG, komorowski L, melzer N,2016,Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration,Neurol Neuroimmunol Neuroinflamm; 4 (1): e 307)). Each biochip mosaic (biochip mosaic) was incubated with 70. Mu.L of PBS-diluted samples for 30 min at room temperature, washed with PBS-Tween and immersed in PBS-Tween for 5 min. In the second step, alexa 488-labeled goat anti-human IgG (Jackson Research, suffolk, united Kingdom) or Fluorescein Isothiocyanate (FITC) -labeled goat anti-human IgG (EUROIMMUN Medizinische Labordiagnostika AG, lubeck) was applied and incubated for 30 minutes at room temperature. The slides were again washed with PBS-Tween rinse and then immersed in PBS5 min in Tween. Slides were embedded in PBS buffered DABCO containing glycerol (approximately 20. Mu.L per field) and examined by fluorescence microscopy. Positive and negative controls were included. Samples were classified as positive or negative based on the fluorescence intensity of transfected cells, as compared directly to untransfected cells and control samples. Endpoint potency refers to the last dilution that is capable of exhibiting visible fluorescence.

Results were evaluated by two independent observers using an EUROSTARII microscope (EUROIMMUN Medizinische Labordiagnostika AG, lubeck, germany). Reagents were obtained from Merck, darmstadt, germany and Sigma-Aldrich, heidelberg, germany, if not otherwise specified.

Preparation of porcine cerebellum lysate

The cerebellum of the pig was dissected after slaughtering the animal and rapidly frozen at-80 ℃ until further use. Lysate preparation was performed at 4℃as described by Miske et al (Miske et al, 2017). Briefly, the tissue was weighed in the frozen state, thawed and cut into small pieces with a scalpel. The triturated tissue was transferred to a centrifuge tube (farcon tube) (Sarstedt AG & Co, germany) and 5 volumes of detergent-based solubilization buffer and protease inhibitor cocktail (cocktail) were added per gram of tissue. The mixture was then homogenized to a slurry using a MICCRA D-9 homogenizer. The homogenization step was carried out twice (stage C) with a pause of 5 minutes between each cycle. Subsequently, the cells were further lysed using a Dounce homogenizer with two different pestle (plunger) sizes, each performed 10 times. The pestle size is "large" for producing a homogeneous mixture and the pestle size is "small" for producing maximum friction and cell disruption. Care was taken to avoid excessive foaming when homogenizing the cerebellum lysate mixture. The homogenate was then incubated on a bench rotor for 3 hours at 4 ℃. After incubation, the homogenate was centrifuged at 21,000Xg for 15 min at 4 ℃, the clarified supernatant containing the dissolved proteins was carefully removed and transferred to a fresh Eppendorf microcentrifuge tube (Eppendorf GmbH, germany) and stored at-80 ℃ until further use.

Total lysate immunoprecipitation

Following IFA, total lysate immunoprecipitation was performed to identify the unknown antigen targeted by patient AAb. The test was performed as previously described by Miske et al (Miske et al, 2017).

Frozen immunity

Another variant of the immunoprecipitation assay, termed "frozen immunoprecipitation", proceeds as a complement to the previous method. This protocol is a slight modification of Hist-immunoprecipitation as described by Scharf et al (Scharf et al 2015). Similar to the prior art, the pig cerebellum was dissected and flash-frozen in liquid nitrogen (shock-frezen). The cerebellum was then serially cut into 25X 25 μm frozen sections using a Leitz 1720 Crosostat microtome and added to a four-well plate (Sarstedt AG) containing 3ml of freeze-IP-PBS buffer per well&Co, germany). Together 30 μl of serum from the patient and control was added to each well and incubated for 3 hours at 4deg.C on a shaker. Subsequently, the contents of each well were transferred to fresh 2ml Eppendorf tubes and centrifuged at 2500rpm for 5 minutes at 4 ℃. The supernatant was discarded and the pellet was subsequently washed three times with 1ml PBS/Tween, with a similar centrifugation step between each cycle. The pellet was then solubilized with 1ml of solubilization buffer plus protease-inhibitor cocktail and thoroughly homogenized with a pipette. The mixture was incubated at 4℃for 1 hour and centrifuged at 16,000Xg for 20 minutes at 4 ℃. The supernatant was collected in a separate Eppendorf tube and the pellet was stored at-20 ℃ for analysis. As performed in the previous method, 50. Mu.l will be Protein G beads were incubated with the supernatant overnight at 4 ℃ to capture immune complexes. After incubation, the beads were washed and eluted as described previously.

Identification of antigens

The brains of rats were dissected and flash frozen in liquid nitrogen. Miccra D-8 (Roth, karlsruhe, germany) and a manual homogenizer (Sartorius,germany) in a solubilization buffer (100 mmol/L tris-HCl pH 7.4, 150mmol/L sodium chloride, 2.5mmol/L ethylenediamine tetraacetic acid, 0) containing a protease inhibitor (Complete mini, roche Diagnostics, penzberg, germany).The tissue was homogenized at 4℃in 5% (w/v) sodium deoxycholate, 1% (w/v) Triton X-100. Tissue lysates were centrifuged at 21,000Xg for 15 min at 4℃and the clarified supernatant incubated overnight with patient serum (1:16.7 dilution) at 4 ℃. The samples were then incubated with protein G Dynabeads (ThermoFisher Scientific, dreieich, germany) at 4 ℃ for 3 hours to capture immune complexes. The beads were washed 3 times with PBS and eluted with NuPage LDS sample buffer (ThermoFisher Scientific, schwerte, germany) containing 25mmol/L dithiothreitol for 10 min at 70 ℃. Urea methylation (carbamidomethyl) with 59mM iodoacetamide (Bio-Rad, hamburg, germany) followed by SDS-PAGE (NuPAGE, thermoFisher Scientific, schwerte, germany). Isolated proteins were visualized with coomassie brilliant blue (G-250) (Merck) and identified by mass spectrometry.

Mass spectrometry

The visible protein bands were excised from the coomassie brilliant blue G-250 stained gel. After decolorization and trypsin digestion, peptides were extracted and spotted onto MTP Anchor chip with alpha-cyano-4-hydroxycinnamic acid ^TM 384 TF target.

MALDI-TOF/TOF measurements were performed using a Autoflex III smartbeam TOF/TOF200 system using flexControl 3.4 software. Mass spectra of Peptide Mass Fingerprint (PMF) were recorded with a mass range of 600Da-4,000Da and with a positive ion reflector pattern with 4,000-10,000 bombardes (shots). Mass spectra were externally calibrated with commercially available Peptide Calibration Standard II, treated with flexAnalysis 3.4 and peak lists analyzed with BioTools 3.2.

Mascot search engine Mascot Server 2.3 (Matrix Science, london, UK) is used for protein identification by searching NCBI or SwissProt databases limited to mammalian (Mammalia). The search parameters were as follows: mass tolerance was set at 80ppm, one missed cleavage site was accepted, and urea methylation of cysteine residues and oxidation of methionine residues were set as fixed and variable modifications, respectively. To evaluate protein hits, a significance threshold of p <0.05 was selected.

To further confirm PMF hits, 2 to 5 peptides per identified protein were selected for MS/MS measurement using the WARP feedback mechanism of BioTools. The parent and fragment masses were recorded with 400 and 1000 shots, respectively. Mass spectra were processed and analyzed as described above with a fragment mass tolerance of 0.7 Da.

Patient serum immunoprecipitation of dynamin 1 from cerebellum

Immunoprecipitation analysis was performed using patient serum and porcine cerebellum lysate to identify additional target autoantigens. The total protein concentration of the porcine cerebellum lysate determined by BCA assay during each preparation was ≡20-23mg/ml.

Analysis was performed by immunoprecipitation of total lysates. The immunoprecipitated proteins were then resolved by gel electrophoresis and stained with blue silver stain to identify bands specific to patient serum compared to control, followed by MS. An image of the blue silver stained gel after immunoprecipitation of the total lysate is shown in fig. 1.

In this experiment, sera from 6 anti-GAD 65 and anti-GAD 67 AAb positive patients were included compared to 2 healthy controls. After gel staining, the main target antigens GAD65 and GAD67 were observed to pull down at positions of ≡65kDa and ≡67kDa, respectively, in serum lanes of all patients (fig. 1, line arrow), but not in control serum. In addition, another band specific to the patient's serum lane was identified as dynamin 1 (DNM 1) at position ≡97kDa (FIG. 1, elbow arrow).

The results were confirmed by the second immunoprecipitation method, frozen immunoprecipitation. In this method, porcine cerebellum frozen sections were used instead of tissue lysates. In contrast to the above method, immunoprecipitated proteins were resolved in a gel and stained with blue silver stain (fig. 2). In this experiment, for representative purposes, anti-GAD 65 and anti-GAD 67Abb positive serum and one anti-GAD 65 and anti-GAD 67Abb negative serum were included for four patients in addition to three sera from healthy controls. The results of the two immunoprecipitation methods were comparable. In addition to the one in which the pull down was weaker (fig. 2, lane 5), a strong pull down (. Apprxeq.97 kDa) of DNM1 was observed in the serum of all anti-GAD AAb positive patients (fig. 2, arrow). In addition, the healthy control group had no pull down of DNM 1. In this test, patient serum that was negative for DNM1 pull-down in total lysate immunoprecipitation (FIG. 1, lane 6) was also negative in this test.

Detection of AAb against cerebellum enriched DNM1 with patient serum immunoblots

The IMAC-enriched SNARE protein fraction was separated by gel electrophoresis and transferred onto nitrocellulose membranes. The experimental results are shown in FIG. 3. The membranes were cut vertically into strips and incubated with a mixture of antibodies (Ab, lane 2) containing anti-DNM 1 (1:1000), anti-NSF (1:1000) and anti-STX 1B (1:2000), internal reference patient serum (1:350) positive for AAb against GAD, NSF and STX1B from Eurommun AG as positive control (PC, lane 3) and a panel of serum (1:350) from the patient (lanes: 4-8) with nervous system controls (lanes: 9-12) and healthy (lanes: 13-15) controls. In cell-based assays, the internal reference serum was immunoreactive against NSF and STX1B (data not shown). Reactivity against DNM1 (. Apprxeq.97 kDa), NSF (. Apprxeq.82 kDa) and STX1B (. Apprxeq.33 kDa) was observed with antibodies and reference serum (lanes: 2 and 3, red arrow). In addition, patient serum depicts reactivity against DNM1 (lanes: 4-8). No nervous system control (lanes: 9-12) or healthy control, except one (lane 14), delineated any reactivity against DNM 1.

The enriched DNM1 fractions were resolved in a gel and immunoblotted with patient serum (n=100) against nervous system (n=65) and healthy (n=70) controls. The relative intensity of each band was normalized to the reference serum and expressed as a percentage of the relative intensity obtained. These values were compared by performing a Kruskal-Wallis test using Graph Pad prism 5 software followed by multiple comparisons of Dunn. The reference serum (second upper point) was assigned a value of 100 and a cutoff value of 3SD above the mean value of healthy controls (dotted line; relative intensity: -15%) was calculated for screening purposes only. Overall, 23 patient sera in the patient cohort, 0 patient sera in the nervous system control (NC) and 1 subject in the Healthy Control (HC) exhibited relative intensity values above the DNM1 cutoff value (15%). Thus, the prevalence of AAb against DNM1 in patient cohorts was significantly higher (< 0.0001) compared to the control group. The graph represents the mean ± SD of each group.

Among the AAb-positive patient sera against DNM1, the number of anti-GAD AAb-positive patient sera was 15. Notably, 8 anti-GAD AAb negative patients were positive for AAb against DNM1 in serum. In summary, anti-GAD AAb positive patients may have a higher prevalence of AAb against NDM1 than anti-GAD AAb negative patients. The values from patients without anti-GAD AAb were lower but not negative. AAb prevalence against DNM1 is higher in patients with SPS, PERM and encephalitis than in patients with other related movement disorders, in terms of individual disorders.

Sequence listing

<110> Hangzhou European Mongolian medical examination all Limited

<120> diagnosis of neuroautoimmune disease

<130> 17PP067CN2

<160> 2

<170> PatentIn version 3.5

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Gln Asp Ala Phe Ser Ala Ile Gly Gln Asn Ala Asp Leu Asp Leu Pro

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35 40 45

Glu Asn Phe Val Gly Arg Asp Phe Leu Pro Arg Gly Ser Gly Ile Val

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Thr Arg Arg Pro Leu Val Leu Gln Leu Val Asn Ala Thr Thr Glu Tyr

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Ala Glu Phe Leu His Cys Lys Gly Lys Lys Phe Thr Asp Phe Glu Glu

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Val Arg Leu Glu Ile Glu Ala Glu Thr Asp Arg Val Thr Gly Thr Asn

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Lys Gly Ile Ser Pro Val Pro Ile Asn Leu Arg Val Tyr Ser Pro His

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Val Leu Asn Leu Thr Leu Val Asp Leu Pro Gly Met Thr Lys Val Pro

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Val Gly Asp Gln Pro Pro Asp Ile Glu Phe Gln Ile Arg Asp Met Leu

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Met Gln Phe Val Thr Lys Glu Asn Cys Leu Ile Leu Ala Val Ser Pro

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Ala Asn Ser Asp Leu Ala Asn Ser Asp Ala Leu Lys Val Ala Lys Glu

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Val Asp Pro Gln Gly Gln Arg Thr Ile Gly Val Ile Thr Lys Leu Asp

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Leu Met Asp Glu Gly Thr Asp Ala Arg Asp Val Leu Glu Asn Lys Leu

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Asp Ile Asp Gly Lys Lys Asp Ile Thr Ala Ala Leu Ala Ala Glu Arg

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Gly Thr Pro Tyr Leu Gln Lys Val Leu Asn Gln Gln Leu Thr Asn His

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Leu Ser Ile Glu Lys Glu Val Glu Glu Tyr Lys Asn Phe Arg Pro Asp

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Asp Pro Ala Arg Lys Thr Lys Ala Leu Leu Gln Met Val Gln Gln Phe

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Ala Val Asp Phe Glu Lys Arg Ile Glu Gly Ser Gly Asp Gln Ile Asp

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Thr Tyr Glu Leu Ser Gly Gly Ala Arg Ile Asn Arg Ile Phe His Glu

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Arg Phe Pro Phe Glu Leu Val Lys Met Glu Phe Asp Glu Lys Glu Leu

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Arg Arg Glu Ile Ser Tyr Ala Ile Lys Asn Ile His Gly Ile Arg Thr

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Gly Leu Phe Thr Pro Asp Met Ala Phe Glu Thr Ile Val Lys Lys Gln

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Val Lys Lys Ile Arg Glu Pro Cys Leu Lys Cys Val Asp Met Val Ile

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Tyr Pro Arg Leu Arg Glu Glu Met Glu Arg Ile Val Thr Thr His Ile

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Ile Glu Leu Ala Tyr Met Asn Thr Asn His Glu Asp Phe Ile Gly Phe

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Gly Asn Gln Asp Glu Ile Leu Val Ile Arg Lys Gly Trp Leu Thr Ile

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Asn Asn Ile Gly Ile Met Lys Gly Gly Ser Lys Glu Tyr Trp Phe Val

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Leu Thr Ala Glu Asn Leu Ser Trp Tyr Lys Asp Asp Glu Glu Lys Glu

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Lys Lys Tyr Met Leu Ser Val Asp Asn Leu Lys Leu Arg Asp Val Glu

565 570 575

Lys Gly Phe Met Ser Ser Lys His Ile Phe Ala Leu Phe Asn Thr Glu

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Gln Arg Asn Val Tyr Lys Asp Tyr Arg Gln Leu Glu Leu Ala Cys Glu

595 600 605

Thr Gln Glu Glu Val Asp Ser Trp Lys Ala Ser Phe Leu Arg Ala Gly

610 615 620

Val Tyr Pro Glu Arg Val Gly Asp Lys Glu Lys Ala Ser Glu Thr Glu

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Glu Asn Gly Ser Asp Ser Phe Met His Ser Met Asp Pro Gln Leu Glu

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Arg Gln Val Glu Thr Ile Arg Asn Leu Val Asp Ser Tyr Met Ala Ile

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Val Asn Lys Thr Val Arg Asp Leu Met Pro Lys Thr Ile Met His Leu

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Met Ile Asn Asn Thr Lys Glu Phe Ile Phe Ser Glu Leu Leu Ala Asn

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Leu Tyr Ser Cys Gly Asp Gln Asn Thr Leu Met Glu Glu Ser Ala Glu

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Gln Ala Gln Arg Arg Asp Glu Met Leu Arg Met Tyr His Ala Leu Lys

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Glu Ala Leu Ser Ile Ile Gly Asp Ile Asn Thr Thr Thr Val Ser Thr

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Pro Met Pro Pro Pro Val Asp Asp Ser Trp Leu Gln Val Gln Ser Val

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Pro Ala Gly Arg Arg Ser Pro Thr Ser Ser Pro Thr Pro Gln Arg Arg

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Ala Pro Ala Val Pro Pro Ala Arg Pro Gly Ser Arg Gly Pro Ala Pro

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Gly Pro Pro Pro Ala Gly Ser Ala Leu Gly Gly Ala Pro Pro Val Pro

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Ser Arg Pro Gly Ala Ser Pro Asp Pro Phe Gly Pro Pro Pro Gln Val

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Pro Ser Arg Pro Asn Arg Ala Pro Pro Gly Val Pro Ser Arg Ser Gly

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Gln Ala Ser Pro Ser Arg Pro Glu Ser Pro Arg Pro Pro Phe Asp Leu

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gggcgggggc cccgcggcgc aggcagtctg ggcgcgcggc tgcagcggcg gagccggagt 60

cggagccggg agcgctagcg gcagccggat cgcagcctgc ggggcccgcc gcagccatgg 120

gcaaccgcgg catggaagat ctcatcccgc tggtcaaccg gctgcaagac gccttctctg 180

ccatcggcca gaacgcggac ctcgacctgc cgcagatcgc tgtggtgggc ggccagagcg 240

ccggcaagag ctcggtgctc gagaatttcg taggcaggga cttcttgcct cgaggatctg 300

gcattgtcac ccgacgtccc ctggtcttgc agctggtcaa tgcaaccaca gaatatgccg 360

agttcctgca ctgcaaggga aagaaattca ccgacttcga ggaggtgcgc cttgagatcg 420

aggccgagac cgacagggtc accggcacca acaagggcat ctcgccggtg cctatcaacc 480

tccgcgtcta ctcgccgcac gtgctgaacc tgaccctggt ggacctgccc ggaatgacca 540

aggtcccggt gggggaccaa cctcccgaca tcgagttcca gatccgagac atgcttatgc 600

agtttgtcac caaggagaac tgcctcatcc tggccgtgtc ccccgccaac tctgacctgg 660

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acaagctgct ccccctgcgc agaggctaca ttggagtggt gaaccggagc cagaaggaca 840

ttgatggcaa gaaggacatt accgccgcct tggctgctga acgaaagttc ttcctctccc 900

atccatctta tcgccacttg gctgaccgta tgggcacgcc ctacctgcag aaggtcctca 960

atcagcaact gacgaaccac atccgggaca cactgccggg gctgcggaac aagctgcaga 1020

gccagctact gtccattgag aaggaggtgg aggaatacaa gaacttccgc cctgatgacc 1080

cagctcgcaa gaccaaggcc ctgctgcaga tggtccagca gttcgccgta gactttgaga 1140

agcgcattga gggctcagga gatcagatcg acacctacga actgtcaggg ggagcccgca 1200

ttaaccgaat cttccacgag cgcttccctt tcgagctggt caagatggag tttgatgaga 1260

aggaactccg aagggagatc agctatgcta tcaagaatat ccatggcatt agaacggggc 1320

tgtttacccc agacatggcc tttgagacca ttgtgaaaaa gcaggtgaag aagatccgag 1380

aaccgtgtct caagtgtgtg gacatggtta tctcggagct aatcagcacc gttagacagt 1440

gcaccaagaa gctccagcag tacccgcggc tacgggagga gatggagcgc atcgtgacca 1500

cccacatccg ggagcgcgag ggccgcacta aggagcaggt catgcttctc atcgatatcg 1560

agctggctta catgaacacc aaccatgagg acttcatagg ctttgccaat gctcagcaga 1620

ggagcaacca gatgaacaag aagaagactt cagggaacca ggatgagatt ctggtcatcc 1680

gcaagggctg gctgactatc aataatattg gcatcatgaa agggggctcc aaggagtact 1740

ggtttgtgct gactgctgag aatctgtcct ggtacaagga tgatgaggag aaagagaaga 1800

aatacatgct gtctgtggac aacctcaagc tgcgggacgt ggagaagggc tttatgtcga 1860

gcaagcatat ctttgccctc tttaacacgg agcagaggaa tgtctacaag gattatcggc 1920

agctggagct agcctgtgag acacaggagg aggtggacag ctggaaggcc tccttcctga 1980

gggctggcgt gtaccctgag cgtgttgggg acaaagagaa agccagcgag accgaggaga 2040

atggctccga cagcttcatg cattccatgg acccacagct ggaacggcaa gtggagacca 2100

tccggaatct tgtggactca tacatggcca ttgtcaacaa gaccgtgagg gacctcatgc 2160

ccaagaccat catgcacctc atgattaaca ataccaagga gttcatcttc tcggagctgc 2220

tggccaacct gtactcgtgt ggggaccaga acacgctgat ggaggagtcg gcggagcagg 2280

cacagcggcg cgacgagatg ctgcgcatgt accacgcact gaaggaggcg ctcagcatca 2340

tcggcgacat caacacgacc accgtcagca cgcccatgcc cccgcccgtg gacgactcct 2400

ggctgcaggt gcagagcgta ccggccggac gcaggtcgcc cacgtccagc cccacgccgc 2460

agcgccgagc ccccgccgtg cccccagccc ggcccgggtc gcggggccct gctcctgggc 2520

ctccgcctgc tgggtccgcc ctgggggggg cgccccccgt gccctccagg ccgggggctt 2580

cccctgaccc tttcggccct ccccctcagg tgccctcgcg ccccaaccgc gccccgcccg 2640

gggtccccag ccgatcgggt caggcaagtc catcccgtcc tgagagcccc aggcccccct 2700

tcgacctcta aacagatccc tcctcttctc ggagacctcc ctttccaagc ctgcctggac 2760

ggctgttctg tgacttgaca gtggctcccc cagccccaaa gccagccccc ttcatctgtg 2820

acttaatctg ttgtagtggt gagctgatac attcaggtgt gaccgttggt gaaaacttgt 2880

gccccttctg tggtatgccc ttgccctgtt ctataaatat ctataaatac tcatatatat 2940

acacacctac acatggccaa ccgcctcgcc tctagcgctg ggaatcagtc actgtgctat 3000

ccttgtggag tcttgtggcc caactaccag agaacgctgt cccccgacat cccactccaa 3060

agtgtgccac ctccagtgag cctccttgtc atgcccggcc tgtggacagc cagcccccgc 3120

catccctccc accccctacc aagcatgggg gtgctgtgca ggcagccgtg tggcctgaca 3180

gtttctacca gtcctgctgt ccctcggctg agaataaaac ccatttctgg atgatgggga 3240

atgtcaaaaa aaaaaaaaaa 3260

Claims (17)

1. Use of an agent that detects an autoantibody to DNM1 in the manufacture of a formulation for diagnosing an autoimmune disease in a subject, wherein the autoimmune disease is selected from the group consisting of stiff human syndrome and encephalitis.

2. Use of an agent that detects an autoantibody to DNM1 in the manufacture of a formulation for diagnosing an autoimmune disease in a subject, wherein the autoimmune disease is selected from the group consisting of paraneoplastic stiff human syndrome, progressive Encephalomyelitis (PERM) with tonic and myoclonus, and encephalitis.

3. Use according to claim 1 or 2, wherein the formulation is for contact with a sample from a subject.

4. Use according to claim 3, characterized in that the agent is selected from a DNM1 polypeptide or a fragment thereof, and the preparation is used to contact said sample from the subject with a DNM1 polypeptide or a fragment thereof, and to detect the binding of an antibody to the polypeptide or fragment thereof.

5. Use according to claim 3, characterized in that the sample is a body fluid comprising antibodies, selected from the group consisting of whole blood, serum, cerebrospinal fluid and saliva.

6. The use according to claim 4, characterized in that the DNM1 polypeptide or fragment thereof is derived from a cerebellum lysate.

7. The use according to claim 4, characterized in that a sample from the subject is contacted with a polypeptide according to claim 4 or a fragment thereof having at least 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acids of the sequence according to SEQ ID No. 1 and binding of an antibody from the sample to the polypeptide or fragment thereof is detected.

8. Use according to any one of claims 4 to 7, characterized in that the polypeptide or fragment thereof comprises further amino acids, which are N-terminally or C-terminally attached and facilitate purification of the polypeptide or fragment thereof.

9. Use according to any one of claims 4 to 7, characterized in that the polypeptide or fragment thereof is linked to a reporter molecule or solid phase.

10. Use of a DNM1 polypeptide or fragment thereof according to any one of claims 4 to 9 in the manufacture of a medicament for diagnosing an autoimmune disease, characterized in that a sample from a subject is contacted with a polypeptide or fragment thereof according to any one of claims 4 to 9 and binding of antibodies from the sample to the polypeptide or fragment thereof is detected, wherein the autoimmune disease is selected from the group consisting of stiff human syndrome and encephalitis.

11. Use of a DNM1 polypeptide or fragment thereof according to any one of claims 4 to 9 for the manufacture of a medicament for diagnosing an autoimmune disease, characterized in that a sample from a subject is contacted with a polypeptide or fragment thereof according to any one of claims 4 to 9 and binding of antibodies from the sample to the polypeptide or fragment thereof is detected, wherein the autoimmune disease is selected from the group consisting of paraneoplastic stiff human syndrome, progressive encephalomyelitis (pam) with tonic and myoclonus, and encephalitis.

12. Use according to claim 10 or 11, characterized in that the binding of antibodies from the sample to the polypeptide or fragments thereof is detected by immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays, protein arrays, luminescence tests or spot assays.

13. Use according to claim 10 or 11, characterized in that the binding of antibodies from the sample to the polypeptide or fragment thereof is detected by a radiolabeled immunoassay, an enzyme immunoassay, a chemiluminescent immunoassay, immunofluorescence, western blotting or dot blotting.

14. Use of a DNM1 polypeptide or fragment thereof according to any one of claims 4 to 9 in the manufacture of a kit or medical device for diagnosing an autoimmune disease in a subject, wherein the autoimmune disease is selected from the group consisting of stiff human syndrome and encephalitis.

15. Use of a DNM1 polypeptide or fragment thereof according to any one of claims 4 to 9 in the manufacture of a kit or medical device for diagnosing an autoimmune disease in a subject, wherein the autoimmune disease is selected from the group consisting of paraneoplastic stiff human syndrome, progressive Encephalomyelitis (PERM) with tonic and myoclonus, and cerebellar encephalitis.

16. Use according to claim 14 or 15, wherein the medical device is selected from the group consisting of a slide, a biochip, a microtiter plate, a test strip, a membrane, a chromatographic column and a bead.

17. Use according to claim 14 or 15, wherein the medical device is selected from the group consisting of slides and magnetic beads for microscopy.