[go: up one dir, main page]

WO2011085081A2 - Procédés de sélection de patients souffrant de sclérose en plaques (ms) pour une thérapie spécifique à un antigène - Google Patents

Procédés de sélection de patients souffrant de sclérose en plaques (ms) pour une thérapie spécifique à un antigène Download PDF

Info

Publication number
WO2011085081A2
WO2011085081A2 PCT/US2011/020345 US2011020345W WO2011085081A2 WO 2011085081 A2 WO2011085081 A2 WO 2011085081A2 US 2011020345 W US2011020345 W US 2011020345W WO 2011085081 A2 WO2011085081 A2 WO 2011085081A2
Authority
WO
WIPO (PCT)
Prior art keywords
myelin
mbp
patient
protein
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/020345
Other languages
English (en)
Other versions
WO2011085081A3 (fr
Inventor
William Robinson
Amena Rahman
Robert King
Hideki Garren
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayhill Therapeutics Inc
Original Assignee
Bayhill Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayhill Therapeutics Inc filed Critical Bayhill Therapeutics Inc
Publication of WO2011085081A2 publication Critical patent/WO2011085081A2/fr
Publication of WO2011085081A3 publication Critical patent/WO2011085081A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to a method for selecting patients afflicted with multiple sclerosis (MS) for antigen specific therapy when a blood sample from the patient has been determined to contain antibodies to MS-associated antigens that exceeds an antibody threshold. .
  • MS multiple sclerosis
  • a method for determining the MS-associated antibodies of a patient sample and antibody threshold using arrays is also described.
  • MS Multiple Sclerosis
  • CNS central nervous system
  • MS affects the white matter of the brain and spinal cord through damaging the myelin sheath of the neurons, and thereby inhibiting the ability of signals to propagate from the brain to the rest of the body.
  • MS is the most common demyelinating disorder of the CNS and affects about 400,000 Americans and 2.5 million people worldwide.
  • MS The course of MS is often progressive, from the most common symptoms of depression, fatigue, visual impairments, gait disorders, limb weaknesses, trouble walking, sensory disturbances/vertigo, numbness in extremities pain, sexual dysfunction, spasticity, as well as bowel and bladder dysfunctions, and ending with symptoms as severe as paralysis.
  • demyelination and recurrent lesions on the brain white matter progressively lead to disabling of an individual in a stepwise fashion.
  • methods to diagnose early are important.
  • early diagnosis combined with assays for predicting efficacy, lack of efficacy, or adverse effects of therapy are critical to treating the disease while symptoms are mild and/or reversible and before severe disability results.
  • MS Multiple sclerosis
  • CSF cerebrospinal fluid
  • glatiramer acetate is a synthetic protein comprising components of myelin protein that reduces the frequency of relapses in relapsing-remitting MS; Tysabri, an anti- alpha4-integrin antibody that also reduces relapse rate; and, steroids (medrol and decadron) that reduce inflammation and are typically used to treat an acute attack of MS.
  • None of the current therapies for MS are antigen specific, and none of the current therapies include an assay to identify MS patients who would benefit from antigen specific therapy. [0006]
  • Several clinical trials of antigen specific therapies in multiple sclerosis have recently been undertaken. An altered peptide ligand derived from MBP did not meet its primary endpoint.
  • a seventeen amino acid peptide derived from MBP did not meet its Attorney Docket No.: 0 1686-0023 lUPC primary endpoint of delaying disease progression as measured by Expanded Disability Status Scale (EDSS) in patients with secondary progressive multiple sclerosis (SPMS).
  • EDSS Expanded Disability Status Scale
  • SPMS secondary progressive multiple sclerosis
  • Antibodies have been measured in serum and cerebrospinal fluid (CSF) of MS patients. Antibodies against myelin associated proteins have been measured previously by Western Blot detecting antibodies to MOG and MBP in serum and CSF (Reindl, M et al, Brain 1999; 122: 2041-2056 and Egg, R., et al., Mult Scler. 2001 ; 7: 285-9). Berger, et al, NEJM 349: 139-45, 2003 and Kulhe, et al, NEJM 356: 371-8, 2007 used Western Blot to correlate levels of anti-MOG and anti-MBP antibodies with disease progression in MS patients.
  • CSF cerebrospinal fluid
  • Mantegazza et al utilized ELISA to detect anti-MOG antibodies in serum and CSF to evaluate antibody levels in MS patients (Mantegazza et al; International Immunology Vol 16, No. 3, 559-565 (2004). Zadro etal (Clinical Neurology and Neurosurgery 109 (2007) 23- 26 measured levels of anti-MOG antibodies in relapsing remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and in non-inflammatory neurological diseases (NIND).
  • RRMS relapsing remitting multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • NIND non-inflammatory neurological diseases
  • the present invention relates, at least in part, to the discovery that if antibodies to myelin antigens in the serum of a patient afflicted with multiple sclerosis exceeds an antibody threshold that patient would derive clinical benefit from antigen specific therapy.
  • the measure of MS-associated antibodies and whether it exceeds an antibody threshold is a surrogate measure of an immune-mediated attack on one or more component(s) of the central nervous system myelin that could be treated with antigen specific therapy. Screening patients to determine whether a patient's biological sample contains antibodies to myelin antigens exceeding an antibody threshold enables the identification of a subpopulation of MS patients who can derive greater clinical benefit from antigen specific therapy such as BHT 3009.
  • the invention provides a method for treating multiple sclerosis comprising administering to a patient antigen specific therapy in an amount effective to treat multiple sclerosis, wherein a blood sample from the patient has been determined to contain antibodies to MS-associated antigens that exceeds an antibody threshold.
  • the MS-associated antigens can be myelin antigens, including but not limited to myelin auto-antigens such as proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), myelin-associated glycoprotein (MAG), or myelin- associated oligodendrocytic basic protein (MBOP).
  • myelin antigens are used in combinations. Examples include, MBP+PLP, MBP+MOG, MBP+MAG, MBP+PLP+MOG, MBP+PLP+MOG+MAG, MBP+ cyclic nucleotide phosphodiesterase (CNPase), and MBP+MBOP.
  • the MS-associated antigens may be alpha-B-crystallin; viral and bacterial mimicry peptides (e.g., influenza, herpes viruses, hepatitis B virus, and the like), cyclic nucleotide phosphodiesterase (CNPase),
  • viral and bacterial mimicry peptides e.g., influenza, herpes viruses, hepatitis B virus, and the like
  • CNPase cyclic nucleotide phosphodiesterase
  • the antigen specific therapy typically includes administration of a polynucleotide encoding a myelin antigen, such as myelin basic protein.
  • a myelin antigen such as myelin basic protein.
  • the polynucleotide is BHT 3009.
  • the antigen specific therapy may also comprise administration of a myelin polypeptide or peptide autoantigen.
  • the antibody threshold can be determined using a variety of methods.
  • an array usually a microarray, is used.
  • the invention provides a method for treating multiple sclerosis comprising administering to a patient an antigen specific therapy in an amount effective to treat the MS, wherein a patient blood sample has been determined to contain antibodies to myelin basic protein (MBP) that exceeds an antibody threshold.
  • MBP myelin basic protein
  • the invention concerns a method for treating multiple sclerosis, comprising administering to a patient antigen specific therapy in the form of a polynucleotide encoding an MS-associated protein in an amount effective to treat MS, wherein a patient blood sample has been determined to contain antibodies to myelin antigens that exceeds an antibody threshold.
  • the invention also relates to a method of assessing whether a patient would benefit from antigen specific therapy comprising determining whether the MS-associated antibodies in a patient's sample exceeds an antibody threshold, wherein exceeding an antibody threshold to the MS-associated antigens identifies an MS patient who can derive greater clinical benefit from antigen specific therapy such as BHT 3009.
  • the invention provides a method of selecting a patient afflicted with multiple sclerosis for antigen specific therapy comprising determining the MS- associated antibodies to MS-associated antigens in a blood sample from the patient, wherein MS-associated antibodies to the MS-associated antigens in the blood sample that exceeds an antibody threshold indicates the patient is likely to respond to therapy with antigen specific therapy, such as BHT 3009.
  • the method of selecting the patient comprises the following steps:
  • the foregoing method of selecting the patient will be carried out using an MS-associated protein array.
  • the MS-associated antigen elements on the array are typically myelin antigens, such as proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), myelin-associated glycoprotein (MAG), and myelin-associated myelin-associated
  • oligodendrocytic basic protein The array may also comprise MS-associated antigens, proteins such as alpha-B-crystallin; viral and bacterial mimicry peptides (e.g., influenza, herpes viruses, hepatitis B virus, and the like), cyclic nucleotide phosphodiesterase (CNPase); and TNF receptor superfamily 5 (CD40).
  • MS-associated antigens proteins such as alpha-B-crystallin
  • viral and bacterial mimicry peptides e.g., influenza, herpes viruses, hepatitis B virus, and the like
  • CNPase cyclic nucleotide phosphodiesterase
  • CD40 TNF receptor superfamily 5
  • the MS-associated protein elements comprise myelin protein elements, such as myelin basic protein.
  • myelin protein elements can be MASQKRPSQRHGSKYLATAS, HGSKYLATASTMDHARHGFL,
  • FKLGGRDSRSGSPMARR ENPVVHFFKNIVTPRTP, LSRFSWGAEGQRPGFGYGG, ASDYKSAHKGLKGVDAQGTLSKIFK, and AQGTLSKIFKLGGRDSRSGSPMARR.
  • the methods can include the additional step of treating the patient with antigen specific therapy.
  • the antigen specific therapy comprises administration of a polynucleotide encoding an MS-associated antigen such as an autoantigen targeted in MS, such as proteolipid protein (PLP); myelin basic protein (MBP); myelin oligodendrocyte protein (MOG); myelin-associated glycoprotein (MAG), or myelin- Attorney Docket No.: 021686-0023 lOPC associated oligodendrocytic basic protein (MBOP).
  • the polynucleotide is often a DNA plasmid such as BHT 3009.
  • the invention also relates, in yet another aspect, to a method for treating multiple sclerosis comprising administering to a patient antigen specific therapy, such as BHT 3009, in an amount effective to treat multiple sclerosis, wherein a blood sample from the patient has been determined to " contain antibodies to MS-associated proteins, such as antibodies to myelin basic protein, that exceeds an antibody threshold.
  • a patient antigen specific therapy such as BHT 3009
  • an “immunoassay” is any assay that can be used to measure antibodies against a desired antigen.
  • the antigens are associated with multiple sclerosis (MS) (referred to herein as “MS-associated antigens").
  • MS-associated antigens typically, the immunoassays of the invention employ a capture and detection reagent.
  • the capture reagent is a MS-associated antigen, typically a MS-associated protein, e.g., a component of the myelin sheath.
  • the detection reagent can either be the same autoantigen crosslinked to a marker (iso-sandwich format, or bridging ELISA) or alternatively a secondary Ab (e.g. a non-human Ab such as mouse, rat, or goat) against human antibody.
  • a marker iso-sandwich format, or bridging ELISA
  • a secondary Ab e.g. a non-human Ab such as mouse, rat, or goat
  • the detection reagent can itself be tagged with an instrument readable marker such as a fluorescent dye, or can be crosslinked to a specific marker for amplification such as HRP, chemiluminescence or DIG.
  • ELISA enzyme-linked immunosorbent assays
  • solution phase ELISAs radioimmunoassays
  • MSD enzyme-linked immunosorbent assays
  • Luminex Luminex
  • DELFIA Dissociation-Enhanced Lanthanide Fluorescent Immunoassay
  • Arrays enable the use of multiple MS-associated antigens for medium to large scale multiplexing.
  • Antigen refers to any molecule that is recognized and bound by an antibody. Such a molecule can be a protein, polypeptide, peptide, lipid, nucleic acid, carbohydrate, aptamer, peptoid, or macromolecule.
  • array refers to any collection of addressable elements. Such elements can be spatially addressable, such as arrays contained within microtiter plates or printed on planar surfaces where each element is present at distinct X and Y coordinates. Alternatively, elements can be addressable based on tags, beads, nanoparticles, or physical properties.
  • the microarrays can be prepared according Attorney Docket No.: 021686-0023 lOPC to the methods known to the ordinarily skilled artisan (See for example, U.S. Pat. No.
  • Arrays as used herein refer to any biologic assay with multiple addressable elements.
  • the addressable elements are proteins associated with multiple sclerosis.
  • the addressable elements are epitopes from proteins, for example, autoantigens associated with multiple sclerosis.
  • a microarray is a miniaturized form of an array
  • the arrays of this invention are MS-associated antigen arrays.
  • the arrays comprise MS-associated antigens.
  • the arrays comprise a set of discrete molecular entities, e.g. MS-associated peptides, capable of binding to auto-antibodies associated with multiple sclerosis which are arranged in a manner that allows identification of the presence of the auto-antibodies contained within the patient sample.
  • the array comprises a set of MS-associated antigens having distinct sequences, three dimensional shapes, or molecular structures, where each target is coded for identification.
  • MS-associated antigens may be used in an array, for example, proteins, polypeptides, peptides, RNA, DN A, lipid, glycosylated molecules, polypeptides with phosphorylation modifications, and polypeptides with citrulline modifications, aptamers, peptoids and similarly shaped molecules.
  • the elements are from an antigen associated with multiple sclerosis, for example a protein (i.e., a "MS-associated protein," that can be bound by an antibody in a patient sample.
  • MS-associated protein proteins, peptides or other molecules associated with MS.
  • MS-associated antigen or MS-assoicated protein is an autoantigen that binds antimyelin antibodies associated with an active immune response that gives rise to an MS lesion.
  • MS-associated antigens can be, but are not limited to, proteins, polypeptides, peptides, RNA, DNA, lipids, glycosylated molecules, carbohydrates, polypeptides with phosphorylation modifications, polypeptides with citrulline modifications, aptamers, oxidated molecules, and other molecules.
  • the ordinarily skilled artisan would appreciate that one could combine various types of molecules in a single array, for example, peptides, proteins, polypeptides, glycosylated or citrulline modified peptides, proteins, or polypeptides using the appropriate assay conditions but not other molecules.
  • a wide variety of linkers and spacers can be used to attached peptides, proteins or other biomolecules for optimal binding to antibodies in immunoassays.
  • linkers and spacers include Attorney Docket No.: 021686-0023 lOPC polyethylene glycol (for example PEG6 linked peptides), C6 or CI 2 (6 carbon or 12 carbon linkers), biotin-avidin linkage, and other molecules that optimally bind the peptide or protein for use in the immunoassay.
  • Peptides can be spotted at various concentrations in the range of (50-1000 ⁇ g/ml), as would be known to the ordinarily skilled artisan, including for example, two different concentrations of 200 and 500 g/ml.
  • antibody threshold refers to a level of antibodies in a patient sample which if exceeded identifies a subpopulation of MS patients who can derive greater clinical benefit from antigen specific therapy. Antibody threshold is determined by measuring antibodies in a sample in two respects: the first measures the antibody titer or magnitude, that is the concentration of antibodies, e.g., auto-antibodies, in a patient sample that measurably bind to one MS-associated antigen, e.g., a peptide, comprising an epitope, of a myelin antigen; and, the second measures antibody breadth or range, that is the number of MS- associated antigens to which there is a measurable antibody titer.
  • MS-associated antibodies to MS-associated antigens in a patient's sample exceed an antibody threshold is determined comparatively using either: 1) clinical reference samples; 2) standardized antibody sample(s); and/or, 3) standard curve(s) developed to determine the titer/magnitude and breadth of antibodies in a series of control samples.
  • the MS-associated antigens are auto-antigens, and the antibodies are auto-antibodies.
  • the antibody threshold is typically expressed as a single number taking into account the auto-antibody titer and breadth in a patient sample.
  • a standard curve can be generated using antibodies from a variety of sources.
  • Example of antibody sources include: a) samples from MS patients; b) samples from patients with other diseases; c) samples from animals immunized with an antigen; or, d) samples spiked with antibodies (from various sources including for example, recombinant or hybridoma).
  • an antibody threshold is determined. Patient samples containing MS-associated antibodies to MS-associated antigens that exceed an antibody threshold enable the identification of patients afflicted with multiple sclerosis who can derive greater clinical benefit from antigen specific therapy such as BHT 3009.
  • MS-associated antibodies refers to the level of antibody reactivity in the patient sample against the MS-associated antigen, for example each MBP peptide antigen on the array, and is determined, based on comparison to antibody-binding levels to that same peptide antigen in a reference or standard.
  • MS-associated antibodies of a Attorney Docket No.: 021686-0023 lOPC human MS patient sample can be determined using an antigen microarray containing peptides derived from MBP.
  • the breadth of the antibody response in the patient sample is determined based on the number of individual peptide antigens with reactivity above a level defined by the reference or standard run on the same myelin array. Further means and methods for determining the antibody threshold are described in more detail below.
  • exceeding an antibody threshold refers to the MS-associated antibodies to myelin antigens that has been determined, according to this invention, to exceed an antibody threshold thus enabling the identification of individuals afflicted with multiple sclerosis who can derive greater clinical benefit from antigen specific therapy such as BHT 3009.
  • Means for determining the antibody threshold for a particular assay are described in more detail below.
  • Patients with MS-associated antibodies greater than the predetermined antibody threshold are selected for antigen specific therapy as those patients can derive greater clinical benefit from antigen specific therapy such as BHT 3009 in comparison to patients with MS-associated antibodies less than the predetermined threshold level.
  • addressability refers to the location, position, tags, cleavable tags or markers, identifiers, spectral properties, electrophoretic properties, or other physical properties that enable identification of the element.
  • addressability also known as coding
  • spatial addressability where the position of the molecule is fixed, and that position is correlated with the identity. This type of spatial array is generally synthesized or spotted onto a planar substrate, producing, for example, microarrays, where a large number of different molecules are densely laid out in a small area, e.g.
  • An alternative to this type of spatial coding array is the use of molecular "tags," where the target autoantigens or epitopes are attached to a detectable label, or tag, which provides coded information about the autoantigen or epitope. In certain cases these tags can be cleaved from the element, and subsequently detected to identify the element.
  • a set of autoantigens or epitopes may be synthesized or attached to a set of coded beads, where each bead is linked to a distinct autoantigen or epitope, and where the beads are themselves coded in a manner that allows identification of the attached autoantigen or epitope.
  • MS-associated antigens include molecules such as nucleic acids, lipids, and others.
  • MS -associated antigens include, for example, those autoantigens associated with multiple sclerosis. For ⁇ each autoantigen associated with multiple sclerosis, there exists a panel of epitopes that represent the immunologic determinants of that autoantigen. Autoantigens associated with multiple sclerosis comprise immunologic epitopes.
  • Targets of the autoimmune response in autoimmune demyelinating diseases may comprise epitopes from proteolipid protein (PLP); myelin basic protein (MBP); myelin oligodendrocyte protein (MOG); myelin-associated glycoprotein (MAG), and myelin-associated oligodendrocytic basic protein (MBOP); OSP
  • PBP proteolipid protein
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte protein
  • MAG myelin-associated glycoprotein
  • MBOP myelin-associated oligodendrocytic basic protein
  • oligodendrocyte specific-protein citrulline-modified MBP (the C8 isoform of MBP in which 6 arginines have been de-imminated to citrulline), etc.
  • the integral membrane protein PLP is a dominant autoantigen of myelin. Determinants of PLP antigenicity have been identified in several mouse strains, and include residues 139-151, 103-116, 215-232, 43-64 and 178- 191. At least 26 MBP epitopes have been reported (Meinl et al., J Clin Invest,
  • MS-associated proteins may also include, for example, alpha-B-crystallin (a heat shock protein); viral and bacterial mimicry peptides, e.g., influenza, herpes viruses, hepatitis B virus, etc.; cyclic nucleotide phosphodiesterase (CNPase); or TNF receptor superfamily 5 (CD40).
  • alpha-B-crystallin a heat shock protein
  • viral and bacterial mimicry peptides e.g., influenza, herpes viruses, hepatitis B virus, etc.
  • CNPase cyclic nucleotide phosphodiesterase
  • CD40 TNF receptor superfamily 5
  • MBP myelin basic protein
  • MBP 87-99 the amino acid Attorney Docket No.: 021686-0023 lOPC motif HFFK is a dominant target of both the T and B cell response (Wucherpfennig et al., J Clin Invest, 100:1114-22 (1997)).
  • Another study observed lymphocyte reactivity against myelin-associated oligodendrocytic basic protein (MOBP), including residues MOBP 21-39 and MOBP 37-60 (Holz et al., J Immunol, 164:1103-9 (2000)).
  • MOBP myelin-associated oligodendrocytic basic protein
  • Zadro etal (Clinical Neurology and Neurosurgery 109 (2007) 23-26 measured levels of anti- MOG antibodies in relapsing remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and in non-inflammatory neurological diseases (NIND).
  • RRMS relapsing remitting multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • NIND non-inflammatory neurological diseases
  • oligopeptides derived from an MS-associated protein are used. Oligopeptides are generally less than about 100 residues in length. In the present invention, oligopeptides are usually at least about 7 amino acids in length and may be as many as about 50 amino acids in length. In some embodiments, the oligopeptides are about 10 to about 30 residues in length. In still others, the oligopeptides are about 12 to about 25 residues in length. The peptides may be overlapping by 7-10 amino acids, and can encompass the whole sequence of the protein of interest.
  • the oligopeptide can also be a mimic of a native peptide shape, for example a cyclic peptide, nucleic acid aptamer, or can be another molecule, drug, or organic molecule that mimics the three dimensional shape recognized by the antibody.
  • Epitopes are portions of MS-associated antigens that are recognized by B
  • lymphocytes and specifically by the antibodies expressed on the cell surface and secreted by B cells. Epitopes can also be recognized by specific receptors on T lymphocytes.
  • An individual antigen typically contains multiple epitopes, although there are instances in which Attorney Docket No.: 021686-0023 lOPC an antigen contains a single epitope.
  • peptide fragments derived from a whole protein autoantigen are used to represent individual epitope(s) targeted by the autoantibodies produced by B cells associated with multiple sclerosis.
  • fragments of molecules representing post-translational modifications, carbohydrates, lipids and other molecules can be used to represent individual epitopes.
  • Epitopes represent shapes recognized by immune B and T cells associated with multiple sclerosis, and can also be represented by non-antigen derived peptides and other molecules that possess the same epitope shape that is present within the native antigen.
  • An example of an element with an epitope shape is an aptamer.
  • An aptamer is a molecule that provides a shape that can mimic an immunologic epitope. Using a plurality of aptamers a library of epitope shapes can be generated.
  • a variety of assay types can be used to measure antibodies against MS-associated antigen, e.g., the myelin sheath or other antigens associated with MS, employing a capture and detection reagent.
  • MS-associated antigen e.g., the myelin sheath or other antigens associated with MS
  • Commonly used immunoassays include Western blot (semi- quantitative), ELISA, and solution phase ELISAs.
  • Various assays have been described for the identification of protein-peptide interaction antagonists. (Dev Biol (Basel). 2005:122:155-70)
  • arrays of autoantigens and autoantigen-derived epitopes can be used to determine whether a: 1. patient is likely to develop multiple sclerosis; 2.
  • An antigen array comprises the various antigens either known to be associated with multiple sclerosis, suspected to be associated with multiple sclerosis, or a library of potential antigens associated with multiple sclerosis.
  • An antigen array consisting of panels of autoantigens may be used for screening purposes, where the panel reflects the different epitopes associated with multiple sclerosis.
  • Autoantigen epitope panels of interest include panels optimized for multiple sclerosis, including one or more whole proteins, peptide and overlapping peptides within the sequence of the myelin based proteins, and peptides representing dominant epitopes.
  • polypeptide designates any of myelin based proteins and peptides. Where short peptides are used, peptides are at least about 7 amino acids in length, may be at least about 15 amino acids in length, and as many as 22 amino acids in length.
  • peptides or protein fragments may also be used, with peptides of at least about 30 amino acids in length, as many as 50 amino acids in length, as many as 100, 200 or 500 amino acids Attorney Docket No.: 021686-0023 lOPC in length or longer.
  • Protein fragments, containing immunologic epitopes from different regions of the protein may also be used.
  • the peptides or protein fragments may be overlapping by 7-10 amino acids, and can encompass the whole amino acid sequence of the protein of interest.
  • the peptide can also be a mimic of a native peptide shape, for example a cyclic peptide, nucleic acid aptamer, or can be another molecule, drug, or organic molecule that mimics the 3 dimensional shape recognized by the autoantibody or T cell receptor molecule.
  • Peptide or protein fragments may be synthesized using techniques well known to the skilled artisan, or may be derived from purified myelin-associated molecules, for example by enzymatic proteolysis.
  • Treating,” “treat,” “treatment,” or “therapy” of multiple sclerosis shall mean slowing, stopping or reversing the progression of MS, as evidenced by decreasing, cessation, maintaining (i.e., no change from prior testing) or elimination of either clinical or diagnostic symptoms, e.g., MRIs evaluating T2 lesions and/or gadolinium-enhancing lesions.
  • Treating,” “treatment,” or “therapy” also means a decrease in the severity of signs or symptoms in acute or chronic MS or a decrease in the relapse rate as for example in the case of a relapsing or remitting disease course.
  • treating a disease means reversing or stopping the progression of MS, ideally to the point of eliminating the disease itself.
  • treating a disease means reversing or stopping progression of the Multiple Sclerosis Functional Composite (MSFC) MSFC or any of its components.
  • MSFC Multiple Sclerosis Functional Composite
  • prophylactic refers to any activity that slows, stops or reverses the progression of MS, as evidenced by decreasing, maintaining (i.e., no change as compared to prior tests) cessation or elimination of either clinical or diagnostic symptoms or a combination of both clinical and diagnostic symptoms.
  • Preventative treatment can also effect a decrease in the severity of signs or symptoms in acute or chronic MS or disorder or a decrease in the relapse rate of MS.
  • Preventative measures or treatment can take place at primary, secondary and tertiary prevention levels. Primary prevention avoids the development of the disease.
  • the present invention enables the diagnosis of patients with MS with subsequent treatment directed to primary, secondary or tertiary prevention levels.
  • Primary prevention activities are aimed at delaying the onset of disease signs or symptoms in subjects who are at risk of developing MS, and reducing the severity of the symptoms when they first appear. Secondary prevention activities are aimed Attorney Docket No. : 021686-00231 OPC at early disease detection and treatment, thereby increasing opportunities for interventions to prevent progression . of the disease and emergence of symptoms. Tertiary prevention reduces the negative impact of an already established disease by preventing progression, restoring function and reducing disease-related complications. [0042]
  • responsive or “receptive" to therapy interchangeably refer to a prediction of whether a particular treatment will be efficacious when administered to an MS patient.
  • an efficacious treatment will, e.g., improve, reduce or ameliorate the signs or symptoms of MS or reduce, slow or prevent the progression of MS.
  • the determination of responsiveness or receptiveness is relative in that certain subpopulations of patients are predicted to respond better than other subpopulations.
  • a patient can be predicted to have improved or increased responsiveness or receptiveness to a particular therapy, for example, as compared to a patient from a different subpopulation.
  • the improved responsiveness is detectable by a selected parameter, and can be significant or substantial.
  • the improved or increased responsiveness may be at least 50%, 60%, 70%, 80%, 90%, 1 -fold, 2-fold, 3-fold greater, according to the measurement of a selected parameter (e.g., demyelination lesions, relapse rate, etc), in the patients of a selected subpopulation in comparison to patients of a non-selected subpopulation.
  • a selected parameter e.g., demyelination lesions, relapse rate, etc
  • relapse refers to the appearance or reappearance of one or more significant neurological abnormalities persisting for at least 48 hours and immediately preceded by a period of relatively stable or improving disease for a period of time, such as 30 days. Normal fluctuations in a subject's MS signs or symptoms do not themselves constitute a relapse. MS relapses are typically confirmed by a physician, and are usually accompanied by objective changes on the neurological examination and an increase in EDSS.
  • a relapse can develop over a matter of days, remain constant for three to four weeks, and then slowly resolve over a period of about a month.
  • Symptoms of a relapse include visual impairment; muscle weakness; weak, absent or excessive reflexes;
  • Figure 2 shows the percentage of each positive peptide (i.e., peptide bound by serum antibody) with partition function for all MS samples tested (87 individual and 10 MS pooled serum samples). 7 peptides were positive (i.e., peptide bound by serum antibody) in at least 35% of the MS samples and collectively these peptides achieved a 90% sensitivity level for diagnosing MS. 12 peptides were positive in at-least 30% of the MS samples and collectively a higher sensitivity of 95% was achieved.
  • Figure 3 shows the distribution of 7 selected peptides in patient population.
  • the Y axis shows the number of positive peptides in any given patient.
  • the X axis shows the number of patients for each positive peptide level (i.e., peptides bound by serum antibody) and the percentage is shown near each bar. In the eighty-seven individual serum samples that were tested, only 11.5% of patients did not test positive for any serum antibody to the seven selected peptides.
  • the mosaic plot illustrates the distribution of positive peptides, with most falling between 2 and 4 positive peptides.
  • Figure 4 shows the distribution of twelve selected peptides in the patient population.
  • the Yaxis shows the number of positive peptides (i.e., peptide bound by serum antibody) in any given patient.
  • the X axis shows the number of patients for each positive peptide level and the percentage is shown near each bar. In the 87 individual serum samples tested, only 4.6% of patients did not test positive for any of the 12 selected peptides.
  • the mosaic plot on the right, illustrates the distribution of positive peptides (i.e., peptides bound by serum antibody) with most falling between 3 and 6 positive peptides.
  • Figure 5 shows the beneficial effect on brain Gd+ lesions as demonstrated in a phase 2 clinical trial of the 0.5 mg dose of BHT-3009 as a function of the number of peptides for MBP detected by patient plasma samples at baseline (NUM).
  • the y-axis represents the percent reduction in new brain MRI Gd+-enhancing lesions per each 4-week period between visits 10 and 15 (weeks 28 to 48) of the treatment period as compared to placebo treated patients.
  • the x-axis represents the number (NUM) of peptide epitopes of MBP detected by plasma antibodies from patient samples collected at baseline before dosing.
  • Figure 6 shows the beneficial effect on brain T2 lesions as demonstrated in a phase 2 clinical trial of the 0.5 mg dose of BHT-3009 as a function of the number of peptides for Attorney Docket No.: 021686-0023 l OPC
  • the y-axis represents the percent reduction in total new and enlarging MRI T2 lesions between visits 10 and 15 (weeks 28 to 48) of the treatment period as compared to placebo treated patients.
  • the x-axis represents the number (NUM) of peptide epitopes of MBP detected by plasma antibodies from patient samples collected at baseline before dosing.
  • Figure 7 shows the beneficial effect on MS relapses as demonstrated in a phase 2 clinical trial of the 0.5 mg dose of BHT-3009 as a function of the number of peptides for MBP detected by patient plasma samples at baseline (NUM).
  • the y-axis represents the annualized relapse rate (ARR) of confirmed clinical MS relapses during the 48 week treatment period.
  • the x-axis represents the number (NUM) of peptide epitopes of MBP detected by plasma antibodies from patient samples collected at baseline before dosing.
  • the present invention is based in part on the surprising and unexpected discovery that analysis of the MS-associated antibodies in a sample from a patient, for example a serum or plasma sample, enables: 1) the selection of a patient afflicted with multiple sclerosis for antigen specific therapy; and, 2) the diagnosis of patients with multiple sclerosis.
  • the MS- associated antibodies in a patient sample that exceeds the antibody threshold in particular a serum or plasma sample, is a marker for selecting multiple sclerosis patient(s) for antigen specific therapy and identifying a patient with multiple sclerosis.
  • the MS- associated antibodies in a patient sample that exceed the antibody threshold provide a pharmacodynamic marker to assist in dose and regimen optimization.
  • the means and methods of the invention enable the determination of whether the MS-associated antibodies in a patient sample (e.g., a serum or plasma sample) exceeds the antibody threshold.
  • the MS-associated antibodies in the patient sample are determined by measuring the antibody titer or the level or number of antibodies, e.g., autoantibodies in the patient sample that bind to an epitope of a myelin antigen and the antibody breadth or the number of myelin epitopes to which there is a measurable antibody titer.
  • ARRAYS Attorney Docket No.: 021686-0023 lOPC
  • arrays refers to a collection of addressable elements. Such elements can be spatially addressable, such as arrays contained within microtiter plates or printed on planar surfaces where each element is present at distinct X and Y coordinates. Alternatively, elements can be addressable based on tags, beads, nanoparticles, or physical properties.
  • a microarray is a miniaturized form of an array. The arrays can be prepared according to the methods known to the ordinarily skilled artisan (See for example, U.S. Pat. No. 5,807,522; Robinson et al. (2002) Nature Medicine 8:295-301 ; Robinson et al. (2002) 46:885-93). In the methods of the present invention, the addressable elements are
  • autoantigens associated with multiple sclerosis usually peptide antigens.
  • the autoantigens can be, but are not limited to, proteins, polypeptides, peptides, peptide mimetics, RNA, DNA, lipids, glycosylated molecules, carbohydrates, polypeptides with phosphorylation
  • addressability refers to the location, position, tags, cleavable tags or markers, identifiers, spectral properties, electrophoretic properties, or other physical properties that enable identification of the element.
  • addressability also known as coding
  • spatial addressability where the position of the molecule is fixed, and that position is correlated with the identity.
  • This type of spatial array is generally synthesized or spotted onto a planar substrate, producing, for example, micro arrays, where a large number of different molecules are densely laid out in a small area, e.g. comprising at least about 400 different sequences per cm 2 , and may be 1000 sequences per cm , or as many as 5000 sequences per cm , or more.
  • Less dense arrays such as may be found in ELISA or RIA plates where wells in a plate each contain a distinct antigen, may comprise from about 96 sequences per plate, up to about 100 sequences per cm 2 , up to the density of a micro array.
  • Other spatial arrays utilize fiber optics, where distinct antigens are bound to fibers, which can then be formed into a bundle for binding and analysis. Methods for the manufacture and use of spatial arrays of polypeptides are known in the art.
  • Exemplary articles include Joos et al. (2000) Electrophoresis 21 (13): 2641-50 describing a microarray-based immunoassay containing serial dilutions of antigens; Roda et al. (2000) Biotechniques 28(3):492-6 describing a system obtained by adapting a commercial ink-jet Attorney Docket No.: 021686-0023 lOPC printer and used to produce mono- and bidimensional arrays of spots containing protein on cellulose paper; and Ge (2000) Nucleic Acids Res 28(2):e3 describing a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein ligand interactions. See also, Mendoza et al.
  • the supports used in the preparation of the arrays can be any of those known to those of skill in the art.
  • the support may be made of any composition to which the autoantigens can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape.
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • the particular manner of binding of the autoantigen to the surface is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the immunoreaactivity of the autoantigen and is non-diffusible.
  • An alternative to this type of spatial coding array is the use of molecular "tags," where the target MS-associated proteins, e.g., autoantigens, are attached to a detectable label, or tag, which provides coded information about the sequence of the autoantigen. In certain cases these tags can be cleaved from the element, and subsequently detected to identify the element.
  • a set of MS-associated proteins may be synthesized or attached to a set of coded beads, where each bead is linked to a distinct autoantigen, and where the beads are themselves coded in a manner that allows identification of the attached autoantigen.
  • the use of a multiplexed microsphere set for analysis of clinical samples by flow cytometry is described in International Patent application no. 97/14028; and Fulton et al. (1997) Clinical Chemistry 43: 1749-1756). It is also possible to use other addressable particles or tags (reviewed in Robinson et al, al. (2002) Arthritis Rheumatism 46:885-93). Attorney Docket No.: 021686-0023 l OPC
  • the autoantigens of the array will comprise epitopes recognized by (or potentially recognized by) the immune system of the patient. These include epitopes recognized by B lymphocytes, and specifically by the antibodies expressed on the cell surface and secreted by B cells. Epitopes can also be recognized by specific receptors on T lymphocytes. An individual antigen typically contains multiple epitopes, although there are instances in which an antigen contains a single epitope. In one embodiment of this invention, peptide fragments derived from a whole protein antigen (e.g., MBP) are used to represent individual epitope(s) targeted by the antibodies produced by B cells.
  • MBP whole protein antigen
  • fragments of molecules representing post-translational modifications, carbohydrates, lipids and other molecules can be used to represent individual epitopes.
  • Epitopes represent shapes recognized by immune B and T cells, and can also be represented by non-antigen derived peptides and other molecules (e.g., peptide mimetics) that possess the same epitope shape that is present within the native antigen.
  • An example of an element with an epitope shape is an aptamer.
  • An aptamer is a molecule that provides a shape that can mimic an immunologic epitope. Using a plurality of aptamers a library of epitope shapes can be generated.
  • An MS-associated antigen array e.g., autoantigen array
  • An MS-associated antigen array will typically include autoantigens optimized to provide predictive or pharmacodynamic biomarkers for MS.
  • Autoantigens are any molecule produced by the organism that can be the target of an immunologic response.
  • such molecules are peptides, polypeptides, and proteins encoded within the genome of the organism.
  • such molecule are post-translationally-generated modifications of these peptides, polypeptides, and proteins, such as cleavage, phosphorylation, deimination of arginine to citrulline, and other modifications generated through physiologic and non-physiologic cellular processes.
  • such molecules include carbohydrates, lipids and other molecules produced by the organism.
  • auto antigens examples include endogenous proteins or fragments thereof that elicit a pathogenic immune response. Of particular interest are auto antigens that induce a T cell mediated pathogenic response. Autoimmune diseases characterized by the involvement of T cells include multiple sclerosis, experimental autoimmune encephalitis, rheumatoid arthritis, insulin dependent diabetes mellitus, etc.
  • the arrays of the invention can be used to determine a patient's MS-associated antibodies for a number of purposes, including the determination or identification of: 1) a patient who can derive greater clinical benefit from antigen specific therapy such as BHT 3009; 2) whether a patient has multiple sclerosis (MS); 3) whether a patient is likely to develop more or less severe MS; 4) whether a particular antigen specific treatment has been successful, unsuccessful, or detrimental; and, 5) a patient who is likely to have a disease flare or relapse.
  • the methods of the invention are typically used for identifying a patient afflicted with MS and selecting an MS patient for antigen specific therapy. The patients may or may not have already been diagnosed as having MS.
  • the patient is exhibiting signs or symptoms of MS and is subject to the blood test of this invention followed by antigen specific therapy.
  • a patient who has signs or symptoms of MS may or may not have active or overt signs or symptoms, particularly if the patient is in a remission.
  • symptoms of MS are well-known and can include acute or subacute attack of unilateral visual impairment, muscle weakness, paresthesias, ataxia, vertigo, urinary incontinence, dysarthria, or mental disturbance (in order of decreasing frequency).
  • Such symptoms result from focal lesions of demyelination which cause both negative conduction abnormalities due to slowed axonal conduction, and positive conduction abnormalities due to ectopic impulse generation (e.g., Lhermitte's symptom).
  • the patient has been diagnosed with MS by a physician.
  • MRI magnetic resonance imaging
  • CSF cerebral spinal fluid
  • IgG abnormal evoked responses
  • the assay of this invention can also be used for the diagnosis of MS by determining whether the magnitude of myelin autoantibodies in a patient's blood sample is equal to or greater than a threshold magnitude.
  • a biological sample from the patient is contacted with an array under conditions that permit specific binding of antibodies in the sample to the antigens on the array.
  • Biological samples may be any fluid or tissue from the patient that contains antibodies.
  • Such sample could include, for example, blood, plasma, serum, sputum, urine, tears, mucus, cerebrospinal fluid, and the like.
  • the sample will be a serum or plasma sample.
  • the sample is contacted with the antigen array and the MS-associated antibodies (i.e., the titer and breadth of serum antibodies binding myelin autoantigens in a particular array) is determined.
  • the MS-associated antibodies i.e., the titer and breadth of serum antibodies binding myelin autoantigens in a particular array
  • Such assays are carried out according to well known immunoassay methods.
  • such assays are typically sandwich assays, in which the sample antibodies are bound between the antigen (typically bound to a solid support) and a second labeled binding agent such as a goat, sheep and other non-human antibody that recognizes human immunoglobulins (anti-human antibody).
  • the label is then detected to measure or detect the resultant complex by visual or instrument means.
  • the label may be coupled directly or indirectly to any desired component of the assay according to methods well known in the art.
  • Non-radioactive labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
  • Enzymes of interest as labels will primarily be hydrolases, particularly
  • phosphatases phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3- dihydrophthalazinediones, e.g., luminol.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti- ligand (e.g., streptavidin) molecule that is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
  • Those samples having MS-associated antibodies above a control antibody threshold by comparing patient samples to appropriate controls as described herein, identify a patient who would benefit from antigen specific therapy.
  • the antibody threshold will depend upon the nature of the sample, the assay conditions, the MS- associated antigens used, and the like.
  • the determination of threshold magnitude samples for a particular assay can be readily determined according to a number of methods. For example, a standard curve can be generated by determining the number of antibodies bound to one or more of the antigens on the array (defined, for example, by having a signal to background ratio greater than 1) using samples from MS patients, as well as normal, healthy subjects as well as a variety of other non-MS neurological diseases.
  • highly reactive samples can be designated as those samples having MS-associated antibodies that are at least two standard deviations above the average antibody threshold of normal, healthy subjects.
  • highly reactive samples can be designated as those samples having MS-associated antibodies that are above the antibody threshold measured in other samples derived from patients with the disease.
  • the antibody threshold of a sample can be expressed as a ratio of the number of antigens bound by antibodies to the total number of antigens on the array that could be bound by antibodies. For example, in the assays described in Example 2, samples showing an immunoreactivity of 14 out of 23 were determined to be above the antibody threshold. Thus, samples with MS-associated antibodies that exceed the antibody threshold can be identified as those showing greater than 60% MS-associated antibodies as compared to control samples. Alternatively, samples with MS-associated antibodies that exceed the antibody threshold can be identified as those having 40%, 50%, 60%, 70%, or greater MS- assoicated antibodies as compared to control samples depending on the assay.
  • Patients with MS-associated antibodies greater than the predetermined antibody threshold would be selected for antigen specific therapy as they would be more likely to respond to such treatment than patients with MS-associated antibodies less than the predetermined threshold level.
  • the response of a patient selected for antigen specific therapy would then be monitored using the assay of this invention as well as using any assays and tests known to the clinician treating MS by measuring the signs or symptoms, combination of signs or symptoms, or progression of MS, e.g., decreased number and extent of
  • demyelination lesions decreased antibody or T cell responsiveness against autoantigens targeted in MS (i.e., increased immune tolerance); decreased relapse frequency, intensity or rate; improvement or stabilization of disability as measured by standard scales such as the Expanded Disability Status Scale or EDSS; Multiple Sclerosis Functional Composite (MSFC) MSFC or any of its individual components; decreased muscle weakness or increased muscle strength; reflexes; improved vision; and patient self-assessment.
  • Assays and methods for measuring and scoring clinical and physical symptoms of MS are well known in the art. See, e.g., Barkof, Mult Scler. (1999) 5(4):283-6; Amato and Ponziani, Mult Scler. (1999)
  • the determination of MS-associated antibodies in a patient's sample enables the selection of a patient for antigen specific therapy.
  • the response of a MS patient selected for antigen specific therapy will be monitored using the assay of this invention, as well as by monitoring a patient's signs or symptoms. It is an object of the present invention to improve the response of a MS patient selected for therapy by measuring the MS-associated antibodies and determining whether it exceeds the antibody threshold Attorney Docket No.: 021686-0023 l OPC using the assay of this invention and treating those patients with antigen specific therapy as compared to patients treated without first measuring the MS-associated antibodies in a patient sample.
  • the measurable or detectable improvement is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more based on the measurable parameter of the sign/symptom, or signs/symptoms being compared (e.g. , number and extent of demyelination lesions; relapse rates), in comparison to patients receiving treatment without measuring the anti-MS-associated antibodies in a patient sample and determining whether it exceeds the antibody threshold using the assay of this invention.
  • the measurable or detectable improvement is at least about 1 -fold, 2-fold, 3-fold, 4-fold or more based on the measurable parameter of the symptom or symptoms being compared (e.g. , number and extent of demyelination lesions; relapse rates).
  • the methods of the invention can be performed on a patient without comparison to another patient.
  • the responsiveness or decrease in measurable symptoms of MS in patients receiving antigen specific therapy after first measuring the MS-associated antibodies in a patient sample and determining whether it exceeds the antibody threshold can be made in comparison to the same patient before treatment or during the course of treatment to monitor the dose and/or treatment regimen.
  • ANTIGEN SPECIFIC THERAPY [0076] Upon determining the MS-associated antibodies in an MS patient's sample using the assay of this invention, a clinician treating MS patients can then determine whether the MS- associated antibodies in the patient's sample is greater than an antibody threshold and if so can then treat the patient using antigen specific therapy.
  • the means and methods of this invention provide for measuring MS-associated antibodies in a patient's sample and if such MS-associated antibodies is greater than an antibody threshold and then selecting a MS patient for antigen specific therapy.
  • antigen specific therapies include polynucleotide therapy, peptide vaccination, protein vaccination, administration of immunoinhibitory oligonucleotides and immunoinhibitory antibodies when used in conjunction with DNA antigen specific therapy, described in greater detail below.
  • Polynucleotide therapy is the administration (e.g., intramuscular, subcutaneous, intranasal) of a polynucleotide to an MS patient to modulate the immune response.
  • administration e.g., intramuscular, subcutaneous, intranasal
  • DNA vaccination DNA immunization
  • DNA immunization or
  • polynucleotide therapy refers to the administration of polynucleotides encoding one or more self-polypeptides that include one or more autoantigenic epitopes associated with a MS.
  • the "DNA vaccination” serves the purpose of modulating an initial or ongoing immune response in MS to suppress autoimmune destruction for the treatment or prevention of MS. Modulation of an immune response in reaction to "DNA vaccination” may include shifting self-reactive lymphocytes from a Thl- to a Th2-type response. The modulation of the immune response in MS may occur systemically or only locally in the brain and CNS that is under autoimmune attack.
  • a polynucleotide may be either DNA or RNA encoding one or more self-protein(s), -polypeptide(s), -peptide(s) associated with MS may be administered by intramuscular, subcutaneous, intravenous or by oral delivery.
  • Exemplified autoantigens are discussed herein, and include without limitation, proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), cyclic nucleotide phosphodiesterase (CNPase), myelin-associated glycoprotein (MAG), myelin-associated oligodendrocytic basic protein (MBOP), and alpha-B-crystallin.
  • Additional autoantigens include MBP peptides 83-97 and 82-98.
  • the autoantigen is MBP, MBP peptide 83-97 or MBP 82-98.
  • autoantigens associated with MS may be post-translationally modified autoantigens with addition of lipid groups, dephosphorylation by phosphatases, addition of dimethylarginine residues, alpha-B-crystallin phosphorylation; citrullination of MBP, and other posttranslational modifications.
  • DNA plasmid vectors suitable for inhibiting an autoimmune response are known in the art.
  • DNA plasmid vectors that find use can have one or more immunostimulatory motifs removed or altered.
  • Exemplified immunostimulatory nucleic acid motifs include the formulae 5'-purine-pyrimidine-C-G-pyrimidine-pyrimidine-3' or 5 '-purine -purine-C-G- pyrimidine-pyrimidine-3'.
  • immunostimulatory motifs can be rendered non-stimulatory or less stimulatory by introducing a cytosine to non-cytosine substitution is within the CpG dinucleotide. See, U.S. Patent No. 7,030,098 and U.S. Patent Publication No. 2005/0261215, the entire disclosures of which are hereby incorporated herein by reference in their entirety for all purposes.
  • DNA plasmid vectors that find use can also have one or more immunoinhibitory motifs incorporated. Such immunoinhibitory sequences are described below and in U.S. Patent Publication No. 2003/0148983, the entire disclosure of which is hereby incorporated herein by reference in its entirety for all purposes.
  • One exemplified DNA plasmid vector suitable for use in immunizing with an autoantigen to inhibit an autoimmune response is the DNA vector backbone BHTl, described in PCT Publication No. WO 2007/147011, the entire disclosure of which is hereby incorporated herein by reference in its entirety for all purposes.
  • the BHTl vector is useful to deliver a nucleic acid encoding any autoantigen targeted in MS, as described herein.
  • An exemplified BHTl vector contains a nucleic acid encoding for MBP; it is also known as BHT-3009. The BHT-3009 vector is also described in PCT Publication No.
  • WO 2007/14701 Another example of a DNA plasmid vector suitable for use in antigen specific therapy is the vector described in PCT Publication No. WO2007/044394.
  • Polynucleotide therapy methods typically includes administration of "Immune Modulatory Sequences (IMSs) or "Immune Inhibitory Sequences” (IISs).
  • IMSs or IISs can be incorporated into the vector or administered as separated oligonucleotides.
  • the immune inhibitory sequences have the following structure:
  • the core hexamer of IISs can be flanked 5' and/or 3' by any composition or number of nucleotides or nucleosides.
  • IISs can range between 6 and 100 base pairs in length, for example, 16-50 base pairs in length.
  • IISs can also be delivered as part of larger pieces of DNA, ranging from 100 to 100,000 base pairs.
  • IISs can be incorporated in, or already occur in, DNA plasmids, viral vectors and genomic DNA.
  • IISs can also range from 6 (no flanking sequences) to 10,000 base pairs, or larger, in size. Sequences present which flank the hexamer core can be constructed to substantially match flanking sequences present in any known immunoinhibitory sequences (IIS). For example, the flanking sequences
  • flanking sequences incorporates a series of pyrimidines (C, T, and U), either as an individual pyrimidine repeated two or more times, or a mixture of different pyrimidines two or more in length. Different flanking sequences have been used in testing inhibitory modulatory sequences. Further examples of flanking sequences for inhibitory oligonucleotides are contained in the following references: U.S. Pat. Nos.
  • IMSs of the invention include oligonucleotides or plasmid vectors containing the following hexamer sequences:
  • IIS immunostimulatory sequences
  • ISS immunostimulatory sequences
  • This IIS in the absence of an ISS, was shown for the first time by this invention to prevent and treat autoimmune disease either alone or in combination with DNA polynucleotide therapy.
  • This IIS contained the core hexamer AAGGTT. That sequence is referred to herein as an immune modulatory sequence or IMS.
  • Other related IISs with a similar motif include:
  • Autoantigens targeted in MS can also be delivered as a polypeptide or peptide.
  • Exemplified routes of administration include intravenous,
  • amino acid sequence or subsequence of any autoantigen or combination of autoantigens targeted in MS can find use in inducing tolerance in a patient exhibiting symptoms of MS.
  • Exemplified amino acid sequences suitable for inhibiting an autoimmune response in MS include MBP peptides 83-97 and 82-98. Tolerance can be induced by administering the polypeptides or peptides at a sufficiently high doses
  • Myelin proteins can also be injected to induce immune tolerance in MS. Injection of MBP protein in mice with EAE has attenuated autoimmune responses and treated the EAE model for multiple sclerosis (Critchfield JM et al, Science. 1994 Feb 25;263(5150): 1 139-43).
  • Other myelin proteins, such as MOG, and MS associated proteins, such as alpha-B-crystallin (Cryab) have also been administered to attempt to induce immune tolerance (Genain CP et al, Science. 1996 Dec 20;274(5295):2054-7; Ousman SS et al, Nature. 2007 Jul
  • Myelin proteins delivered orally can induce tolerance and prevent EAE in mice.
  • a human trial was also conducted to test delivery of oral bovine myelin to humans with MS, and although the initial findings were positive, subsequent larger trials did not demonstrate clear efficacy (Weiner HL et al, Science. 1993 Feb 26;259(5099): 1321-4; Hohol MJ et al, Ann N Y Acad Sci. 1996 Feb 13;778:243-50.).
  • Antibodies also find use in antigen specific therapies to treat MS. Antibodies may be directed against specific peptides/HLA complexes presented on the surface of antigen presenting cells and may be used in blocking the antigen presenting cell T-cell interaction. [0097] Cell-Based Antigen Specific Therapy
  • cell-based therapies Another immune-based therapy that find use in the treatment of MS patients include cell-based therapies.
  • Contemplated cell-based therapies involve the administration of whole cells to a patient to specifically inhibit an autoimmune response.
  • Cells that find use include T cells and dendritic cells, among others.
  • cell-based therapies influence pathogenic T cells in an autoimmune response, for example by specifically eliminating the pathogenic T cells or by shifting the T cell response in a deleterious immune response from a Thl-type to a less pathogenic Th2-type response.
  • the cell-based therapy is T cell vaccination.
  • T cell vaccination involves administering to a patient autologous attenuated autoreactive T cells to induce an immune response specifically targeted against the autoreactive T cells.
  • the anti- clonotypic T cell response results in the specific depletion or suppression of the autoreactive T cells.
  • T cell vaccination has been applied clinically in human patients.
  • autoreactive or pathogenic T cells can be suppressed or deleted by immunization with T cell receptor (TCR) peptides or nucleic acids encoding TCR peptides.
  • TCR T cell receptor
  • This approach also finds use in shifting a pathological Thl-type response to a less pathological Th2-type response.
  • Peptide vaccination with epitopes from TCR has also been tested clinically in human patients. See, e.g. , Vandenbark, Curr Drug Targets Inflamm Allergy (2005) 4(2):217-29; Vandenbark, et al, Neurochem Res (2001) 26(6):713-30; and Vandenbark, et al., Immunology (2008) 123(l):66-78.
  • Vaccination with nucleic acid Attorney Docket No.: 021686-0023 l OPC sequences encoding epitopes from TCR is described, for example, in Buch and Waisman, Methods Mol Med (2006) 127:269-80 and U.S. Patent No. 5,939,400.
  • the cell-based therapy is the administration of tolerogenic dendritic cells.
  • This strategy is described, for example, by Link, et al., J Neuroimmunol (2001) 1 14(l-2):l-7; Steinman, et al, Annu Rev Immunol (2003) 21 :685-71 1 ; and Rutella and Lemoli, Immunol Lett (2004) 94(1 -2): 1 1-26.
  • peptides with an N-terminal PEG6 linker were synthesized at 95% purity level by reverse phase HPLC.
  • the peptides are shown in Table 1. These peptides were selected as a subset comprising autoantigens implicated in the etiology of MS. In some cases, for example when AMI and Luminex microarrays were used, PEG6 linked peptides were used instead of naked peptides.
  • HIVgp41 (667-680)/4E 10 ASLWN WFNITN WLW
  • Microarray slide substrates [0105] Microarray slide substrates:
  • Peptides can be spotted at various concentrations in the range of (50-1000 ⁇ g/ml), and the concentrations used in this example were 200 and 500 ⁇ ⁇ . Peptide dilutions were performed with DPBS buffer (no Ca++ or Mg++) to produce the final spotting solution. Various slide types were used. The arrays were spotted utilizing custom Piezo jet non- contact dispensing instruments. The target spot size was -120 ⁇ . Dispense parameters were tested to control the spot size, optimal spot morphology, and spot reproducibility. The dispensing of all slide substrates for the initial comparisons was completed in one dispensing batch to minimize variation and to allow for efficient substrate selection methods.
  • Assayed peptide slides were scanned in an Axon 4000B fluorescence scanner with scanning parameters optimized for the intensity and background of each slide type and sample applied. For the final three substrate slides with serum samples, the scanning parameters used were PMT 600 and Power 100%, all at 635 nm.
  • spot mean signal with median background subtraction on an individual spot basis (Raw intensity).
  • spot mean spot mean
  • Bkgd median concentric spot background ring outside of the buffer zone for which the median intensity is calculated
  • Signal strength is also calculated as the ratio of the spot mean divided by the local background noise level (median).
  • Serum samples were diluted 1 : 150. The three slide types described above were used with a 2hr incubation with samples. The IgM alone secondary Ab (Cy5 Donkey anti-hu).
  • IgM Fc5m fragment Jackson Immunoresearch 709-175-073 was used for detection.
  • a partition function analysis was run on the SB of each peptide between normal and MS serum to identify a group of peptides that evaluates the breadth component of MS-associated antibodies. Performing the partition function iteratively on individual samples enables the selection of peptides from the test population.
  • the breadth component of MS-associated antibodies can be determined by analyzing antibodies against peptide components of MS-associated proteins, e.g., the myelin sheath. Initially 48 peptides were selected based on scientific relevance to multiple sclerosis. Subsets of the 48 myelin-associated peptides were also evaluated. The peptides for a particular subset were selected based on the titer, i.e. the SB ratio of individual MS samples and the Pooled MS control sample compared to normal for both the 200 and 500 ⁇ g/ml spotting
  • the mean number of peptides that were positive was 2.68 with a 95% confidence interval ranging from 2.36-3.
  • the distribution of the peptides in the sample population is shown in Figure 3. Approximately 90% of the MS samples tested were positive for at least 1 out of the 7 peptides, demonstrating a high level of sensitivity (see Figure 3). A still higher level of sensitivity can be achieved by adding more peptides. With a prevalence rate of at least 30% per individual peptides, 12 peptides can be selected with a sensitivity of 95%. The mean number of peptides (out of 12) that were positive for the tested MS patient population was 4.29 with a 95% confidence interval ranging from 3.81 to 4.76. 95% of the MS patients tested positive for at least 1 out of 12 peptides, (see Figure 4) [0118] The results provided here show that a selection of peptides can predict MS correctly in approx 90-95% of the patients tested.
  • the methodology used in performing the microarray analysis and analyzing the corresponding results are described in detail in the foregoing Examples.
  • the arrays comprised the 23 MBP peptides identified in Table 1. When AMI microarrays were used forty-eight peptides and three HIV peptides were used. See Table 2.
  • MS-associated antibodies e.g., autoantibodies to epitopes of human myelin basic protein (MBP) in each patient sample exceeded an antibody threshold was determined using the plasma anti-myelin peptide microarray (antibody threshold/NUM) and was calculated by tallying the total number of myelin peptides on the array to which antibodies bound at a titer of 10,000 digital fluorescence units (median value for all replicate features of each peptide on the array).
  • antibody threshold/NUM plasma anti-myelin peptide microarray
  • Subjects were then divided into subgroups based on whether the titer and breadth (i.e., the number of peptides on the array to which antibodies bound at a sufficient titer) of antibodies exceeded the antibody threshold or NUM/antibody threshold categorization (e.g. >10, >12 or >14 peptides bound with serum antibody with the foregoing titer to epitopes of MBP) for subsequent correlation with clinical data.
  • the NUM/antibody threshold categorization is not an absolute number but will vary based on the assay conditions and reagents used and the standardization control samples based on which the assay is calibrated.
  • Figures 5 through 7 show the findings from the response prediction analysis. More specifically, these figures present data for all subjects in the patient's studied and the subgroups of subjects with NUM/antibody threshold >9, >10, >1 1 , >12, >13 or >14 peptides with plasma antibody binding to epitopes of human MBP (See Table 2 for patient numbers in each group):
  • a study was carried out to identify a serum predictive marker of BHT-3009 efficacy using a MS-associated protein. Multiplex assays were performed with a panel of CLIA certified antigens in HumanMap version 1.6 (133 antigens) comprising 89 antigens and 44 antigens from autoimmune serology panel. Initially 52 baseline serum samples from the immune sub-group of BHT 3009 PII clinical trial were assayed to identify potential markers of interest. One marker that showed significant promise is soluble CD40 in serum. At levels over 0.68 soluble CD40, patients receiving 0.5 mg of BHT3009, a DNA antigen specific therapy, had fewer Gd+-enhancing lesion compared to placebo group. Change over time in CD40 levels were also assessed for all serum samples tested by comparing baseline values with values at the end of treatment (wk 44). Serum CD40 levels were observed to be constant over a one year duration (R2 0.81) for all patients sampled.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Rehabilitation Therapy (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Rheumatology (AREA)
  • Genetics & Genomics (AREA)
  • Diabetes (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention porte sur un procédé pour sélectionner des patients souffrant de sclérose en plaques (MS) pour une thérapie spécifique à un antigène sur la base d'un échantillon biologique provenant d'un patient ayant été déterminé comme contenant des anticorps aux antigènes associés à la sclérose en plaques (MS) qui dépasse un seuil d'anticorps d'un échantillon témoin. L'invention porte également sur un procédé pour déterminer les anticorps associés à la sclérose en plaques (MS) d'un échantillon de patient et le seuil d'anticorps à l'aide de dispositifs.
PCT/US2011/020345 2010-01-08 2011-01-06 Procédés de sélection de patients souffrant de sclérose en plaques (ms) pour une thérapie spécifique à un antigène Ceased WO2011085081A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29354410P 2010-01-08 2010-01-08
US61/293,544 2010-01-08

Publications (2)

Publication Number Publication Date
WO2011085081A2 true WO2011085081A2 (fr) 2011-07-14
WO2011085081A3 WO2011085081A3 (fr) 2011-11-10

Family

ID=44306135

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/020345 Ceased WO2011085081A2 (fr) 2010-01-08 2011-01-06 Procédés de sélection de patients souffrant de sclérose en plaques (ms) pour une thérapie spécifique à un antigène

Country Status (1)

Country Link
WO (1) WO2011085081A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013020914A1 (fr) * 2011-08-10 2013-02-14 Celares Gmbh Peptides peg-conjugués
US20130071392A1 (en) * 2011-06-23 2013-03-21 Lawrence Steinman Small Heat Shock Proteins and Active Fragments Thereof as a Therapy for Inflammation and Ischemia
EP3109257A1 (fr) * 2015-06-26 2016-12-28 Roberto Paterno' Diagnostic et thérapie de la sclérose en plaques
WO2018083124A1 (fr) * 2016-11-01 2018-05-11 Matn Scientific Limited Détection et traitement de maladies démyélinisantes
CN112362877A (zh) * 2020-10-27 2021-02-12 江苏先声医学诊断有限公司 一种mbp重组蛋白以及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7090982B2 (en) * 1991-10-22 2006-08-15 The Governors Of The University Of Alberta Methods of predicting therapeutic efficacy of treatment of a multiple sclerosis patient
US8323963B2 (en) * 1996-05-29 2012-12-04 University Of Southern California Construction and use of genes encoding pathogenic epitopes for treatment of autoimmune disease
IL158316A0 (en) * 2001-04-10 2004-05-12 Univ Leland Stanford Junior Therapeutic and diagnostic uses of antibody specificity profiles
US20030092089A1 (en) * 2001-11-14 2003-05-15 Moscarello Mario Anthony Method for diagnosing multiple sclerosis and an assay therefore

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130071392A1 (en) * 2011-06-23 2013-03-21 Lawrence Steinman Small Heat Shock Proteins and Active Fragments Thereof as a Therapy for Inflammation and Ischemia
US10034915B2 (en) * 2011-06-23 2018-07-31 The Board Of Trustees Of The Leland Stanford Junior University Small heat shock proteins and active fragments thereof as a therapy for inflammation and ischemia
WO2013020914A1 (fr) * 2011-08-10 2013-02-14 Celares Gmbh Peptides peg-conjugués
EP3109257A1 (fr) * 2015-06-26 2016-12-28 Roberto Paterno' Diagnostic et thérapie de la sclérose en plaques
US10633427B2 (en) 2015-06-26 2020-04-28 Prindex S.R.L. Diagnosis and therapy of multiple sclerosis
EP3831844A1 (fr) * 2015-06-26 2021-06-09 Prindex S.r.l. Diagnostic et thérapie de la sclérose en plaques
US11505592B2 (en) 2015-06-26 2022-11-22 Prindex S.R.L. Diagnosis and therapy of multiple sclerosis
WO2018083124A1 (fr) * 2016-11-01 2018-05-11 Matn Scientific Limited Détection et traitement de maladies démyélinisantes
US11435345B2 (en) 2016-11-01 2022-09-06 Matn Scientific Limited Detection and treatment of demyelinating diseases
CN112362877A (zh) * 2020-10-27 2021-02-12 江苏先声医学诊断有限公司 一种mbp重组蛋白以及其应用
CN112362877B (zh) * 2020-10-27 2024-04-12 江苏先声医学诊断有限公司 一种mbp重组蛋白以及其应用

Also Published As

Publication number Publication date
WO2011085081A3 (fr) 2011-11-10

Similar Documents

Publication Publication Date Title
Mathian et al. Monitoring disease activity in systemic lupus erythematosus with single‐molecule array digital enzyme‐linked immunosorbent assay quantification of serum interferon‐α
Hueber et al. Antigen microarray profiling of autoantibodies in rheumatoid arthritis
Tozzoli et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases
US8148084B2 (en) Diagnosis of autoimmune disease
US20090291456A1 (en) Method for Diagnosing Multiple Sclerosis
Lewis et al. Autoantibodies targeting TLR and SMAD pathways define new subgroups in systemic lupus erythematosus
US20080026485A1 (en) Antibody profiling for determination of patient responsiveness
JP5706817B2 (ja) ループスのためのバイオマーカー
Yeste et al. Antigen microarrays for the study of autoimmune diseases
EP2606356A1 (fr) Méthodes et moyens pour le diagnostic de la spondylarthrite
Mostafa et al. The role of measurement of serum autoantibodies in prediction of pediatric neuropsychiatric systemic lupus erythematosus
CN109337974B (zh) 一种检测银屑病的诊断标志物的试剂及其应用
WO2011085081A2 (fr) Procédés de sélection de patients souffrant de sclérose en plaques (ms) pour une thérapie spécifique à un antigène
EP2336769A1 (fr) Test pour différencier les troubles rhumatismaux des troubles non rhumatismaux
US11609237B2 (en) Marker sequences for diagnosing and stratifying systemic sclerosis patients
CN110850096B (zh) 生物标记物组及其应用和蛋白芯片、蛋白芯片试剂盒和elisa试剂盒
Alghamdi et al. Advances in the diagnosis of autoimmune diseases based on citrullinated peptides/proteins
Wang et al. Arraying autoantibodies in SLE–lessons learned
Guthridge et al. Expanded autoantibody profiles for subsetting of native American, African American, and European American patients with systemic lupus erythematosus
US12174187B2 (en) Methods for detecting multiple sclerosis (MS) diagnostic autoantibodies
EP2380027B1 (fr) Prédiction de diagnostic de l'arthrite rhumatoïde et lupus érythémateux systémique
Fujii et al. Central nervous system-specific antinuclear antibodies in patients with multiple sclerosis
US20140227303A1 (en) Methods for diagnosing and monitoring diseases or conditions using disease modified biomolecules and measurement of a functional immune response
Dotzlaw et al. Spots, blots, peaks and chips: proteomic approaches in autoimmune diseases
WO2020198299A1 (fr) Compositions et procédés de caractérisation et de traitement de la maladie d'alzheimer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11732131

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 25/10/2012)

122 Ep: pct application non-entry in european phase

Ref document number: 11732131

Country of ref document: EP

Kind code of ref document: A2