JP7033840B2 - Diagnosis of neuroautoimmune diseases - Google Patents

Description

(Detailed description of the invention)
The present invention presents a polypeptide comprising, in a sample derived from a patient, an autoantibody that binds to Flotillin 1 and / or Flotillin 2, Flotillin 1 and / or Flotillin 2 or a variant thereof (which is preferably immobilized). Methods for diagnosing a disease, including the steps of detection, use of the above-mentioned polypeptide for diagnosing a disease, autoantibodies that bind to Flotillin 1 and / or Flotillin 2, methods for isolating the above-mentioned autoantibodies, Also related to pharmaceutical compositions, medical devices or test kits containing the above polypeptides.

The development of diagnostic systems for neurological disorders remains difficult in biomedical science, and many of the symptoms encountered are genetically-inherited diseases, drug abuse, malnutrition, infections, injuries, psychiatric disorders, etc. This is all the more so as it can be due to a wide variety of causes, including immunological defects and cancer.

Because neurological disorders are rarely associated with the characteristic pattern of clinical symptoms, it is often difficult to provide a reliable diagnosis based solely on the findings and examinations of affected patients or their medical history. Is.

The importance of early diagnosis cannot be overemphasized. Many neurological disorders (most notably Alzheimer's disease and Parkinson's disease, as well as multiple sclerosis) cannot be cured, but drugs that can be used to slow their progression are available. The earlier the diagnosis, the greater the chance that the range of available drugs will be in the best interests of the patient.

This is even more true in cases of neurological disorders associated with autoantibodies. In some cases, the association between specific detectable autoantibodies and the condition is strong enough to allow rapid diagnosis.

But even if this is not the case, detection of autoantibodies may indicate to the attending physician a therapeutic measure that can be used to improve the patient's condition. There are a variety of widely used immunosuppressive agents that can be used independently of the target nature of autoantibodies. Alternatively, apheresis can be used to remove autoantibodies from the patient's blood. In many cases, patients have come to live a normal life after early diagnosis and treatment of neurological autoimmune disorders.

Diagnostic assays based on the detection of autoantibodies can also confirm the diagnosis of diseases other than those associated with autoantibodies. If the blood sample is found to be free of specific autoantibodies, this will probably help narrow down the range of conditions that the attending physician may consider, as it excludes a range of possibilities.

Examples of neurological conditions that occur with the development of autoantibodies are neuromyelitis optica (a disease characterized by loss of vision and spinal cord function) and anti-NMDA receptor encephalitis (which is autonomic dysfunction, hypoventilation,). Cerebral ataxia, unilateral paralysis, loss of consciousness, or associated with catatony). Thus, while the involvement of autoantibodies and the nature of these conditions has long been poorly understood, many of the diseases are now thanks to the availability of assays based on the detection of autoantibodies. Can be efficiently diagnosed and treated.

Multiple sclerosis is another disease that is widely thought to be associated with the autoimmune destruction of life-sustaining body structures. About 20% of the above patients suffer from visual impairment, more specifically optic neuritis. Specific diagnosis is difficult due to the wide range of symptoms and the nature of the disease, and sometimes complete disappearance of the symptoms occurs after the attack.

Therefore, it is of utmost importance to develop new approaches to distinguish neurological conditions associated with autoantibodies from those not associated with autoantibodies.

The underlying challenges of the present invention provide novel reagents, devices and methods that can be used to assist in the diagnosis and treatment of neurological disorders, particularly those associated with visual impairment, headache and / or brain lesions. It is to be.

Another task underlying the present invention is the most promising treatment regimen, more specifically immunosuppression, that distinguishes autoimmune diseases, especially neurological autoimmune diseases, from diseases other than autoimmune diseases. It is to provide novel reagents, devices and methods that can be used even more to determine if a procedure is appropriate.

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

In the first aspect, the underlying task of the invention is by a method for diagnosing a disease, comprising the step of detecting an autoantibody that binds to Flotillin 1 and / or Flotillin 2 in a sample derived from a patient. It will be resolved.

In the second aspect, the underlying task of the invention is by a polypeptide comprising flotilin 1 and / or flotilin 2 or a variant thereof, which is preferably immobilized, more preferably immobilized on a solid carrier. It will be resolved.

In a third aspect, the underlying task of the invention is preferably the polypeptide of the invention for diagnosing a disease comprising the step of detecting an autoantibody that binds to flotilin 1 and / or flotilin 2 in a sample. It is solved by using.

In a fourth aspect, the underlying problem of the invention is solved by the polypeptide of the invention for use in the treatment of disease.

In a fifth aspect, the underlying task of the invention is an autoantibody that binds to flotilin 1 and / or flotilin 2, preferably to a complex comprising flotilin 1 and flotilin 2, preferably an isolated autoantibody. However, the autoantibody is preferably solved by the one in the state of a complex with the polypeptide 2 of the present invention.

In the sixth aspect, the underlying problem of the invention is solved by a method for isolating an autoantibody that binds to flotilin 1 and / or flotilin 2, preferably a complex containing flotilin 1 and flotilin 2. And the above method is
a) A step of contacting a sample containing the autoantibody with the polypeptide of the present invention under conditions suitable for forming a complex, wherein the autoantibody binds to the polypeptide.
b) Step of isolating the complex formed in step a),
c) includes the steps of dissociating the complex isolated in step b) and d) separating the autoantibody from the polypeptide.

In the seventh aspect, the underlying problem of the invention is solved by a pharmaceutical composition or medical device, preferably a diagnostic device, comprising the polypeptide of the invention.

In the eighth aspect, the underlying problem of the present invention is solved by a test kit for diagnosing a disease, which test kit comprises the polypeptide of the present invention, wherein preferably the test kit is further described. , A means for detecting a complex comprising the polypeptide according to claim 2 and an autoantibody that binds to flotilin 1 and / or flotilin 2.

In a preferred embodiment, the patient has elevated cell count in CSF, intrathecal IgG synthesis, oligoclonal band in CSF, MRZ (M for measles, R for ruts, Z for varicella) reaction in CSF, visual impairment, One or more symptoms selected from the group comprising a group including preferably visual impairment, optic neuritis, headache, spinal cord lesions and brain lesions, preferably visual impairment, more preferably visual impairment, headache, spinal cord lesions and brain lesions, preferably 2 The disease has one or more of the species or more, or the disease is associated with the one or more, preferably two or more.

In a preferred embodiment, the disease is a neurological disease, preferably a demyelinating disease, preferably a CNS disease, preferably a brain disease, preferably a disease associated with optic nerve inflammation.

In a preferred embodiment, the polypeptide is provided in the form of a cell containing the nucleic acid encoding the polypeptide, or in the form of a tissue containing the polypeptide.

In a preferred embodiment, the polypeptide is a recombinant polypeptide and / or an isolated polypeptide.

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

In a preferred embodiment, both a polypeptide comprising Flotillin 1 or a variant thereof and a polypeptide comprising Flotillin 2 or a variant thereof are present and are preferably part of the complex.

In a preferred embodiment, the autoantibody binds to a complex containing flotilin 1 and flotilin 2.

The present invention relates to autoantibodies to Flotillin 1 and / or Flotillin 2, as well as increased cell count in CSF, intrathecal IgG synthesis, oligoclonal bands in CSF, MRZ response in CSF, visual impairment, optic neuritis, headache, spinal cord lesions and Based on our surprising findings that there are neurological autoimmune diseases associated with symptoms from the group including brain lesions.

In addition, the invention is a sample obtained from a healthy subject, although autoantibodies to flotilin 1 and / or flotilin 2 are present and can be detected in samples derived from many patients suffering from neurological symptoms. It is based on the surprising finding of the present inventors that it is not detected.

Furthermore, the present invention is based on the surprising findings of the present inventors that known neurological diseases of unknown etiology, in particular demyelinating diseases, are associated with the presence of autoantibodies to flotilin 1 and / or flotilin 2. Based on.

Without wishing to be bound by any theory, the presence of such autoantibodies causes neurological symptoms, more specifically demyelinating diseases, especially those associated with the importance of vision. To the effect, it suggests that the activity and function of Flotillin 1 and / or Flotillin 2 and / or downstream effectors are impaired in patients with such autoantibodies.

Flotillins 1 and -2 (also known as reggie-2 and -1) were originally found to be upregulated in the regeneration of goldfish retinal ganglion axons after traumatic injury of the optic nerve. However, they are well conserved in many eukaryotes, including mammals. In vertebrates, both proteins are ubiquitously expressed and are most abundant in striated muscle, adipose tissue and lung tissue. At the molecular level, flotilin is bound to the cytoplasmic side of lipid rafts and is therefore often used as a marker for lipid microdomains. Similar to other lipid raft components, they are predominantly insoluble in Triton X-100, but float after sucrose density centrifugation. Functionally, they are involved in protein interactions, cell signaling, clustering of amyloid precursor protein and processing of amyloid-producing in neurons (Sutermer CA, Plattner H. The'lipid raft'microdomain protein reggie-1). and reggie-2 (flotillins) are scaffolds for protein intervention and signaling. Biochem Soc Symmp 2005; (72): 109-18). Overexpression of flotilin-2 is found in some cancers and is generally associated with more severe progression (Arkhipova KA, Sheyderman AN, Laktionov KK, Mochalnikova VV, Zborovskaya ssilosyl-IB. flotillin-2, stomatin and caveolin-1 in non-small cell lung cancer cancer and soft tissue sarcomas. BMC Cancer 2014; 14: 100).

The present invention is an immunogenic variant that is reactive to mammalian, preferably human Flotillin 1 or a variant thereof and / or Flotillin 2 or a variant thereof, preferably Flotillin 1 and Flotillin 2 or a variant thereof that binds to an autoantibody. With respect to polypeptides containing. Examples of mammalian Flotilin 1 and / or Flotilin 2 include those derived from humans, monkeys, mice, rats, rabbits, guinea pigs or pigs. In the most preferred embodiment, Flotilin 1 is a polypeptide or variant thereof encoded by database code O75955, and / or Flotilin 2 is a polypeptide or variant thereof encoded by database code Q14254. Throughout this application, any database code cited refers to the Uniprot database, more specifically the version accessible online on May 29, 2015. NP_005794 and NP_004466 represent the nucleotide sequences encoding Flotillin 1 and Flotillin 2, respectively.

In a more preferred embodiment, the polypeptides of the invention comprise Flotillin 1 or a variant thereof and Flotillin 2 or a variant thereof fused directly or via a linker to each other. In the most preferred embodiment, a complex comprising both a polypeptide comprising Flotrin 1 or a variant thereof and a polypeptide comprising Flotillin 2 or a variant thereof, which are attached to each other directly or via other molecules, is used. Preferably, the complex is free of proteins other than the polypeptide comprising Flotillin 1 or a variant thereof and the polypeptide comprising Flotillin 2 or a variant thereof. Such complexes can be used to capture autoantibodies that bind to the complex for the diagnosis of disease.

When a complex containing a first polypeptide containing Flotyline 1 or a variant thereof and a second polypeptide containing Flotyline 2 or a variant thereof is used, the first and second polypeptides are in the same cell. It can be expressed to form such a complex. Alternatively, the first and second polypeptides can be expressed separately in different cells and, after expression, can be reconstituted in purified form as needed.

The teachings of the present invention are expressly or implicitly referred to herein by a polypeptide, in particular a polypeptide comprising a natural sequence of Flotillin 1 and / or Flotillin 2, or, for example, by function, name, sequence or accession number. Not only may it be performed using a nucleic acid having the exact sequence to be made, but it may also be performed using such a polypeptide or variant of nucleic acid.

In a preferred embodiment, the term "variant", as used herein, refers to at least one fragment of the full-length sequence referred to, more specifically, one or more amino acids as compared to the full-length sequence. May refer to one or more amino acid or nucleic acid sequences that are shortened at one or both ends. Such fragments are at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 of the original sequence. Contains or encodes peptides or variants thereof with contiguous amino acids. The total length of the above variants is at least 6, 7, 8, 9, 10, 11, 12, 20, 20, 25, 30, 40, 50, 60, 70. It can be 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 the referenced amino acid sequence or fragment thereof referred to at least 40%, 50%, 60%, 70%, 75%, 80. %, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% also with respect to a polypeptide or fragment thereof comprising an amino acid sequence that is identical, where biological activity. Amino acids other than those essential for the ability of an antigen to bind to a (self) antibody, or for the folding or structure of the polypeptide, are deleted or substituted, and / or one or more such essential amino acids. Is replaced in a conservative manner and / or amino acids are added, so that the biological activity of the polypeptide is preserved. Technical levels include various methods that can be used to align two predetermined nucleic acid or amino acid sequences and calculate the degree of identity (eg, Arthur Lesk (2008), Industry to bioinformatives, Oxford University Press). , 2008, 3rd ^edition ). In a preferred embodiment, the ClustalW software (Larkin, MA, Blackschilds, G., Brown, N.P., Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F. , IM, Wilm, A., Lopez, R., Thomasson, J.D., Gibson, T.J., Higgins, D.G. (2007). Clustal Wand Crystal X version 2.0. Bioinformatics, 23, 2947-2948) is used with default settings.

In a preferred embodiment, the polypeptide or variant thereof is chemically modified, eg, isotope-labeled or covalently modified (eg, glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation). Etc.) can be further included. Those of skill in the art are familiar with how to modify a polypeptide. Any modifications are designed so as not to disrupt the biological activity of the above variants.

In addition, variants can also be produced by fusion with other known polypeptides or variants thereof and are at least 70%, 75%, 80 when aligned with the active moiety or domain, preferably the active moiety of the reference sequence. %, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% having sequence identity, wherein the term "active moiety" is used herein. As used in the book, the code refers to an amino acid sequence shorter than the full-length amino acid sequence, or in the case of a nucleic acid sequence, a code shorter than the full-length amino acid sequence, and / or a variant of the natural sequence, but the biological activity described above. Hold at least some of them.

In a preferred embodiment, the term nucleic acid "variant" comprises a complementary strand nucleic acid that hybridizes to a reference nucleic acid or wild-type nucleic acid, preferably under stringent conditions. The stringency of the hybridization reaction is readily determined by one of ordinary skill in the art and is generally an empirical calculation that depends on probe length, wash temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require less heat. Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: the degree of homology desired between the probe and the hybridizable sequence is high. The higher the relative temperature that can be used. As a result, the higher the relative temperature, the more stringent the reaction conditions tend to be, while the lower the temperature, the less stringent it tends to be. For further details and explanation of stringency of hybridization reactions, see Ausubel, F. et al. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. checking ... In addition, one of ordinary skill in the art will discuss how to identify DNA sequences by means of hybridization in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for For Filter HYBRIDIZEN, Boehringer .. , Ehrmann, M. et al. , Ludwig, W.M. , And Schleifer, K.K. H. (1991) International Journal of Systematic Bacteriology 41: 255-260 may be followed. In a preferred embodiment, stringent conditions apply to any hybridization, that is, hybridization occurs only if the probe is 70% or more identical to its target sequence. Probes with a low degree of identity with respect to their target sequence can hybridize, but such hybrids are unstable and in washing steps under stringent conditions, eg, salt concentration to 2 x SSC, or If necessary, the temperature is then reduced to 0.5 × SSC and removed, while the temperatures are about 50 ° C. to 68 ° C., about 52 ° C. to 68 ° C., and about 54 ° C. to 68 in order of increasing priority. ° C., about 56 ° C. to 68 ° C., about 58 ° C. to 68 ° C., about 60 ° C. to 68 ° C., about 62 ° C. to 68 ° C., about 64 ° C. to 68 ° C., and about 66 ° C. to 68 ° C. In a particularly preferred embodiment, the temperature is from about 64 ° C to 68 ° C or from about 66 ° C to 68 ° C. It is also possible to adjust the salt concentration to 0.2 × SSC or even 0.1 × SSC. At least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% degree of identity with respect to the reference sequence or wild-type sequence. Nucleic acid sequences with can be isolated. In a preferred embodiment, a variant of the term nucleic acid sequence, as used herein, refers to the same amino acid sequence as the reference nucleic acid sequence and any nucleic acid sequence encoding the variant thereof, in accordance with the reduction of the genetic code. ..

Variants of the above polypeptides have biological activity. In a preferred embodiment, such biological activity is the ability to specifically bind to the autoantibody of interest, preferably flotilin 1 and found in patients suffering from the disease identified by us. / Or the ability to bind to flotilin 2.

Polypeptides of the invention or self-antibodies of the invention comprising Flotrin 1 and / or Flotillin 2 or variants thereof include said polypeptides in an endogenous form when used to carry out the teachings of the invention. Liquid samples, tissues or cells, more preferably cells overexpressing the above polypeptides, and / or optionally essentials, purified polypeptides from crude or enriched lysates of such cells. Even isolated polypeptides that are pure in nature can be provided in any form and at any degree of purification. In a preferred embodiment, the polypeptide is a natural polypeptide, wherein the term "natural polypeptide" as used herein is a folded polypeptide, more preferably tissue or. Folded polypeptide purified from cells (more preferably from mammalian cells or tissues, and optionally from non-recombinant tissues or cells). In another preferred embodiment, the polypeptide is a recombinant protein, wherein the term "recombinant" as used herein is a genetically engineered approach at any stage of the production process. Is produced, for example, by fusing the nucleic acid encoding the polypeptide to a strong promoter for overexpression in cells or tissues, or by manipulating the sequence of the polypeptide itself. Refers to a polypeptide. One of ordinary skill in the art is a method for manipulating nucleic acids and encoded polypeptides (eg, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning-an nucleic acid, described in Hapman & Hall), and methods for producing and purifying natural or recombinant polypeptides (eg, Handbooks “Strategies for Protein Purification”, “A). Purification "," Purifying Cloning Proteins "(2009/2010), public by GE Healthcare Life Sciences, and in Burgess, R. R., Deutscher. .. In a preferred embodiment, the polypeptide is SDS polyacrylamide gel electrophoresis followed by at least 60%, 70%, 80%, 90%, 95% or 99% of the polypeptide in each sample. It is pure if it consists of the above polypeptides, as judged by Coomassie blue staining and visual inspection.

When the polypeptides of the invention are provided in the form of tissues, the tissues are mammalian tissues such as human, rat, primate, donkey, mouse, goat, horse, sheep, pig or bovine tissue, and more. It is preferably the brain tissue, most preferably the donkey. When cytolytic lysates are used, it is preferred that the cytolytic lysates include a membrane that associates with the surface of the cells. When the polypeptide is provided in the form of recombinant cells, the recombinant cells are eukaryotic cells, such as yeast cells, more preferably multicellular eukaryotic cells (eg, plants, mammals, etc.). Frogs or insects), most preferably cells derived from mammals (eg, rats, humans, primates, donkeys, mice, goats, horses, sheep, pigs or cows).

Polypeptides (including any variant) used to carry out the teachings of the invention are preferably recognized by and / or specific to the autoantibody that binds to Flotillin 1 and / or Flotilin 2. Designed to contain epitopes that bind specifically. In one embodiment, such polypeptides are 6, 7, 8, 9, 10, 11, 11, 12, 20, 25, 30 derived from Flotillin 1 and / or Flotillin 2. Includes a range of conservative amino acids of 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9 but 16 or less. One of ordinary skill in the art will use guidelines for designing peptides with sufficient immunogenicity, such as Jackson, D. et al. C. , Fitzmaurice, C.I. J. , Brown, L. E. , Zeng, W. (1999), Preparation and properties of totaly immunogenics, Vaccine Volume 18, Issues 3-4, September 1999, Page 355-361; and Black, M. , Trend, A. , Tirrel, M. et al. and Olive, C.I. (2010), Advances in the design and peptide of peptide vaccines with a focus on Toll-like receptor agonist; Briefly, the peptides should meet as many of the following requirements as possible: (a) have a high degree of hydrophilicity, (b) than the group containing aspartic acid, proline, tyrosine and phenylalanine. It contains one or more residues to be selected, (c) has no or little homology to other known peptides or polypeptides due to its higher specificity, (d) needs to be sufficiently soluble. There is, and (e) does not include glycosylation sites or phosphorylation sites unless required for specific reasons. Alternatively, a bioinformatics approach can be followed (eg, Moreau, V., Fleury, C., Piquer, D., Nguyen, C., Novali, N., Villard, S., Laune, D., Granier, C. and. Molina, F. (2008), PEPOP: Computational design of immunogenic peptides, described by BMC Bioinformatics 2008, 9:71).

The polypeptides of the invention are one or two or more polypeptides comprising Flotillina 1 and / or Flotillin 2 or variants thereof, preferably Flotillina and Flotillina 2. And when used in accordance with the present invention, can be provided in any kind of conformation. For example, the polypeptide can be an essentially unfolded polypeptide, a partially or completely folded polypeptide. In a preferred embodiment, the polypeptide takes the folding that the natural protein takes in its natural environment as a whole, the epitope essential for binding to the autoantibodies of the invention, or the protein or variant thereof. In that sense, it is folded. Those skilled in the art are suitable methods for determining whether a polypeptide is folded and, if so, what structure it has (eg, protein limited degradation, NMR spectroscopy, etc.). Familiar with CD spectroscopy or X-ray crystallography (eg, Banaszak L. J. (2008), Foundations of Structural Biology, Academia Press, or Tenge Q. (2013), Protein Q. (2013), Protein (See), preferably CD spectroscopy is used.

The polypeptide of the invention can be a fusion protein comprising an amino acid sequence other than that derived from Flotillin 1 and / or Flotillin 2, in particular a C-terminal or N-terminal tag, preferably a C-terminal tag. , In a preferred embodiment, as used herein, is an additional sequence motif or polypeptide having some biological or physical function, eg, purifying, fixing the polypeptide of the invention. , Can be used to precipitate or identify. In a more preferred embodiment, the tag is more than a group comprising a ligand, eg, a tag (His tag, thioredoxin, maltose binding protein, glutathione-S-transferase, fluorescent tag (eg, derived from the group comprising green fluorescent protein). A sequence or domain that can specifically bind to (selected). SEQ ID NOs: 1 and 2 and SEQ ID NOs: 5 and 6 represent exemplary fusion polypeptides containing Flotillin 1 and Flotillin 2, respectively.

The polypeptide of the invention can be a immobilized polypeptide. In a preferred embodiment, the term "fixed", as used herein, is more preferably a covalent bond, electrostatic interaction, encapsulation or capture of an insoluble solid carrier in an aqueous solution. Molecules bound via, for example, by denaturing a spherical polypeptide in a gel, or via hydrophobic interactions, most preferably via one or more covalent bonds. Such carriers are preferably artificial carriers, which are largely non-biological materials (eg, tissue sections). Various suitable carriers such as paper, polystyrene, metal, silicon or glass surfaces, microfluidic channels, membranes, beads (eg, magnetic beads), column chromatography media, biochips, polyacrylamide gels, etc. are described in the literature. (For example, Kim, D., and Herr, AE (2013), Proteinimmobilization technologies for siliconifluidic responses, Biomicrofluidics 7 (4), 041501). In this way, the immobilized molecule, along with the insoluble carrier, can be separated from the aqueous solution in a convenient manner, for example by filtration, centrifugation or transfer to another container. The immobilized molecule may be immobilized in a reversible manner or in an irreversible manner. For example, the fixation is when the molecule interacts with the carrier via an ionic interaction that can be blocked by the addition of a high concentration of salt, or when the molecule is a cleavable covalent bond (eg, the addition of a thiol-containing reagent). It is reversible when bound via a disulfide bridge that can be cleaved by. In contrast, the fixation is due to the reaction of epoxide and amine groups in which the molecule is frequently used to couple covalent bonds that cannot be cleaved in aqueous solution (eg, coupling lysine side chains to affinity columns). It is irreversible when it is connected to the carrier via a bond formed). The protein is indirectly immobilized, for example, by immobilizing an antibody or other entity having an affinity for the molecule, followed by the formation of a complex to the effect that the molecule-antibody complex is immobilized. obtain. Various methods for immobilizing molecules are described in the literature (eg, Kim, D., Herr, and AE (2013), Protein immobilization techniques for microfluidic assays, Biomiculos, 04 .. In addition, various reagents and kits for the immobilization reaction are commercially available, for example, from Pierce Biotechnology.

It is essential that the sample used for diagnosis according to the present invention contains an antibody (also referred to as immunoglobulin). Typically, a sample of body fluid comprises a representative set of the entire immunoglobulin of the subject. However, once provided, the sample may affect the relative distribution of various classes of immunoglobulins, fractionation, centrifugation, enrichment of the entire immunoglobulin or any immunoglobulin class of the subject. Alternatively, it may be subjected to further processing which may include isolation.

The reagents, devices, methods and uses described throughout this application can be used for the diagnosis of disease. In a preferred embodiment, the disease is a neurological disease. In a more preferred embodiment, the term "neurological disorder", as used herein, is associated with the lack of essential elements of the nervous system, more preferably the visual sense, more preferably the optic nerve. Refers to any disease.

In a preferred embodiment, the disease, more preferably the neurological disease, is cancer, increased cell count in CSF, intrathecal IgG synthesis, oligoclonal band in CSF, MRZ response in CSF, visual impairment, preferably vision. One or more symptoms from the group including disorders, optic neuritis, headache, spinal cord lesions and brain lesions, more preferably two or more, most preferably three or more. Preferably, the disease responds to immunomodulatory therapy, preferably immunosuppressive therapy.

In another preferred embodiment, the disease is Alzheimer's disease, autism, Asperger's syndrome, apathy, dementia, cerebral syndrome, encephalitis, butoh disease, encephalitis, movement disorder, spinal cerebral imbalance (preferably non-progressive). Morphology), paralysis, anti-paralysis, Gauche disease, myopathy, severe myasthenia, polysclerosis, Parkinson's disease, polyneuropathy and dementia, preferably cerebral syndrome, cerebral inflammation, polysclerosis, movement disorders And dementia, more preferably a neurological disorder selected from the group comprising multiple sclerosis. In a more preferred embodiment, the disease is a demyelinating disease, more preferably a demyelinating disease that affects the CNS, more preferably multiple sclerosis, most preferably multiple sclerosis associated with optic neuritis. I have a disease.

In another preferred embodiment, the disease is cancer, or preferably paraneoplastic cerebral syndrome, which is associated with one or more neurological symptoms, preferably increased cell count in CSF, CSF. (Derived from the group including internal IgG synthesis, oligoclonal band in CSF, MRZ response in CSF, visual impairment, optic neuritis, headache, spinal cord lesions and brain lesions) and is further associated with cancer. Detection of autoantibodies to Flotillin 1 and / or Flotillin 2 may indicate an increased likelihood that cancer cannot be detected using other methods or that there is cancer that emerges as the disease progresses. In a preferred embodiment, the cancer is a lung tumor, a thoracic gland tumor, a thoracic gland tumor, a testis tumor, a head and neck cancer tumor, a breast cancer tumor, an anal genital cancer tumor, a melanoma, a sarcoma, a carcinoma, a lymphoma, a leukemia, Cyclicoma, glioma, embryonic cell tumor, chorionic villus cancer, pancreatic cancer, ovarian cancer, gastric cancer, cancerous lesion of pancreas, lung adenocarcinoma, colon-rectal adenocarcinoma, pulmonary squamous adenocarcinoma , Gastric adenocarcinoma, neoplasm of superficial ovarian epithelium (eg, various benign, proliferative or malignant neoplasms about it), oral squamous epithelial cancer, non-small cell lung cancer, endometrial cancer, bladder cancer, prostate cancer, Acute myeloid leukemia (AML), myelodystrophy syndrome (MDS), non-small cell lung cancer (NSCLC), Wilms tumor, leukemia, lymphoma, fibrogenic small round cell tumor, mesopharyngeal tumor (eg, malignant mesotheloma) , Gastric cancer, colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian cancer, uterine cancer, thyroid cancer, hepatocellular carcinoma, thyroid cancer, liver cancer, kidney cancer, Kaposi sarcoma and other cancers Alternatively, it is a cancer of a tumor selected from the group including sarcoma.

In a preferred embodiment, the term "diagnosis", as used herein, affects or is likely to affect a patient or is more likely to be affected than an average or comparative subject. Obtaining information useful in assessing whether (the latter preferably has similar symptoms), having suffered from certain diseases or disorders in the past, at the time of diagnosis or in the future, and how the above diseases progress. Any type of procedure aimed at finding out if or may progress in the future, or assessing a patient's responsiveness to certain treatments (eg, administration of immunosuppressive drugs). To mention. In other words, the term "diagnosis" includes not only diagnosing, but also prognosing and / or monitoring the course of a disease or disorder.

In many cases, mere detection, in other words, determining whether or not a detectable level of the antibody is present in the sample is sufficient for diagnosis. If the autoantibodies can be detected, this is useful information for the clinician's diagnosis and indicates an increased likelihood that the patient will suffer from the disease. In a preferred embodiment, the relative concentration of the antibody in serum can be determined relative to the levels that can be found in the average healthy subject. While in many cases it may be sufficient to determine whether autoantibodies are present or detectable in the sample, the methods used to obtain information useful for diagnosis are at their concentrations. At least 0.1 times, preferably 0.2 times, 0.5 times, 1 times, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times the concentration found in the average healthy subject. It may include determining whether it is fold, 100 fold, 200 fold, 500 fold, 1000 fold, 10000 fold or 100 000 fold higher.

Clinicians usually cannot conclude whether a patient suffers from or is likely to suffer from a disease, condition or disorder only on the basis of a single diagnostic parameter, but other aspects, eg A definitive diagnosis is reached taking into account the presence of other autoantibodies, markers, blood parameters, clinical evaluation of patient symptoms, or the results of medical imaging or other non-invasive methods (eg, polysomnography). Recognize the need. Baenkler H. W. (2012), General assistants of autoimmune diagnostics, in Renz, H. et al. , Autoimmune diseases, 2012, de Gruyter, page 3. Since the value of a diagnostic agent or diagnostic method may lie in the possibility of excluding one disease, another indirect diagnosis may also be possible. In a preferred embodiment, the meaning of any symptom or disease referred to throughout this application is in line with the understanding of one of ordinary skill in the art as of May 29, 2015, as evidenced by textbooks and scientific publications. In a preferred embodiment, whether the polypeptide according to the invention or the method described above suffers from a disease characterized by a symptom similar to that of MS, more preferably with respect to the distinction between MS and NMO. Can be used to determine.

Thus, the term "diagnosis" preferably implies that a diagnostic method or agent according to the invention is definitive and sufficient to make a diagnostic decision based on a single test, as well as parameters. Rather, it may refer to a contribution to what is referred to as "differential diagnosis" (ie, a systematic diagnostic procedure that considers the possibility of a range of possible conditions based on a range of diagnostic parameters). In conclusion, the method, polypeptide or use of the invention to determine if a patient suffers from a disease, as needed, is the step of obtaining a sample from a patient (preferably a human patient), flotilin 1 and /. Alternatively, it is a step of determining whether or not an autoantibody that binds to flotilin 2 is present in the sample, and the step of determining here is a step performed by contacting the sample with the polypeptide of the present invention. , And whether or not binding has occurred between the polypeptide and the autoantibody is preferably a step of detecting using a labeled secondary antibody, wherein the autoantibody is present in the sample. If it is determined that the step of binding to the polypeptide and the autoantibodies are present in the sample, then the neurological disorder or cancer is or is likely to be affected. And may include the steps of diagnosing the patient. In a preferred embodiment, the methods of the invention may comprise a) aquaporin-4, and b) a step of detecting antibodies to flotilin 1 and / or flotilin 2 (preferably in that order). The term "diagnosis" may also refer to methods or agents used to distinguish between two or more conditions associated with similar or identical symptoms.

The term "diagnosis" may also refer to a method or agent used to select the most promising treatment regimen for a patient. In other words, the method or agent may relate to selecting a treatment regimen for the subject. For example, detection of autoantibodies may indicate that immunosuppressive therapy will be selected, which therapy may include administering one or more immunosuppressive agents to the patient.

The present invention relates to a complex comprising an antibody that binds to the polypeptide of the invention, preferably an autoantibody. Such complexes can be used or detected as part of a method for diagnosing a disease. Liquid samples containing antibody from the subject can be used to carry out the above method. Such a liquid sample can be any body fluid containing a representative set of antibodies derived from the subject, preferably a sample containing an IgG immunoglobulin class antibody derived from the subject. For example, the sample can be cerebrospinal fluid (CSF), blood or serum, lymph, interstitial fluid, preferably cerebrospinal fluid or CSF, more preferably serum.

The step of contacting a liquid sample containing an antibody with the polypeptide of the invention binds the immobilized form of the polypeptide to the polypeptide and the polypeptide of the invention in the presence of the sample containing the antibody. This can be done by incubating under conditions that are compatible with the formation of the complex containing the antibody, preferably the autoantibody. The liquid sample, which is then bound to the polypeptide of the invention and depleted of the antibody, can be subsequently removed, followed by one or more washing steps. Finally, a complex containing the antibody and the polypeptide can be detected. In a preferred embodiment, the term "conditions for complex formation and compatibility" allows for a specific antigen-antibody interaction, the complex comprising the polypeptide an the. It is a condition for forming antibody). In a preferred embodiment, such conditions may include incubating the polypeptide in a sample diluted 1: 100 with PBS buffer for 30 minutes at 25 ° C. In a preferred embodiment, the term "autoantibody", as used herein, is an antibody that specifically binds to an endogenous molecule of the animal (preferably a mammal) that produces the autoantibody. The level of such an antibody is more preferably an antibody that is elevated relative to the average of any other antibody that specifically binds to such an endogenous molecule. In the most preferred embodiment, the autoantibody is an autoantibody that binds to Flotillin 1 and / or Flotillin 2.

In a preferred embodiment, the detection of a complex for a prognosis, diagnosis, method or test kit according to the present invention is an immunodiffusion method, immunofluorescence method, light scattering immunoassay, light scattering immunoassay, aggregation method, labeled immunoassay (eg, eg). Includes the use of methods selected from the group including radioimmunoassays, enzyme immunoassays, chemiluminescent immunoassays, and radioimmunoassays). Those skilled in the art are familiar with these methods, and they are, at the art level, eg, Zane, H. et al. D. (2001), Immunology-Theoretical & Laboratory Conducts in Laboratory Medicine, W. et al. B. The Sanders Company, in particular, is described in Chapter 14.

Alternatively, a sample containing a tissue containing the polypeptide of the present invention other than the liquid sample may be used. The tissue sample is preferably derived from a tissue expressing endogenous Flotillin 1 and / or Flotillin 2. Such a sample, which may then be in the form of a carrier, eg, a tissue section immobilized on a glass slide for microscopic analysis, is an antibody of the invention that binds to the polypeptide of the invention, preferably. Can be contacted with autoantibodies. The antibodies are preferably labeled to allow differentiation from endogenous antibodies that bind to the polypeptides of the invention, resulting in the detection and optionally of newly formed complexes. Can be quantified. If the amount of complex formed is less than the amount found in a sample taken from a healthy subject, the subject tested for the sample is probably afflicted with the disease.

Any data indicating the presence or absence of the complex comprising the antibody and the polypeptide of the invention can be correlated with the reference data. For example, detection of the complex indicates that the patient who provided the sample to be analyzed is already suffering from, is suffering from, or may possibly be affected in the future. If the patient was previously diagnosed, the method for obtaining information related to the diagnosis is performed again, correlating the amount of complex detected in both runs, of disease progression and / or treatment. You can find out about success. For example, if the amount of complex is found to be increasing, this means that the disorder is progressing, is probably manifested in the future, and / or none of the attempted treatments are successful. Suggest.

In a preferred embodiment, a microplate, membrane ELISA, dot blot, or line blot is used to perform diagnostic methods according to the invention. Those skilled in the art are familiar with experimental setups, which are described in the art level (Radault, D., and Dash, G.A. (1989), The line blot: an immunoassay for Monoclonal antibody. It's application to the serotyping of gram-negative bacteria. J. Immunol. Methods, 125 (1-2), 57-65; WO2013017540).

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 of skill in the art are familiar with such techniques and the preparation of suitable samples, which are of the relevant state of the art (US4647543; Voice, J., Krause, C., Rohwaeder, E, Saschenbrecker, S., Han, M., Dankwardt, M., Feirer, C., Ens, K, Fechner, K, Barth, E, Martinetz, T., and Stoecker, W. (2012), Immunofluorescence Cells, "Clinical and Development Immunology, vol. 2012, doi: 10.1155 / 2012/65105; Bonilla, E., Frances, L., Alllam, F., et al., Immunofluorescence- For detection of antibody antibody reaction in systemic fluorescence erythematosus patients, Clinical Immunology, vol. 124, NO. 1, pp. It is commercially available from Leebeck, Technology).

Samples are subjected to tests to determine only the presence of autoantibodies that bind to Flotillin 1 and / or Flotilin 2, but diagnostic methods, tests, devices, etc. vary with neurological autoimmune disorders. Antigens or variants thereof (preferably Hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER / Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, Neurochrondrin, CARPVIII, Zic4, Sox1, Ma, MAG, MP0, MBP, GAD65, Amphificin, Recoverin, GABA A Receptor, GABA B Receptor, Glycin Receptor, Gephyrin, IgLON5, DPPX, Aquaporin-4, MOG, NMDA Receptor, AMPA Receptor, GRM1, GRM5, LGC1, VGCC and mgluR1 Also selected from the group containing CASPR2, it is preferred to attempt to determine the presence of autoantibodies to, for example, those antigens that are immobilized on a medical device (eg, a line blot). Diagnostically relevant markers neurochondrin, (EP1500118), ITPR1 (EP14003503.7), NBC1 (EP14003588.7) and ATP1A3 (human neuron Na (+) / K (+) also referred to as the α3 subunit of the ATPase. (EP141711561.5)) is described at the technical level.

According to the teachings of the invention, antibodies, preferably autoantibodies that bind to the polypeptides of the invention, are used and provided for the diagnosis of disease. Those of skill in the art are familiar with methods for purifying antibodies (eg, Hermanson, GT, Mallia, AK, and Smith, PK (1992), Immobilized Affinity Ligand Techniques, S. Diego: As described in Academic Press). Briefly, an antigen that specifically binds to the antibody of interest (which is the polypeptide of the invention) is immobilized and the antibody of interest is purified from a suitable source via affinity chromatography. Used for. Liquid samples containing antibodies from patients suffering from neurological disorders identified by us can be used as a source thereof.

According to the invention, antibodies such as autoantibodies are provided, which may specifically bind to the polypeptides of the invention. In a preferred embodiment, the term "antibody" as used herein is any immunoglobulin-based binding moiety, more preferably at least one immunoglobulin heavy chain and one immunoglobulin light chain. (Includes, but not limited to, monoclonal and polyclonal antibodies, and variants of the antibody, in particular fragments), wherein the binding moiety can bind to their respective antigens, more preferably. Those that can specifically bind to each of the antigens. In a preferred embodiment, the term "specifically bound", as used herein, is determined by surface plasmon resonance using a Biacore device in PBS buffer (pH 7) at 25 ° C. If so, 1 × 10 ^-5 M, more preferably 1 × 10 ^-7 M, more preferably 1 × 10 ^-8 M, more preferably 1 × 10 ^-9 M, more preferably 1 × 10 ^-10 M. , More preferably 1 × 10 ^-11 M, more preferably 1 × 10 ^-12 M, meaning stronger than the binding reaction characterized by the dissociation constant. The heterogeneous antibody may be part of a heterologous autoantibody preparation or may be an allogeneic autoantibody, wherein the heterologous preparation is prepared from the serum of a human donor, eg, the antigen. It contains multiple autoantibody species that differ from those that can be obtained by affinity chromatography using immobilized antigens to purify any autoantibodies that may bind to. The antibody may or may not be glycosylated. Those of skill in the art are familiar with the methods that can be used for the identification, production and purification of antibodies and variants thereof (eg, those described in EP 2423 226 A2 and references therein). The antibody can be used as a diagnostic agent itself or in combination, for example, in the form of a complex with the polypeptide of the invention.

The present invention provides a method for isolating an antibody that binds to the polypeptide of the present invention, preferably an autoantibody, wherein the method a) comprises a sample containing the above antibody and the polypeptide of the present invention. The steps of contacting the cells so that they are formed, b) the step of isolating the complex formed in step a), c) the step of dissociating the complex isolated in step b), and d) the above. Includes the steps of separating an antibody from the polypeptide of the invention. Samples from patients suffering from the novel neurological disorders identified by us can be used as a source of antibodies. Appropriate methods are, for example, Handbooks “Affinity Chromatography”, “Strategy for Protein Purification” and? Antibodies Purification ”(2009/2010), GE Health Described in techniques in immunochemistry, 1992, Marcel Decker, Inc.

The present invention provides a pharmaceutical composition comprising the polypeptide of the invention, which is preferably suitable for administration to a subject, preferably a mammalian subject, more preferably to a human. Such pharmaceutical compositions may include pharmaceutically acceptable carriers. The pharmaceutical composition may be administered, for example, orally, parenterally, by inhalation spray, topically, by infusion, rectum, nose, mouth, vagina or via a reservoir implanted. , Where the term "parenteral" as used herein is subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrabursa, intrathoracic, intrathecal. Includes intra-articular and intracranial injection or infusion techniques. The pharmaceutical compositions may be provided in suitable dosage forms, such as capsules, tablets and aqueous suspensions and liquids, preferably in sterile form. It can be used in a method of treating a disease, the method comprising administering to a subject an effective amount of a polypeptide of the invention. In a preferred embodiment, the invention uses a vaccine comprising the polypeptide of the invention, including, optionally, an adjuvant (eg, an adjuvant or buffer), as well as the polypeptide of the invention for the preparation of the vaccine. I will provide a.

Within the scope of the invention, a medical device comprising the (autoantibody) antibody of the invention and / or the polypeptide of the invention (preferably coated with the (autoantibody) antibody of the invention and / or the polypeptide of the invention). Alternatively, a diagnostic device is provided. Preferably, such a medical or diagnostic device is a form that allows the polypeptide of the invention to be brought into contact with the device in a convenient manner with an aqueous solution, more preferably a liquid human sample. Including in. In particular, the polypeptides of the invention included are from the group comprising artificial carriers (preferably glass plates or slides, biochips, microtiter plates, beads such as magnetic beads, affinity devices, chromatography columns, membranes and the like. Can be anchored to the surface of a carrier such as (selected). Exemplary medical devices include line blots, microplates, microscope slides, beads and biochips. In addition to the polypeptides of the invention, the medical or diagnostic device may include additional polypeptides, preferably in enriched, isolated and / or recombinant forms, eg, positive or negative. It may include other known antigens that bind to control or diagnostic autoantibodies, in particular those of other related diseases associated with one or more identical or similar symptoms. The medical device, preferably one or more antigens, preferably one diagnostically useful carrier containing more than one antigen, or more than one diagnostically useful carrier (each one or more). In addition to the polypeptides of the invention, the following: Hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER / Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, Neurochondrin, CARPVIII, Zic4, Sox1, Ma, MAG, MP0, MBP, GAD65, Amphificin, Recoverin, GABA A receptor, GABA B receptor, Glycin receptor, Gephyrin, IgLON5, DPPX, Aquaporin-4, MOG, NMDA It may include one or more antigens from the group comprising a receptor, an AMPA receptor, GRM1, GRM5, LGI1, VGCC and mGluR1 and CASPR2, preferably at least a combination comprising a polypeptide according to the invention, aquaporin-4 and MOG. As a biological activity, variants of each antigen that have the ability to bind to their respective autoantibodies to the antigen can be used in place of the antigens described above.

The technique of the present invention preferably provides a kit for diagnosing a disease. Such a kit provides instructions detailing how to use the kit and means for contacting the polypeptide of the invention with a body fluid sample from a subject (preferably a human subject), eg, a line blot. It may include, where the polypeptide of the invention is immobilized on the line blot. In addition, the kit is detectable for positive controls (eg, batches of autoantibodies or recombinant antibodies known to bind to the polypeptides of the invention) and negative controls (eg, for the polypeptides of the invention). It may contain proteins that have no affinity (eg, bovine serum albumin). Finally, such a kit may include a standard solution of antibody or antigen to create a calibration curve.

In a preferred embodiment, the kit detects a complex comprising an antibody that binds to a polypeptide of the invention, more preferably an autoantibody, preferably a polypeptide of the invention and an antibody that binds to the polypeptide of the invention. By doing so, it includes means for detection. Such means are preferably agents comprising a label that binds to or modifies the complex or makes the complex detectable. For example, the means may be a labeled antibody that binds to the polypeptide or to the constant region of the primary antibody at a binding site other than the one recognized by the primary antibody. Alternatively, the means may be a secondary antibody that binds to the constant region of the autoantibody, preferably a secondary antibody specific for a mammalian IgG class antibody. Numerous methods and means for detecting such complexes are available at the art level, eg, Philips, Terry, M. et al. , Analytical techniques in immunochemistry, 1992, Marcel Dekker, Inc. It is described in.

The polypeptides of the invention comprising Flotillin 1 and / or Flotillin 2 can be produced or provided in the form of cells comprising and / or expressing the nucleic acids encoding the above polypeptides. When nucleic acids containing sequences encoding the polypeptides of the invention or variants thereof are used, such nucleic acids can be unmodified nucleic acids. In a preferred embodiment, the nucleic acid is not naturally present in its own right and has at least one modification compared to the naturally occurring nucleic acid, eg, isotope content or chemical modification (eg, methyl indicating a synthetic origin). Nucleic acid containing chemicals, sequence modification, labeling, etc.). In a preferred embodiment, the nucleic acid is a recombinant nucleic acid or part or a nucleic acid, and in a more preferred embodiment, it is part of a vector, wherein it is an expression of the nucleic acid. , Preferably can be functionally linked to a promoter that allows overexpression. Those of skill in the art are familiar with a variety of suitable vectors, among others, commercially available from Origin, for example. For example, a vector encoding a fusion construct with C-terminal GFP can be used. The cells can be eukaryotic cells or prokaryotic cells, preferably eukaryotic cells (eg, yeast cells), more preferably mammalian cells, more preferably human cells (eg, HEK293 cells). .. Examples of mammalian cells include HEK293 cells, CHO cells or COS-7 cells. The cell containing the nucleic acid encoding the polypeptide of the invention can be a recombinant cell or an isolated cell. The term "isolated" as used herein means that there are few cells of other differentiation or cells of other species, or in fact, such other, as compared to the wild-type environment of the above cells. It means that the above cells are enriched so that there are no cells of the above.

The teachings of the present invention can be used not only for diagnosis but also for prevention or treatment of diseases, and more specifically, methods for preventing or treating diseases are described in a) the above. Steps to reduce the concentration of autoantibodies bound to the polypeptide of the invention in the blood of a subject and / or b) one or more immunosuppressive pharmaceutical substances (preferably rituximab, prednisone, methylprednisone, cyclophosphami). Des, mycophenolate mofetil, intravenous immunoglobulin, tacrolimus, methotrexate, azathioprine and / or selected from the group comprising the above pharmaceutical compositions).

Some sequences are disclosed herein, more specifically, SEQ ID NO: 1 represents the nucleotide sequence of the expression vector pTriEX-1-Flotillin-1 [human] -His, and SEQ ID NO: 2 represents the C-terminal His. SEQ ID NO: 3 represents the nucleotide sequence of the expression vector pTriEX-1-Flotilin-1 [human], and SEQ ID NO: 4 represents the polypeptide sequence of human flotilin 1 bound to the tag. SEQ ID NO: 5 represents the nucleotide sequence of the expression vector pTriEX-1-Flotillin-2 [human] -His, SEQ ID NO: 6 represents the polypeptide sequence of human Flotillin 2 bound to the C-terminal His tag, and SEQ ID NO: 7 represents. , Expression vector pTriEX-1-Flotilin-2 [human], SEQ ID NO: 8 represents the polypeptide sequence of human Flotillin 2.

The present invention provides, for example, the following items.
Item 1. A method for diagnosing a disease comprising the step of detecting an autoantibody that binds to Flotillin 1 and / or Flotillin 2 in a sample derived from a patient.
Item 2. A polypeptide comprising flotilin 1 and / or flotilin 2 or a variant thereof, preferably immobilized, more preferably immobilized on a solid carrier.
Item 3. Use of the polypeptide according to the preceding item for diagnosis of disease, preferably comprising the step of detecting an autoantibody that binds to Flotillin 1 and / or Flotillin 2 in the sample.
Item 4. The polypeptide according to any one of the preceding items for use in the treatment of a disease.
Item 5. An autoantibody that binds to Flotillin 1 and / or Flotilin 2, preferably an isolated autoantibody, preferably in the form of a complex with the polypeptide according to any one of the preceding items. Autoantibodies.
Item 6. A method for isolating an autoantibody that binds to Flotillin 1 and / or Flotilin 2. a) A sample containing the autoantibody is combined with the polypeptide according to any one of the preceding items. A step of contacting under conditions suitable for body formation, wherein the autoantibody binds to the polypeptide.
b) Step of isolating the complex formed in step a),
c) A method comprising the steps of dissociating the complex isolated in step b) and d) separating the autoantibody from the polypeptide.
Item 7. A pharmaceutical composition or medical device, preferably a diagnostic device, comprising the polypeptide according to any one of the preceding items.
Item 8. A test kit for diagnosing a disease, comprising the polypeptide according to any one of the preceding items, preferably the polypeptide according to any one of the preceding items and flotilin 1 and / or flotilin. A test kit further comprising means for detecting a complex comprising an autoantibody that binds to 2.
Item 9. A composition for diagnosing a disease, comprising an agent that detects an autoantibody that binds to Flotillin 1 and / or Flotilin 2 in a sample.
Item 10. A composition comprising the polypeptide according to any one of the preceding items for the diagnosis of a disease, wherein the autoantibody that binds to Flotillin 1 and / or Flotillin 2 is detected in the sample.
Item 11. A composition for use in the treatment of a disease comprising the polypeptide according to any one of the preceding items.
Item 12. A diagnostic agent for diagnosing a disease, comprising a detection agent that detects an autoantibody that binds to Flotillin 1 and / or Flotilin 2 in a sample.
Item 13. A diagnostic agent for diagnosing a disease, comprising the polypeptide according to any one of the preceding items, which detects an autoantibody that binds to Flotillin 1 and / or Flotillin 2 in a sample.
Item 14. A therapeutic agent for use in the treatment of a disease, comprising the polypeptide according to any one of the preceding items.
Item 15. A kit or device for diagnosing a disease, comprising means for detecting the binding of autoantibodies to Flotillin 1 and / or Flotilin 2 in a sample.
Item 16. A kit or device for diagnosing a disease comprising the polypeptide according to any one of the preceding items, wherein the kit is an autoantibody that binds to Flotillin 1 and / or Flotillin 2 in a sample. To detect a kit or device.
Item 17. A kit or device for use in the treatment of a disease, comprising the polypeptide according to any one of the preceding items.
Item 18. The patient was selected from the group including increased cell count in CSF, intrathecal IgG synthesis, oligoclonal band in CSF, MRZ response in CSF, visual impairment, preferably visual impairment, optic neuritis, headache, spinal cord lesions and brain lesions. Any of the preceding items that have one or more, preferably two or more symptoms, or that the disease is associated with the one or more, preferably two or more symptoms. The method, polypeptide, use, autoantibody, pharmaceutical composition, device, test kit, composition, diagnostic agent, or kit according to paragraph 1.
Item 19. The disease is described in any one of the preceding items, wherein the disease is a neurological disease, preferably a demyelinating disease, preferably a CNS disease, preferably a brain disease, preferably a disease associated with optic nerve inflammation. Methods, polypeptides, uses, autoantibodies, pharmaceutical compositions, devices, test kits, compositions, diagnostic agents, or kits.
Item 20. The polypeptide, use, self Antibodies, methods, pharmaceutical compositions, devices, test kits, compositions, diagnostic agents, or kits.
Item 21. The polypeptide, use, autoantibody, method, pharmaceutical composition, device or test according to any one of the preceding items, wherein the polypeptide is a recombinant polypeptide and / or an isolated polypeptide. kit.
Item 22. The method according to any one of the preceding items, wherein the sample is preferably a body fluid containing an antibody selected from the group containing whole blood, serum, cerebrospinal fluid and saliva.
Item 23. The method according to any one of the preceding items, wherein both the polypeptide comprising Flotillin-1 or a variant thereof and the polypeptide comprising Flotillin-2 or a variant thereof are present and preferably are part of the complex. , Polypeptides, uses, autoantibodies, pharmaceutical compositions, devices, or test kits.
Item 24. The method, polypeptide, use, autoantibody, pharmaceutical composition, device, test kit, composition according to any one of the preceding items, wherein the autoantibody binds to a complex containing flotilin 1 and flotilin 2. , Diagnostic agent, or kit.

(Summary of this disclosure)
The present invention detects in a sample derived from a patient a polypeptide comprising an autoantibody that binds to Flotillin 1 and / or Flotillin 2, Flotillin 1 and / or Flotillin 2 or a variant thereof (which is preferably immobilized). Methods for diagnosing a disease comprising the steps, use of the above-mentioned polypeptide for diagnosing a disease, autoantibodies that bind to Flotillin 1 and / or Flotillin 2, methods for isolating the above-mentioned autoantibodies, and the above-mentioned. Concerning pharmaceutical compositions, medical devices or test kits containing polypeptides.

FIG. 1 shows head MRI showing multiple demyelinating lesions in anti-Flotillin-positive patients. The female patient presented with optic neuritis of suspected autoimmune origin. Lesions that remained stable under immunomodulatory treatment for 5 months with interferon β. T2-enhanced magnetic resonance imaging (A, C) and flare images (B, D) of the same 34-year-old female patient at the initial presentation (A, B) and 6 months later (C, D). FIG. 2 shows immunofluorescent staining of central nervous system. Cold sections were incubated with patient serum (1: 100) or CSF (undiluted) in the first step and with FITC-labeled goat anti-human IgG in the second step. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). Fine-granular staining of the molecular layer (ML) and mottled staining of the granular layer were obtained. In the hippocampus, the outer ML was stronger than the inner ML. A) rat hippocampus, B) rat cerebellum, C) monkey cerebellum. FIG. 3 shows tissue-immunoprecipitation and antigen identification. Cold sections of rat or porcine cerebellum were incubated with the patient's serum (1: 200) above, washed with PBS and solubilized with detergent. The above solution was incubated with protein G-coated magnetic beads. Immune complexes were eluted with SDS and subjected to SDS-PAGE analysis and Western blot. Figure 3A: Left: Staining with colloidal Coomassie. Right: Western blot after incubation with anti-flotilin 2. Lane 1: Molecular Weight Marker, Lanes 2 & 3: Patient Serum Tissue from Rat Cerebellum-Immunoprecipitate, Lanes 4 & 5: Control Sample Tissue-Immunoprecipitate. Arrows indicate the location of the immunoprecipitated antigen 50 kDa, while dashed arrows indicate the location of the 52 kDa IgG heavy chain. FIG. 3B: Rat hippocampus (1-3) and cerebellum (4-6), monkey intestine (7-9) and optic nerve (10-12) tissue sections with patient serum (1 & 4) and anti-Flotillin 2 antibody (2 & 5). Immunofluorescent staining. The integrated photographs show the co-localization of both reactions, including stronger staining of the outer molecular layer of the hippocampus (3 & 6). FIG. 4 shows the immunofluorescent staining of recombinant flotilin and its neutralization of antibody reactions on tissues. FIG. 4A: Immunofluorescence analysis of transfected HEK293 cells. Patient serum or control serum (1: 1000) (green) on acetone-fixed recombinant HEK293 cells or mocked overexpressing Flotillin 1 (A), Flotillin 2 (B), Flotillin 1 and Flotillin 2 (C). Transfection control (D) was incubated on acetone-fixed recombinant HEK293 cells. FIG. 4B: Neutralization of immunofluorescent response on neuronal tissue. Patient serum (green) was preincubated with an extract of HEK293 cells transfected with an empty vector (1-4) or Flotilin 1/2 (5-8) as a control. Extracts containing Flotillin 1/2 significantly reduced or abolished the immune response. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). Hippocampal rats (1 & 5), cerebellar rats (2 & 6), cerebellar monkeys (3 & 7), HEK2933-Flotillin 1/2 (4 & 8).

(Overview:)
The following examples were clinically evaluated for patients suffering from optic neuritis, more specifically visual impairment, headache and brain lesions, but the molecular basis of their disease is unknown. Indicates that it remained. Patients' blood was screened and did not contain autoantibodies to known neurological markers. Autoantibodies were isolated based on characteristic staining patterns after reaction with some mammalian tissues, more specifically the cerebellum and hippocampus derived from rats and pigs.

Immunoprecipitation, mass spectrometry and competitive binding studies using recombinant Flotillin 1 and Flotilin 2 have revealed Flotillin 1 and Flotillin 2 as targets for the above antibodies. Serum from 34 patients with various neurological autoantibodies (anti-NMDAR, anti-Hu, anti-Yo, anti-Ri, anti-AQP4, anti-LGI1, anti-CASPR2) as well as sera from 226 healthy controls are combined. No reaction was observed when contacted with the modified flotilin 1/2, confirming that the method of the invention is a specific assay.

In that sense, the above patients responded positively to immunosuppressive treatment. The visual symptoms have completely disappeared.

(Patient characterization)
In other aspects, a healthy female patient (34 years old) first showed blurred vision, localized post-eyeball pain, and the intensity of red vision in the right eye diminished, which began 5 days before the presentation. There was no conspicuous history and no implications for any autoimmune disease in relatives' families, but the maternal grandmother was suspected of having multiple sclerosis. Clinical neurological findings revealed impaired right-eye visual acuity (0.6; left: 1.0). Examination of visual evoked potentials revealed severe impairment of visual afferent processing of optical afferences. In addition, further electrophysiological examinations also revealed that the sensory afferents (sensitive afferences) from the left leg were slightly impaired, but responded normally after transcranial magnetic stimulation to all limbs. Head MRI shows signs of inflammation of the right optic nerve (enhanced contrast) and some small subcortical white matter lesions (no signs of blood-brain barrier damage) typical of demyelinating diseases (eg, multiple sclerosis). It became clear (Fig. 1). Blood tests showed normal values of renal and liver function, normal electrolytes, and normal numbers and distribution of red blood cells and white blood cells. Testing of autoantibodies in serum did not reveal significant findings: rheumatoid factor, pANCA, AMA, antiphospholipids, anti-Borrelia (IgG / IgM) and anti-Treponema were negative; ANA It existed at a titer of 1: 320. CSF revealed mild preocytosis (9 cells / μL), normal protein (269 mg / L), local IgG synthesis (53%), and oligoclonal bands that were not present in serum. Anti-AQP4 was undetectable in both CSF and serum.

The above patients were treated with intravenous glucocorticoid pulse therapy under suspicion of autoimmune neuritis of the optic nerve. Assuming clinically isolated syndrome, immunomodulation with betaferon was started. After 5 months, the visual symptoms were completely gone.

Twenty-two months after the onset of the first sign, the patient presented a stable clinical condition without further progression of neurological symptoms. Control lumbar puncture revealed autologous antibody production, along with oligoclonal band in CSF and mild pleocytosis of 5 cells / μL CSF.

(Indirect Immunofluorescence Assay (IFA))
Slides with biochip mosaics containing cold sections of brain tissue (rat hippocampus, rat, monkey and pig cerebellum) and HEK293 cells individually expressing 30 recombinant brain antigens were used for IFA. The slides were incubated with a 70 μL sample diluted with PBS, 0.2% Tween-20 (IFA buffer) at room temperature for 30 minutes, washed with IFA buffer and immersed in IFA buffer for 5 minutes. Then, polyclonal goat anti-human pan-IgG (EUROIMMUN) or monoclonal mouse anti-human IgG1, IgG2, IgG3, or IgG4 (Sigma-Aldrich) (each labeled with fluorescein isothiocyanate (FITC)) is applied at room temperature for 30 minutes. Incubated. The slides were washed again, embedded in PBS buffered DABCO (about 20 μL / mosaic) containing glycerol, and used by a laser scanning microscope (LSM700, Zeiss, Jena, Germany) by two unrelated observers. Inspected. Included positive and negative controls. Samples were compared directly with non-transfected cells and control samples and classified based on the tissue pattern and fluorescence intensity of the transfected cells. The endpoint titer is the highest dilution that exhibits obvious fluorescence. Live cell IFA in primary hippocampal neurons was performed (Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, et al. Anti-NMDA-receptor analysis: case series. Ranchet Neurol 2008 Oct 11).

A polyclonal rabbit antibody against flotilin 2 (Sigma-Aldrich, diluted 1: 100) was used in the first step in some experiments, followed by anti-rabbit IgG-Cy3 (Jackson Research, Suffolk, United Kingdom). Incubated with. Cell nuclei were visualized by DNA staining with TO-PRO3 iodide (diluted 1: 2000) (Thermo Fisher Scientific, Schwerte, Germany). Recombinant antigens were mixed with diluted serum samples for 1 hour prior to IFA for neutralization experiments (Stoecker W, Otte M, Ulrich S, Normaln D, Finkbeiner H, Stoecker K, et al. to pancreatic juice in Brown's disease.

(Tissue-immunoprecipitation and antigen identification)
The cerebellum of a rat or pig was removed and shock-frozen in -160 ° C. isopentane. The tissue was then frozen sections (4 μm) on a SM2000R microtome (Leica Microtome, Nußloch, Germany), placed on a glass slide, dried and stored at -196 ° C. For HIP, slides were incubated with patient serum (1: 100 dilution) at 4 ° C. for 3 hours, followed by 3 washes with IFA buffer. The above tissue was then subjected to solubilization buffer (100 mmol / L tris-HCl pH 7.4, 150 mmol / L sodium chloride, 2.5 mmol / L EDTA, 0.5% (w / v) deoxycholate, 1%. (W / v) Triton X-100, containing protease inhibitor) was extracted at room temperature for 30 minutes. The resulting suspension was homogenized and centrifuged at 16,000 xg at 4 ° C. for 15 minutes. Immune complexes were precipitated from the clear supernatant in Protein G Dynabeads (Thermo Fisher Scientific, Dreieich, Germany) overnight at 4 ° C., washed 3 times with PBS, PBS 5 mmol / L dithiothreitol, 1%. (W / v) Sodium dodecyl sulfate was eluted at 95 ° C. for 10 minutes. The eluent was analyzed by SDS-PAGE and mass spectrometry or Western blotting.

(Recombinant expression of Flotillin 1 and Flotillin 2 in HEK293)
Cloning of expression vectors (SEQ ID NOs: 1 and 3 encoding polypeptides containing Flotillin 1; SEQ ID NOs: 5 and 7 encoding polypeptides containing Flotillin 2) was performed using standard methods. To prepare the substrate for IFA, HEK293 was seeded on sterile coverglasses, transfected and expressed either Flotillin 1 or Flotillin 2 individually or together for 48 hours. Coverglass was washed with PBS, fixed in acetone at room temperature for 10 minutes, air dried, cut into millimeter-sized biochips and used as a substrate in IFA as described. Alternatively, cells were transfected in a standard T-flask and the cells were harvested 5 days later. The cell sediment was extracted with a solubilizing buffer. The extract was aliquoted and stored at −80 ° C. until further use.

(Study in a larger patient cohort)
Forty-nine patients with various neurological autoantibodies (including 20 with autoantibodies to AQP4 (up to a titer of 1: 3,200)), and 226 healthy control-derived sera. Analyzed by IFA. None of the above sera reacted with HEK2933-Flotyline-1, HEK2933-Flotyrin-2, and HEK2933-Flotyline-1 / 2.

Anti-Flotillin-1 / 2 status was then retroactively determined in 224 samples and a comprehensive extensive neurological autoantibody screening (including the parameters described above) was performed on these samples in the Clinical Immunological Laboratory Louebeck and is known. We have reported a neural tissue-reactive IgG autoantibody that does not have the antigen specificity of. Serum anti-flotilin-1 / 2 was revealed in 4 patients (P2-P5 in Table 1, titers: 1: 1,000, 1: 1,000, 1: 10000, 1: 10,000). Patients P2, P3, and P5 also showed anti-flotilin-1 / 2 in CSF (titer: 1: 3.2, 1: 100, 1: 1,000). For P4, CSF was not available. Specific antibody index> 4 was calculated for P1, P3, and P5. Further follow-up sera of the above patients were analyzed when available and the anti-flotilin-1 / 2 titers of P1, P2, and P4 were stable over a period of 18, 24, and 72 months, respectively. It was shown to be. Serum of P5 showed a decrease to 1: 320 7 weeks after plasmapheresis.

HEK2933-Flotillin-1 / 2 was then integrated into the extensive autoantibody screening regimen of the reference measurement facility. In a subsequent cohort of 521 patients who were required to determine anti-AQP4, 18 were positive for anti-AQP4, while 3 showed anti-flotilin-1 / 2. Medical records were accessible for one of the latter (serum: 1: 1,000, no CSF, P6 in Table 1). Additional patients, during the subsequent diagnostic examination of 150 unselected neurological patients (sera: 1:10, CSF negative, P7 in Table 1), and additional patients, 57 anonymous patients, simply. It was identified by screening anti-AQP4 negative and anti-MOG negative sera from patients with separated ON (1:10, CSF was not available).

In summary, all 7 patients whose medical records could be evaluated had radiological signs of disseminated demyelination, mild preocytosis and OCB in CSF, consistent with MS or CIS suggestive of MS. .. Six of them showed optic neuritis. In all cases, the autoantibodies were subclass IgG1 and bound to flotilin-1 / 2, but neither individual flotilin-1 nor flotilin-2. None of the above patients showed anti-AQP4 antibody or anti-MOG antibody.

Claims (8)

A method for diagnosing a neurological autoimmune disease comprising the step of detecting an autoantibody that binds to a complex containing flotilin 1 and flotilin 2 in a sample derived from a patient. Flotillin 1 or a variant thereof and Flotillin 2 or a variant thereof for the diagnosis of neurological autoimmune disease, comprising the step of detecting autoantibodies that bind to a complex containing Flotillin 1 and Flotillin 2 in a sample derived from a patient. Use of the complex with. An autoantibody that binds to a complex containing Flotillin 1 and Flotillin 2. A method for isolating an autoantibody that binds to a complex containing Flotillin 1 and Flotillin 2.
a) A step of contacting a sample containing the autoantibody with a first complex of Flotillin 1 or a variant thereof and Flotillin 2 or a variant thereof under conditions suitable for the formation of a further second complex. Here, the autoantibody binds to the first complex and
b) Step of isolating the second complex formed in step a),
c) Step b) Dissociating the isolated second complex, and d) Separating the autoantibody from the first complex.
A method of including. A test kit for the diagnosis of neurological autoimmune diseases, which comprises a complex of Flotillin 1 or a variant thereof and Flotillin 2 or a variant thereof. 1 according to claim 1, wherein the disease is associated with one or more symptoms selected from the group including increased cell count in CSF, intrathecal IgG synthesis, oligoclonal band in CSF, MRZ response in CSF, and visual impairment. the method of. The method of claim 4, wherein the Flotillin 1 or a variant thereof and Flotillin 2 or a variant thereof are recombinant polypeptides and / or isolated polypeptides. The method according to any one of claims 1, 4 and 7, wherein the sample is a body fluid containing an antibody selected from the group containing whole blood, serum, cerebrospinal fluid and saliva.

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