WO2020163794A1 - Detection of brain-derived debris in recirculating phagocytes - Google Patents

Detection of brain-derived debris in recirculating phagocytes Download PDF

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Publication number
WO2020163794A1
WO2020163794A1 PCT/US2020/017332 US2020017332W WO2020163794A1 WO 2020163794 A1 WO2020163794 A1 WO 2020163794A1 US 2020017332 W US2020017332 W US 2020017332W WO 2020163794 A1 WO2020163794 A1 WO 2020163794A1
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Prior art keywords
disease
biomarker
patient
phagocytes
nervous system
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PCT/US2020/017332
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French (fr)
Inventor
Ramesh C. Nayak
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Msdx, Inc.
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Publication of WO2020163794A1 publication Critical patent/WO2020163794A1/en

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • 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
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • ⁇ 00031 in general, when tissue damage occurs, it incites inflammation, which usually aids in wound healing.
  • inflammation usually aids in wound healing.
  • one of the normal functions of inflammation is to recruit phagocytes to clear away the cellular debris and prepare the injured site for repair and rebuilding.
  • phagocytes may be resident in the brain (e.g., dendritic cells, microglial cells) or recruited from the blood strea (e.g, monocytes).
  • Cells that engulf debris are thought to ente the brain by crossing the blood-brain barrier but are not believed to retur to the biood stream. For example, whe phagocytes engulf tissue debris and exit the tissue, it is thought to be via the lymph nodes,
  • the present invention describes a phagocytic shuttle method (PSM) wherein the phagocytes that re-enter the blood strea from the central nervous system (CNS) are shuttles for CNS-derived debris, and the debris can be indicative of processes occurring in the CNS (e.g.. brain),
  • the presence and/or amount of the debris may be associated with various states of the brain, e g., active central nervous system tissue damage, active central nervous system repair, active neurodegeneration, etc.
  • the presence and/or amount of the debris may be used for monitoring a brain condition or neurological disease (e.g., monitoring worsening or improvement of a particular brain condition or neurological disease).
  • the presence of the debris may be associated with particula disease states or injury.
  • the presence and/or amount of the debris may be used for detecting neurological damage (e.g., neuroiogical damage associated with a disease or injury).
  • neurological damage e.g., neuroiogical damage associated with a disease or injury.
  • the presence and/or amount of the debris may be used to detect active neurodegeneratlve diseases, active centra! nervous system tissue damage, and/or active central: nervous system repair.
  • the present invention features detection and/or measurement of the neural-derived cargo
  • the present invention may provide close to reaS-time data on what is happening in the brain since that particular cargo may oniy be present i the recirculating phagocytes for a certain length of time (e.g., a few days) before it is completely digested.
  • the present invention features methods for creating patient cohorts (e g,, of one or more patients) for a clinical trial, e g,, a clinicai trial for testing a therapeutic composition or therapeutic intervention for an effect on central nervous system (CNS) tissue damage, CNS repair, or neurodegeneration, etc.
  • CNS tissue damage, CNS repair, neurodegenerati ' on, etc. may be associated with a particular disease, condition, trauma, etc., as described herein.
  • the method features isolating or retrieving a biomarker or antigen from the central nervous system (e.g,, a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector.
  • the method comprises isolating circulating phagocytes from a fluid sample from each patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a level of a biomarker in the phagocytes from each patient, th biomarker being associated with CNS tissue damage, CNS syste repair, or neurodegeneration, wherein if the level of the biomarker in the phagocytes is abnormal, then the patient has active CNS disease; and selecting only patients with active CNS disease to create the patient cohort for the clinical trial.
  • a central nervous system tissue e.g., outside of the brain tissue
  • the step of isolating circulating phagocytes from the fluid sample from the patient may further comprise concentrating the circulating phagocytes, in some embodiments, the circulatin phagocytes are isolated or concentrated using forward-scattered light or side-scaitered light in flow cytometry. In some embodiments, the circulating phagocytes are isolated or concentrated base on immunotype.
  • the circulating phagocytes have a specific immunotype, the immunotype is determined using a marker selected from; GDI 1a, GUI 1 b, CD1 1c, CD14, Cp1 S i, T CP33, CD84, CD68, CD8G, CDS6, GDI OS/eridOgith, CD 115, CD163, CD195/CCR5, CD282/TLR2, OD284/TLR4, HLA-DR/MHG Class
  • the level of the biomarker Is compared to a predetermined threshold to determine if it is abnormal
  • the predetermined threshold is an industry standard
  • the !evef of the biomarke is compared to a predetermined threshold to determine if it is abnormal
  • the predetermined threshold is a laboratory standard in some embodiments
  • the level of the biomarker is compared to a predetermined threshold to determine if It is abnormal* the predetermined threshold Is a level of the biomarker in phagocytes isolated fro a fluid sample obtained from the patient before administration of the therapeutic compositions.
  • the biomarker or antigen may be one or more of the following biomarkers.
  • the present invention also features methods of identifying a therapeutic composition or therapeutic intervention: having positive effect on centra! nervous system (CMS) tissue damage, CMS repair, or neurodegeheratioo.
  • CMS tissue damage, CMS repair, neurodegeneration, etc. may be associated with a particular disease, condition, trauma, etc,, as described herein.
  • the method features isolating or retrieving a biomarker or antigen fro the central nervous syste (e.g., a brain antigen, etc,) utilizing re-circulating phagocytes as a shuttle vector.
  • the method comprises: selecting at least one patient with active CMS disease by isolating circulating phagocytes front a fluid sample from the patient, the fluid sample being from: outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detesting a level of a biomarker in the phagocytes from the patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneration, etc. in some embodiments, if the level of the biomarker in the phagocytes is abnormal, then the patient has active CNS disease.
  • a central nervous system tissue e.g., outside of the brain tissue
  • the method may further comprise; administering to the at least one patient with active CMS disease the therapeutic composition or therapeutic intervention; isolating circulating phagocytes from a fluid sample from the patient after a predetermined amount of tim following administration of the therapeutic composition or therapeutic intervention, the fluid sample being fro outside of a centra!
  • a biomarker in the phagocytes from each patient is associated with; CNS tissue damage, CMS system repair, or neurodegenefafien, etc, in some embodiments, (a) if the level of the biomarker in the phagocytes fro a patient is normal, then the therapeutic composition or therapeutic intervention has a positive effect on CMS tissue damage, CNS repair, or neurodegeneration; (b) if the level of the biomarker in the phagocytes from a patient is within a predetermined threshold associate with improved CNS disease, then the therapeutic composition or therapeutic intervention has a positive effect on CNS tissu damage, CNS repair, or neurodegeneration; or (c) if the level of the biomarker In the phagocytes from a patient is improved compared to a level of the biomarker in phagocytes in a sample obtained fro the patient at a time point prior to administration of the therapeutic composition or therapeutic intervention;
  • the predetermined threshold may be industry standards in some embodiments, the predetermined threshold is a laboratory standard. In some embodiments, the predetermined threshold is a level of the biomarker In phagocytes isolated from a fluid sample obtained from the patient before administration of the therapeutic compositions. Each particular biomarker of interest may have its own predetermined threshold, in some embodiments, a level of biomarker above the predetermined threshold is an abnormal level of the biomarker. In some embodiments, a level of biomarker below the predetermined threshold is an an norma I level of the biomarker.
  • the therapeutic composition or therapeutic intervention may include but is not limited to; Mastinab, ; Dadizumab, Zinbryta, ER-Seta agonist, cyclophosphamide, rHlgM22, ponesimod, alpha-4 integrin, AMP-110,: an antisense oligonucleotide, immune tolerizing agent, MuitiStem, vi .3 Blocker, Alemtuzumab, !FNb ⁇ 1 b, BHT-3009-01 , IF b-la, dimethyl fumarate, IF b-la (PEGyiated); Natalizumab, MT130T, Abatacept, RCP1083, a compound related to !amitrigene, CN -AU8, Mesenchymal stem cell transplant, apolipoprotein E ⁇ based, modified pepiidomimetic, Aimspro, Anti-BAFF Human Ab, NDC-13G8, GNbACt, Vatellzum
  • the present invention also features methods of selecting a candidate for monitoring mild cognitive impairment or Parkinson’s disease.
  • Th method features retrieving a biomarker or antigen from the central nervous syste fe.g., a brain antigen, etc,) utilizing re-circulating phagocytes a a shuttle vector.
  • the method comprises isolating circulating phagocytes from a fluid sample from a patient, the fluid sample being from outside of a central nervous system tissue (e.g.. outside of the brain tissue) of the patient; detecting a level of a biomarke in the phagocytes, the biomarker being associated with CMS tissue damage, CMS system repair, or neurodegeneration. in some embodiments, if the level of the blomarker in the phagocytes is abnormal, then the patient has active central nervous system disease.
  • the method may comprise selecting the patient for monitoring for mild cognitive impai ment or Parkinson’s disease based on the patient having active centra! nervous system disease,
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes,
  • the candidate has REM sieep behavior disorder (RBD).
  • the biomarker may include but is not limited to dopamine-beta-hydroxyiase (DBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FU78071, structuriipoprotein C-i!, immunoglobulin heavy constant gamma 3, alpha-1 -acid glycoprotein 1, alpha-1 -acid glycoprotein 2, haptoglobin-related protein, leucine-rich 3 ⁇ 4lpha-2 ⁇ g!ycoprotein t C-reactive protein, Tay, phosphoryiated Tau, hippocalcin-1 , or 14-3-3 protein, MBP, UCH-L1 , TDP-43.
  • the present invention is not limited the aforementioned biomarkers or antigens.
  • the biomarker may be selected based on its association with a particular disease or condition,
  • the present invention aiso features methods of treating a patient suspected of having centra i nervous system tissue damage, central nervous system repair, or neurodegeneration.
  • the method features isolating or retrieving a biomarker or antigen from the centra! nervous system e,g. t a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector in some embodiments, the method comprises: isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system ti sue (e.g..).
  • the step of Isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes
  • the central nervous system tissue damage, central nervous system repair, or neurodegeneration is a result of Multiple Sclerosis, Alzheimer’s disease, mild cognitive impairment, Parkinson ' s disease, Multiple System Atrophy, Progressive Supranuclear Atrophy, Lewy body disease, Coriicobasal Degeneration, Amyotrophic Lateral Sclerosis, Huntington’s Disease, REM sleep behavior disorder, concussion, Traumatic Brain injury, or a disease causing secondary centra! nervous system damage in some embodiments * the central nervous system tissue damage, central nervous system repair, or neurodegeneraiion causes cognitive impairment, motor disturbances, or doth.
  • the biomarker is Tau, phosphorylaied Tau, hsppocalcin-1 , or 14-3-3 protein, MBP.
  • the therapeutic composition or therapeutic intervention is Mastinab, Baeiizumab, Zinbryta, ER-Beta agonist, cyclophosphamide, rBigM22, pohesimod, aipha-4 Ihtegrin, AMP-110, an antisense oligonucleotide, immune toierizing agent, MuiiSStem, Kvt,3 Blocker, Alemtuzumab, iFNbdb, BHT- 3003-01 , iFNb ⁇ 1a, dimethyi fu arate, !FNb-1a (PEGyiaied), Nataiizumah, MU 301 , Abatacept, RCP1063, a compound related to !amitrigene, CNM-Au8, Mesenchymal stem ceil transplant, apQiipoprotein E-based, modified peptldomimetic, A
  • RG145Q, RG74 2, POE4E, PDE4B Mefhyithiohinium chloride, T3D-959, MultiTEP, ABT957, Anii-Tau antibodies, Aducanumab, Soianezu ab, or Inbrija,
  • the present invention is not limited to the aforementioned therapeutic compositions or therapeutic interventions.
  • the present invention also features a method of treatin a atient suspected of having an autoimmune disease or condition associate with neurodegeneration.
  • the metho features isolating or retrieving a biomarker or antigen from the centra! nervous syste (e,g though a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector.
  • the method comprises isolating circulating phagocytes from a fluid sampie from the patient, the fluid sample being from; outside of a centra!
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the autoimmune disease or condition associated with neurodegeneration is multiple sclerosis,
  • the present invention also features a method of treating a patient suspected of having disease or condition causing cognitive impairment.
  • the method features isolating or retrieving a biomarker or antigen from the central nervous system (e.g,, a brain antigen, etc.) utilizing re- circulating phagocytes as a shuttle vector.
  • the method comprises isolating circulating phagocytes from a fluid sample from the patient* the fluid sample being from outside of a central nervous system tissue (e.g..
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the disease o condition causing cognitive impairment is Alzheimer’s disease, or Lewy body disease.
  • the present invention also features a method of treating a patient suspected of having a disease or a condition causing motor disturbances.
  • the method features isolafing o retrieving a biomarker or antigen from the central nervous system (e.g.. a brain antigen, etc.) utilizing recirculating phagocytes as a shuttle vector.
  • the central nervous system e.g. a brain antigen, etc.
  • the method comprises isolating circulating phagocytes from a fluid sample from the patient, the flui sample being from outside of a cental nervous system tissue (e.g, , outside of the brain tissue ⁇ of the patient; detecting a level of a fsiomarke in the phagocytes, the biomarker being associated with the diseas or condition, wherein if the ievei of the biomarker is abnormal then the patient has active central nervous system disease; and administering to the patient having active central nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the disease or condition or a symptom thereof.
  • a cental nervous system tissue e.g, outside of the brain tissue ⁇ of the patient
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes.
  • the disease or condition causing motor disturbances is Parkinson’s disease, Multiple Sclerosis, Supranuclear Atrophy, or Coifieobasai Degeneration, h
  • the present invention also features a method of treating patient suspected of having brain trauma.
  • the method features isolating or retrieving a biomarker or antigen from the central nervous system (e,g., a brain antigen, etc.) utilising re-eircuSating phagocytes as a shuttle vector !n some embodiments, the method comprises Isolating: circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a level of a biomarker in the phagocytes, the biomarker being associated with brain trauma, wherein if the level of the biomarker is abnormal then the patient has active central nervous system disease; an administering to the patient having active centra!
  • a central nervous system tissue e.g., outside of the brain tissue
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes in some embodiments, the method is performed within a time frame from 24 to 48 hours after a head injury. In some embodiments * the metho is performed within a time frame from 1 to 48 hours after a head injury,
  • the brain trauma is a traumatic brain Injury or a concussion.
  • the present invention also features a method of treating a patient having a genetic disorder associated with neurodegeneration (e.g., Huntington’s disease, amyotrophic lateral sclerosis (AL$), etc,).
  • the method features isolating or retrieving a biomarke or antigen from the central nervous system (e.g., a brain antigen, etc.) utilizing re-circulating phagocytes as a shutle vector, in some embodiments, the method comprises isolating circulatin phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a ievel of a biomarker in the phagocytes, the biomarker being associated with the genetic disorder, wherein If the level of the biomarker is abnormal then the patient has activ central nervous system disease; and administering to the patient having active centra!
  • a genetic disorder associated with neurodegeneration e.g., Huntington’s disease, am
  • the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes .
  • the present invention also features method of treating a patient suspected of having a relapse-remiting disease or infection,
  • the method features isolating or retrieving a biomarker or antigen utilizing re-circulating phagocytes as a shuttle vector, in some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from the patient; detecting a level of a biomarker in the phagocytes the biomarker being associated with the relapse-remitting disease, wherein if the levei of the biomarker is abnormal then the patient has active disease; and administering the patient having active disease relate to the relapse-remitting disease or infection a therapeutic composition or therapeutic intervention effective tor treating or preventing worsening of the relapse-remitting disease, or a symptom thereof in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the fluid sample is from outside of a
  • the relapse-remitting disease is multipie sclerosis (MS), Lyme disease, rheumatoid arthritis (RA). systemic lupus erythematosus (SLE), inflammatory bowel disease, human immunodeficiency virus (HIV), neurosareoidosis, or psoriasis,
  • the methods herein may also feature using a ratio of a biomarker to another parameter such as a plasma marker, an imaging marker, a different marker associated with a disease state or condition, a second biomarker in the circulating phagocytes, other particular diagnostic or investigative marker, etc.
  • a ratio of a circulating phagocyte biomarker to a plasma marker may be used to detect CHS damage or repair.
  • a ratio of a circulating phagocyte biomarker to an imaging marker or diagnostic indicator is used to detect active disease in a patient (e g though multiple sclerosis patient) in some embodiments, a ratio of a circulating phagocyte to a secondary marker associated with REM sleep disorder is used to detect REM sleep disorder. In some embodiments, a ratio of a first circulating phagocyte biomarker to a second circulating phagocyte biomarker is used to detect active CMS damage.
  • the present invention is not limited to the aforementioned examples of ratios of biomarkers or diagnostic indicators.
  • the ratio as it related to the disease process may be a predetermined value, e.g., an industry standard, a laboratory standard, etc. For example, for some cases, a high ratio may be associated with active disease, and in some cases a low ratio may be associated with active disease.
  • the present invention also features preservation of samples for preserving the amount and/or structure and/or location of the biomarker(s) of interest (e.g., for preserving the amount and/or structure and/or location of the epitope(s) of interest).
  • the present invention provides methods for treating samples for the purposes of preserving the biomarker, e g., via heat denaturation (wherein proteolytic enzymes or other factors are inhibited without affecting the biomarker, e.g,, the epitope of the biomarker, to a large extent).
  • Other methods of preservatio may include freeze drying or other rapid freezing processes, application of heparin or other factors, modifying the pH of the sample, etc.
  • the present invention i not limited to the aforementioned methods or compositions,
  • the phagocytes containing the biomarkers of interest be characterized and isolated based on immunaphenatyping, This process be used for investigative purposes, for example to help determine if there is a subpopulation of cells with the particular biomarker of interest. Further, the process, once a particular immunophenotyped identified for a biomarker of interest, may be used as a technique for concentrating tSie phagocytes during sample preparation and analysis. The association of a particular immunophenotyped ceil and a biomarker of interest may be achieved by any appropriate method, e,g.
  • the results may identify Known phagocytic celi types (CD 14+ monocytes and/or macrophages (CD 68/CD11b)) to be the source of particular biomarkers ⁇ e.g,, neural antigens) in PBMCs.
  • Non- limiting examples of lineage antigens for !mmunophenotyping and Immunosefeetion may include CD14, CD 18, CD 71 , GDI la, GDI 1 b, GDI 1c, CD15 iew , CD33, CD84, CP88, CDSO, CQ88, CDlOS/endogiin, CD115, CD163, CDl95/CeR5, GD282/TLR2, CD284/TLR4, HLA-DR/MHC Class SI, ILT 1 ILT3.
  • the subject or animal is a mouse
  • the lineage antigens may include but are not limited to CDl l a, GDI I D, CD13, CDi4 « «r» CD18/CD32, CD84, CD68, CD8Q, CD86, CD107/Mac3, CPU S, CD282/TLR2, CD2S4/TLR4, F4/SG, Gaiactsn-S/MaoG, GITRL, MMC Class il, 33D1 , CD4, CDS, CD1 ib iiw , CD1 1c, OD40, CD45R/B220 pE>c , CD83, 00123 ⁇ , C01S7/CGR7, GD20S/DEC-205, GD207/Langerin t C D209/DC-S!
  • biomarkers that are associated with particular disease states of interest e. ⁇ ., biomarkers found in the re-circulating phagocytes as described herein
  • wifi continue to be discovered.
  • the methods herein are not necessarily limited by the particular biomarker but instead features the phagocytic shuttle method (e.g., wherein the phagocytes are shuttles for GNS-derived debris indicative of processes occurring in the CHS) and steps for isolating the biomarkers within theshuttle phagocytes, the present invention includes those biomarkers that will be discovered in the future,
  • FIG 1 shows the binding of the 4S4 peptide to neuromelanin in extracts of retinal pigment epithelium immobilized on ELISA plates in two-fold dilution series
  • FIG. 2 shows curve of varying amounts of pure Tau protein isolated using a Tau HT? antibody an an anti ⁇ Tau rabbit poiyelonai antibody.
  • FiG. 3 shows Tau and Hippocalcin 1 tike i ieve!s in PBMC lysates in 12 apparently healthy controls and 18 MS subjects.
  • FIG. 4 shows T au in PBMCs of cuprizone fed mice.
  • FIG 5 shows hippocalcin 1 like 1 in PBMCs of cuprizone fed mice
  • FIG. 6 shows myelin basic protein in PBMCs of cuprizone fed mice.
  • FIG, ? shows a macrophage with engulfed photoreceptor debris in the process of diapedesis through an optic nerve head capillary, the cell nucleus is inside the capillary whereas parts of the debris-containing cytoplasm Is still externally located (arrow) (from Joty S et aL Resident Microglia and Bone Marrow Immigrants Remove Dea Photoreceptors in Retinal Lesions, The American Journal of Pathology, Vol, 174, No, 6, June 2009),
  • FIG. S shows the relationship between several biomarkers, such as Tau, GFAP, NFL, UCH-L1 , etc., and the active disease processes associated with diseases such as Multiple Sclerosis, Alzheimer’s disease, Parkinson’s disease » etc.
  • biomarkers such as Tau, GFAP, NFL, UCH-L1 , etc.
  • active disease processes associated with diseases such as Multiple Sclerosis, Alzheimer’s disease, Parkinson’s disease » etc.
  • debris-laden phagocytes a may re-enter the blood stream from the rain and if sufficient numbers are present, it may be possible to detect or measure the brain-derived debris (e g., debris or blpmafkers that would only normally be found in brain or centra! nervous system tissue) that is inside the phagocytic cells.
  • the brain-derived debris e g., debris or blpmafkers that would only normally be found in brain or centra! nervous system tissue
  • the presence and/or amount of the debris may be associated with various states of the brain, e.g., active central nervous syste tissue damage, active central nervous system repair, active neurodegeneraiion, etc.; thus, obtaining these neora!
  • -derived circulating phagocytes can be used for monitoring a brain condition or neurological disease (e.g., monitoring worsening of improvement of a particular brain condition or neurological disease), detecting neurological damage (e.g., neurological damage associated with a disease or injury), detectin active neurodegenerative diseases, active central nervous syste tissue damage, and/or active central nervous system repair, etc.
  • a brain condition or neurological disease e.g., monitoring worsening of improvement of a particular brain condition or neurological disease
  • neurological damage e.g., neurological damage associated with a disease or injury
  • detectin active neurodegenerative diseases e.g., active central nervous syste tissue damage, and/or active central nervous system repair, etc.
  • the present invention can provide close to real-time data on what is happening in the brain since that particular cargo may only be present in the recirculating phagocytes for a certai length of time (e.g,, a few days) before it is completely digested,
  • T e present invention also features methods for stratifying patients suspected of having an active neurodegenerative disease process, for example for selecting patients with active disease as part of a cohort for a clinical trial or for treatment. This enables patients not
  • stratification methods may also be used to identify and monitor those suspected of having a neurodegenerative disease (e,g., patients experiencing mild cognitive impairment or other symptoms) before the disease progresses too far. This may be particularly valuable In certain disease processes, such: as Parkinson’s disease, wherein patients are typically identified too late.
  • a neurodegenerative disease e.g., patients experiencing mild cognitive impairment or other symptoms
  • Various neural-derived debris antigens or biemafkers may be found in recirculating phagocytes in the peripheral blood.
  • Such debris antigens may be used to detect (or monitor) neurodegenerative or neuroinfiammatory diseases (e.g., diseases as described herein).
  • Debris from other diseases, such as relapse-remitting diseases may be retrieved specificaiiy from circulatin phagocytes as well.
  • the biomarker may be present in a circulating phagocyte, e.g., peripheral phagocyte,
  • peripheral phagocyte refers to anything outside of brain tissue.
  • a peripheral phagocyte may be obtained from cerebrospina! fluid (CSF)
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • Such circulatin phagocytes may be found in tissues, cells, and/or fluids in the body , for example in biood, peripheral blood mononueiear cel is (PSfVICs), synovial fluid, cerebrospinal fluid fCSF), central nervous system tissues, synovia!
  • PSfVICs peripheral blood mononueiear cel is
  • synovial fluid cerebrospinal fluid fCSF
  • central nervous system tissues synovia!
  • the biomarker is an intracellular component
  • the biomarker may be obtained from within a macrophage.
  • the macrophage sample is permeabi!teed.
  • the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
  • the fluid obtained for probing the phagocytes may be dependent on the disease or condition of interest,
  • a mammal includes a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow.
  • the present invention Is not limited to the aforementioned subjects or patients.
  • the sample is obtained from the subject, e.g., mammal, immediately following a relapse (e,g,, exacerbation of symptoms) before a therapy (e,g., a steroid or other drug or other therapeutic intervention) treatment has begun, in some embodiments, the sample is obtained fro the roammai before a relapse, In some embodiments, the sample is obtained during the course of the therapy treatment .
  • a relapse e.g, exacerbation of symptoms
  • a therapy e,g., a steroid or other drug or other therapeutic intervention
  • the disease or interest associated with central nervous system tissue damage, central nervous system repair, neurodegeneration, etc. includes but is not limited to: autoimmune diseases, e.g., multiple sclerosis rheumatoid arthritis, lupus, Sjogren’s syndrome, thyroiditis, uveitis, Crohn's disease, ulcerative colitis, psoriasis, type 1 diabetes mei!itus, autoimmune Addison's disease, autoimmune hepatitis, celiac disease, pempbigous, chronic inflammatory demyelinating polyneuropathy, acute disseminated encephaiomyeiopathy, sarcoidosis, dermatomyositis and behcet’s disease; neuroSogicai diseases, e.g., stroke, concussion, chronic traumatic encephalopathy, neuromyelitis optica, transverse myelitis, intractable epilepsy and CNS infections; Parkinson’s disease; primary tumor growth,
  • autoimmune diseases e.g
  • Other diseases may include but are not limited to mil cognitive impairment, traumatic brain injury, Huntington’s disease, amyotrophic lateral sclerosis, progressive supranuciear atrophy, corticobasai degeneration, multipie system atrophy, Lyme disease, and systemic lupus erythematosus, Neuromyelitis Optica, transverse myelitis, .Acute and chronic Stroke, etc.
  • the present invention is not limited to the aforementioned diseases,
  • Table 2 shows non-limiting examples of biomarkers that may be associated with disease states ⁇ e.g., degenerative disease states) with various organs.
  • the present invention may be used to detect a diabetes condition by detecting somatostatin in a similar manner as described herein, e.g.. similar to methods for detecting neuromeianin for Parkinson’s disease.
  • me biomarkers or antigens (or nsurodegenefative disease- associated proteins or biomarkers) (or fragments thereof) indude but are not limited to those in Table 1 , a Tau protein ⁇ or fragment thereof), a Tau protein or fragment thereof comprising a phosphorylated residue (e.g,, a phosphorySated serine reside, a phosphorylated threonine reside; e.g,, serine 214, serine 235, serine 282, serine 358, serine 396, serine 404, serine 413, serine 46, serine 515, serine 518, serine 519, serine 531 , serine 552, serine 610, serine 822, serine 641 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threon
  • fO055j FiG. 8 shows the relationship between severai blamarkers, such as Tau, GFAP, NFL, UCH-L1 , etc., and the active disease processes associated with diseases such as Multiple Sclerosis, Alzheimer's disease, Parkinson's disease, etc,
  • neurorneianin may be detected In the debris of degenerated dopaminergic neurons in recirculating phagocytes. Abnormai neurorneianin levels may be
  • Neuromelanin can be measured: in several ways, e,g., via thebinding gf labeled: melanin selective peptides (e,g., 484 peptide), e g,, biotihyiaied 4B4 peptide; a control peptide F601G may be use as a control); the binding of monoclonal or polyclonal antibodies to melanin; measuremen of metal binding to melanin; measurement of the semiconductor properties of melanin; measurement of the fluorescence properties of melanin; and extraction of melanin fro recirculating phagocytes and subsequent quantification of melanin, it’s components or adducts (both natural or synthetic); physical methods such as gas chromatography, liquid chromatography or mass spectrometry; and combinations of these methods,
  • FIG. 1 shows the binding of the 4S4 peptide. Extracts of retinal pigment epithelium were immobilized on ELISA plates in two-foid dilution series of the extract and incubated with biotinylate 4B4 peptide. Unbound peptide was washed off and bound peptide was detected with streptavidin-HRP. in comparison, PB!VICs fro healthy human subjects show little binding of 4S4 peptide.
  • the biomarker is a Tau protein (or a fragment thereof) or a Tau protein (or fragment thereof) comprising a phosphoryiated residue ⁇ e.g., a phosphoryiated serine residue, a phosphoryiated threonine residue).
  • the phosphoryiated residue is serine 214, serine 235, serine 262, serine 358, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531 , serine 552, serine 610, serine 622, serine 841 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threonine 492, threonine 49S, threonine 522, threonine 529, threonine 534, threonine 548, the like, or a combination thereof.
  • phosphorylation of Tau can decrease its solubility in some embodiments, the method comprises detecting a level of insoluble Tau protein in the sample. In some embodiments, an increased level of insoluble Tau protein as compared to a control level of insoluble au protein is indicative of the disease or condition of interest or a risk thereof.
  • the biomarker of interest may be Tau or a fragment thereof.
  • Table 3 shows the amino acid sequence of full-length human Tau protein.
  • Tau biomarfcer may refer to a particular epitope of Tau, e.g,, an epitope within a particular region of amino adds in some embodiments, the epitope is in a region fro aa 240-441 in some embodiments, the epitope is in a region from aa 243-441. i some embodiments, the epitope is in a region from aa 244-274. In some embodiments, th epitope is in a region from aa 275-305. in some embodiments, th epitope is in a region from aa 308-336. in some embodiments, the epitope is in a region from aa 337-368.
  • the epitope is in a region from aa 388-411 , Non-timiiing examples of ranges of amino acids in which the epitope may be contained is shown in Table 5. The present invention is not limited to these regions. Further, the epitope may be in shorter regions of amino adds, e.g., aa 244-260, aa 270-280, aa 290-310, aa 330-360, etc.
  • the methods of the present invention may feature isolating from the phagocytes (e.g,, re-cfrcuiating phagocytes) and/or detecting and/or measuring and/or evaluating the biomarker of interest, wherein the biomarker of interest is a particular epitope of a biomarker such as Tau.
  • the method may comprise detecting an epitope of Tau in the region of aa 201-441.
  • the method comprises detecting an epitope of Tau in the region from aa 243-441 , in some embodiments, the metho comprises detecting an epitope of Tau in the region from aa 244-274, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 275-306, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 306-336, in some embodiments, the method comprises detectin an epitope of Tau In the region from aa 337- 368, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 388-411.
  • a combination e.g,, a pair
  • biomarker-specific antibodies are used for Isolating and detecting the biomarker of interest.
  • an ELISA assay may use a first biomarker-specific antibody as a capturing antibody and a second biomarker-specifie antibody as a defection antibody.
  • FIG, 2 shows an example of such an assay, wherein both an anti-Tau HT7 antibody and an anti-Tau rabbit polyclonal antibody were used in an ELiSA.
  • the present inventio is not limited to the use of the pair of aforementioned antibodies.
  • the present invention includes a combination of any of the antibodies disclosed herein or antibodies specific to the biomarker of interest not necessarily iisted herein, e,gNeill those that may be produced in the future, those that are commercially available, etc.
  • the biomarkers of interest are being cjeaved or modified in a way that makes detection more difficult than isolating or detectin recombinant versions of the biomarker.
  • a commercial antibody that is able to detect a recombinant biomarker may not work well to detect the biomarker in a serum sample.
  • the present invention also features producing or identifying binding molecules (e.g., antibodies, fragments, etc.) that may be used to detect particular antigens or biomarkers of interest in the sample (e.g,. serum, etc.),
  • biomarker is detected in the sampie(s).
  • the biomarkeifs) is a neural-derived biomarker.
  • the biomar er(s) is not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neurai-derlyed biomarkers, or a combination thereof.
  • the biomarker may be of various lengths.
  • the biomarker is from 5 to 20 amino acids, in seme embodiments, the biomarker is from 20 to 40 amino acids, in some embodiments, the biomarker is from 40 to 80 amino acids, in some embodiments, the biomarker is from 80 to 150 amino acids, in some embodiments, the biomarker is from 150 to 20 amino acids , in some embodiments, the biomarker is from 2Q0 to 300 amino acids, in some embodiments, the biomarker is from 300 to 400 amino acids, in some embodiments, the biomarker is from 400 fo 500 amino acids in some embodiments, the biomarker is from 500 to 800 amino acids,
  • the biomarker may comprise various regions of the full-length protein for example, in some embodiments, the biomarker comprises the amino-terminus (e.g., N-terminus, NH2- terminus, N-terminai end, amine-terminus).
  • the amino-terminus refers to the amino acid at the end of a protein or polypeptide that has a free amine group ( ⁇ NH2).
  • the biomarker comprises about the first 15 amino acids, in some embodiments, the biomarker comprises about the first 25 amino acids in some embodiments, the biomarker composes about the first 50 amino acids, in some embodiments, the biomarker comprises about the first 75 amino acids, in some embodiments, the biomarker comprises about the first 100 amino acids, in some embodiments, the biomarker comprises about the first 126 amino acids.
  • the biomarker or fragment thereof comprises the carboxy-terminus (e g,, C-terminus, COON-terminus, C-terminai end, carboxyl-terminus) :
  • the carboxy-terminus refers to the amino acid at the end of a protein or polypeptide that has a free carboxylic acid group (-COOH), in some embodiments, the biomarker comprises the last 100 amino acids,
  • the step of detecting the biomarker in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the biomarker.
  • the step of detecting the biomarker in the sample comprises subjecting the sample to a western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an fmmunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a mass spectrometry assay, a flow cytometry assay (e.g., fSorescence- aefivated ceil sorting (FACS)), or a combination thereof and the like.
  • ELISA enzyme-linked immunosorbent assay
  • a lateral flow assay e.g., a radioimmunoassay, an fmmunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a mass spectrometry assay, a flow cytometry assay (e.g., fSorescence- aefivated ceil sorting
  • the step of detecting the biomarker further comprises contacting the sample with an antibody that binds to the biomarker and detecting an antibody-biomarker complex :
  • the step of detecting an antibody-biomarker complex may comprise subjecting the sample to a micro array, western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an immunohistochemsstry assay, a bioluminescent assay, a chemiluminescent assay, a flow cytometry assay (e.g,, florescence-activated ceil sotting (FAGS)), or a combination thereof and the like, in some embodiments, detecting the antibody-biomarker complex indicates the presence of the particular disease or condition of investigation or a ris of the particular disease or condition of investigation,
  • the step of detecting the biomarker may comprise subjecting the sample florescence-activated cell sorting (FACS).
  • FACS Fluorescence-activated cell sorting
  • FACS Fluorescence-activated cell sorting
  • a current of a rapidly Sowing stream of liquid carries a suspension of cells through a nozzle. The flow Is selected such that there is a large separation between cells relative to their diameter.
  • Vibrations at the tip of the nozzle cause the strea of cells to break into individual droplets, and the system is adjusted so that there is a low probability of more than one ceil being in a droplet,
  • a monochromatic laser beam illuminates the droplets, which are electronically monitored by fluorescent detectors.
  • the droplets that emit the proper fluorescent wavelengths are electrically charged between deflection plates in order to be sorted into collection tubes.
  • the present invention features method of detecting and/or treating diseases described herein.
  • the present invention features methods for detecting multiple sclerosis or a risk of multiple sclerosis, or methods for monitoring disease activity, drug efficacy, methods for treating multiple sclerosis, etc.
  • the present invention also features methods for detecting Alzheimer's disease or a risk of Alzheimer's disease, or methods for monitoring disease activity, drug efficacy, etc.
  • the present invention also features methods for treating Alzheimer's disease.
  • the present invention also features methods for detecting Parkinson ' s disease or risk of Parkinson’s disease, or methods for monitoring disease activity, drug efficacy, etc.
  • the present invention also features methods for treating Parkinson’s disease.
  • the present invention also features methods for detecting concussion or a risk of concussion, or methods for monitoring disease activity, drug efficacy, etc.
  • the present invention also features methods for treating concussion.
  • the present invention also features methods for detecting central nervous system tissue damage or a risk of central nervous syste tissue damage, or methods for monitoring disease activity, drug efficacy, etc.
  • the present invention also features methods for treating centrai nervous system tissue damage
  • the present invention also features methods for detecting centrai nervous system repair, or methods for monitoring disease activity, drug efficacy, etc.
  • the present Invention also features methods for treating centrai nervous system repair.
  • the present invention also features methods for defecting a relapse-remitting disease or a risk of a relapse-remitting disease, or methods for monitoring disease activity, drug efficacy, etc.
  • the present invention also features methods for treating a relapse-remitting disease.
  • the relapse- remitting disease may be, for exampie, multiple sclerosis (MS), Lyme disease, rheumatoid arthritis (RA), systemic !upus erythematosus (SLE), Ghron’s disease, etc.
  • the present Jrwentfon also features a method of detecting art inflammatory condition in a similar manner, wherein the biomarker is associated with an inflammatory condition.
  • the fluid obtained does not necessarily directly come into contact with the inflamed tissue being detected.
  • there may be a barrier between the fluid and the source of the biomatker in other words, the fluid obtained may have once directly come into contact with the inflamed tissue, but at the time that it is being extracted in accordance with the present invention, it is being separated from the inflamed tissue by a barrier.
  • samples are obtained for subjects or patients (e,g., a mammal such as a human, rat, mouse, primate, etc. ⁇ .
  • the sample is a plasma sample
  • the sampl is a blood sample.
  • the sample Is DNS fluid
  • the biomarkers are detected in the recirculating phagocytes in the collected fluid sample, in some embodiments, two or more biomarkers are detected in a sample, e.a,, a pattern of biomarkers may be detected in the sample, in some embodiments, the bsomarkerfs) is a neural- derived biomarker.
  • the biomarkeifs) Is not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein th biomarkers are neural-derived, non-neurabderived biomarkers, or a combination thereof,
  • the biomarker(s) may be detected using a variety of methods, Methods may include an immunoassay, a histological assay, a flow cytometry assay, the like, or a combination thereof.
  • Methods may include an immunoassay, a histological assay, a flow cytometry assay, the like, or a combination thereof.
  • the step of detecting the biomarkers) in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the biomarker or is specific for the blomarker,
  • Tbs method may feature comparing the amount of blomarker In the sample to a predetermined threshold for determining whether or not th level of biomarker in the sample is abnormal, in some embodiments, the amount of blomarker in the sample (a first sample) is compared to a level of biomarker In a control.
  • the control may be, for example, a sample (a second sample) obtained from the patient at a time prior to obtaining the first sample, in some embodiments, the predetermined threshold is a laboratory standard. In some embodiments, the predetermined threshold is an industry standard.
  • detecting an abnormal level of the blomarker as compared to th level of the biomarker of a control sample or threshold indicates the presence of the neurological condition, the disease, central nervous system tissue damage, CNS repair, etc,, or a risk thereof/
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes. Such circulating phagocytes may be found in tissues, cells, andior fluids in the body, for example in blood, peripheral blood mononuclear cells (PB Cs), cerebrospinal fluid (CSF). central nervous syste tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular
  • the biomarker is an intracellular component,
  • the biomarker may be obtained from within a macrophage, in some embodiments, the macrophage sample is permeabiiized.
  • the macrophage is lysed via various means, e.g, , . hypotonic solution treatment, detergent solution treatment, mechanical stress, etc,
  • the present invention features methods for treating patients suspected of having diseases or conditions uch as Multiple Sclerosis, brain ceil damage, brain cel! death, brain ce!i repair, Parkinson's Disease, Alzheimer's disease, etc.
  • the method may comprise isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a brain tissue of the patient; detecting a level of a biomarker i the phagocytes, the biomarker being associated with the disease or condition ,, wherein if the level of the biomarker is abnormal then the patient has active centra! nervous system disease; and administering to the patient having active centra! nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the disease or condition or a symptom thereof.
  • compositions, treatments, drugs, or therapeutic interventions may include but are not limited to: crenezumab, mastinab, daclizumab, zinbryta, ER-Beta agonist, cyclophosphamide, rHSgM22, ponesimod, alpha-4 integrin, AMP-110, an antisense oligonucleotide, Immune toierizing agent, MultiStem, Kv1.3 Blocker, Alemtuzumab, IFNb-lb, BHT- 3009-01 , IFNb-1a, dimethyl fumarate, IFNb- ⁇ PEGyiated ⁇ , Nataiizumab, MT1301, Abatacept, RCP1063, a compound related to !amitrigene, CNM-AuS, Mesenchymal stem cell transplant apolipoprotein E-based, modified peptidomsmetic, Aimspro, Anti-BAFF Human Ab, NDC-1308, GRbACt
  • the present invention also features methods of selecting a patient for a clinical trial for testing a drug for an effect on centra! nervous system (CNS) tissue damage, CNS repair, or neurodegeneration in some embodiments, the method comprises isolating circulating phagocytes from a fluid sampie from the patient, the fluid sample being fro outside of a brain tissue of the patient; detecting a level of a biomarker in the phagocytes, the biomarker being associated wit CNS tissue damage, CNS system repair, or neurodegeneration, wherein if the level of the b marker in the phagocytes is abnormal, then the patient has active central nervous system disease; and selecting the patient for the clinical trial if the patient has active CNS disease.
  • CNS centra! nervous system
  • the cohorts of patients for clinical trials created using method herein will be better suited to test a particular therapeutic intervention or composition of interest. Further, these cohorts may be used to reevaluate drugs that may have previously failed in ciinicai trials (e.g. , crenezumab), because the drugs may have failed due to the cohort of patients not being appropriate for the testing. Patients exhibiting low current disease activity may be poor responders or non -responder to therapeutic treatments.
  • ciinicai trials e.g. , crenezumab
  • the present invention also features methods for discovering a therapeutic composition having a positive effect on central nervous system (CNS) tissue damage, CNS repair, or neurodegeneration.
  • the method comprises selecting only patients with active CNS disease according to the present invention; administering to the patients with active CNS disease the therapeutic composition; isolating circulating phagocytes from a fluid sample fro each patient after a predetermined amount of time following administration of the therapeutic composition, the fluid sampie being from outside of a brain tissue of the patient; and detecting a level of a biomarker in the phagocytes from each patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneralion.
  • the therapeutic composition if the level of the biomarker in the phagocytes from a patient is normal, then the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration. In some embodiments, if the level of the biomarker in the phagocytes from a patient is within a predetermined threshold associated with improved CNS disease, then the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration.
  • the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration * 10088]
  • 3 ⁇ 4 ⁇ $ ⁇ features methods of determining the status of a disease or condition (e.g., a neuroiogicai condition, an inflammatory condition, multiple sclerosis, etc, ⁇ or determining the status of drug efficacy.
  • the present invention may also feature methods of monitoring disease activity and efficacy of a drug, therapeutic composition, and/or or therapeutic intervention.
  • biomarkers can be used to detect a disease or condition and the biomarkers may be used to determin severity of the disease or condition (e.g. relapse, remission, etc. ⁇ ,
  • the method comprises providing a sample ( d ⁇ 9 ⁇ n a fluid, a brain tissue), the sample comprising a circulating phagocyte.
  • the sample may be derived from a mammal (e.g,, a patient, a mouse, a rat, etc,),
  • a blomarker or level thereof associated with a disease or condition e.g., a multiple sclerosis-associated biomarker
  • the biomarker detected may be compared to the ievei or presence of the biomarker in a second sample, the second sample having been collected prior to the first sample.
  • the monitoring of disease activity may be use to determine drug, therapeutic composition, and/or o therapeutic intervention efficacy, in some embodiments, the monitoring of disease activity ma be used to determine therapeutic failure and/or breakthrough disease in some embodiments, the monitoring of disease activity may be used to determine patient complianc with a therapy. In some embodiments, the monitoring of disease activity may be used to determine therapeutic non-responders in some embodiments, the monitoring of disease activity may be used to aid drug development.
  • the present invention features a method of monitoring disease activity of a neurological condition, the method comprises obtaining from a mammal a first fluid sample from outside of a brain tissue of the mammal, the first fluid sample comprises a first circulating phagocyte; detecting a level of a blomarker associated with the neurological condition in the first sample; and comparing the level of the blomarker In the first sample with a Ievei of the biomarker in a second sample, the second sample bein either a control sample or a second fluid sample from outside of a brain tissue of the mammal, the second fluid sample comprising a second circulating phagocyte, the second fluid sample having been taken prio to the first fluid sample.
  • the step of detecting the biomarker may comprise subjecting the sample to an enzyme-jinked immunosorbent assay (ELiSA), ELfSA is an assay used to detect the presence of an antibody or a biomarker in a sample.
  • ELiSA enzyme-jinked immunosorbent assay
  • a sample containing an unknown amount of biomarker e.g., an antigen
  • a surface e.g., a polystyrene microtiter plate.
  • an antibody that binds to the antigen of Interest is washed over the surface so that it can bind the antigen and fom an antibody/antlgen complex in some cases, this antibody is covalently linked to an enzyme in some cases, the antibody is not covalently linked to an enzyme but can b defected by a secondary antibody that is linked to an enzyme i the final step, a substance (e.g,, substrate) that the enzyme is capable of converting to a detectable visible signal (e.gang flick color -signal) is added to the reaction, Thus, if the antibody/antigen complex is present, the substrate will be converted to the detectable visible signal, and then amount of antigen in the sarnpie can be measured.
  • a substance e.g, substrate
  • the present invention also features method of detecting brain ceil damage, brain cert death, brain cell repair, or the like.
  • the method comprises obtaining from a mammal a first fluid sarnpie from outside of a brain tissue of the mammal and introducin to the first fluid sample magnetic beads, whereupon phagocytes (e.g,, monocytes, macrophages, combinations thereof, etc.) in the first fluid sample engulf the magnetic beads yielding magnetic phagocytes.
  • the first fluid sample may comprise a biood sample (e.g,, in a container).
  • the method may further comprise separating the magnetic phagocytes using a magnetic separation mechanism and making detectable and quantifying a neural-derived biomarker in the separated magnetic phagocytes,
  • the neural-derived biomarker comprises a molecule derived from the brain ⁇ e.g., a biomarker normally just found in the brain or central nervous system tissue ⁇ ,
  • the detection of the neural-derived biomarker may be compared to other sampies derived from the mammal, e,g. sampies taken previously.
  • the detection of the neural-derived biomarker may be compared to standards or controls in some embodiments, the neural-derived biomarker may be used as a means of monitoring a patient, e.g., monitoring brain ceil damage, brain cert death, brain ceil repair, etc, in some embodiments, elevated levels of the biomarker may be indicative of brain cell death or damage or worsening of a condition in some embodiments, elevated levels of the biomarker may be indicative of repair or improvement of a condition.
  • Sower levels of the biomarker may be indicative of brain ceil death o damage or worsening of a condition, in some embodiments, Sower levels of the biomarker may be indicative of repair or improvement of a condition,
  • detecting the neural-derived biomarke in the first fluid sample at a level that is different than that of a control or a second fluid sample obtained from the mamma! is indicative that the mammal has experienced a change in brain cell damage, brain cell death, or brain cell repair, !h some embodiments, detecting the neural-derived biomarker in the first fluid sample at a level that is higher than that of a control or a second fluid sample obtained from the mammai (the second fluid sample being identical to the first fluid sarnpie except that If is collected prior to the first fluid sarnpie) is indicative that the mammal has experienced a change in brain ceil damage, brain cell death, or brain ceil repair, in some embodiments, detecting th neural-derived biomarker in the first fluid sample at a level that is iower than that of a control or a second fluid sample obtained from the
  • the method may' further comprise introducing to the first fluid sample during or after the first fluid sample is obtained a molecule for inhibiting further degradation of the neural-derived bio arker in the phagolysosome of the phagocytes.
  • any component that increases the pH of the phagolysosomes, which would inhibit the enzymes in the phagolysosomes, would help reduce the degradation of peptides (e.g , the biomarkers of interest) In the phagolysosomes.
  • the molecule for inhibiting further degradation of the neural-derived biomarker in the phagolysosome of the phagocytes comprises one or a combination of phagoiysosomal protease inhibitors.
  • the proteas inhibitor comprises leupeptin
  • the molecule for inhibiting further degradation of the neural-derived biomarker in the phagolysosome of the phagocytes comprises a molecule that increases the pH of the phagolysosomes of the phagocytes in the first fluid sample.
  • the molecule for increasing the pH of the phagolysosomes of the phagocytes in the first fluid sample comprises an alkaline buffer Alkaline buffers are well known to one of ordinary skill in the art, e.g, chioroguin, carbonate/bicarbohate buffer, buffers of pH 9.2 or above, weak base buffers, quinine, etc.
  • both a phagoiysosomal inhibitor an alkaline buffer are added in some embodiments, the magnetic beads/pariicies are coated with a phagoiysosomal inhibitor (e.g., leupeptin), in some embodiments, the magnetic beads/particles are coated with a mix of compounds, e.g,, a phagoiysosomal inhibitor (e.g., leupeptin), an antibody (e g,, SgG, IgG(fc), etc,).
  • a phagoiysosomal inhibitor e.g., leupeptin
  • an antibody e.g, SgG, IgG(fc), etc,
  • the method further comprises introducing to the first fluid sample a stimulator to stimulate phagocytosis of the magnetic beads by the phagocytes, in some embodiments, the magnetic beads are conjugated with an aci hydrolase inhibitor in some embodiments, the magnetic beads are conjugated with an antibody or antibody component to stimulate phagocytosis. In some embodiments, the magnetic beads are introduced to the first fluidsample within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes of when the first fluid sample is obtained.
  • the step of making detectable the neural -derived biomarker comprises subjecting the separated phagocytes to an immunoassay.
  • the immunoassay comprises introducing an antibody to the sample, wherein the antibody binds to the neural-derived biomarker.
  • the antibody Is a polyclonal antibody.
  • the container for the blood sample comprises an anticoagulant.
  • the anticoagulant comprises citrate, heparin, or a combination thereof
  • the method comprises isolating peripheral blood mononuclear cells (P8MCs) from th blood sample prior to introduction of the phagocytosis of the magnetic beads
  • the magnetic separation mechanism comprises a magnetic column or magnetic rack
  • the container (for th blood sample) comprises ficoif. in some embodiments, the container ( for the blood sample) does not comprise Ficoil or is free of Fico!i.
  • the magnetic phagocytes are separated using the magnetic separation mechanis within 1 hour of harvesting of the first fluid sample in some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism within 12 hours of harvesting of the first fluid sample, in some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism within 24 hours of harvesting of the first fluid sa pie. in some embodiments, the magnetic phagocytes are separate using the magnetic separation mechanism within 48 hours of harvesting of the first fluid sample,
  • the biomarker is a synaptic antigen.
  • Synaptic antigens are weii known to one of ordinary skill in the art, e.g., PSD-9S protein, neorogranin, SNAP-25, and TDP-43, etc,
  • the biomarker is one that helps define subgroups of a particular disease state or disease process.
  • the biomarker is TOP-43, which may be used to help define subgroups of ALS.
  • a protease inhibitor e.g.. a phagolysosomai inhibitor ⁇ is introduced to the sample within 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes, or within 10 minutes, 15 minutes, 20 minutes, etc,, of when the samp!e is obtained.
  • kits for detecting the presenc or a risk of a disease or interest ⁇ e,g though those described herein ⁇ in a circulating phagocyt sample derived from amammal comprises an antibody that binds to a specified appropriate biomarker in some embodiments, the kit further comprises a means for detecting the binding of the antibody to the biomarker/antigen in the sample (e,g., an antibody-antigen complex ⁇ .
  • the kit comprises an antibody specific for the biomarker of interest, wherein the antibod is a monociohaS or a poiycionai antibody, in some embodiments, the antibody is derived from a human, a mouse, a rat, a llama, a rabbit, a dog, a pri ate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow, in some embodiments, the antibody is humanized in some embodiments, the antibody is a chimera.
  • kits may be purified, e.g., affinity purified for the antigen of interest.
  • kits further comprise reagents for detecting additional biomarkers, e,g Berry additional biomarkers in the circulating phagocytes, serum biomarkers, plasma biomarkers, eto.
  • additional biomarkers e.g., for preserving the sample, e.g., for preserving the amount, structure, and/or location of the biomarker of interest ⁇ e.g,, the epitope of the biomarker of interest),
  • the kit may further comprise appropriate reagents, manuals, equipment, etc.
  • the kit may comprise reagents for automated assays.
  • the kid comprises reagents for multiplex assays .
  • the disease or condition of interest Is multiple sclerosis and the present invention features methods of detecting multiple sclerosis, methods for detectin a risk of multiple sclerosis, methods for selecting a patient with multiple sclerosis for monitoring, methods of selecting a patient with multiple sclerosis for a clinical trial, etc.
  • the methods of the present invention may allow for monitoring, detecting and/or predicting a reiapse or a remission of multiple sclerosis.
  • the method of detecting multiple sclerosis is used in combination with one or more methods of detecting multiple sclerosis.
  • the present methods may be used in conjunction with othermodalities to monitor, detect or predicting a relapse or a remission of multiple sclerosis.
  • the present Invention is not limited to multiple sclerosis.
  • the methods for detecting multiple sclerosis comprises (1) providing a first sample (e.g., a fluid sample) that comprises a peripheral (e.g., circulating) phagocyte.
  • the first sample may be derived from outside of a brain tissue, and (2) detecting a multiple sclerosis-associated biomarker in the phagocyte.
  • the sample may be provided from a mamma ⁇ (e.g., a patient, a mouse, a rat, etc,).
  • one or more biomarkers is detected in the sample in some embodiments, for example if the diseas of interest is multiple sclerosis, the biomarkers are associated with multiple sclerosis,
  • the metho may further comprise comparing the level of the biomarker In the first sample with a predetermined threshold or other particular threshold, e,g., a level of the biomarker in a second sample (e.g., a fluid sample), wherei the second sample may be a control sample or other sample for comparison purposes in some embodiments , the second sample is a fluid sample from outside of a brain tissue comprising a peripheral ⁇ e g., circulating phagocyte), The second sample may be provided from a mamma! ⁇ e.g,, a patient, a mouse, a rah etc.). The second sample may have been collected prior to the first fluid sample. Detecting the biomarker may indicate the presence of the disease or condition of interest (or a risk thereof), e.g., muitipie sclerosis.
  • the level of the biomarker or antigen may be evaluated (compared to the predetermined threshoid or control level or other comparison) and determined to be an abnormal level.
  • Abnormal levels may be previously determined based on industry standard or laboratory standard, etc.
  • a high level of the jo afker In comparison to the threshold or control is an abnormal level in some embodiments, a low level of the biomarker in comparison to the threshoid or control is an abnormal level.
  • detecting a increased level of the biomarker as compared to the ieyei of the biomarker of a control sample indicates the presence of the disease or condition of interest or a risk thereof in some embodiments, detecting a decreased level of the biomarker as compared to the !evei of the biomarker of a control sample indicates the presence of the disease or condition of Interest or a risk thereof.
  • an antibody is used to detect the presence of the multiple sclerosis-associated biomarker.
  • the multiple sclerosis-associate biomarker may be detecte with a variety of antibodies, in some embodiments , the antibody is a monocionai or a polyclonal antibody, in some embodiments, the antibody is a humanized antibody, in some embodiments, the antibody is a chimera.
  • the antibody is derived from a human, a mouse, a rat, a lla a, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow, in some embodiments, the antibody is synthetic, in some embodiments, the antibody is a recombinant antibody .
  • antibodies are labeled either covalently or non-cova!entiy by combining the antibody with a second substance that provides for detectable signal.
  • labels and conjugation techniques are known in the art and are reported extensively In both the scientific and patent literature. Examples of labels include but are not limited to radioisotopes, enzymes, substrates, cofactors, inhibitors, fSuorescers, chemiSuminescers, magnetic particles, and the like in some embodiments of the present invention, the antibody comprises a label,
  • a unit may be an absorbance unit (e.g., from an EUSA), a percent positive (e.g., from a flow cytometry or FACS assay ⁇ , of a fluorescence unit,
  • the present invention also provides a method of monitoring the progression of multiplesclerosis and/or monitoring the treatment of multiple sclerosis.
  • the present invention may be used to measure the level of the multiple sclerosis- associated biomarker in order to detect a change in the level (e g,, an increase in the level, a decrease in the level, a maintaining of the level),
  • a change In the level of the multiple sclerosis-associated biomarker may correlate With a change in the patient’s status (e.g., remission, progression, worsening).
  • a decrease in the ievel of the multiple sclerosis-associated biomarker may Indicate the patient has entered or will enter a remission period.
  • the present invention may be used to monitor the level of the multiple sclerosis-associated biomarker in a patient while the patient is o a treatment regimen (e.g,, a drug).
  • a treatment regimen e.g., a drug
  • a treatment regimen that is effective at inhibiting the progression of multiple sclerosis and/or reducing the symptoms of multiple sclerosis may decrease the level of the multiple sclerosis-associated biomarker in the patient.
  • the method of the present invention for detecting multiple sclerosis is used in combination with one or more different methods for detecting multiple sclerosis. For example, in some cases, a combination of family history, a physical exam, and magnetic resonance imaging (MRI) findings are used to diagnose multiple sclerosis.
  • MRI magnetic resonance imaging
  • MR! is the most sensitive radiographic technique for the imaging of multiple sclerosis.
  • Multiple sclerosis plaques are commonly seen as round or void discrete lesion in the periventricular white matter. Other common locations fo multiple sclerosis plaques include the corpus callosum, corona radiate, internal capsule, and centrum semiovafe.
  • the present invention i used to measure a multiple sclerosis -associated biomarker, and the level of the multiple sclerosis-associated biomarker is correlated with a magnetic resonance imaging (MR! measurement.
  • MR magnetic resonance imaging
  • the method of the present invention for detecting multiple sclerosis may be used in combination with one or more methods for detecting a different condition.
  • the method of the present invention may also help to distinguish multiple sclerosis from other diseases with similar clinical manifestations.
  • neuromyeiitis optica also known as Devic e syndrome
  • the eharacteristic inflammatory demyeiinating lesions of fsSlViO selectively and repeatedly affect the optic nerves and the spinal cord, causing blindness and paralysis.
  • a marker e.g., aquaporin-4 antibodies
  • an NMO marker e.g., aquaporin-4 antibodies
  • the method of detecting the presence of multiple sclerosis or a risk of multiple sclerosis comprises detecting the presence or absence of at least two biomarkers (e.g., proteins, antigens, or the like) wherein at least one biomarker is detected in order to distinguish ltiple sclerosis from a disease with similar clinical manifestations.
  • the present invention also provides a method of diagnosing multiple sclerosis at an eariy stage of the disease before all clinical criteria are fulfilled, thus justifying early initiation of a multiple sclerosis-appropriate therapy.
  • the following example describes the detection of multiple sclerosis in a patient according to two methods disclosed In the present invention.
  • a 24 ⁇ year-o!d male patient presents to his primary care physician complaining of changes in vision, limb weakness, and extreme fatigue. He mentions his symptoms have been recurring over the last 3 months.
  • the physician suspects the possibility of a tumor in the central nervous system (CMS) or a CNS disease, as well as multiple sclerosis.
  • CMS central nervous system
  • the physician obtains a blood sample to be sent to a diagnostic laboratory for multiple sclerosis testing, and also refers the patient to a neurologist.
  • PSMCs are obtained from a SD VacutainerTM CRT tube using a cell separation procedure.
  • the cells are washed three times in IX PBS and centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1500 RCF (Relative Centrifugal force).
  • the supernatant is removed, and the cells are resuspended In iX PBS, After the final wash, extracts of the PBMCs are prepared by lysing with a hypotonic solution o othe method.
  • the lysate is subjected to assay Involving an antibody that binds to Tau, e g., a protein fragment comprising the phosphoryiated serine residue Ser*404, Th assay indicates that an abnormal level of said Tau protein fragment is present in the PBMCs.
  • the results of the assay indicate that the patient has active CNS damage, which may be an indication that the patient has multiple sclerosis. The physician notifies the patient, who then begin treatment immediately,
  • the following example describes a direct ELISA assay used for detecting an antigen in a sample of peripheral phagocytes.
  • the protein concentration of the sample is determined using the Bio RadTM (Bradford method) assay, MicroEL!SA plates are coated by addition of 100 pL of a 5- 20 pg/mL solution of the sample, which is then incubated for 1 hour at 2 O.
  • the wells are washed out with phosphate buffere solution (PBS) with 0 + QS% poiysorhate ⁇ Tween 20TM ⁇ , The wells are then filled with 0 1 M glycine in PBS and incubated for 1 hour at 21 "C to block unoccupied binding sites.
  • PBS phosphate buffere solution
  • the following example describes an indirect ELISA assay used for detecting an antigen (isolated from: peripheral phagocytes) in various samples.
  • This assay is constructed using polyclonal and monoclonal antibodies.
  • ELISA wells are coated with polyclonal antibody at an appropriate concentration and the wells are washed and blocked as described above.
  • Various dilutions of antigen containing samples are added to the wells and incubated for 1 hour at 2 O, after which the wells are washed 3 times with FBS-0.05 TweenTM 20.
  • the monoclonal antibody is then added at an appropriate dilution in P8S-0.Q5% TweenTM 20 - 1 % BSA and incubated for 1 hour at 2T'C, The wells are then washed 3 times and an appropriately diluted horse radis peroxidase conjugated anti-mouse igM in PBS-0,05% Tween T? 20-1 % BSA is then added to each well and incubated for 1 hour at 2 V C The wells are then washe twice wit FBS-0.05% TweenTM 20 and finally with PBS. One hundred pL of soluble MTB substrate so!ution is added to each well an incubated for 38 minutes at 21 ⁇ after which 100 pL of MTB stop reagent is added and the color intensity is measured at 450n using an ELISA plate reader.
  • PSMCs from multiple sclerosis S subjects and control subjects are stained with fluorescent antibodies to the multiple sclerosis-associated antigen (e.g., Tau protein) and also with fluorescent labeled antibodies to cluster designation (CD) 3 T-iymphocyte marker or CD 19 8- Lymphocyte marker, CD6S intracellular monocyte marker and CD 14 monocyte/ macrophage cell surface marker.
  • CD cluster designation
  • CD6S intracellular monocyte marker
  • CD 14 monocyte/ macrophage cell surface marker
  • PBSVICs are obtained from a BD VacutalherTM OPT tube using a ceil separation procedure.
  • the ceils are washed three times in IX PBS an centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1300 RCF ⁇ Relative Centrifugal force).
  • the supernatant is removed, and the cells are resuspended in 1 X PBS.
  • the ceils are resuspended to approximatel 4.0 ml in IX PBS.
  • Approximatel 50 pi, of the cell suspension to be analyzed is transferred into tubes for double staining with selected antibody pairs, Ten pL of 40mg/mL normal human SgG (Sigma-Aldrich) for a total of 4QQ pg is added to each tube to block FC binding. The appropriate ceil surface monoclonal antibodies CD3 PE, CD19 PE or CD14 PE are added at this time and incubated for 20 minutes at room temperature.
  • the appropriate amount of the antibody specific for the multipie sclerosis-associate antige is added to the appropriate tubes.
  • the tubes are mixed gently to ensure that the cells are in suspension and incubated at room temperature for 15-60 minutes, Two ml of IX PBS working solution is added to each test tube and mixed gent!y.
  • the tubes are centrifuged at 300 X g for 5 minutes, and then th supernatant Is aspirated, leaving approximately 50 pi of fluid.
  • the fluid is mixed thoroughly to ensure that the cells are in suspension,
  • the pellet is resuspended in an appropriate volume of fiuid for flow cytometry analysis.
  • the sample is analyzed on a Sow cytometer within 24 -48 hours.
  • the gate is on the monocyte population and the data is collected in list mode, Qualitative and or quantitative differences are determined between normal and MS patients using th analysi software. Optimization steps include varying incubation time with antibodies, fixation time and permeabilization time.
  • the present invention features the use of a new source of circulating biomarkers for use in diagnosis and monitoring of disease activity by exploiting the ability of monocytes/macrophages to enter the brain and engulf debris coupled with the paradigm shifting hypothesis that at least some of these ceils re-enter the circulation.
  • the present invention isolates these re-circulating cells that have retrieved an igens from the brain ce!!s and looks for new and established CMS tissue markers.
  • antigens engulfed b phagocytes are degraded within a few days. This means antigens are detected, they are reflective of resent central nervous system tissue Injury (e.o . “active” neurodeqenerative process). Given that 70-80% of slnato-nigrai neurons are lost before clinical symptoms arise, the clearance of the dead neurons by recruited and re-cireuiating mononuclear phagocytes should he an early indicator of neurons! damage.
  • the present invention allows for rapidly identifying and develop potential biomarkers to improve the efficiency and outcome of Phase !i clinical trials and advance therapeutic development for PD or other appropriate diseases or conditions.
  • PBMCs peripheral blood mononuclear cells
  • the present invention probes for these proteins in PBMCs from PD subjects, as well as alpha-synucSein, glutamic acid decarboxylase (GAD) and ubiqostin carboxyl-terminal esterase 11 (UCHL1) proteins.
  • the methods of the present invention may also be used to discover potentially informative CMS antigens in PD PBMCs using a shotgun proieomic analysis. Hippoca!cin like 1 was discovered to be a phagocytosed biomarker using this approach,
  • the prevalence of each marker can be determined. For example, the prevalence of these markers may be determined in a number of recently diagnosed ⁇ 3 years) PP subjects and apparently healthy subjects.
  • the cells containing the neural markers are characterized b immunophenotyping PBMCs.
  • Coexistence of neural antigens with antigens specific for leukocyte subpopulations may be determined by use of specific antibodies, This may b achieved by flow cytometry, immunofluorescent microscopy and/or ce!S type specific enrichment/depletion using magnetic beads.
  • the results may identify known phagocytic ceil types (CD14+ monocytes and/or macrophages (CO 88/CD11b)) to be the source of the neural antigens in PBMCs.
  • PBMCs peripheral blood from 18 subjects with MS and 12 apparently healthy individuals was obtained.
  • PBMCs were isolated and osmoticaliy lysed. The lysates were coaled onto EUSA wells at 5ug/ml and probed for Tau antigen or HippocaScin iike-1 antigen using standard protocols, Tau is a neuron specific microtubule associated protein that is best known as the substrate of neurofibrillary tangles in Alzheimer’s disease.
  • Hsppoealeih i ike-1 is a neuron specific eafeiu sequestering protein that is most abundant In the hippocampus.
  • the ELISA assay results for these antigens i this study are shown in FIG. 3.
  • PBMCs from normal C57BI/0 mice and cuprizone fe C57B1/8 mice were examined for CNS antigens.
  • Cuprizone causes De ye!ination at 3-4 weeks and also neurodegeneration at 4-5 weeks.
  • Discontinuation of Cuprizone feeding at 6 weeks or earlier results in repair of the CNS damage.
  • Five mice were fed with regular lab chow, two groups of five mice were fed with Cuprizone (0,2%) chow for 3 weeks and 4 weeks and one group of 4 mice were fed with Cuprizone (0.2%) chow for 5 weeks.
  • the mice were bled and PBMCs isolated by Real! density gradient centrifugation.
  • FIG. 4 FiG. 5, and FIG. 6 show there is no difference in control and Cuprizone treated mice until 5 weeks of Cuprizone feeding, a time point when there is extensive histological evidence of demyeitnation and naurodagenaratiGm
  • the methods of the present invention may still be applied to monitoring, detecting :, treating, managing, etc., Parkinson’s disease.
  • the present invention may also be used to help determine the antigens most relevant to PD. For example, one strategy is to obtain PBMGs from Parkinson’s disease subjects and test them for the antigens that have found to be relevant in other CNS diseases, e.g., Tau, Hippocaicin iike-i and myelin basic protein.
  • the PBMCs may also be probed for alpha synuciein, glutamic acid decarboxylase (GAD) and ubiquifln carboxyl-terminal esterase LI ,
  • Alpha synuciein is associated with PD pathology and GAD Is associated with "stiff man” syndrome, which may have some overla with symptoms of PD.
  • Ubiqui!in earboxy! terminai esterase LI UGHL1) Is found in all neurons of the brain and should be present in recirculating phagocytes when there is active neurodegeneration present.
  • UCHtl represents a safety net biomarker.
  • UCHli polymorphisms have been reported to influence risk of development of PD.
  • a shotgun proteomic approach may be employed for discovering ne antigens that may associate with PD. This method has been used to identify Hippocaicin like-1 as an antigen of interest.
  • PBMGs from short duration PD subjects may be utilized for new phagocytosed antigen discovery.
  • a lysate of PD PBMCs may be subjected to 1-D PAGE and the gel can be coohiassie stained.
  • Coo assie stained protein bands may be cut out and subjected to in gel trypsin digestio followed by protein identification by LC-MS-MS. The list of proteins present will be examined for the presence of CNS proteins.
  • Candidate biomarkers foun will be studied using either commercial antibodies if available or by contracted production of anti- peptide antibodies.
  • the potential biomarkers selected for initial testing may be screened on lysates from a furthe short duration PD PBMC lysates by ELISA assay.
  • any potential biomarker that is positive on two lysates will be a candidate for further screening A minimum of three such candidate antigens will be screened.
  • Candidate biomarkers may be screened on lysates from recently diagnosed ⁇ 3 years) PD subjects to determine prevalence. Comparisons may also be made to apparently healthy control subjects. A single high prevalence (e.g., ⁇ 80%) antigen or a combination of antigens providing high prevalence in aggregate will be sought.
  • the ceil typefs) carrying neural antigens in the PBMCs have not been specifically identified. Without wishing to li it the present invention to any theory or mechanism, it is assumed they are bone marrow derived mononuclear cells
  • the cells containing the neural markers may be characterized by immunophenotyping PBMCs, Coexistence of neural antigens with antigens specific for !eukocyte subpopulations may be determined by use of specific antibodies. This may be achieved by flow cytometry, immunof!uorescent microscopy and/or celt type specific enrichment/depletion using magnetic beads.
  • the resuits may identify known phagocytic ceil types (CD14+ monocytes and/or macrophages (CD 6S CD11 b)> to be the source of the neurai antigens in PBfv!Cs. Identifying the cell type carrying neurai antigens mat enable additional sample preparation steps that will enric the relevant cells and wiii be expected to increase sensitivity of the assays.
  • 2 x 8 b tubes of blood may be collected from PD subjects and the PBMCs prepared. Half of the PBMC are used to make a lysates and the other half temporarily cn/opreserved. The lysates are assayed to determine which neurai biomarkers are present and then the cryopresefved cells are resuscitated for analysis with CD markers for leukocyte sub- populations and the relevant neural antigens.
  • Statistical analysis may be required to interpret the results, e g., the unmatched t-test may be used to determine whether the means of each group are significantly different from each other. A p-vaSiie of 0 05 or less will indicate statistical significance. Apparently healthy controls may be used to determine a cut off value (mean +2SD) in order to create categorical data that can be analyzed with 2 x contingency tables an the chi squared test. This may also enable expression of the results as positive and negative predictive values.
  • Proteomic studies may also be performed on PP PBMCs to detect potentially relevant CNS antigens for PD (e.g,, UCHL1 , Tau, etc.). Some antigens may have regional differences in abundance that may affect detect ability and ma therefore only be seen in particular ciinicai phenotypes.
  • CNS antigens for PD e.g, UCHL1 , Tau, etc.
  • Some antigens may have regional differences in abundance that may affect detect ability and ma therefore only be seen in particular ciinicai phenotypes.
  • the present invention provides methods for antigen retrieval fro the brain utilizing re-circuiating phagocytes as a shuttle vector. Because these antigens are short lived i phagocytes, the detection of their presence wi!l indicate current active neurodegeneration.
  • the prevent invention provides for methods for monitoring disease activity in Parkinson's disease or other diseases wherein detecting certain antigens In re-circulating phagocytes can be indicative of active disease processes. As drugs that decrease disease activity wi!i be expecte to decrease CNS antigen laden phagocytes in the circulation, this approach may facilitate evaluation of response to therapy and eariy evaluation of likelihood of success of phase I! trials, etc.
  • Equipment/Reagents Needed Centrifuge; SOmL conical tube; Storage vials for aliquots, - S0d capable, iOQuL-lmL (e.g, polypropySene mierocerstnfuge tubes); PBS - Phosphate Buffered Saline, pH 7.4; RBC lysis buffer ⁇ AGK ⁇ ; Protease inhibitor (e g. G Bioscienees ProteaseArrest flQdXj); Magnetic rtanopartic!es/beads.
  • iOQuL-lmL e.g, polypropySene mierocerstnfuge tubes
  • PBS - Phosphate Buffered Saline pH 7.4
  • RBC lysis buffer ⁇ AGK ⁇ RBC lysis buffer ⁇ AGK ⁇
  • Protease inhibitor e g. G Bioscienees ProteaseArrest flQdXj
  • I00T54 Blood may be collected into an anticoagulant containing tube (Becton Dickinson) that also contains 1 mg of magnetic nano particies. A single 8 mL or 4 mL tube per subject may be received. Samples may contain the minimu volume specified by Table 8 (below). Samples for research may be collected under an !RB protocol. Phagocytes may be harvested within 38 hours of the blood draw. After receipt of sample, phagocytes are harvested by sedimentation on a magnet. While the phagocytes are held by the magnet, ail other blood components may be poured or aspirated off. The phagocytes are then washed twice with PBS.
  • the pellets from two tubes may be recombined in the total volume specified for one tube.
  • )0O15?j Lysates may be stored at -80C, in a freezer monitored by an external monitor and labeled with the correct contact information to cat! if a failure is noted.
  • Assays lysates may be conducted within 30 days of harvesting. Sample preparations may be frozen at least 24 hours prior to assaying. Label primary container (box or conical tube containing aliquots) with sample identifier, type of sample (plasma or PBMC lysate), date processed, and processor ’ s initials. After experimentation, samples may either be stored or destroyed according to the !RB protoco! under which they were collected
  • An alternative process includes the use of magnetic nanoparticSes coated with ieupeptin, magnetic nanopartieles coated with a mix of ieupeptin and human igG(Fc), etc,
  • a carbonate/bicarbonate buffer may be used in some embodiments, in lieu of ehioroquin any weak base may be used, in some embodiments, in lieu of or in addition to a base, ieupeptin (e.g,, 0.25 mM) may be added.
  • ieupeptin e.g, 0.25 mM
  • Synaptic biomarkers may inciude but are not limited to PSD-9S protein, neurogranln, SNAP-25, and TDP-43.
  • This example describes a non-limiting procedure of an assay that may be used when assaying Peripheral Stood Phagocyte lysates for Tau, Myelin Basic Protein (MBP), UCH-L1 , or Hlppocalcin-iike ⁇ l (HPCAL1 ) debris.
  • MBP Myelin Basic Protein
  • HPCAL1 Hlppocalcin-iike ⁇ l
  • [00161 i Eq u ipment/reagenis that may be needed may inciude: Clear poiystyrene Enzyme-linked immunosorbent assay (E-LISA) plate, protein binding (Pierce 15041); Specifophotametrie ELiSA plate reader capable of absorbance measurement at 450nm (e.g.
  • BioTek ELxSOO Human Tau- 441 isoform (2M4R) (e.g, rPeptide T- 1001-2); MBP peptide, derived from human brain ⁇ e.g, Erizo2OO-6O6-MQ01); HPCAL1 peptide (Aviva AAP34682); lysate samples; PBS - Phosphate Buffered Saline, pH 7.4; BSA - Bovine Serum Albumin (e.g.
  • Anti-human Tau rabbit poiycionai SgG (e.g, Dako A0024); Anti-human MBP, mouse monoclonal IgG (e.g., Santa Cruz Biotechnology sc-2 1524); Anti-human UCH-L1 , mouse monoclonal IgG (e.g. Santa Cruz Biotechnology sc-271639); Anti-human HPCAL1 , rabbit poiycionai (e.g, Aviva ARP34682; Proteinteeh Group 1098S-i-AP); Anti-rabbit SgG, peroxidase labeled (e.g.
  • the known concentrations should be plotted on the X-axis, with the corresponding O.D. reading on the Y-axis). Fit the curve (or linear region) with a linear trendline. Determine the sample concentration by calculating the X value from substituting the corresponding O.D, into the trendline equation obtained in the standard curve,
  • Lyme disease is contracted by bein bitten by a deer tick Infected with the bacterium Bomirn burgdorferi. Typically, people who are bitten develop a characteristic rash and develop flu like symptoms, which resolve within three weeks of antibiotic treatment. Some people have a recurrence of symptoms months or years after the first episode, The reasons for relapse remain controversial Two theories have been put forward to explain recurrence of symptoms; relapse due to treatment failure and reinfection through a second tick bite. Note that if recurrence were to be caused by a second bite, the bacteria may be genetically different In both instances but If recurrence is caused by treatment failure, the bacteria will be genetically the same.
  • One study evaluated 22 pairs of infected blood samples and determined that all of the bacteria in these samples were geneticaiiy identical, making it unlikely that recurrence was due to a second tick bite,
  • the present invention features methods for diagnosing Lyme disease, detecting a relapse of Lyme disease, monitoring Lyme disease, etc.
  • the methods herein can be used to retrieve hidden bacteria in disseminated relapsing Lyme disease [e.g., retrieve the bacteria fro macrophages in the blood), Briefly » blood samples may be isolated from patients, and the PBMCs (source of macrophages) may be isolated, extracted, and/or concentrated. Lysates of the cells of Interest will be analyzed for particular antigens or biomarkers of Interest associated with Ly e disease.
  • the figures are representative only and the ciaims are not limited by the dimensions of the figures, in some embodiments, descriptions of the inventions described herein using the phrase“comprising'’ includes embodiments that could be described as“consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase“consisting of is met.

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Abstract

Methods for detecting or monitoring active disease processes, such as neurodegenerative disease processes or central nervous system damage related to Multiple Sclerosis, Alzheimer's disease, Parkinson's disease, mild cognitive impairment, traumatic brain injury, amyotrophic lateral sclerosis, or the like, wherein biomarkers associated with said disease processes are detected in circulating phagocytes outside of the central nervous system, The ability to detect active disease in patients may be useful for building cohorts of patients for drug trials, wherein only patients with active disease.

Description

DETECTION OF BRAIN -DERIVED DEBRIS IN RECIRCULATING PHAGOCYTES CROSS-REFERENCES TO RELATED APPLICATIONS
fiKMJI j This application claims priority to U.S Patent Application No, 16/2.71 ,186 fifed February 3, 2019, the specification's) of which is/are incorporated herein in their entirety by reference.
REFERENCE TO A SEQUENCE LISTING
I00Q21 .Applicant asserts that the information recorded in the form of an Annex C/ST.25 text file submitted: under Rule ISfenlfa), entitled MSDX 15 01 CIP3 PCI Sequence : Listing, is identical to that forming part of the international application as filed. The content of the sequence listing is incorporated herein by referenc in its entirety,
BACKGROUND OF THE INVENTION
{00031 in general, when tissue damage occurs, it incites inflammation, which usually aids in wound healing. For example, one of the normal functions of inflammation is to recruit phagocytes to clear away the cellular debris and prepare the injured site for repair and rebuilding, These phagocytes may be resident in the brain (e.g., dendritic cells, microglial cells) or recruited from the blood strea (e.g,, monocytes). Cells that engulf debris are thought to ente the brain by crossing the blood-brain barrier but are not believed to retur to the biood stream. For example, whe phagocytes engulf tissue debris and exit the tissue, it is thought to be via the lymph nodes,
{00(541: We have surprisingly discovered that debris-laden phagocytes may re-enter the blood stream: from the brain and if sufficient numbers are present, it may be possible to detect or measure the brain-derived debris that is inside the phagocytic cells,
100051 The present invention describes a phagocytic shuttle method (PSM) wherein the phagocytes that re-enter the blood strea from the central nervous system (CNS) are shuttles for CNS-derived debris, and the debris can be indicative of processes occurring in the CNS (e.g.. brain), For example, the presence and/or amount of the debris may be associated with various states of the brain, e g., active central nervous system tissue damage, active central nervous system repair, active neurodegeneration, etc, The presence and/or amount of the debris may be used for monitoring a brain condition or neurological disease (e.g., monitoring worsening or improvement of a particular brain condition or neurological disease). The presence of the debris may be associated with particula disease states or injury. The presence and/or amount of the debris may be used for detecting neurological damage (e.g., neuroiogical damage associated with a disease or injury). The presence and/or amount of the debris ma be used to detect active neurodegeneratlve diseases, active centra! nervous system tissue damage, and/or active central: nervous system repair.
{0006 j The present invention features detection and/or measurement of the neural-derived cargo
I ( g,, brain-derived, e.g,, the debris or biomar ers that woul only normally be found in brain or CNS tissue) in the circulating phagocytes. Without wishing to limit the present invention to any theory or mechanism, the present invention may provide close to reaS-time data on what is happening in the brain since that particular cargo may oniy be present i the recirculating phagocytes for a certain length of time (e.g., a few days) before it is completely digested.
SUMMARY OF THE iNVE TION
|00Q7| The present invention features methods for creating patient cohorts (e g,, of one or more patients) for a clinical trial, e g,, a clinicai trial for testing a therapeutic composition or therapeutic intervention for an effect on central nervous system (CNS) tissue damage, CNS repair, or neurodegeneration, etc. The CNS tissue damage, CNS repair, neurodegenerati'on, etc., may be associated with a particular disease, condition, trauma, etc., as described herein. The method features isolating or retrieving a biomarker or antigen from the central nervous system (e.g,, a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector.
{0008j In some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from each patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a level of a biomarker in the phagocytes from each patient, th biomarker being associated with CNS tissue damage, CNS syste repair, or neurodegeneration, wherein if the level of the biomarker in the phagocytes is abnormal, then the patient has active CNS disease; and selecting only patients with active CNS disease to create the patient cohort for the clinical trial.
fOOOfSj in some embodiments, for each of the methods herein, the step of isolating circulating phagocytes from the fluid sample from the patient may further comprise concentrating the circulating phagocytes, in some embodiments, the circulatin phagocytes are isolated or concentrated using forward-scattered light or side-scaitered light in flow cytometry. In some embodiments, the circulating phagocytes are isolated or concentrated base on immunotype. In some embodiments, the circulating phagocytes have a specific immunotype, the immunotype is determined using a marker selected from; GDI 1a, GUI 1 b, CD1 1c, CD14, Cp1 Si, T CP33, CD84, CD68, CD8G, CDS6, GDI OS/eridOgith, CD 115, CD163, CD195/CCR5, CD282/TLR2, OD284/TLR4, HLA-DR/MHG Class |l, SLT1 , SLT3, iLT4, SLT5, Mature Macrophage afker®****, CO 1a, CDIb, CD 1c, CD40, CD49d, CD83, CD85g/SLT7 KC, CD123, CD197/CCR7, CD205/DEC-205, CD207/Langerin, CP209/DC-SIGN, CD273/B7-DC, CD2S9/TLR9, CD303, CD304, CMKLR-1pDC, or a combination thereof. In some embodiments, the level of the biomarker Is compared to a predetermined threshold to determine if it is abnormal, the predetermined threshold is an industry standard, in some embodiments, the !evef of the biomarke is compared to a predetermined threshold to determine if it is abnormal, the predetermined threshold is a laboratory standard in some embodiments, the level of the biomarker is compared to a predetermined threshold to determine if It is abnormal* the predetermined threshold Is a level of the biomarker in phagocytes isolated fro a fluid sample obtained from the patient before administration of the therapeutic compositions.
10016} The biomarker or antigen may be one or more of the following biomarkers. Tau, phosphorySated Tau, hippocaicin-1 , or 14-3-3 protein, N1BP, UGH-L1 , TDP-43, superoxide dismutase (SOD), neurornelanin, GFAP, neurGitiaments light chains (NFL), UCH-11 , amyloid beta, alpha-syndciein, apo A=i, Apo E, Apo J, JC viral antibody titers, TGF-beta, VEGF, dopamine-beta- bydroxyiase (D8H), vitamin D binding protein, histidine-rich glycoprotein, cDNA FU78071;, apolipoprotein C-iS, immunoglobulin heavy constant gamma 3, aipha-l-acid glycoprotein 1, alpha's -acid glycoprotein 2, haptogiobin-reiated protein, leucine-rich alpha-2-giycoproteln, superoxicSe dismutase (SOD), TDP-43, or G-reactive protein, The presen invention is not limited the aforementioned biomarkers or antigens. The biomarker may be selected based on its association with a particular disease or condition,
jOOIJj The present invention also features methods of identifying a therapeutic composition or therapeutic intervention: having positive effect on centra! nervous system (CMS) tissue damage, CMS repair, or neurodegeheratioo. The CMS tissue damage, CMS repair, neurodegeneration, etc., may be associated with a particular disease, condition, trauma, etc,, as described herein. The method features isolating or retrieving a biomarker or antigen fro the central nervous syste (e.g., a brain antigen, etc,) utilizing re-circulating phagocytes as a shuttle vector.
I (HU 21 In some embodiments, the method comprises: selecting at feast one patient with active CMS disease by isolating circulating phagocytes front a fluid sample from the patient, the fluid sample being from: outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detesting a level of a biomarker in the phagocytes from the patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneration, etc. in some embodiments, if the level of the biomarker in the phagocytes is abnormal, then the patient has active CNS disease. The method may further comprise; administering to the at least one patient with active CMS disease the therapeutic composition or therapeutic intervention; isolating circulating phagocytes from a fluid sample from the patient after a predetermined amount of tim following administration of the therapeutic composition or therapeutic intervention, the fluid sample being fro outside of a centra! nervous system tissue (e„g„ outside of the brain tissue) of the patient; and detecting a level of a biomarker in the phagocytes from each patient, the biomarker being associated with; CNS tissue damage, CMS system repair, or neurodegenefafien, etc, in some embodiments, (a) if the level of the biomarker in the phagocytes fro a patient is normal, then the therapeutic composition or therapeutic intervention has a positive effect on CMS tissue damage, CNS repair, or neurodegeneration; (b) if the level of the biomarker in the phagocytes from a patient is within a predetermined threshold associate with improved CNS disease, then the therapeutic composition or therapeutic intervention has a positive effect on CNS tissu damage, CNS repair, or neurodegeneration; or (c) if the level of the biomarker In the phagocytes from a patient is improved compared to a level of the biomarker in phagocytes in a sample obtained fro the patient at a time point prior to administration of the therapeutic composition or therapeutic intervention; then the therapeutic composition or therapeutic intervention has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration.
(0013} The predetermined threshold may be industry standards in some embodiments, the predetermined threshold is a laboratory standard. In some embodiments, the predetermined threshold is a level of the biomarker In phagocytes isolated from a fluid sample obtained from the patient before administration of the therapeutic compositions. Each particular biomarker of interest may have its own predetermined threshold, in some embodiments, a level of biomarker above the predetermined threshold is an abnormal level of the biomarker. In some embodiments, a level of biomarker below the predetermined threshold is an an norma I level of the biomarker.
|0014j The therapeutic composition or therapeutic intervention may include but is not limited to; Mastinab,; Dadizumab, Zinbryta, ER-Seta agonist, cyclophosphamide, rHlgM22, ponesimod, alpha-4 integrin, AMP-110,: an antisense oligonucleotide, immune tolerizing agent, MuitiStem, vi .3 Blocker, Alemtuzumab, !FNb~1 b, BHT-3009-01 , IF b-la, dimethyl fumarate, IF b-la (PEGyiated); Natalizumab, MT130T, Abatacept, RCP1083, a compound related to !amitrigene, CN -AU8, Mesenchymal stem cell transplant, apolipoprotein E~based, modified pepiidomimetic, Aimspro, Anti-BAFF Human Ab, NDC-13G8, GNbACt, Vatellzumab, Ofatumumab, ozanezumab(Nono-A mAb), Firategrasf (88-683699, T-0047), interferon Alfa N3, MStoxantrone, !budilast, Simvastatin, Raitegravir. Plovamer Acetate, Vedo!izumab, Siponimod, Secukinumab, Fingoiimod, Copaxone, LSD 1 -MAOS, Amiloride, adrenocorticotropic hormone, Rituximab, Ocrelizumab, Teriflunomlde, gSatiramer acetate, laquinimod, TG-11Q1 (ubiituximab), Clemastine Fumarate, Lipoic Acid, Minocycline, Riluzoie, IVIiS416, Fibrin, ATX-MS1467, ARX424, Tceina, T8C4746, Feidetre , PV-267, RNS60, R1295 VSN16R, Angiotensin AT2 agonist, GEH 120714, PDD1A, PDD3a, Zonisamide, defenprone, NTCELL, Caffine, Ssradlpioe, Rivastigmine, Inosine, Rasagine, GSVI1 gangiioside, Nicotine, Exenaiide, Piogiitazone, Glycerol Phenylbutyrate, EPE743, an antiinflammatory drug, ANCΌ05, AADvacl , 5HT-6, Verubestat, PXT864, Donepezil, NPT08S, RG1450, RG7412, PDE4E, PDE4B, Methylthioninium chloride, T3D-969, MultiTEP, ABT9S7, Anti-Tau antibodies, Aducanumab, Solanezumab, or lnbrija.
jOOlS;| The present invention also features methods of selecting a candidate for monitoring mild cognitive impairment or Parkinson’s disease. Th method features retrieving a biomarker or antigen from the central nervous syste fe.g., a brain antigen, etc,) utilizing re-circulating phagocytes a a shuttle vector.
( 016} In some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from a patient, the fluid sample being from outside of a central nervous system tissue (e.g.. outside of the brain tissue) of the patient; detecting a level of a biomarke in the phagocytes, the biomarker being associated with CMS tissue damage, CMS system repair, or neurodegeneration. in some embodiments, if the level of the blomarker in the phagocytes is abnormal, then the patient has active central nervous system disease. The method may comprise selecting the patient for monitoring for mild cognitive impai ment or Parkinson’s disease based on the patient having active centra! nervous system disease, In some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes,
10017] in some embodiments, the candidate has REM sieep behavior disorder (RBD). The biomarker may include but is not limited to dopamine-beta-hydroxyiase (DBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FU78071, apoiipoprotein C-i!, immunoglobulin heavy constant gamma 3, alpha-1 -acid glycoprotein 1, alpha-1 -acid glycoprotein 2, haptoglobin-related protein, leucine-rich ¾lpha-2~g!ycoproteint C-reactive protein, Tay, phosphoryiated Tau, hippocalcin-1 , or 14-3-3 protein, MBP, UCH-L1 , TDP-43. superoxide dismutase (SOD), neuro elanin, GFAP, neurofiia ents light chains (NFL), UCH-L1 , amyloid beta, alph -synuciein, apo AN, Apo E, Apo J, JC viral antibody titers, IGF -beta, VEGF, the like, or a combination thereof. The present invention is not limited the aforementioned biomarkers or antigens. The biomarker may be selected based on its association with a particular disease or condition,
(0018) The present invention aiso features methods of treating a patient suspected of having centra i nervous system tissue damage, central nervous system repair, or neurodegeneration. The method features isolating or retrieving a biomarker or antigen from the centra! nervous system e,g.t a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector in some embodiments, the method comprises: isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system ti sue (e.g.. outside of the brain: tissue) of the patient; detecting a feveS of a biomarker in the phagocytes, the biornarker being associated with central nervous syste tissue damage, central nervous system repair, or neurodegeneration, wherein if the level of the biomarker is abnormal then the patient has active central nervous system disease; and administering to the patient having active centra! nervous system disease a therapeutic composition o therapeutic intervention effective for treating or preventing worsening of the central nervous system tissue damage, central nervous syste repair, or neurodegeneration, or a symptom thereof, in some embodiments, the step of Isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes,
(0019) in so e embodiments, the central nervous system tissue damage, central nervous system repair, or neurodegeneration is a result of Multiple Sclerosis, Alzheimer’s disease, mild cognitive impairment, Parkinson's disease, Multiple System Atrophy, Progressive Supranuclear Atrophy, Lewy body disease, Coriicobasal Degeneration, Amyotrophic Lateral Sclerosis, Huntington’s Disease, REM sleep behavior disorder, concussion, Traumatic Brain injury, or a disease causing secondary centra! nervous system damage in some embodiments* the central nervous system tissue damage, central nervous system repair, or neurodegeneraiion causes cognitive impairment, motor disturbances, or doth.
|0020| in some embodiments (for the method above and/or for the methods herein), the biomarker is Tau, phosphorylaied Tau, hsppocalcin-1 , or 14-3-3 protein, MBP. UCH-L1 , neurome!anin, GFAP, neurofilamenis Sight chains (NFL), UCH-L1 , a yioid beta, aipha-synuclein, apo A=i, Apo B, ApO J„ JC viral antibody titers, TGF-beta, VEGf, doparnsne-beia-hydroxyiase DBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FU78071 , apolipoproteib C-ll, immunoglobulin heavy constant gamma 3, alpha- 1 -acid glycoprotein i , aipha-l-acid glycoprotein 2, haptoglobin-related protein, leucine-rich a!pha-2-giycoproiein, TDP-43, superoxide dlsmutase (SOD), G-reactive protein, a combination thereof, etc. The present invention is not limited to the aforementione biomarkers,
10021 i in some embodiments (for the method above and/or for the methods herein), the therapeutic composition or therapeutic intervention is Mastinab, Baeiizumab, Zinbryta, ER-Beta agonist, cyclophosphamide, rBigM22, pohesimod, aipha-4 Ihtegrin, AMP-110, an antisense oligonucleotide, immune toierizing agent, MuiiSStem, Kvt,3 Blocker, Alemtuzumab, iFNbdb, BHT- 3003-01 , iFNb~1a, dimethyi fu arate, !FNb-1a (PEGyiaied), Nataiizumah, MU 301 , Abatacept, RCP1063, a compound related to !amitrigene, CNM-Au8, Mesenchymal stem ceil transplant, apQiipoprotein E-based, modified peptldomimetic, Aimspro, Anti-BAFF Human Ab, NDC-1308, GNbACi , Vateiizumab, Ofatumumab, ozanezuma (Nono-A m,Ab), Firategrsst (SB-683699, T- 0047), interferon Alfa N3, Mitoxantrone, ibudi!ast, Simvastatin, Raitegravir, Piovamer Acetate, Vedolizumab, Slponimod, Secukinumab, F!ngofimod, Copaxone, LSDi-MAGB, Ami!oride, adrenocorticotropic ho mi one, Riiuximab, Gcreiizumab, Terif!unomide, glatiramer acetate, Laquinimod,, TG-11G1 (ubiituximab), Clemastine Fumarate, Upoic Add, Minocycline, Riiuzofe, MIS416, Fibrin, ATX-MS1467, ARX424, Tceina, TBC-4746, Feldeirex, PV- 207, RNS60, R129S, VShil8R, Angiotensin AT2 agonist, GEH 120714, PDOIA, PGDSa, Zo sa ide, deferiprone, NTCELL, Gaffine, isradipine, Rivastigmine, inosine, Rasagine, 6M1 gangiioside, Nicotine, Exenatide, Piogiitazone, Giyceroi Phenyibutyrate, EPl-743, an anti-infiammatory drug, ANX0Q8, AADvaci , 5HT-6, Veru bests†, PXT364, Donepezii, NPT0S6. RG145Q, RG74 2, POE4E, PDE4B, Mefhyithiohinium chloride, T3D-959, MultiTEP, ABT957, Anii-Tau antibodies, Aducanumab, Soianezu ab, or Inbrija, The present invention is not limited to the aforementioned therapeutic compositions or therapeutic interventions.
(0022] The present invention also features a method of treatin a atient suspected of having an autoimmune disease or condition associate with neurodegeneration. The metho features isolating or retrieving a biomarker or antigen from the centra! nervous syste (e,g„ a brain antigen, etc.) utilizing re-circulating phagocytes as a shuttle vector. In some embodiments, the method comprises isolating circulating phagocytes from a fluid sampie from the patient, the fluid sample being from; outside of a centra! nervous system tissue (e*g,* outside of the brain tissue) of the patient; defecting a level of a btomarker in the phagocytes, the blomar er being associated with the disease or condition, wherein if the level of the biomarker is abnormal then the patient has active central nervous system disease; and administering to the patient having active central nervoussystem disease a therapeutic composition or therapeutic interventio effective for treating or preventing worsenin of the disease or condition or a symptom thereof in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the autoimmune disease or condition associated with neurodegeneration is multiple sclerosis,
{0025} The present invention also features a method of treating a patient suspected of having disease or condition causing cognitive impairment. The method features isolating or retrieving a biomarker or antigen from the central nervous system (e.g,, a brain antigen, etc.) utilizing re- circulating phagocytes as a shuttle vector. In some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from the patient* the fluid sample being from outside of a central nervous system tissue (e.g.. outside of the brain tissue) of the patient; detecting a level of a bio marker in the phagocytes, the biomarker being associated with the disease or condition, wherein if the level of the biomarker is abnormal then the patient has acti ve central nervous system disease; and administering to the patient having active central nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsenin of the disease or condition or a symptom thereof, in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the disease o condition causing cognitive impairment is Alzheimer’s disease, or Lewy body disease.
{01)24} The present invention also features a method of treating a patient suspected of having a disease or a condition causing motor disturbances. The method features isolafing o retrieving a biomarker or antigen from the central nervous system (e.g.. a brain antigen, etc.) utilizing recirculating phagocytes as a shuttle vector. In some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from the patient, the flui sample being from outside of a cental nervous system tissue (e.g, , outside of the brain tissue} of the patient; detecting a level of a fsiomarke in the phagocytes, the biomarker being associated with the diseas or condition, wherein if the ievei of the biomarker is abnormal then the patient has active central nervous system disease; and administering to the patient having active central nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the disease or condition or a symptom thereof. In some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes. In some embodiments, the disease or condition causing motor disturbances is Parkinson’s disease, Multiple Sclerosis, Supranuclear Atrophy, or Coifieobasai Degeneration, h |0O2S) The present invention also features a method of treating patient suspected of having brain trauma. The method features isolating or retrieving a biomarker or antigen from the central nervous system (e,g., a brain antigen, etc.) utilising re-eircuSating phagocytes as a shuttle vector !n some embodiments, the method comprises Isolating: circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a level of a biomarker in the phagocytes, the biomarker being associated with brain trauma, wherein if the level of the biomarker is abnormal then the patient has active central nervous system disease; an administering to the patient having active centra! netvous system disease a therapeutic compositio or therapeutic intervention effective for treating or preventing worsening of the brain trauma or a symptom thereof, in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes in some embodiments, the method is performed within a time frame from 24 to 48 hours after a head injury. In some embodiments* the metho is performed within a time frame from 1 to 48 hours after a head injury, In some embodiments, the brain trauma is a traumatic brain Injury or a concussion.
f0026| The present invention also features a method of treating a patient having a genetic disorder associated with neurodegeneration (e.g., Huntington’s disease, amyotrophic lateral sclerosis (AL$), etc,). The method features isolating or retrieving a biomarke or antigen from the central nervous system (e.g., a brain antigen, etc.) utilizing re-circulating phagocytes as a shutle vector, in some embodiments, the method comprises isolating circulatin phagocytes from a fluid sample from the patient, the fluid sample being from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient; detecting a ievel of a biomarker in the phagocytes, the biomarker being associated with the genetic disorder, wherein If the level of the biomarker is abnormal then the patient has activ central nervous system disease; and administering to the patient having active centra! nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the genetic disorder or a symptom thereof, in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes .
|0O27| The present invention also features method of treating a patient suspected of having a relapse-remiting disease or infection, The method features isolating or retrieving a biomarker or antigen utilizing re-circulating phagocytes as a shuttle vector, in some embodiments, the method comprises isolating circulating phagocytes from a fluid sample from the patient; detecting a level of a biomarker in the phagocytes the biomarker being associated with the relapse-remitting disease, wherein if the levei of the biomarker is abnormal then the patient has active disease; and administering the patient having active disease relate to the relapse-remitting disease or infection a therapeutic composition or therapeutic intervention effective tor treating or preventing worsening of the relapse-remitting disease, or a symptom thereof in some embodiments, the step of isolating circulating phagocytes from the fluid sample from the patient further comprises concentrating the circulating phagocytes, in some embodiments, the fluid sample is from outside of a central nervous system tissue (e,g„ outside of the brain tissue).
[0028] in some embodiments, the relapse-remitting disease is multipie sclerosis (MS), Lyme disease, rheumatoid arthritis (RA). systemic lupus erythematosus (SLE), inflammatory bowel disease, human immunodeficiency virus (HIV), neurosareoidosis, or psoriasis,
(0029) The methods herein may also feature using a ratio of a biomarker to another parameter such as a plasma marker, an imaging marker, a different marker associated with a disease state or condition, a second biomarker in the circulating phagocytes, other particular diagnostic or investigative marker, etc. For example, in some embodiments, a ratio of a circulating phagocyte biomarker to a plasma marker may be used to detect CHS damage or repair. In some embodiments, a ratio of a circulating phagocyte biomarker to an imaging marker or diagnostic indicator (e,g., MR!) is used to detect active disease in a patient (e g„ multiple sclerosis patient) in some embodiments, a ratio of a circulating phagocyte to a secondary marker associated with REM sleep disorder is used to detect REM sleep disorder. In some embodiments, a ratio of a first circulating phagocyte biomarker to a second circulating phagocyte biomarker is used to detect active CMS damage. The present invention is not limited to the aforementioned examples of ratios of biomarkers or diagnostic indicators. The ratio as it related to the disease process may be a predetermined value, e.g., an industry standard, a laboratory standard, etc. For example, for some cases, a high ratio may be associated with active disease, and in some cases a low ratio may be associated with active disease.
(0030) The present invention also features preservation of samples for preserving the amount and/or structure and/or location of the biomarker(s) of interest (e.g., for preserving the amount and/or structure and/or location of the epitope(s) of interest). For example, the present invention provides methods for treating samples for the purposes of preserving the biomarker, e g., via heat denaturation (wherein proteolytic enzymes or other factors are inhibited without affecting the biomarker, e.g,, the epitope of the biomarker, to a large extent). Other methods of preservatio may include freeze drying or other rapid freezing processes, application of heparin or other factors, modifying the pH of the sample, etc. The present invention i not limited to the aforementioned methods or compositions,
(0031) The phagocytes containing the biomarkers of interest be characterized and isolated based on immunaphenatyping, This process be used for investigative purposes, for example to help determine if there is a subpopulation of cells with the particular biomarker of interest. Further, the process, once a particular immunophenotyped identified for a biomarker of interest, may be used as a technique for concentrating tSie phagocytes during sample preparation and analysis. The association of a particular immunophenotyped ceil and a biomarker of interest may be achieved by any appropriate method, e,g. flow cytometry, immunofiuorescent microscopy, etc:, The results may identify Known phagocytic celi types (CD 14+ monocytes and/or macrophages (CD 68/CD11b)) to be the source of particular biomarkers {e.g,, neural antigens) in PBMCs. Non- limiting examples of lineage antigens for !mmunophenotyping and Immunosefeetion may include CD14, CD 18, CD 71 , GDI la, GDI 1 b, GDI 1c, CD15iew, CD33, CD84, CP88, CDSO, CQ88, CDlOS/endogiin, CD115, CD163, CDl95/CeR5, GD282/TLR2, CD284/TLR4, HLA-DR/MHC Class SI, ILT 1 ILT3. 1LT4, SLT5, Mature Macrophage Marker50^', CD1a, CD1b, CP1e, CD40, CD490,CD83, CD85g/SLT7fi!¾', CD123, CD197/CCR7, CD2O5/DEC-205, CD207/Langerio, CD209/DC- SIGN, CD273/S7-DC, CD289rTLR9, CD303, CD304, CMKLR- *00, the like, or a combination thereof. The present invention is not limited to the aforementioned antigens, Further, the lineage antigens are not limited to human antigens and may include any appropriate corresponding cellular antigen in a different species. For example, in some embodiments, the subject or animal is a mouse, and the lineage antigens may include but are not limited to CDl l a, GDI I D, CD13, CDi4««r» CD18/CD32, CD84, CD68, CD8Q, CD86, CD107/Mac3, CPU S, CD282/TLR2, CD2S4/TLR4, F4/SG, Gaiactsn-S/MaoG, GITRL, MMC Class il, 33D1 , CD4, CDS, CD1 ibiiw, CD1 1c, OD40, CD45R/B220pE>c, CD83, 00123^, C01S7/CGR7, GD20S/DEC-205, GD207/Langerint C D209/DC-S! GN“!wa!UFe, CD273/87-DC, CD289/TLR9, 00317/R00A-1rίCΪ, 84/80^', MHC Class it, Sigiec HpDC, the like, or a combination thereof. Table 1 below lists various markers for human and mouse cell types,
TABLE 1
Figure imgf000012_0001
[0032] Without wishing to limit the present invention to any theory or mechanism» it is believed that biomarkers that are associated with particular disease states of interest [e.§., biomarkers found in the re-circulating phagocytes as described herein) wifi continue to be discovered. Since the methods herein are not necessarily limited by the particular biomarker but instead features the phagocytic shuttle method (e.g., wherein the phagocytes are shuttles for GNS-derived debris indicative of processes occurring in the CHS) and steps for isolating the biomarkers within theshuttle phagocytes, the present invention includes those biomarkers that will be discovered in the future,
(0033} Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutuallyinconsistent as wifi be apparent from the context, this specification, and the Knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and ciaims.
BRIEF DESCRIPTION OF THE DRAWINGS
10 34] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:
10035] FIG 1 shows the binding of the 4S4 peptide to neuromelanin in extracts of retinal pigment epithelium immobilized on ELISA plates in two-fold dilution series,
10036] FIG. 2 shows curve of varying amounts of pure Tau protein isolated using a Tau HT? antibody an an anti~Tau rabbit poiyelonai antibody.
1003 ]: FiG. 3 shows Tau and Hippocalcin 1 tike i ieve!s in PBMC lysates in 12 apparently healthy controls and 18 MS subjects.
[0038] FIG. 4 shows T au in PBMCs of cuprizone fed mice.
10039] FIG 5 shows hippocalcin 1 like 1 in PBMCs of cuprizone fed mice
[0040] FIG. 6 shows myelin basic protein in PBMCs of cuprizone fed mice.
[0041] FIG, ? shows a macrophage with engulfed photoreceptor debris in the process of diapedesis through an optic nerve head capillary, the cell nucleus is inside the capillary whereas parts of the debris-containing cytoplasm Is still externally located (arrow) (from Joty S et aL Resident Microglia and Bone Marrow Immigrants Remove Dea Photoreceptors in Retinal Lesions, The American Journal of Pathology, Vol, 174, No, 6, June 2009),
[0042] FIG. S shows the relationship between several biomarkers, such as Tau, GFAP, NFL, UCH-L1 , etc., and the active disease processes associated with diseases such as Multiple Sclerosis, Alzheimer’s disease, Parkinson’s disease» etc.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0043] As previously discussed, it has been sufprisingfy discovered: that debris-laden phagocytes a may re-enter the blood stream from the rain and if sufficient numbers are present, it may be possible to detect or measure the brain-derived debris (e g., debris or blpmafkers that would only normally be found in brain or centra! nervous system tissue) that is inside the phagocytic cells.{0044] The presence and/or amount of the debris may be associated with various states of the brain, e.g., active central nervous syste tissue damage, active central nervous system repair, active neurodegeneraiion, etc.; thus, obtaining these neora! -derived circulating phagocytes (that were previously in the central nervous system) can be used for monitoring a brain condition or neurological disease (e.g., monitoring worsening of improvement of a particular brain condition or neurological disease), detecting neurological damage (e.g., neurological damage associated with a disease or injury), detectin active neurodegenerative diseases, active central nervous syste tissue damage, and/or active central nervous system repair, etc.
[0045] Without wishing to limit the present invention to any theory or mechanism, it is believed that the present invention can provide close to real-time data on what is happening in the brain since that particular cargo may only be present in the recirculating phagocytes for a certai length of time (e.g,, a few days) before it is completely digested,
{00461 T e present invention also features methods for stratifying patients suspected of having an active neurodegenerative disease process, for example for selecting patients with active disease as part of a cohort for a clinical trial or for treatment. This enables patients not
experiencing active disease to be eliminated from those clinical trials or treatments, since their tack of disease activity may have a negative effect on the outcome of the clinical trial. ;
[0047] These stratification methods may also be used to identify and monitor those suspected of having a neurodegenerative disease (e,g., patients experiencing mild cognitive impairment or other symptoms) before the disease progresses too far. This may be particularly valuable In certain disease processes, such: as Parkinson’s disease, wherein patients are typically identified too late.
Biomarkers and Diseases
10040] Various neural-derived debris antigens or biemafkers (e.g,, debris from brain tissue or other central nervous system tissue, antigens that would not normally be found outsid of the brain, etc.) may be found in recirculating phagocytes in the peripheral blood. Such debris antigens may be used to detect (or monitor) neurodegenerative or neuroinfiammatory diseases (e.g., diseases as described herein). Debris from other diseases, such as relapse-remitting diseases, may be retrieved specificaiiy from circulatin phagocytes as well.
|0049j The biomarker (e.g., antigen, neurological condition-associated protein, etc.) may be present in a circulating phagocyte, e.g., peripheral phagocyte, As used herein, the term "peripheral” refers to anything outside of brain tissue. For example, a peripheral phagocyte may be obtained from cerebrospina! fluid (CSF), Phagocytes may include monocytes, macrophages, and/or lymphocytes. Such circulatin phagocytes may be found in tissues, cells, and/or fluids in the body , for example in biood, peripheral blood mononueiear cel is (PSfVICs), synovial fluid, cerebrospinal fluid fCSF), central nervous system tissues, synovia! fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreaf fluid, urine the like, or a combination thereof in some embodiments, the biomarker is an intracellular component For example, the biomarker may be obtained from within a macrophage. In some embodiments, the macrophage sample is permeabi!teed. In some embodiments, the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc. The fluid obtained for probing the phagocytes may be dependent on the disease or condition of interest,
10050] As used herein related to the methods herein, a mammal includes a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow. The present invention Is not limited to the aforementioned subjects or patients. jOOSlj in some embodiments, the sample is obtained from the subject, e.g., mammal, immediately following a relapse (e,g,, exacerbation of symptoms) before a therapy (e,g., a steroid or other drug or other therapeutic intervention) treatment has begun, in some embodiments, the sample is obtained fro the roammai before a relapse, In some embodiments, the sample is obtained during the course of the therapy treatment .
|0052j in some embodiments, the disease or interest associated with central nervous system tissue damage, central nervous system repair, neurodegeneration, etc., includes but is not limited to: autoimmune diseases, e.g., multiple sclerosis rheumatoid arthritis, lupus, Sjogren’s syndrome, thyroiditis, uveitis, Crohn's disease, ulcerative colitis, psoriasis, type 1 diabetes mei!itus, autoimmune Addison's disease, autoimmune hepatitis, celiac disease, pempbigous, chronic inflammatory demyelinating polyneuropathy, acute disseminated encephaiomyeiopathy, sarcoidosis, dermatomyositis and behcet’s disease; neuroSogicai diseases, e.g., stroke, concussion, chronic traumatic encephalopathy, neuromyelitis optica, transverse myelitis, intractable epilepsy and CNS infections; Parkinson’s disease; primary tumor growth, metastasis Of tumors; etc. Other diseases may include but are not limited to mil cognitive impairment, traumatic brain injury, Huntington’s disease, amyotrophic lateral sclerosis, progressive supranuciear atrophy, corticobasai degeneration, multipie system atrophy, Lyme disease, and systemic lupus erythematosus, Neuromyelitis Optica, transverse myelitis, .Acute and chronic Stroke, etc. The present invention is not limited to the aforementioned diseases,
(0053] Table 2 shows non-limiting examples of biomarkers that may be associated with disease states {e.g., degenerative disease states) with various organs. For example, the present invention may be used to detect a diabetes condition by detecting somatostatin in a similar manner as described herein, e.g.. similar to methods for detecting neuromeianin for Parkinson’s disease.
Table 2
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
|(10$4| in some embodiments, me biomarkers or antigens (or nsurodegenefative disease- associated proteins or biomarkers) (or fragments thereof) indude but are not limited to those in Table 1 , a Tau protein {or fragment thereof), a Tau protein or fragment thereof comprising a phosphorylated residue (e.g,, a phosphorySated serine reside, a phosphorylated threonine reside; e.g,, serine 214, serine 235, serine 282, serine 358, serine 396, serine 404, serine 413, serine 46, serine 515, serine 518, serine 519, serine 531 , serine 552, serine 610, serine 822, serine 641 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threonine 492, threonine 498, threonine 522, threonine 529, threonine 534, threonine 548); a protein or a fragment thereof selected from the group consisting of UCHL-1 , neurorneianin or a fragment thereof, M8P, transketolase, NS1 associated protein 1 , major vault protein, synaptojanin, enoiase, alpha synuciein, glial fibrillary acidic protein, S-1QG protein, Neu-N, 26S proteasome subunit 9, ubiquitin activating enzyme ZE1 , ubiquitin 8 precursor, vimentin, 14-3-3 protein (e.g., zeta Isoform), NQGO-A, neumriai-specific protein gen product 9,5; proteoiipid protein; rilyeiin oligodendrocyte glycoprotein, SFAP, neurofSSamenis light chains (NFL), UCH-L1 , amyloi beta, a!pha-sy nuclein, ape A-S, Apo E, Apo J, JO viral antibody titers, TGF-beta, VEGF, dopamine-befa- bydroxyfase (D8H), vitamin P binding protein, histidine-rieh giycoprotein, cD A FU78071 , apoiipoprotein C-ii, immunoglobulin heavy constant gamma 3, aipha-1~add glycoprotein 1, alpha- i-aeid giycoprotein 2, haptog!obsn-reiated protein, ieucine-rich aipha-2-giycoprotein, or C-reactive protein, heurogiobin, valosin-contalning protein, brain hexoksnase, hippocafcin-1 , nestin, synaptotagmin, myelin associated giycoprotein, myelin basic protein, myelin oligodendrocyte giycoprotein, myelin proteQiipid protein, tfansketplase, NS1 associated protein 1 , majo vault protein, synaptojanin, enoiase, alpha synuciein, gliai fibrillary acidic protein, $-100 protein Neu-N, 28$ proteasome subunit 9, annexin A2, annexin A3, annexin A5, annexin A6, annexin Al t, ubiquitin activating enzyme ZE1, ubiquitin 8 precursor, vimentin, gJyceraldehyde-S-phosphate dehydrogenase, 13-3-3 protein, 14-4-4 protein, neurofi!ament heavy chain and neurofiia meat light chain, TDP-43, superoxide dismutase (SOD), etc. The present invention is not limited to the aforementioned biomarkers,
fO055j FiG. 8 shows the relationship between severai blamarkers, such as Tau, GFAP, NFL, UCH-L1 , etc., and the active disease processes associated with diseases such as Multiple Sclerosis, Alzheimer's disease, Parkinson's disease, etc,
fOOSeij As non-limiting example, neurorneianin may be detected In the debris of degenerated dopaminergic neurons in recirculating phagocytes. Abnormai neurorneianin levels may be
IS associated with Parkinson’s disease, Neuromelanin can be measured: in several ways, e,g., via thebinding gf labeled: melanin selective peptides (e,g., 484 peptide), e g,, biotihyiaied 4B4 peptide; a control peptide F601G may be use as a control); the binding of monoclonal or polyclonal antibodies to melanin; measuremen of metal binding to melanin; measurement of the semiconductor properties of melanin; measurement of the fluorescence properties of melanin; and extraction of melanin fro recirculating phagocytes and subsequent quantification of melanin, it’s components or adducts (both natural or synthetic); physical methods such as gas chromatography, liquid chromatography or mass spectrometry; and combinations of these methods,
fO057| As an example of a means of measurement of melanin within ceils, FIG. 1 shows the binding of the 4S4 peptide. Extracts of retinal pigment epithelium were immobilized on ELISA plates in two-foid dilution series of the extract and incubated with biotinylate 4B4 peptide. Unbound peptide was washed off and bound peptide was detected with streptavidin-HRP. in comparison, PB!VICs fro healthy human subjects show little binding of 4S4 peptide.
|0058| In some embodiments, the biomarker is a Tau protein (or a fragment thereof) or a Tau protein (or fragment thereof) comprising a phosphoryiated residue {e.g., a phosphoryiated serine residue, a phosphoryiated threonine residue). In some embodiments, the phosphoryiated residue is serine 214, serine 235, serine 262, serine 358, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531 , serine 552, serine 610, serine 622, serine 841 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threonine 492, threonine 49S, threonine 522, threonine 529, threonine 534, threonine 548, the like, or a combination thereof.
10059] in some embodiments, phosphorylation of Tau can decrease its solubility in some embodiments, the method comprises detecting a level of insoluble Tau protein in the sample. In some embodiments, an increased level of insoluble Tau protein as compared to a control level of insoluble au protein is indicative of the disease or condition of interest or a risk thereof.
|006q| As a nomlimiting example, the biomarker of interest may be Tau or a fragment thereof. Table 3 shows the amino acid sequence of full-length human Tau protein.
TABLE 3
Figure imgf000018_0001
Figure imgf000019_0002
10061} Table 4 shows nooliraiting exa mpies of possible Tm fragments, TABLE 4
Figure imgf000019_0001
!7
Figure imgf000020_0001
n
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
2:5
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
3i:
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
3%
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
4ί
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
|00621 The term Tau biomarfcer may refer to a particular epitope of Tau, e.g,, an epitope within a particular region of amino adds in some embodiments, the epitope is in a region fro aa 240-441 in some embodiments, the epitope is in a region from aa 243-441. i some embodiments, the epitope is in a region from aa 244-274. In some embodiments, th epitope is in a region from aa 275-305. in some embodiments, th epitope is in a region from aa 308-336. in some embodiments, the epitope is in a region from aa 337-368. in some embodiments, the epitope is in a region from aa 388-411 , Non-timiiing examples of ranges of amino acids in which the epitope may be contained is shown in Table 5. The present invention is not limited to these regions. Further, the epitope may be in shorter regions of amino adds, e.g., aa 244-260, aa 270-280, aa 290-310, aa 330-360, etc.
TABLE S
Figure imgf000046_0002
|006Jj With respect to the epitopes described and/or contemplated herein, the methods of the present invention (e.g,, detecting CMS damage, repair, or particular disease states as described herein) may feature isolating from the phagocytes (e.g,, re-cfrcuiating phagocytes) and/or detecting and/or measuring and/or evaluating the biomarker of interest, wherein the biomarker of interest is a particular epitope of a biomarker such as Tau. For example, the method may comprise detecting an epitope of Tau in the region of aa 201-441. in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 243-441 , in some embodiments, the metho comprises detecting an epitope of Tau in the region from aa 244-274, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 275-306, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 306-336, in some embodiments, the method comprises detectin an epitope of Tau In the region from aa 337- 368, in some embodiments, the method comprises detecting an epitope of Tau in the region from aa 388-411.
|O064j: in some embodiments, a combination (e.g,, a pair) of biomarker-specific antibodies are used for Isolating and detecting the biomarker of interest. For example, an ELISA assay may use a first biomarker-specific antibody as a capturing antibody and a second biomarker-specifie antibody as a defection antibody. FIG, 2 shows an example of such an assay, wherein both an anti-Tau HT7 antibody and an anti-Tau rabbit polyclonal antibody were used in an ELiSA. The present inventio is not limite to the use of the pair of aforementioned antibodies. The present invention includes a combination of any of the antibodies disclosed herein or antibodies specific to the biomarker of interest not necessarily iisted herein, e,g„ those that may be produced in the future, those that are commercially available, etc.
100651 Without wishin to limit the present invention to any theory* or mechanism, it is believed that in at least some cases, the biomarkers of interest are being cjeaved or modified in a way that makes detection more difficult than isolating or detectin recombinant versions of the biomarker. For example, in some embodiments, a commercial antibody that is able to detect a recombinant biomarker may not work well to detect the biomarker in a serum sample. The present invention also features producing or identifying binding molecules (e.g., antibodies, fragments, etc.) that may be used to detect particular antigens or biomarkers of interest in the sample (e.g,. serum, etc.),
10066] in some embodiments, more than one biomarker is detected in the sampie(s). in some embodiments, the biomarkeifs) is a neural-derived biomarker. However, the biomar er(s) is not limited to neural-derived biomarkers. In some embodiments, one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neurai-derlyed biomarkers, or a combination thereof.
10067] The biomarker may be of various lengths. For example, in some embodiments, the biomarker is from 5 to 20 amino acids, in seme embodiments, the biomarker is from 20 to 40 amino acids, in some embodiments, the biomarker is from 40 to 80 amino acids, in some embodiments, the biomarker is from 80 to 150 amino acids, in some embodiments, the biomarker is from 150 to 20 amino acids , in some embodiments, the biomarker is from 2Q0 to 300 amino acids, in some embodiments, the biomarker is from 300 to 400 amino acids, in some embodiments, the biomarker is from 400 fo 500 amino acids in some embodiments, the biomarker is from 500 to 800 amino acids,
(0068) The biomarker may comprise various regions of the full-length protein for example, in some embodiments, the biomarker comprises the amino-terminus (e.g., N-terminus, NH2- terminus, N-terminai end, amine-terminus). The amino-terminus refers to the amino acid at the end of a protein or polypeptide that has a free amine group (~NH2). in some embodiments, the biomarker comprises about the first 15 amino acids, in some embodiments, the biomarker comprises about the first 25 amino acids in some embodiments, the biomarker composes about the first 50 amino acids, in some embodiments, the biomarker comprises about the first 75 amino acids, in some embodiments, the biomarker comprises about the first 100 amino acids, in some embodiments, the biomarker comprises about the first 126 amino acids.
(0069) in some embodiments, the biomarker or fragment thereof comprises the carboxy-terminus (e g,, C-terminus, COON-terminus, C-terminai end, carboxyl-terminus): The carboxy-terminus refers to the amino acid at the end of a protein or polypeptide that has a free carboxylic acid group (-COOH), in some embodiments, the biomarker comprises the last 100 amino acids,
Ί 00701 in some embodiments, the step of detecting the biomarker in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the biomarker.
100711 in some embodiments, the step of detecting the biomarker in the sample comprises subjecting the sample to a western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an fmmunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a mass spectrometry assay, a flow cytometry assay (e.g., fSorescence- aefivated ceil sorting (FACS)), or a combination thereof and the like. Such assays are well known in the art,
(00721 in some embodiments, the step of detecting the biomarker further comprises contacting the sample with an antibody that binds to the biomarker and detecting an antibody-biomarker complex: The step of detecting an antibody-biomarker complex may comprise subjecting the sample to a micro array, western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an immunohistochemsstry assay, a bioluminescent assay, a chemiluminescent assay, a flow cytometry assay (e.g,, florescence-activated ceil sotting (FAGS)), or a combination thereof and the like, in some embodiments, detecting the antibody-biomarker complex indicates the presence of the particular disease or condition of investigation or a ris of the particular disease or condition of investigation,
|0073J As describe above, in some embodiments, the step of detecting the biomarker may comprise subjecting the sample florescence-activated cell sorting (FACS). Fluorescence-activated cell sorting (FACS) is a type of flow cytometry that sorts a mixture of biological ceils, one at a time, into separate containers based upon the specific light scattering and fluorescent characteristics of each ceil it provides quantitative recording of fluorescent signals from individual cells as well as physical separation of ceils of particular interest, Generally, a current of a rapidly Sowing stream of liquid carries a suspension of cells through a nozzle. The flow Is selected such that there is a large separation between cells relative to their diameter. Vibrations at the tip of the nozzle cause the strea of cells to break into individual droplets, and the system is adjusted so that there is a low probability of more than one ceil being in a droplet, A monochromatic laser beam illuminates the droplets, which are electronically monitored by fluorescent detectors. The droplets that emit the proper fluorescent wavelengths are electrically charged between deflection plates in order to be sorted into collection tubes.
Detecting centra/ nervous system damage via analysis of circulating phagocytes
10074] The present invention features method of detecting and/or treating diseases described herein. For exampie, the present invention features methods for detecting multiple sclerosis or a risk of multiple sclerosis, or methods for monitoring disease activity, drug efficacy, methods for treating multiple sclerosis, etc. The present invention also features methods for detecting Alzheimer's disease or a risk of Alzheimer's disease, or methods for monitoring disease activity, drug efficacy, etc. The present invention also features methods for treating Alzheimer's disease. The present invention also features methods for detecting Parkinson's disease or risk of Parkinson’s disease, or methods for monitoring disease activity, drug efficacy, etc. The present invention also features methods for treating Parkinson’s disease. The present invention also features methods for detecting concussion or a risk of concussion, or methods for monitoring disease activity, drug efficacy, etc. The present invention also features methods for treating concussion. The present invention also features methods for detecting central nervous system tissue damage or a risk of central nervous syste tissue damage, or methods for monitoring disease activity, drug efficacy, etc. The present invention also features methods for treating centrai nervous system tissue damage The present invention also features methods for detecting centrai nervous system repair, or methods for monitoring disease activity, drug efficacy, etc. The present Invention also features methods for treating centrai nervous system repair.
fOOTS] The present invention also features methods for defecting a relapse-remitting disease or a risk of a relapse-remitting disease, or methods for monitoring disease activity, drug efficacy, etc. The present invention also features methods for treating a relapse-remitting disease. The relapse- remitting disease may be, for exampie, multiple sclerosis (MS), Lyme disease, rheumatoid arthritis (RA), systemic !upus erythematosus (SLE), Ghron’s disease, etc. |0O76} The present Jrwentfon also features a method of detecting art inflammatory condition in a similar manner, wherein the biomarker is associated with an inflammatory condition. The fluid obtained does not necessarily directly come into contact with the inflamed tissue being detected. For example, there may be a barrier between the fluid and the source of the biomatker, in other words, the fluid obtained may have once directly come into contact with the inflamed tissue, but at the time that it is being extracted in accordance with the present invention, it is being separated from the inflamed tissue by a barrier.
|O077j In the methods herein, samples are obtained for subjects or patients (e,g., a mammal such as a human, rat, mouse, primate, etc.}. In some embodiments, the sample is a plasma sample, in some embodiments, the sampl is a blood sample. In some embodiments, the sample Is DNS fluid,
|0078) The biomarkers are detected in the recirculating phagocytes in the collected fluid sample, in some embodiments, two or more biomarkers are detected in a sample, e.a,, a pattern of biomarkers may be detected in the sample, in some embodiments, the bsomarkerfs) is a neural- derived biomarker. However, the biomarkeifs) Is not limited to neural-derived biomarkers. In some embodiments, one or more biomarkers are detected in the sample, wherein th biomarkers are neural-derived, non-neurabderived biomarkers, or a combination thereof,
{0079} The biomarker(s) may be detected using a variety of methods, Methods may include an immunoassay, a histological assay, a flow cytometry assay, the like, or a combination thereof. For example, in some embodiments, the step of detecting the biomarkers) in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the biomarker or is specific for the blomarker,
IO08Q] Tbs method may feature comparing the amount of blomarker In the sample to a predetermined threshold for determining whether or not th level of biomarker in the sample is abnormal, in some embodiments, the amount of blomarker in the sample (a first sample) is compared to a level of biomarker In a control. The control may be, for example, a sample (a second sample) obtained from the patient at a time prior to obtaining the first sample, in some embodiments, the predetermined threshold is a laboratory standard. In some embodiments, the predetermined threshold is an industry standard.
{0 81} in some embodiments, detecting an abnormal level of the blomarker as compared to th level of the biomarker of a control sample or threshold indicates the presence of the neurological condition, the disease, central nervous system tissue damage, CNS repair, etc,, or a risk thereof/
{0082} Phagocytes may include monocytes, macrophages, and/or lymphocytes. Such circulating phagocytes may be found in tissues, cells, andior fluids in the body, for example in blood, peripheral blood mononuclear cells (PB Cs), cerebrospinal fluid (CSF). central nervous syste tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular
4k fluids, vltreai fluid, urine, the like, or a combination thereof, in some embodiments, the biomarker is an intracellular component, For example, the biomarker may be obtained from within a macrophage, in some embodiments, the macrophage sample is permeabiiized. In some embodiments, the macrophage is lysed via various means, e.g,,. hypotonic solution treatment, detergent solution treatment, mechanical stress, etc,
|00S3| As previously discussed, the present invention features methods for treating patients suspected of having diseases or conditions uch as Multiple Sclerosis, brain ceil damage, brain cel! death, brain ce!i repair, Parkinson's Disease, Alzheimer's disease, etc, The method may comprise isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a brain tissue of the patient; detecting a level of a biomarker i the phagocytes, the biomarker being associated with the disease or condition,, wherein if the level of the biomarker is abnormal then the patient has active centra! nervous system disease; and administering to the patient having active centra! nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the disease or condition or a symptom thereof.
10084] Therapeutic compositions, treatments, drugs, or therapeutic interventions may include but are not limited to: crenezumab, mastinab, daclizumab, zinbryta, ER-Beta agonist, cyclophosphamide, rHSgM22, ponesimod, alpha-4 integrin, AMP-110, an antisense oligonucleotide, Immune toierizing agent, MultiStem, Kv1.3 Blocker, Alemtuzumab, IFNb-lb, BHT- 3009-01 , IFNb-1a, dimethyl fumarate, IFNb- {PEGyiated}, Nataiizumab, MT1301, Abatacept, RCP1063, a compound related to !amitrigene, CNM-AuS, Mesenchymal stem cell transplant apolipoprotein E-based, modified peptidomsmetic, Aimspro, Anti-BAFF Human Ab, NDC-1308, GRbACt, Vafelizumab, Ofatumumab, ozanezumab (Nono-A mAh), Firategrast {SB-883899, T- 0047], Interferon Alfa N3, Mitoxantrone, ibudilast, Simvastatin, Ra!tegravir, Plovamer Acetate, Vedolizumab, Siponimod, Secu inumafe, Fingolimott, Copaxone, ISDT-MAQB, Amiloride, adrenocorticotropic hormone, Rituximab, Dcreiizumab, Teriflunomide, glatiraroer acetate, Laguinimod, TG-1 01 {ub!ltuximab}, Clemastine Fumarate, Lipoie Acid, Minocycline, Riiuzoie, Fibrin, ATX-MS1467, ARX434, Tceioa, TBG-4748, Feldetfex, PV-267 RNS60, R1295, VS 16R, Angiotensin AT2 agonist, GEH 120714, PODIA, PDDSa, Zonisamfde, deferiprone, NTCELL:, Caffine, !sradipine, Rivastigmine, Inosine, Rasagiline, GM1 gangiioslde, Nicotine, Exenatide, PiogSitazone, Glycerol Phenyibutyrate, EPI-743, an anti-inflammatory drug, ANX005, AADvacl , 8HT-6, Verubestat, PXTB64, Dooepezil, NPT088, RG1450, RG7412, PDE4E, PPE4S, eibyithioniniu chloride, T3D-959, MuIίITER, ABT9S7, Anti-Tau antibodies, Aducanumab, Soianezumab, tnbrija, flurbiprofen, ketorolac, ketoprofen, tolmetin, aspirin, ibuprofen, naproxen, indomethacin, suiindae, piroxicam, mefenamic acid, meloxicarn, diclofenac, celecoxib, etodolac, etoricoxib, iumiracoxib, rofecoxlb, prodydlidine, diphenhydramine, benztropine, trihexyphenidyl, biperidin, bromocriptine, safmamide, selegiline, rotigotine, pramipexoie, apomorphine, rasagiline, rapiniroie, ©ntacapone, amantadine, pergolide, carbsdopa, levodopa, tolcapcsoe, metformin, a!ogiiptih, canagiifSozin, dapagiifiozin, empagisflozih, giipizide, or any other appropriate dru or combination of drugs or other therapeutic interventions. The present invention is not limited to the aforementioned therapeutic compositions or therapeutic interventions,
[0085] The present invention also features methods of selecting a patient for a clinical trial for testing a drug for an effect on centra! nervous system (CNS) tissue damage, CNS repair, or neurodegeneration in some embodiments, the method comprises isolating circulating phagocytes from a fluid sampie from the patient, the fluid sample being fro outside of a brain tissue of the patient; detecting a level of a biomarker in the phagocytes, the biomarker being associated wit CNS tissue damage, CNS system repair, or neurodegeneration, wherein if the level of the b marker in the phagocytes is abnormal, then the patient has active central nervous system disease; and selecting the patient for the clinical trial if the patient has active CNS disease.
[O086] Without wishing to limit the present invention to any theory or mechanism, it is believed that the cohorts of patients for clinical trials created using method herein will be better suited to test a particular therapeutic intervention or composition of interest. Further, these cohorts may be used to reevaluate drugs that may have previously failed in ciinicai trials (e.g. , crenezumab), because the drugs may have failed due to the cohort of patients not being appropriate for the testing. Patients exhibiting low current disease activity may be poor responders or non -responder to therapeutic treatments.
|0087j The present invention also features methods for discovering a therapeutic composition having a positive effect on central nervous system (CNS) tissue damage, CNS repair, or neurodegeneration. In some embodiments, the method comprises selecting only patients with active CNS disease according to the present invention; administering to the patients with active CNS disease the therapeutic composition; isolating circulating phagocytes from a fluid sample fro each patient after a predetermined amount of time following administration of the therapeutic composition, the fluid sampie being from outside of a brain tissue of the patient; and detecting a level of a biomarker in the phagocytes from each patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneralion. In some embodiments, if the level of the biomarker in the phagocytes from a patient is normal, then the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration. In some embodiments, if the level of the biomarker in the phagocytes from a patient is within a predetermined threshold associated with improved CNS disease, then the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration. in some embodiments, if the level of the biomarker in the phagocytes from a patient is improved compared to a level of the biomarker in phagocytes in ¾ sample obtained from the patient at a time point prior to administration of the drug, then the therapeutic composition has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration* 10088] T!ie present invention ¾}$ø features methods of determining the status of a disease or condition (e.g., a neuroiogicai condition, an inflammatory condition, multiple sclerosis, etc,} or determining the status of drug efficacy. The present invention may also feature methods of monitoring disease activity and efficacy of a drug, therapeutic composition, and/or or therapeutic intervention. For example, biomarkers can be used to detect a disease or condition and the biomarkers may be used to determin severity of the disease or condition (e.g. relapse, remission, etc.},
[0089) in some embodiments, the method comprises providing a sample (d· 9·n a fluid, a brain tissue), the sample comprising a circulating phagocyte. The sample may be derived from a mammal (e.g,, a patient, a mouse, a rat, etc,), A blomarker or level thereof associated with a disease or condition (e.g., a multiple sclerosis-associated biomarker) may be detected in the sample (e.g., in the phagocyte) and compare to the ievei or presence of the biomarker In control samples or to predetermined thresholds, In some embodiments, the biomarker detected may be compared to the ievei or presence of the biomarker in a second sample, the second sample having been collected prior to the first sample. By comparin the level or presence of the blomarker in th sample to either a control sample or a patient’s previous sample, disease activit may b determined,
10098] in some embodiments, the monitoring of disease activity may be use to determine drug, therapeutic composition, and/or o therapeutic intervention efficacy, in some embodiments, the monitoring of disease activity ma be used to determine therapeutic failure and/or breakthrough disease in some embodiments, the monitoring of disease activity may be used to determine patient complianc with a therapy. In some embodiments, the monitoring of disease activity may be used to determine therapeutic non-responders in some embodiments, the monitoring of disease activity may be used to aid drug development.
{0091] in some embodiments, the present invention features a method of monitoring disease activity of a neurological condition, the method comprises obtaining from a mammal a first fluid sample from outside of a brain tissue of the mammal, the first fluid sample comprises a first circulating phagocyte; detecting a level of a blomarker associated with the neurological condition in the first sample; and comparing the level of the blomarker In the first sample with a Ievei of the biomarker in a second sample, the second sample bein either a control sample or a second fluid sample from outside of a brain tissue of the mammal, the second fluid sample comprising a second circulating phagocyte, the second fluid sample having been taken prio to the first fluid sample.
{0092] As described above, in some embodiments, the step of detecting the biomarker may comprise subjecting the sample to an enzyme-jinked immunosorbent assay (ELiSA), ELfSA is an assay used to detect the presence of an antibody or a biomarker in a sample. Generally, in EL!SA, a sample containing an unknown amount of biomarker, e.g., an antigen, is affixed/immobfeed to a surface (e.g., a polystyrene microtiter plate). Then, an antibody that binds to the antigen of Interest is washed over the surface so that it can bind the antigen and fom an antibody/antlgen complex in some cases, this antibody is covalently linked to an enzyme in some cases, the antibody is not covalently linked to an enzyme but can b defected by a secondary antibody that is linked to an enzyme i the final step, a substance (e.g,, substrate) that the enzyme is capable of converting to a detectable visible signal (e.g„ color -signal) is added to the reaction, Thus, if the antibody/antigen complex is present, the substrate will be converted to the detectable visible signal, and then amount of antigen in the sarnpie can be measured.
(0093) The present invention also features method of detecting brain ceil damage, brain cert death, brain cell repair, or the like. In some embodiments, the method comprises obtaining from a mammal a first fluid sarnpie from outside of a brain tissue of the mammal and introducin to the first fluid sample magnetic beads, whereupon phagocytes (e.g,, monocytes, macrophages, combinations thereof, etc.) in the first fluid sample engulf the magnetic beads yielding magnetic phagocytes. The first fluid sample may comprise a biood sample (e.g,, in a container). The method may further comprise separating the magnetic phagocytes using a magnetic separation mechanism and making detectable and quantifying a neural-derived biomarker in the separated magnetic phagocytes, The neural-derived biomarker comprises a molecule derived from the brain {e.g., a biomarker normally just found in the brain or central nervous system tissue},
|00‘ | The detection of the neural-derived biomarker may be compared to other sampies derived from the mammal, e,g. sampies taken previously. The detection of the neural-derived biomarker may be compared to standards or controls in some embodiments, the neural-derived biomarker may be used as a means of monitoring a patient, e.g., monitoring brain ceil damage, brain cert death, brain ceil repair, etc, in some embodiments, elevated levels of the biomarker may be indicative of brain cell death or damage or worsening of a condition in some embodiments, elevated levels of the biomarker may be indicative of repair or improvement of a condition. In some embodiments, Sower levels of the biomarker may be indicative of brain ceil death o damage or worsening of a condition, in some embodiments, Sower levels of the biomarker may be indicative of repair or improvement of a condition,
(0 951 id some embodiments, detecting the neural-derived biomarke in the first fluid sample at a level that is different than that of a control or a second fluid sample obtained from the mamma! (the second fluid sample being identical to the first fluid sarnpie except that it is collected prior to the first fluid sample) is indicative that the mammal has experienced a change in brain cell damage, brain cell death, or brain cell repair, !h some embodiments, detecting the neural-derived biomarker in the first fluid sample at a level that is higher than that of a control or a second fluid sample obtained from the mammai (the second fluid sample being identical to the first fluid sarnpie except that If is collected prior to the first fluid sarnpie) is indicative that the mammal has experienced a change in brain ceil damage, brain cell death, or brain ceil repair, in some embodiments, detecting th neural-derived biomarker in the first fluid sample at a level that is iower than that of a control or a second fluid sample obtained from the mamrnai (the second fluid sample being identical to the first fluid sample except that it is collected prior to the first field sample) is indicative that the mammal lias experienced a change in brain cell damage, brain cell death, or brash cell repair.
|0096j The method may' further comprise introducing to the first fluid sample during or after the first fluid sample is obtained a molecule for inhibiting further degradation of the neural-derived bio arker in the phagolysosome of the phagocytes. Generally , any component that increases the pH of the phagolysosomes, which would inhibit the enzymes in the phagolysosomes, would help reduce the degradation of peptides (e.g , the biomarkers of interest) In the phagolysosomes, In some embodiments, the molecule for inhibiting further degradation of the neural-derived biomarker in the phagolysosome of the phagocytes comprises one or a combination of phagoiysosomal protease inhibitors. In some embodiments, the proteas inhibitor comprises leupeptin, In some embodiments, the molecule for inhibiting further degradation of the neural-derived biomarker in the phagolysosome of the phagocytes comprises a molecule that increases the pH of the phagolysosomes of the phagocytes in the first fluid sample. So some embodiments, the molecule for increasing the pH of the phagolysosomes of the phagocytes in the first fluid sample comprises an alkaline buffer Alkaline buffers are well known to one of ordinary skill in the art, e.g,, chioroguin, carbonate/bicarbohate buffer, buffers of pH 9.2 or above, weak base buffers, quinine, etc. In some embodiments, both a phagoiysosomal inhibitor an alkaline buffer are added in some embodiments, the magnetic beads/pariicies are coated with a phagoiysosomal inhibitor (e.g., leupeptin), in some embodiments, the magnetic beads/particles are coated with a mix of compounds, e.g,, a phagoiysosomal inhibitor (e.g., leupeptin), an antibody (e g,, SgG, IgG(fc), etc,).
(0097f In some embodiments, the method further comprises introducing to the first fluid sample a stimulator to stimulate phagocytosis of the magnetic beads by the phagocytes, in some embodiments, the magnetic beads are conjugated with an aci hydrolase inhibitor in some embodiments, the magnetic beads are conjugated with an antibody or antibody component to stimulate phagocytosis. In some embodiments, the magnetic beads are introduced to the first fluidsample within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes of when the first fluid sample is obtained.
fOOdkj In some embodiments, the step of making detectable the neural -derived biomarker comprises subjecting the separated phagocytes to an immunoassay. In some embodiments, the immunoassay comprises introducing an antibody to the sample, wherein the antibody binds to the neural-derived biomarker. In some embodiments, the antibody Is a polyclonal antibody.
(0099] in some embodiments, the container for the blood sample comprises an anticoagulant. In some embodiments, the anticoagulant comprises citrate, heparin, or a combination thereof, in some embodiments, the method comprises isolating peripheral blood mononuclear cells (P8MCs) from th blood sample prior to introduction of the phagocytosis of the magnetic beads i some embodiments, the magnetic separation mechanism comprises a magnetic column or magnetic rack, in some embodiments, the container (for th blood sample) comprises ficoif. in some embodiments, the container ( for the blood sample) does not comprise Ficoil or is free of Fico!i. in some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanis within 1 hour of harvesting of the first fluid sample in some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism within 12 hours of harvesting of the first fluid sample, in some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism within 24 hours of harvesting of the first fluid sa pie. in some embodiments, the magnetic phagocytes are separate using the magnetic separation mechanism within 48 hours of harvesting of the first fluid sample,
{00100) in some embodiments , the biomarker is a synaptic antigen. Synaptic antigens are weii known to one of ordinary skill in the art, e.g., PSD-9S protein, neorogranin, SNAP-25, and TDP-43, etc,
{00101) in some embodiments, the biomarker is one that helps define subgroups of a particular disease state or disease process. In some embodiments, the biomarker is TOP-43, which may be used to help define subgroups of ALS.
100102] in some embodiments, a protease inhibitor (e.g.. a phagolysosomai inhibitor} is introduced to the sample within 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes, or within 10 minutes, 15 minutes, 20 minutes, etc,, of when the samp!e is obtained.
Kits
|00tO3) The present invention also features kits for detecting the presenc or a risk of a disease or interest {e,g„ those described herein} in a circulating phagocyt sample derived from amammal. The kit comprises an antibody that binds to a specified appropriate biomarker in some embodiments, the kit further comprises a means for detecting the binding of the antibody to the biomarker/antigen in the sample (e,g., an antibody-antigen complex}.
100104) in some embodiments, the kit comprises an antibody specific for the biomarker of interest, wherein the antibod is a monociohaS or a poiycionai antibody, in some embodiments, the antibody is derived from a human, a mouse, a rat, a llama, a rabbit, a dog, a pri ate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow, in some embodiments, the antibody is humanized in some embodiments, the antibody is a chimera.
{00105) The antibodies in the kits may be purified, e.g., affinity purified for the antigen of interest.
{00106) in some embodiments, the kits further comprise reagents for detecting additional biomarkers, e,g„ additional biomarkers in the circulating phagocytes, serum biomarkers, plasma biomarkers, eto. [00107] in some embodiments, the kits further comprise reagents for preserving the sample, e.g., for preserving the amount, structure, and/or location of the biomarker of interest {e.g,, the epitope of the biomarker of interest),
[00108} The kit may further comprise appropriate reagents, manuals, equipment, etc. For example, the kit may comprise reagents for automated assays. In some embodiments, the kid comprises reagents for multiplex assays .
Multiple Sclerosis
100109} For the purpose of discussion, in some embodiments, the disease or condition of interest Is multiple sclerosis and the present invention features methods of detecting multiple sclerosis, methods for detectin a risk of multiple sclerosis, methods for selecting a patient with multiple sclerosis for monitoring, methods of selecting a patient with multiple sclerosis for a clinical trial, etc. In some embodiments, the methods of the present invention may allow for monitoring, detecting and/or predicting a reiapse or a remission of multiple sclerosis. So some embodiments, the method of detecting multiple sclerosis is used in combination with one or more methods of detecting multiple sclerosis. For example, the present methods may be used in conjunction with othermodalities to monitor, detect or predicting a relapse or a remission of multiple sclerosis. The present Invention is not limited to multiple sclerosis.
[00110} A previously discussed, the methods herein, for example, the methods for detecting multiple sclerosis comprises (1) providing a first sample (e.g.,, a fluid sample) that comprises a peripheral (e.g., circulating) phagocyte. The first sample may be derived from outside of a brain tissue, and (2) detecting a multiple sclerosis-associated biomarker in the phagocyte. The sample may be provided from a mamma} (e.g., a patient, a mouse, a rat, etc,). In some embodiments, one or more biomarkers is detected In the sample in some embodiments, for example if the diseas of interest is multiple sclerosis, the biomarkers are associated with multiple sclerosis,
[00111} The metho may further comprise comparing the level of the biomarker In the first sample with a predetermined threshold or other particular threshold, e,g., a level of the biomarker in a second sample (e.g., a fluid sample), wherei the second sample may be a control sample or other sample for comparison purposes in some embodiments,, the second sample is a fluid sample from outside of a brain tissue comprising a peripheral {e g., circulating phagocyte), The second sample may be provided from a mamma! {e.g,, a patient, a mouse, a rah etc.). The second sample may have been collected prior to the first fluid sample. Detecting the biomarker may indicate the presence of the disease or condition of interest (or a risk thereof), e.g., muitipie sclerosis.
[00112} For the aforementioned methods related to muitipie sclerosis or any of the methods herein, in some embodiments, the level of the biomarker or antigen may be evaluated (compared to the predetermined threshoid or control level or other comparison) and determined to be an abnormal level. Abnormal levels may be previously determined based on industry standard or laboratory standard, etc, In some embodiments, a high level of the jo afker In comparison to the threshold or control is an abnormal level in some embodiments, a low level of the biomarker in comparison to the threshoid or control is an abnormal level. In some embodiments, detecting a increased level of the biomarker as compared to the ieyei of the biomarker of a control sample indicates the presence of the disease or condition of interest or a risk thereof in some embodiments, detecting a decreased level of the biomarker as compared to the !evei of the biomarker of a control sample indicates the presence of the disease or condition of Interest or a risk thereof.
{001.131 As mentioned above, in some embodiments, an antibody is used to detect the presence of the multiple sclerosis-associated biomarker. The multiple sclerosis-associate biomarker may be detecte with a variety of antibodies, in some embodiments,, the antibody is a monocionai or a polyclonal antibody, in some embodiments, the antibody is a humanized antibody, in some embodiments, the antibody is a chimera. In some embodiments, the antibody is derived from a human, a mouse, a rat, a lla a, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow, in some embodiments, the antibody is synthetic, in some embodiments, the antibody is a recombinant antibody .
100114] Frequently, antibodies are labeled either covalently or non-cova!entiy by combining the antibody with a second substance that provides for detectable signal. A wide variety of labels and conjugation techniques are known in the art and are reported extensively In both the scientific and patent literature. Examples of labels include but are not limited to radioisotopes, enzymes, substrates, cofactors, inhibitors, fSuorescers, chemiSuminescers, magnetic particles, and the like in some embodiments of the present invention, the antibody comprises a label,
(00115] As an example, to identify the level of the biomarker that is the normal level samples arepooled from about, for example, SSQ patients (or an appropriate number of patients that would be statistically meaningful) who do not experience any symptoms of the disease and who do not test positive for the disease as detected by MR! or other appropriate methods. From those pooled samples, the average level of the biomarker can be quantified and then defined as being the normal level of the biomarker. if the norma! level of the biomarker is about zero, then an elevated level refers to any level that is greater than zero, for example, about 5 units, about 25 units, about SO units, about 100 units, about 500 units, about 1000 units, about 10,000 units, about 100,000 units, about 1 ,000,000 units. In some embodiments, a unit may be an absorbance unit (e.g., from an EUSA), a percent positive (e.g„ from a flow cytometry or FACS assay}, of a fluorescence unit,
100110] The present invention also provides a method of monitoring the progression of multiplesclerosis and/or monitoring the treatment of multiple sclerosis. For example, in some embodiments, the present invention may be used to measure the level of the multiple sclerosis- associated biomarker in order to detect a change in the level (e g,, an increase in the level, a decrease in the level, a maintaining of the level), Without wishing to limit the present Invention to an theory or mechanism, a change In the level of the multiple sclerosis-associated biomarker may correlate With a change in the patient’s status (e.g., remission, progression, worsening). For example, a decrease in the ievel of the multiple sclerosis-associated biomarker may Indicate the patient has entered or will enter a remission period. In some embodiments, the present invention may be used to monitor the level of the multiple sclerosis-associated biomarker in a patient while the patient is o a treatment regimen (e.g,, a drug). Without wishing to limit the present invention to any theory or mechanism a treatment regimen (e.g., a drug) that is effective at inhibiting the progression of multiple sclerosis and/or reducing the symptoms of multiple sclerosis may decrease the level of the multiple sclerosis-associated biomarker in the patient.
[Oil 17} As mentioned above, in some embodiments, the method of the present invention for detecting multiple sclerosis is used in combination with one or more different methods for detecting multiple sclerosis. For example, in some cases, a combination of family history, a physical exam, and magnetic resonance imaging (MRI) findings are used to diagnose multiple sclerosis. Currently, MR! is the most sensitive radiographic technique for the imaging of multiple sclerosis. Multiple sclerosis plaques are commonly seen as round or void discrete lesion in the periventricular white matter. Other common locations fo multiple sclerosis plaques include the corpus callosum, corona radiate, internal capsule, and centrum semiovafe. in some embodiments, the present invention i used to measure a multiple sclerosis -associated biomarker, and the level of the multiple sclerosis-associated biomarker is correlated with a magnetic resonance imaging (MR!) measurement. Without ishing to limit the present invention to any theory or mechanism, it is believed that elevated levels of the multiple sclerosis-associaied biomarker correlate with an MR! scan showing the presence of multiple sclerosis plaques in the brain.
|O0H8) The method of the present invention for detecting multiple sclerosis may be used in combination with one or more methods for detecting a different condition. For example, the method of the present invention may also help to distinguish multiple sclerosis from other diseases with similar clinical manifestations. For example, neuromyeiitis optica (NMD), also known as Devic e syndrome, is a neurological disorder regarded as a severe variant of multiple sclerosis. The eharacteristic inflammatory demyeiinating lesions of fsSlViO selectively and repeatedly affect the optic nerves and the spinal cord, causing blindness and paralysis. A marker (e.g., aquaporin-4 antibodies) has been identified in serum and cerebrospinal fluid of patients with NMO, and the presence of an NMO marker (e.g., aquaporin-4 antibodies) may be used to distinguish NMO fro multiple sclerosis. In some embodiments, the method of detecting the presence of multiple sclerosis or a risk of multiple sclerosis comprises detecting the presence or absence of at least two biomarkers (e.g., proteins, antigens, or the like) wherein at least one biomarker is detected in order to distinguish ltiple sclerosis from a disease with similar clinical manifestations.
|00H9) The present invention also provides a method of diagnosing multiple sclerosis at an eariy stage of the disease before all clinical criteria are fulfilled, thus justifying early initiation of a multiple sclerosis-appropriate therapy.
EXAMPLE 1
10012 1 The following example describes the detection of multiple sclerosis in a patient according to two methods disclosed In the present invention. A 24~year-o!d male patient presents to his primary care physician complaining of changes in vision, limb weakness, and extreme fatigue. He mentions his symptoms have been recurring over the last 3 months. The physician suspects the possibility of a tumor in the central nervous system (CMS) or a CNS disease, as well as multiple sclerosis. The physician obtains a blood sample to be sent to a diagnostic laboratory for multiple sclerosis testing, and also refers the patient to a neurologist.
|0012i I The laboratory receives the patient’s blood sample collected in a CRT tube, PSMCs are obtained from a SD Vacutainer™ CRT tube using a cell separation procedure. The cells are washed three times in IX PBS and centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1500 RCF (Relative Centrifugal force). The supernatant is removed, and the cells are resuspended In iX PBS, After the final wash, extracts of the PBMCs are prepared by lysing with a hypotonic solution o othe method. Then the lysate is subjected to assay Involving an antibody that binds to Tau, e g., a protein fragment comprising the phosphoryiated serine residue Ser*404, Th assay indicates that an abnormal level of said Tau protein fragment is present in the PBMCs. Thus, the results of the assay indicate that the patient has active CNS damage, which may be an indication that the patient has multiple sclerosis. The physician notifies the patient, who then begin treatment immediately,
EXAMPLE S
(00122] The following example describes a direct ELISA assay used for detecting an antigen in a sample of peripheral phagocytes. The protein concentration of the sample is determined using the Bio Rad™ (Bradford method) assay, MicroEL!SA plates are coated by addition of 100 pL of a 5- 20 pg/mL solution of the sample, which is then incubated for 1 hour at 2 O. The wells are washed out with phosphate buffere solution (PBS) with 0+QS% poiysorhate {Tween 20™}, The wells are then filled with 0 1 M glycine in PBS and incubated for 1 hour at 21 "C to block unoccupied binding sites. After rewashing the wells, 100 pL Of an appropriate dilution of antibody in PBS-0.05% Tween™ 20 with 1 % bovine serum albumin (BSA) is added and incubated for 1 hour at 2GO. The unboun antibody is then washed out with three exchanges of PBS-0.05% Tween™ 20. One hundred pL of an appropriately diluted horse radish peroxidase conjugated ants -immunoglobulin G (IgG) in PBS-0.05% Tween™ 20-1% BSA is then added to each well and incubated fo 1 hour at 2T"C. The wells are then washed twice with PBS~0.05% Tween™ 20 and finally with PBS. One hundred pL of soluble TB substrate solution Is added to each well and incubated for 30 minutes at 2TΌ after which 100 pL of MTB stop reagent is added and the color intensify is measured at 4S0nm using an ELISA plat reader. (00123] Appropriate dilutions of the antigen and antibody are established by performing checkerboard titrations. Antigen concentrations in samples are interpolated from standard curves.
EXAMPLE 3
100124] The following example describes an indirect ELISA assay used for detecting an antigen (isolated from: peripheral phagocytes) in various samples. This assay is constructed using polyclonal and monoclonal antibodies. ELISA wells are coated with polyclonal antibody at an appropriate concentration and the wells are washed and blocked as described above. Various dilutions of antigen containing samples are added to the wells and incubated for 1 hour at 2 O, after which the wells are washed 3 times with FBS-0.05 Tween™ 20. The monoclonal antibody is then added at an appropriate dilution in P8S-0.Q5% Tween™ 20 - 1 % BSA and incubated for 1 hour at 2T'C, The wells are then washed 3 times and an appropriately diluted horse radis peroxidase conjugated anti-mouse igM in PBS-0,05% TweenT? 20-1 % BSA is then added to each well and incubated for 1 hour at 2 V C The wells are then washe twice wit FBS-0.05% Tween™ 20 and finally with PBS. One hundred pL of soluble MTB substrate so!ution is added to each well an incubated for 38 minutes at 21 Ό after which 100 pL of MTB stop reagent is added and the color intensity is measured at 450n using an ELISA plate reader.
{00125] Appropriate dilutions of antigen and antibody are established by performing checkerboard titrations. Antigen concentrations in samples are interpolated from standard curves.
EXAMPLE 4
SOOl 26] The following example describes a flow cytometry assay used for detecting a antigen in various samples. PSMCs from multiple sclerosis ( S subjects and control subjects are stained with fluorescent antibodies to the multiple sclerosis-associated antigen (e.g., Tau protein) and also with fluorescent labeled antibodies to cluster designation (CD) 3 T-iymphocyte marker or CD 19 8- Lymphocyte marker, CD6S intracellular monocyte marker and CD 14 monocyte/ macrophage cell surface marker. The labeled cells are analyzed by flo cytometry for qualitative or quantitative differences.
100127] PBSVICs are obtained from a BD Vacutalher™ OPT tube using a ceil separation procedure. The ceils are washed three times in IX PBS an centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1300 RCF {Relative Centrifugal force). The supernatant is removed, and the cells are resuspended in 1 X PBS. After the final wash, the ceils are resuspended to approximatel 4.0 ml in IX PBS. Approximatel 50 pi, of the cell suspension to be analyzed is transferred into tubes for double staining with selected antibody pairs, Ten pL of 40mg/mL normal human SgG (Sigma-Aldrich) for a total of 4QQ pg is added to each tube to block FC binding. The appropriate ceil surface monoclonal antibodies CD3 PE, CD19 PE or CD14 PE are added at this time and incubated for 20 minutes at room temperature.
{00128] One hundred pi of Dako Intrastain™ Reagent A (fixative) is added to each tube and then mixed gently with a vortex mixer to ensure that the ceils are in suspension. Ceils are incubated at room temperature for 15 minutes. Two ml of IX PBS working solution is added to each test tube and mixed gently. The tubes are centrifuged at 300 X g for 5 minutes. Supernatant Is aspirated leaving about 50 pi of fluid. The fluid is mixed thoroughly to ensure that the ceils are in suspension, jOO129] One hundred pi of Dako Intrastain™ Reagent B (permeahiiization) is added to each tube. The appropriate amount of the antibody specific for the multipie sclerosis-associate antige is added to the appropriate tubes. The tubes are mixed gently to ensure that the cells are in suspension and incubated at room temperature for 15-60 minutes, Two ml of IX PBS working solution is added to each test tube and mixed gent!y. The tubes are centrifuged at 300 X g for 5 minutes, and then th supernatant Is aspirated, leaving approximately 50 pi of fluid. The fluid is mixed thoroughly to ensure that the cells are in suspension,
[00130 One hundred pL of Dako intrastain™ Reagent B (permeabization) is added to each tube. The appropriate vo!ume of the 2nd step antibod conjugated to FiTC (specific to the multiple sclerosis-associated antigen) is added to the appropriate tubes. The tubes are mixed gently to ensure that the ceils are in suspension and incubated at room temperature for 15-60 minutes. To each tube, 2.0 mis of 1XPBS working soSution is added. The tubes are mixed gently then centrifuged at 300 X for 5 minutes. The supernatant is aspirated, leaving approximately 50 pi of fluid, The tubes are mixed thoroughly to ensure that the ceils are i suspension.
[00131 j The pellet is resuspended in an appropriate volume of fiuid for flow cytometry analysis. The sample is analyzed on a Sow cytometer within 24 -48 hours. For analysis, the gate is on the monocyte population and the data is collected in list mode, Qualitative and or quantitative differences are determined between normal and MS patients using th analysi software. Optimization steps include varying incubation time with antibodies, fixation time and permeabilization time.
EXAMPLE 5
[0013 j Several factors currently impede therapy development and clinical study design for neuroprotective agents for Parkinson's disease. These include the limited ability to detect early stage PD prior to the onset of motor signs, inadequate measures of processes or pathways re !aied to disease pathogenesis, and the Sack of biomarkers that define disease progression. The present invention features the use of a new source of circulating biomarkers for use in diagnosis and monitoring of disease activity by exploiting the ability of monocytes/macrophages to enter the brain and engulf debris coupled with the paradigm shifting hypothesis that at least some of these ceils re-enter the circulation. The present invention isolates these re-circulating cells that have retrieved an igens from the brain ce!!s and looks for new and established CMS tissue markers. Unlike circulating antibody markers, which can remain in the circulation for years after the tissue insult, antigens engulfed b phagocytes are degraded within a few days. This means antigens are detected, they are reflective of resent central nervous system tissue Injury (e.o . “active" neurodeqenerative process). Given that 70-80% of slnato-nigrai neurons are lost before clinical symptoms arise, the clearance of the dead neurons by recruited and re-cireuiating mononuclear phagocytes should he an early indicator of neurons! damage. The present invention allows for rapidly identifying and develop potential biomarkers to improve the efficiency and outcome of Phase !i clinical trials and advance therapeutic development for PD or other appropriate diseases or conditions.
100133] Previous studies in multiple sclerosis and Cuprizone fed mice have shown that Tau, HippocaScin like 1 , myelin basic protein and proteolipid protein can be found by ELISA assay in !ysates of peripheral blood mononuclear cells (PBMCs). The present invention probes for these proteins in PBMCs from PD subjects, as well as alpha-synucSein, glutamic acid decarboxylase (GAD) and ubiqostin carboxyl-terminal esterase 11 (UCHL1) proteins. The methods of the present invention may also be used to discover potentially informative CMS antigens in PD PBMCs using a shotgun proieomic analysis. Hippoca!cin like 1 was discovered to be a phagocytosed biomarker using this approach,
100134] Using an EUSA employing specific antibodies to Identified candidate CHS antigens in PD PB C lysates, the prevalence of each marker can be determined. For example, the prevalence of these markers may be determined in a number of recently diagnosed {< 3 years) PP subjects and apparently healthy subjects.
JO0J 351 The cells containing the neural markers are characterized b immunophenotyping PBMCs. Coexistence of neural antigens with antigens specific for leukocyte subpopulations may be determined by use of specific antibodies, This may b achieved by flow cytometry, immunofluorescent microscopy and/or ce!S type specific enrichment/depletion using magnetic beads. The results may identify known phagocytic ceil types (CD14+ monocytes and/or macrophages (CO 88/CD11b)) to be the source of the neural antigens in PBMCs.
|00136 j in support of this concept, peripheral blood from 18 subjects with MS and 12 apparently healthy individuals was obtained. PBMCs were isolated and osmoticaliy lysed. The lysates were coaled onto EUSA wells at 5ug/ml and probed for Tau antigen or HippocaScin iike-1 antigen using standard protocols, Tau is a neuron specific microtubule associated protein that is best known as the substrate of neurofibrillary tangles in Alzheimer’s disease. Hsppoealeih i ike-1 is a neuron specific eafeiu sequestering protein that is most abundant In the hippocampus. The ELISA assay results for these antigens i this study are shown in FIG. 3.
100137] in the control group one individual gave anomalously high results in both the Tau assay and the HippocaSein Like-1 assay, Consequently, the second highest control was arbitrarily selected as a cut-off point for each assay. For each assay this resulted in 7/18 positive MS subjects and 1/12 positive controls. The results were not stafisticali significant for either antigen alone. However, analyzing the results for positivity for either of the two antigens resulted in 9/18 positive MS subjects end 1/12 positive controls (Table 0), which is a statistically significant difference. The results are shown in the 2x2 contingency table below (Table 6) and are statistically significant by the chi squared test, p=Q.Q235
{00138] Table 6 - 2x2 contingency table of positivity for Tau or Hippocatcin Like-1 (The two-tailed P value equals 0.0235)
Figure imgf000064_0001
100139] PBMCs from normal C57BI/0 mice and cuprizone fe C57B1/8 mice were examined for CNS antigens. Cuprizone causes De ye!ination at 3-4 weeks and also neurodegeneration at 4-5 weeks. Discontinuation of Cuprizone feeding at 6 weeks or earlier results in repair of the CNS damage. Five mice were fed with regular lab chow, two groups of five mice were fed with Cuprizone (0,2%) chow for 3 weeks and 4 weeks and one group of 4 mice were fed with Cuprizone (0.2%) chow for 5 weeks. The mice were bled and PBMCs isolated by Real! density gradient centrifugation. The PBMCs were lysed and the iysates were coated onto ELISA wells and assayed with antibodies to CNS antigens as with the human samples. The mouse iysates were probed for Tau, Hippoea!cin like-1 and myelin basic protein (MSP). FIG. 4, FiG. 5, and FIG. 6 show there is no difference in control and Cuprizone treated mice until 5 weeks of Cuprizone feeding, a time point when there is extensive histological evidence of demyeitnation and naurodagenaratiGm
I00140] Comparison of grouped assay results by unmatche 2 tailed t-test confirmed the visual conclusion that only the mice fed Cuprizone for 5 weeks were different from the control mice (Table 7),
Table 7; Comparison of Cuprizone fed mice with control mice using the t-test (2 tailed P values)
(*** statisticall significant difference)
Figure imgf000064_0002
|00l4 l ! These results are consistent with the hypothesis that some phagocytes that have engulfed debris re-enter the blood circulation and suggest that measurement of the phagocyte's cargo may be informative in measuring disease activity in the CNS, Note that Jo!y et al, {JoSy S et at, £009, Am J of Path, Voi. 174, No, 0) confirmed that resident and recruited phagocytes remove dead photoreceptor ceils from the retina in a rat mode! of retinal degeneration {see electron micrographs shown in FIG. 7, showing a macrophage loaded with photoreceptor ceil debris re-entering the b!ood circulation via a capillary). This cell has to be entering the capillary rather than leaving it as photoreceptor ceils are unique to the retina and therefore cannot have been acquired elsewhere. This study provided evidenc in support of Inventor’s methods herein that phagocytes do indeed re-enter the blood circulation,
|90142j St has not yet been determined which antigens will be most relevant to Parkinson's disease (PD). However, the methods of the present invention may still be applied to monitoring, detecting:, treating, managing, etc., Parkinson’s disease. The present invention may also be used to help determine the antigens most relevant to PD. For example, one strategy is to obtain PBMGs from Parkinson’s disease subjects and test them for the antigens that have found to be relevant in other CNS diseases, e.g., Tau, Hippocaicin iike-i and myelin basic protein. The PBMCs may also be probed for alpha synuciein, glutamic acid decarboxylase (GAD) and ubiquifln carboxyl-terminal esterase LI , Alpha synuciein is associated with PD pathology and GAD Is associated with "stiff man" syndrome, which may have some overla with symptoms of PD. Ubiqui!in earboxy! terminai esterase LI UGHL1) Is found in all neurons of the brain and should be present in recirculating phagocytes when there is active neurodegeneration present. Thus, UCHtl represents a safety net biomarker. UCHli polymorphisms have been reported to influence risk of development of PD. 00143} A shotgun proteomic approach may be employed for discovering ne antigens that may associate with PD. This method has been used to identify Hippocaicin like-1 as an antigen of interest. For example, PBMGs from short duration PD subjects may be utilized for new phagocytosed antigen discovery. Briefly, a lysate of PD PBMCs may be subjected to 1-D PAGE and the gel can be coohiassie stained. Coo assie stained protein bands may be cut out and subjected to in gel trypsin digestio followed by protein identification by LC-MS-MS. The list of proteins present will be examined for the presence of CNS proteins. Candidate biomarkers foun will be studied using either commercial antibodies if available or by contracted production of anti- peptide antibodies.
{001.44} The potential biomarkers selected for initial testing may be screened on lysates from a furthe short duration PD PBMC lysates by ELISA assay. As an example, any potential biomarker that is positive on two lysates will be a candidate for further screening A minimum of three such candidate antigens will be screened.
{00145] Candidate biomarkers may be screened on lysates from recently diagnosed {< 3 years) PD subjects to determine prevalence. Comparisons may also be made to apparently healthy control subjects. A single high prevalence (e.g., ~80%) antigen or a combination of antigens providing high prevalence in aggregate will be sought.
f00!46j As there is no pre-existing data on which to base power calculations, as an example, 50 subjects for each group may be selected as it would enable the detection of a minimum marker prevalence of 2% (1/50)
jO0147] The ceil typefs) carrying neural antigens in the PBMCs have not been specifically identified. Without wishing to li it the present invention to any theory or mechanism, it is assumed they are bone marrow derived mononuclear cells The cells containing the neural markers may be characterized by immunophenotyping PBMCs, Coexistence of neural antigens with antigens specific for !eukocyte subpopulations may be determined by use of specific antibodies. This may be achieved by flow cytometry, immunof!uorescent microscopy and/or celt type specific enrichment/depletion using magnetic beads. The resuits may identify known phagocytic ceil types (CD14+ monocytes and/or macrophages (CD 6S CD11 b)> to be the source of the neurai antigens in PBfv!Cs. Identifying the cell type carrying neurai antigens mat enable additional sample preparation steps that will enric the relevant cells and wiii be expected to increase sensitivity of the assays.
[00! 48] As an example, 2 x 8 b tubes of blood may be collected from PD subjects and the PBMCs prepared. Half of the PBMC are used to make a lysates and the other half temporarily cn/opreserved. The lysates are assayed to determine which neurai biomarkers are present and then the cryopresefved cells are resuscitated for analysis with CD markers for leukocyte sub- populations and the relevant neural antigens.
{00!49j Statistical analysis may be required to interpret the results, e g., the unmatched t-test may be used to determine whether the means of each group are significantly different from each other. A p-vaSiie of 0 05 or less will indicate statistical significance. Apparently healthy controls may be used to determine a cut off value (mean +2SD) in order to create categorical data that can be analyzed with 2 x contingency tables an the chi squared test. This may also enable expression of the results as positive and negative predictive values.
[00150] Proteomic studies may also be performed on PP PBMCs to detect potentially relevant CNS antigens for PD (e.g,, UCHL1 , Tau, etc.). Some antigens may have regional differences in abundance that may affect detect ability and ma therefore only be seen in particular ciinicai phenotypes.
[0015! j As previously discussed, the present invention provides methods for antigen retrieval fro the brain utilizing re-circuiating phagocytes as a shuttle vector. Because these antigens are short lived i phagocytes, the detection of their presence wi!l indicate current active neurodegeneration. Thus, the prevent invention provides for methods for monitoring disease activity in Parkinson's disease or other diseases wherein detecting certain antigens In re-circulating phagocytes can be indicative of active disease processes. As drugs that decrease disease activity wi!i be expecte to decrease CNS antigen laden phagocytes in the circulation, this approach may facilitate evaluation of response to therapy and eariy evaluation of likelihood of success of phase I! trials, etc.
EXAMPLE 6
100152) The following example describes a non-iimiting procedure for pre-anaiyfica! processing of blood samples.
{00153) Equipment/Reagents Needed: Centrifuge; SOmL conical tube; Storage vials for aliquots, - S0d capable, iOQuL-lmL (e.g, polypropySene mierocerstnfuge tubes); PBS - Phosphate Buffered Saline, pH 7.4; RBC lysis buffer {AGK}; Protease inhibitor (e g. G Bioscienees ProteaseArrest flQdXj); Magnetic rtanopartic!es/beads.
I00T54) Blood may be collected into an anticoagulant containing tube (Becton Dickinson) that also contains 1 mg of magnetic nano particies. A single 8 mL or 4 mL tube per subject may be received. Samples may contain the minimu volume specified by Table 8 (below). Samples for research may be collected under an !RB protocol. Phagocytes may be harvested within 38 hours of the blood draw. After receipt of sample, phagocytes are harvested by sedimentation on a magnet. While the phagocytes are held by the magnet, ail other blood components may be poured or aspirated off. The phagocytes are then washed twice with PBS.
Table 8
Figure imgf000067_0001
fOOlSS) Resuspend the pellet in the volume of deionized water specified by Table 8 to lyse the Phagocytes if processing multiple tubes from the same subject and collection time, the pellets from two tubes may be recombined in the total volume specified for one tube.
100156) Bring the magnetic nanoparticies to the side wail of the tube using the magnet then remove the ceil lysate with a microplpette and place in a fresh microfuge tube. Add the volume of protease inhibitor specified by Table 8. Add the volume of Cbioroguin specified by Table 8. Aliquot and store. Lysates are typically collected into 25~100uL aliquots.
)0O15?j Lysates may be stored at -80C, in a freezer monitored by an external monitor and labeled with the correct contact information to cat! if a failure is noted. Assays lysates may be conducted within 30 days of harvesting. Sample preparations may be frozen at least 24 hours prior to assaying. Label primary container (box or conical tube containing aliquots) with sample identifier, type of sample (plasma or PBMC lysate), date processed, and processor’s initials. After experimentation, samples may either be stored or destroyed according to the !RB protoco! under which they were collected
[00158 j An alternative process includes the use of magnetic nanoparticSes coated with ieupeptin, magnetic nanopartieles coated with a mix of ieupeptin and human igG(Fc), etc,
[001591 In some embodiments, in lieu of eh!oroquin, a carbonate/bicarbonate buffer may be used in some embodiments, in lieu of ehioroquin any weak base may be used, in some embodiments, in lieu of or in addition to a base, ieupeptin (e.g,, 0.25 mM) may be added. Synaptic biomarkers may inciude but are not limited to PSD-9S protein, neurogranln, SNAP-25, and TDP-43.
EXAMPLE 7
[00160 j This example describes a non-limiting procedure of an assay that may be used when assaying Peripheral Stood Phagocyte lysates for Tau, Myelin Basic Protein (MBP), UCH-L1 , or Hlppocalcin-iike~l (HPCAL1 ) debris.
[00161 i Eq u ipment/reagenis that may be needed may inciude: Clear poiystyrene Enzyme-linked immunosorbent assay (E-LISA) plate, protein binding (Pierce 15041); Specifophotametrie ELiSA plate reader capable of absorbance measurement at 450nm (e.g. BioTek ELxSOO); Human Tau- 441 isoform (2M4R) (e.g, rPeptide T- 1001-2); MBP peptide, derived from human brain {e.g, Erizo2OO-6O6-MQ01); HPCAL1 peptide (Aviva AAP34682); lysate samples; PBS - Phosphate Buffered Saline, pH 7.4; BSA - Bovine Serum Albumin (e.g. Sigma A7030); Tween 20 or PBS + 0,05% Tween20; Anti-human Tau, rabbit poiycionai SgG (e.g, Dako A0024); Anti-human MBP, mouse monoclonal IgG (e.g., Santa Cruz Biotechnology sc-2 1524); Anti-human UCH-L1 , mouse monoclonal IgG (e.g. Santa Cruz Biotechnology sc-271639); Anti-human HPCAL1 , rabbit poiycionai (e.g, Aviva ARP34682; Proteinteeh Group 1098S-i-AP); Anti-rabbit SgG, peroxidase labeled (e.g. Sigma A6154); Anti-mouse gG, peroxidase labeled (e.g. Sigma A2554); TMB - 3,3t5,5'-tetramethyibenzidine, Soluble substrate (e g. Caibiochem 613544); TM8 Stop Solution (e.g, KPL 50-85-05) or 2N HCt
[00! 62] Next, normalize lysate samples to 5ug/mL protein content, as assessed against RSA standard (in a commercial protein assay kit), in 1x P8S. Dilute standards frt PBS accordin to Table S.
Table 9
Figure imgf000068_0001
Figure imgf000069_0001
[00163] Leave 2-3 wells empty as blank. Add tOOut of standard dilution for each analyte under test in duplicate or triplicate into designated wells. Add lOOut. of samples, in duplicates or triplicates for each analyte under test, into their designated wells. Incubate for two hours af room temperature. Was four times with 1x PBS and blot dry the plate. Add 300ul per well of Block Solution, according to Table 3. Incubate for 2 hours at room temperature or 4<:C overnight. Wash four times with 1x PBS and blot dry the plate. Add 10OuL per well of primary antibody, according to Table 3, diluted in 0.1% B SA/0.05% Tween 20 -P 8 S . Incubate for 2 hours at room temperature. Wash four times with 1x PBS and blot dry the plate. Add fOOuL per well of HRP-iabeled secondary antibody, according to Table 9, dilute in 0,1% 8SA/0.0S% Tweeo20-PBS. Incubate 1 hour at room temperature. Wash four times with lx PBS and blot the plate dry. Add TOQuL per well of TMB sol ution. I ncubate 60 mi nutes at room te mperature. Add tOOul per well of TMB stop solution. Allow to equilibrate for 5 minutes, and then read absorbance 0,0. at 450nm within 30 minutes,
|00164] For calculation of results, average the optical density (O.D.) of duplicate or triplicate wells. Calculate th coefficient of variance %CV) for each set of duplicates or triplicates as; ($t d:ey{weiS-f, well¾ we¾)} * (average(weih, ells, well;:}) x 100, If a group of triplicates has a %CV greater than 1Q%, and two of the three are very similar, the thir value may be discarded from the average. If duplicates have a %CV greater than 10%, the sample should be re-tested. Plot the standard curve on a log-log scale. The known concentrations should be plotted on the X-axis, with the corresponding O.D. reading on the Y-axis). Fit the curve (or linear region) with a linear trendline. Determine the sample concentration by calculating the X value from substituting the corresponding O.D, into the trendline equation obtained in the standard curve,
EXAMPLE 8
[00165] This example describes the application of the phagocytic shuttle method described herein to other diseases such as relapse-remitting diseases (:e,g„ Lyme disease),
[00166] Lyme disease is contracted by bein bitten by a deer tick Infected with the bacterium Bomirn burgdorferi. Typically, people who are bitten develop a characteristic rash and develop flu like symptoms, which resolve within three weeks of antibiotic treatment. Some people have a recurrence of symptoms months or years after the first episode, The reasons for relapse remain controversial Two theories have been put forward to explain recurrence of symptoms; relapse due to treatment failure and reinfection through a second tick bite. Note that if recurrence were to be caused by a second bite, the bacteria may be genetically different In both instances but If recurrence is caused by treatment failure, the bacteria will be genetically the same. One study evaluated 22 pairs of infected blood samples and determined that all of the bacteria in these samples were geneticaiiy identical, making it unlikely that recurrence was due to a second tick bite,
100167] The present invention features methods for diagnosing Lyme disease, detecting a relapse of Lyme disease, monitoring Lyme disease, etc. For example, the methods herein can be used to retrieve hidden bacteria in disseminated relapsing Lyme disease [e.g., retrieve the bacteria fro macrophages in the blood), Briefly» blood samples may be isolated from patients, and the PBMCs (source of macrophages) may be isolated, extracted, and/or concentrated. Lysates of the cells of Interest will be analyzed for particular antigens or biomarkers of Interest associated with Ly e disease.
|00168| Non-limiting examples of antibodies to particular antigens or blomarkers associated with Borr iia burgdorferi (Lyme disease) is shown so Table 10 below.
Table 10
Figure imgf000070_0001
100169] Various modifications of the invention, in addition to those described herein, wili be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporate herein by reference in its entirety.
[00170] Although there has been shown and described the preferred embodiment of the present invention, if will be readily apparent to those skied in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is oniy to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office oniy and are not limiting in any way, in some embodiments, th figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. in some embodiments, the figures are representative only and the ciaims are not limited by the dimensions of the figures, in some embodiments, descriptions of the inventions described herein using the phrase“comprising'’ includes embodiments that could be described as“consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase“consisting of is met.
m

Claims

WHAT IS CLAIMED :»;
1. A method of creating a patient cohort of one or more patients for a clinical trial for testing a therapeutic composition or therapeutic intervention for an effect on centra! nervous system (CMS) tissue damage, CMS repair, or neurodegeneration, said metho comprising;
a. isolating circulating phagocytes from a fluid sample from eac patient, the fluid sample being from outside of a brain tissue of the patient;
b. detecting a level of a biomarker in the phagocytes from each patient, the biomarker being associated with CMS tissue damage, CNS syste repair, or neurodegeneration, wherein if the level of the biomarker in the phagocytes is abnormal, then the patient has active CNS disease: and
c. selecting only patients with active CMS disease to create the patient cohort for the clinical trial.
2. The metho of claim 1 , wherein the step of isolating circulating phagocytes from the fluid sample fro the patient further comprises concentrating the circulating phagocytes.
3, The method of claim 1 , wherein the circulating phagocytes are isolated or concentrated using forward-scattered light or side-scattered tight in flow cytometry.
4. The method of claim 1, wherein the circulating phagocytes are isolated or concentrated base on immunotype,
5. The method of claim 1 , wherein the circulating phagocytes have a specific immunotype, the immunotype is determined using a marker selected from; CDTta, CD110, CDi lc, CD 14, CD15 , CD33, CD84, CD8S, CDSO, CD88, CDlOS/endogiin, CD115.. CD163, GQ195/CCR5, CD2S2/TLR2, CD284/TLR4, HLA-DR/MHC Class II, ILT1 , ILT3, ILT4, ILT5, Mature Macrophage
CD 123, CD19
Figure imgf000072_0001
CD289/TLR9, CD303, CD304, CMKLR-I 00, or a combination thereof.
6. The method of claim i , wherein the biomarke |s Tau, phosphoryiated Tau, a fragment of Tau, hippocaicin-1, or 14-3-3 protein, MBP, UCH-Li , TDP-43, superoxide dismutase (SOD), heuromeianm, ©FAR, neurofiiafrients light chains (NFL), UCH-L1 , amyloid beta, aipha- synuciein, apo A«S, Apo E, Apo J, dC viral antibody titers, TGF~foefa, VEfSF, dopamine-ibeta- hydroxyiase (OBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FLJ78071, apolipoprofein C-lf, immunoglobulin heavy constant gamma 3, alpha-1-acid glycoprotein 1, aipha-1-acid glycoprotein 2, haptoglobin-related protein, leucine-rich aipha-2-giycoprotein, or C-reactlve protein,
7. The method of claim 1 , wherein the level of the biomarker Is compared to a predetermined threshold to determine if it is abnormal, the predetermined threshold is an industry standard.
8. The method: of claim 1 , wherein the level of the biomarker is compared to a predetermine threshold to determine if it is abnormal, the predetermined threshold is a laboratory standard.
9. The method of claim 1 , wherein the level of the biomarker Is compared fo a predetermined threshold to determine if it is abnormal, the predetermined threshold is a level of the biomarker in phagocytes isolated from a fluid sample obtained from the patient before administration of the therapeutic compositions,
10. A method of treating a patient suspected of having central nervous system tissue damage, central nervous system repair, or neurodegeneration, said method comprising;
a, isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a brain tissue of the patient;
b detecting a level of a biomarker in the phagocytes, the biomarker being associated with centra! nervous syste tissue damage, centra! nervous system repair, or oeurodegeneration, wherein if the level of the biomarker is abnormal then the patient has active central nervous system disease; and
c, administering to the patient having active central nervous system disease a therapeutic composition or therapeutic intervention effective for treating or preventing worsening of the centra! nervous system tissue damage, centra! nervous system repair, or neurodegeneration, or a symptom thereof.
11. The method of claim 10, wherein the central nervous system tissue damage, central nervous system repair, or neurodegeneration is associated with Multiple Sclerosis, Alzheimer's disease, mild cognitive impairment, Parkinson's disease, Multiple Syste Atrophy, Lewy body Disease, Progressive Supranuclear Atrophy, Corticobasa! Degeneration, Amyotrophic Lateral Sclerosis, Huntington's Disease, concussion, Traumatic Brain injury, REM sleep behavior disorder, or a disease causing secondary centra! nervous system damage.
12. The method of clai to, wherein the central nervous system tissue damage, centra! nervous system repair, or neurodegeneration causes cognitive impairment, motor disturbances, or both.
13. The method of ciaim 10, whereih the biomarker is Tau, phosphoryiated Tau, a fragment of Tau, hippoca!cin-1 or 14-3-3 protein, M8P, UGH-L1 , TDP-43, superoxide dismutas (SOD), neuromelanin, GEAR, neurofilaments light chains (NFL), UCH-LI, amyloi beta, a!pha- synuciein, apo A~l, Apo E, Apo J, JC viral antibody titers, TGF-beta, VEGF, dopamine-beta- hydroxyiase {DBB}, vitamin D binding protein, histidine-rich glycoprotein, cDJMA FLJ7S071 , apoSipoprotein G-tl, immunogiobuiin heavy constant gamma 3, aipha-1-acid glycoprotein 1, alpha-1-acid glycoprotein 2, haptog!obin-re!ated protein, ieucine-rich aipha-2-giycoprotein, or C-react!ve protein.
14. The method of ciaim 10, wherein the step of isolating circulating phagocytes from the fluid sample fro the patient further comprises concentrating the circulating phagocytes.
15. The method of claim 0, wherein the circulating phagocytes have a specific immunotype,
16. The method of claim 10;, wherein the circulating phagocytes are isolated or concentrate based on immunotype,.
17. The method of claim 10, wherein the therapeutic composition or therapeutic intervention is astinab, Dsciizumab, Zinferyta, ER-8eta agonist, cyciophosphamide, rHigM22, ponesimod, aipba-4 integrin, AMP-110, an antisense oiigonueieolide, immune to!erizing agent, MuitiStem, Kv1 ,3 Blocker, Aiemtuzumab, !FNtJ-Tb, 8HT-300Q-01 , !FN a, dimethyl fumaraie, IFNb-1a (PEGyfafed), Nataizuroab, T1301, Abatacept, RCP1063, a compound related to iamitrigene, CNM-Au8, Mesenchymal stem ceil transplant, apollpoprotein E-based, modified pepfidomsmetic, Aimspro, Anti-BAFF Human Ab, NPG-1308, GN ACI . Vatelizumab, Ofatumumab, ozanezumab (None- A mAb), Fir tegrast (SB-683699, T-0047), Interferon Alfa N3, Mitoxantrone, Ibudsiast, Simvastatin, Raifegravir, Piova er Acetate, Vedolizumab, Siponimod, Secukinuroab, Fsngoiimod, Copaxone, LSD1 -MA08, Amiionde, adrenocorticotropic hormone, Rifuximab, Ocrelizumab, Teof!unomide, giatiramer acetate, Laqumimod, TG-11Q1 (ubiituximah), Clemastine Fumarate, Lipolc Acid, Minocycline, Riiuzoie, MJS416, fibrin, ATX~MS1467, ARX424, Tcelna, TBC-4746, Feidetrex, PV-287, RNS80, R129S, VSN16R, Angiotensin AT2 agonist, GEH 120714, PDD1A, PB03a, Zonisamide, deferiprone, NTGELL, Caffine,, Ssradiptne. Rsvastigmine, Inosine, Rasagine, GM1 gangiioside, Nicotine, Exenaiide, Plogiitazone, Glycerol PhenySbutyraie, EPi-743, an anti-inflammatory drug, ANXQ05, AADvacl , 5HT-8, Verubestat PXT864, Donepezii, NPTOaa, RG1450, RG7412, PDE4E, PDE4B, MetbyltSiioninium chloride, T3D-959, MultiTEP, ABT957, Anti-Tau antibodies, Aducanumab, Soianezumab, or inbdja,
18. The method of claim 10, wherein the method is performed within a time frame from 1 to 48 hours after a head injury.
19, A method of treating a patient suspected of having a relapse-remitting disease or infection, said method comprising:
a. isolating circulating phagocytes from a fluid sample from the patient, the fluid sample being from outside of a brain tissue of the patient;
b. detecting a level of a biomarker in the phagocytes, the biomarker being associated with the relapse-remitting disease, wherein if the level of th biomarker Is abnormal: then the patient has active disease; and
c. administering to the patient having active central nervous syste disease a therapeutic composition or therapeutic interventio effective for treating or preventing worsening of the reiapse-remittiog disease, or a symptom thereof.
29 The method of claim 19, wherein the relapse-remitting disease is multipie sclerosis (MS), Lyme disease, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowei disease, human immunodeficiency virus (Hit/), neurosarcoidosis, or psoriasis.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022221303A1 (en) * 2021-04-12 2022-10-20 Zelosdx, Inc. Detection of neural-derived debris in recirculating phagocytes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140051084A1 (en) * 2010-11-29 2014-02-20 Tormod Fladby Methods and compositions for monitoring phagocytic activity
US20150233904A1 (en) * 2009-11-27 2015-08-20 Msdx, Inc. Detection of neurological diseases via measurement of neuromelanin in recirculating phagocytes
US20160201132A1 (en) * 2013-09-12 2016-07-14 Teva Pharmaceutical Industries Ltd. Gene expression biomarkers of laquinimod responsiveness

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150233904A1 (en) * 2009-11-27 2015-08-20 Msdx, Inc. Detection of neurological diseases via measurement of neuromelanin in recirculating phagocytes
US20140051084A1 (en) * 2010-11-29 2014-02-20 Tormod Fladby Methods and compositions for monitoring phagocytic activity
US20160201132A1 (en) * 2013-09-12 2016-07-14 Teva Pharmaceutical Industries Ltd. Gene expression biomarkers of laquinimod responsiveness

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHANVILLARD, CORALIE AND RAYMOND F JACOLIK; CARMEN INFANTE-DUARTE; RAMESH C NAYAK: "The role of natural killer cells in multiple sclerosis and their therapeutic implications", FRONTIERS IN IMMUNOLOGY, vol. 4, no. 63, 13 March 2013 (2013-03-13), pages 1 - 9, XP055296661 *
WHITE, VANESSA J., C. NAYAK RAMESH: "Re-circulating Phagocytes Loaded with CNS Debris: A Potential Marker of Neurodegeneration in Parkinson's Disease?", AIMS MEDICAL SCIENCE, vol. 2, no. 1, 15 February 2015 (2015-02-15), pages 26 - 34, XP055731144, DOI: 10.3934/medsci.2015.1.26 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022221303A1 (en) * 2021-04-12 2022-10-20 Zelosdx, Inc. Detection of neural-derived debris in recirculating phagocytes

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