WO2022221322A1 - Détection de débris dans des phagocytes recirculants - Google Patents

Détection de débris dans des phagocytes recirculants Download PDF

Info

Publication number
WO2022221322A1
WO2022221322A1 PCT/US2022/024482 US2022024482W WO2022221322A1 WO 2022221322 A1 WO2022221322 A1 WO 2022221322A1 US 2022024482 W US2022024482 W US 2022024482W WO 2022221322 A1 WO2022221322 A1 WO 2022221322A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
biomarker
binding moiety
neutrophils
glycoprotein
Prior art date
Application number
PCT/US2022/024482
Other languages
English (en)
Inventor
Vanessa White
Uwe R. Muller
Original Assignee
Zelosdx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US17/228,416 external-priority patent/US20220236288A1/en
Application filed by Zelosdx, Inc. filed Critical Zelosdx, Inc.
Publication of WO2022221322A1 publication Critical patent/WO2022221322A1/fr
Priority to US18/379,431 priority Critical patent/US20240036065A1/en

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the present invention relates to detection of compounds (e.g., proteins and/or other molecules) derived from neural tissue inside or displayed on the cell surface of recirculating phagocytes, which may be useful for detecting or monitoring or predicting risk for particular disease states or processing, including but not limited to disease states or processes related to brain health, such as a particular neurodegenerative disease, the presence of brain tissue damages, etc.
  • the present invention includes preparation of compounds (e.g., proteins and/or other molecules) derived from neural tissue, wherein the compounds are inside or displayed on the cell surface of recirculating phagocytes.
  • the present invention may include whole sample analysis, single-cell analysis, etc.
  • neural tissue-derived debris e.g., debris from the brain
  • PBMC preparations which excluded neutrophils.
  • neutrophils used PBMC preparations, which excluded neutrophils.
  • neutrophils used PBMC preparations, which excluded neutrophils.
  • neutrophils used PBMC preparations, which excluded neutrophils.
  • neutrophils used in the methods herein is not obvious since the phagocytic response of neutrophils has been primarily associated with pathogens and secretion of inflammatory mediators.
  • the presence, and/or amount of, and/or ratio and/or pattern of debris may be useful for detecting and/or monitoring various disease states or processes (such as but not limited to neurological disease conditions or brain tissue damage), or for determining a risk for particular disease states or processes.
  • various disease states or processes such as but not limited to neurological disease conditions or brain tissue damage
  • the present invention is not limited to biomarkers associated with neurological disease or conditions.
  • the present invention describes methods for detecting neural-derived debris inside or on the cell surface of circulating neutrophils, and methods for detecting and/or monitoring various states of brain health based on the neural-derived debris in said neutrophils.
  • samples from a patient may be subjected to an assay that allows for single cell analysis, e.g., analysis of individual neutrophils.
  • an assay that allows for single cell analysis, e.g., analysis of individual neutrophils.
  • whole blood is used.
  • a sample preparation is used such as but not limited to cell isolates, e.g., isolated neutrophils.
  • the assays may feature specific labeling and detection of neutrophils and the biomarkers associated with said neutrophils (e.g., inside and/or on the cell surface).
  • Methods include but are not limited to flow cytometry based assays, single cell ELISA, microscopy-based assays, etc.
  • the neutrophils are immobilized and stained for one or more brain-derived biomarkers (e.g., a biomarker that would only normally be found in brain or CNS tissue).
  • the present invention is not limited to single cell analysis.
  • neutrophils are isolated and then a lysate prepared for an assay to detect the neural-derived biomarkers.
  • the neutrophils may be obtained from peripheral blood, synovial fluid, cerebrospinal fluid (CSF), or other sources as described herein.
  • the phagocytes are obtained from blood, e.g., drops of blood obtained via venipuncture or a finger prick.
  • the present invention is not limited to the aforementioned samples or methods for obtaining samples.
  • the presence, and/or amount of, and/or ratio and/or pattern of debris may be indicative of various disease states or processes (such as but not limited to neurological disease conditions or brain tissue damage), or indicate risks for particular disease states or processes.
  • the present invention is not limited to biomarkers associated with neurological disease or conditions.
  • the present invention may provide close to real-time data on what is happening in the brain since that particular cargo may only be present inside or on the cell surface of the recirculating phagocytes for a certain length of time (e.g., a few days) before it is completely digested.
  • a patient or subject may refer to an animal such as but not limited to a mammal. Mammals may include but are not limited to primates (e.g., a human, non-human primates), a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a goat, a horse, or a cow.
  • primates e.g., a human, non-human primates
  • a mouse e.g., a human, non-human primates
  • a mouse e.g., a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a goat, a horse, or a cow.
  • the present invention is not limited to the aforementioned subjects or patients.
  • the present invention also includes methods for preparation of cells and/or CNS-derived debris for analysis.
  • the present invention includes methods of preparing neutrophils containing CNS-derived (e.g., brain-derived, neural-derived) compounds (e.g., the debris or biomarkers that would only normally be found in CNS tissue such as but not limited to brain) for analysis.
  • CNS-derived e.g., brain-derived, neural-derived
  • the present invention also features preparation of the CNS-derived compounds found in the circulating neutrophils.
  • the present invention also features preparation of blood samples for analyzing the CNS-derived compounds found in the circulating neutrophils.
  • the present invention is not limited to isolation of circulating neutrophils and creating a lysate.
  • the present invention also includes methods using whole blood.
  • the present invention also includes methods for single-cell analysis.
  • Non-limiting examples of methods described herein include flow cytometry, ELISA, FACS, and fluorescent staining.
  • the analysis is single cell analysis, e.g., using flow cytometry, single cell ELISA, etc.
  • flow cytometry single cell ELISA
  • single cell ELISA cells are captured and immobilized.
  • the cells are permeabilized and antibodies are added directed to biomarkers and subsequently subjected to imaging (e.g., via microscopy, direct imaging, etc.).
  • the cells are immobilized and lysed, and biomarkers are captured via specific antibodies that have also been immobilized on the same surface.
  • the present invention includes the use of systems (e.g., microfluidic devices, etc.) that allow single cell trapping and analysis by ELISA. Details can be found in Yin and Marshall (2012, Cur. Op. Biotechnology 23:110-119), Spiller et al. (2010, Nature 465:736-745), among others.
  • the present invention includes the use of systems (e.g., microfluidic devices, etc.) that allow single cell trapping and analysis by ELISA. The present invention is not limited to these particular methods or systems for single cell analysis.
  • the methods herein for preparing central nervous system (CNS)-derived (e.g., brain-derived) compounds may comprise introducing to a whole blood sample obtained from outside central nervous system (CNS) tissue of a subject a first detectable binding moiety specific for circulating neutrophils and a second detectable binding moiety specific for a CNS-derived molecule, the first detectable binding moiety being differentially detectable from the second detectable binding moiety; subjecting the preparation to single-cell analysis for detecting the first detectable binding moiety and second detectable binding moiety; and analyzing the CNS-derived molecules in the preparation.
  • CNS central nervous system
  • the method comprises producing a preparation comprising CNS-derived molecules by introducing to a whole blood sample obtained from outside central nervous system (CNS) tissue of a subject a first detectable binding moiety specific for circulating neutrophils and a second detectable binding moiety specific for a CNS-derived molecule, the first detectable binding moiety being differentially detectable from the second detectable binding moiety; and analyzing the CNS-derived molecules in the preparation.
  • CNS central nervous system
  • the methods herein for preparing and/or analyzing central nervous system (CNS)-derived (e.g., brain-derived) compounds may comprise single-cell analysis of circulating neutrophils from a fluid sample obtained from outside central nervous system (CNS) tissue of a subject; and analysis of the CNS-derived compounds in the cells.
  • CNS central nervous system
  • the present invention also provides methods for preparing and/or analyzing CNS-derived compounds wherein the CNS-derived compound is displayed on the cell surface of the neutrophils.
  • the method may comprise extracting circulating neutrophils from a fluid sample obtained from outside central nervous system (CNS) tissue of a subject; and producing a fraction of the extracted circulating neutrophils by separating neutrophils with membrane-bound CNS-derived peptides/compounds from neutrophils without membrane-bound CNS-derived peptides/compounds.
  • the fraction of the neutrophils may comprise the neutrophils with membrane-bound CNS-derived peptides/compounds.
  • the method further comprises analyzing the neutrophils in the fraction.
  • the method comprises lysing the whole sample as described herein, rather than first extracting the circulating phagocytes.
  • the method comprises single-cell analysis as described herein.
  • Nanoparticles may be used to determine the presence and/or amount of a particular biomarker (e.g., epitope) in a particular cell or group of cells.
  • the nanoparticles are noble metal nanoparticles or alloys of noble metals.
  • the nanoparticles are gold nanoparticles, silver nanoparticles, or a combination thereof.
  • the nanoparticles are rods, spheres, or a combination thereof.
  • the nanoparticles have a diameter of 2 nm to 250 nm.
  • the biophysical properties refer to the adsorption or emission of electromagnetic waves by the nanoparticles in response to incident electromagnetic waves. In some embodiments, the biophysical properties refer to surface plasmon resonance. In some embodiments, the differential biophysical properties are measured by dynamic light scattering or tunable resistive pulse sensing.
  • the present invention also includes methods for measuring a biomarker inside of or displayed on a cell surface of neutrophils in a sample from a subject.
  • the method comprises labeling neutrophils with a neutrophil-specific binding moiety; the method may further comprise staining at least one target biomarker molecule with a labeled target biomarker-specific binding moiety.
  • the target biomarker is or comprises a compound derived from central nervous system tissue that is within or displayed on the cell surface of the phagocytes.
  • the method may further comprise analyzing the binding moieties, e.g., measuring a ratio, measuring an amount, etc.
  • the binding moiety is a fluorescently labeled binding moiety.
  • the neutrophils are immobilized on a solid surface prior to staining. Solid surfaces may include but are not limited to microscope slides. In some embodiments, the solid surface is coated with one or more phagocyte-specific binding moieties, and neutrophils are immobilized on the solid surface through binding to these moieties. In some embodiments, the neutrophils are immobilized on a solid surface directly from blood, and other cells and blood components are removed from said surface through washing of the surface.
  • the binding moieties are monoclonal or polyclonal antibodies, single-chain antibodies, single-chain proteins, binding peptides, or aptamers.
  • biomarkers or biomarker fragments are detected inside or displayed on the surface of the neutrophils, and detection is possible via attachment of labeled fragment-specific binding moieties.
  • the methods may be used to detect one biomarker.
  • the methods are used to detect two or more target biomarkers simultaneously.
  • the method detects two or more epitopes located on one or more target biomarker molecules.
  • the method comprises applying three or more binding moieties, each specific for a different epitope located on one or more target biomarker molecules.
  • two or more binding moieties are used to detect two or more structural conformations of the target biomarker.
  • a binding moiety used to detect the target biomarker is specific for a particular structural conformation of the target biomarker.
  • the structural conformation is a result of degradation or digestion of the target biomarker. In some embodiments, the structural conformation is a result of degradation or digestion of the target biomarker. In some embodiments, multiple different biomarkers are detected simultaneously by use of differentially labeled biomarker-specific binding moieties.
  • the CNS-derived molecule is GFAP. In some embodiments, the CNS-derived molecule is Tau. In some embodiments, the CNS-derived molecule is GFAP, Tau, or both. In some embodiments, the CNS-derived molecule comprises one or a combination of: Tau, phosphorylated Tau, hippocalcin-1 , 14-3-3 protein, MBP, UCH-L1, TDP-43, superoxide dismutase (SOD), neuromelanin, glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL), neurofilament heavy chain (NFH), neurofilament medium chain (NFM), phosphorylated NFL, phosphorylated NFH, phosphorylated NFM, internexin (Int), peripherin, UCH-L1, amyloid beta, alpha-synuclein, apo A-l, Apo E, Apo J, a viral antigen, a JC viral antigen, TGF
  • the present invention is not limited to the aforementioned biomarkers or antigens.
  • the biomarker may be a partially digested version of the aforementioned biomarkers.
  • the target biomarker molecule is associated with neurologic disease or neurotrauma. The biomarker may be selected based on its association with a particular disease or condition.
  • the level of the biomarker detected inside of or displayed on the cell surface of the neutrophils is compared to a predetermined threshold to determine if it is abnormal.
  • the predetermined threshold may be an industry standard.
  • the predetermined threshold is a laboratory standard.
  • the predetermined threshold is a patient standard.
  • the predetermined threshold is a level of the biomarker (or biomarkers) in neutrophils isolated from a fluid sample obtained from the patient at a particular time point, e.g., before administration of one or more therapeutic compositions.
  • Each particular biomarker of interest may have its own predetermined threshold.
  • the threshold or control may be a level of the particular biomarker or panel of biomarkers in non-diseased control samples. In some embodiments, the predetermined threshold or control is a level of the particular biomarker or panel of biomarkers at a given time point.
  • the present invention is not limited to the aforementioned examples of thresholds or controls.
  • central nervous system tissue damage, central nervous system repair, neurodegeneration or inflammation is associated with Multiple Sclerosis, Alzheimer’s disease, mild cognitive impairment, Parkinson’s disease, Multiple System Atrophy, Lewy body Disease, Progressive Supranuclear Atrophy, Corticobasal Degeneration, Amyotrophic Lateral Sclerosis, Huntington’s Disease, concussion, Traumatic Brain Injury, Chronic Traumatic Encephalopathy, Macrocephaly, Hydrocephalus, Cerebral Hypoxia, REM sleep behavior disorder, or a disease causing secondary central nervous system damage.
  • the central nervous system tissue damage, central nervous system repair, or neurodegeneration causes cognitive impairment, motor disturbances, or both.
  • the present invention also features methods for creating patient cohorts (e.g., of one or more patients) for a clinical trial, e.g., a clinical 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, neurodegeneration, etc. may be associated with a particular disease, condition, trauma, etc., as described herein.
  • the method features detecting a biomarker or antigen from, or produced by, the central nervous system (e.g., a brain antigen, etc.) utilizing the methods described herein.
  • the method comprises: selecting at least one patient with active CNS disease or trauma by detecting a level of a biomarker inside of or displayed on the cell surface of the phagocytes from the fluid sample of the patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneration, etc., using the methods described herein.
  • the fluid sample is from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient.
  • the level of the biomarker inside of or displayed on the cell surface of the phagocytes is abnormal, then the patient has active CNS disease or trauma.
  • the method may comprise: administering to the at least one patient with active CNS disease or trauma the therapeutic composition or therapeutic intervention; and detecting a level of a biomarker inside of or displayed on the cell surface of the phagocytes from the fluid sample of the patient, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneration, etc., using the methods described herein.
  • the fluid sample is from outside of a central nervous system tissue (e.g., outside of the brain tissue) of the patient, e.g., after a predetermined amount of time following administration of the therapeutic composition or therapeutic intervention.
  • the therapeutic composition or therapeutic intervention has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration; (b) if the level of the biomarker inside of or displayed on the cell surface of the phagocytes from a patient is within a predetermined threshold associated with improved CNS disease or trauma, then the therapeutic composition or therapeutic intervention has a positive effect on CNS tissue damage, CNS repair, or neurodegeneration; or (c) if the level of the biomarker inside of or displayed on the cell surface of the phagocytes from a patient is improved compared to a level of the biomarker in phagocytes in a sample obtained from 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.
  • the predetermined threshold may be an industry standard, a laboratory standard, a patient standard, etc.
  • Each particular biomarker of interest may have its own predetermined threshold.
  • a level of biomarker above the predetermined threshold is an abnormal level of the biomarker.
  • a level of biomarker below the predetermined threshold is an abnormal level of the biomarker.
  • the threshold or control may be a level of the particular biomarker or panel of biomarkers in non-diseased control samples.
  • the predetermined threshold or control is a level of the particular biomarker or panel of biomarkers at a given time point. The present invention is not limited to the aforementioned examples of thresholds or controls.
  • the therapeutic composition or therapeutic intervention may include but is not limited to: Mastinab, Daclizumab, Zinbryta, ER-Beta agonist, cyclophosphamide, rHlgM22, ponesimod, alpha-4 Integrin, AMP-110, an antisense oligonucleotide, Immune tolerizing agent, MultiStem, Kv1.3 Blocker, Alemtuzumab, IFNb-1b, BHT-3009-01, IFNb-1a, dimethyl fumarate, IFNb-1a (PEGylated), Natalizumab, MT1301, Abatacept, RCP1063, a compound related to lamitrigene, CNM-Au8, Mesenchymal stem cell transplant, apolipoprotein E-based, modified peptidomimetic, Aimspro, Anti-BAFF Human Ab, NDC-1308, GNbACI, Vatelizumab, Of
  • the present invention also features methods of selecting a candidate for monitoring mild cognitive impairment or Parkinson’s disease.
  • the method comprises detecting one or more biomarkers of interest using the methods described herein.
  • the method may comprise selecting the patient for monitoring for mild cognitive impairment or Parkinson’s disease based on the patient having active central nervous system disease.
  • the candidate has REM sleep behavior disorder (RBD).
  • the biomarker may include but is not limited to dopamine-beta-hydroxylase (DBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FLJ78071, apolipoprotein C-ll, immunoglobulin heavy constant gamma 3, alpha-1-acid glycoprotein 1, alpha-1-acid glycoprotein 2, haptoglobin-related protein, leucine-rich alpha-2-glycoprotein, C-reactive protein, Tau, phosphorylated Tau, hippocalcin-1 , or 14-3-3 protein, MBP, UCH-L1, TDP-43, superoxide dismutase (SOD), neuromelanin, GFAP, neurofilaments light chains (NFL), UCH-L1, amyloid beta, alpha-synuclein, apo A-l, Apo E, Apo J, JC viral antibody titers, TGF-beta, VEGF,
  • DBP dop
  • an autoimmune disease or condition associated with neurodegeneration may be multiple sclerosis.
  • the disease or condition causing cognitive impairment may be Alzheimer’s disease, or Lewy body disease.
  • the disease or condition causing motor disturbances is Parkinson’s disease, Multiple Sclerosis, Supranuclear Atrophy, or Corticobasal Degeneration.
  • 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 preservation 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 is not limited to the aforementioned methods or compositions.
  • biomarkers that are associated with particular disease states of interest e.g., biomarkers found in the re-circulating phagocytes as described herein
  • biomarkers that are associated with particular disease states of interest e.g., biomarkers found in the re-circulating phagocytes as described herein
  • the methods herein are not necessarily limited by the particular biomarker but instead features the phagocytic shuttle wherein the phagocytes are shuttles for CNS-derived debris indicative of processes occurring in the CNS and steps for detecting the biomarkers inside or on the surface of the shuttle phagocytes
  • the present invention includes those biomarkers that will be discovered in the future.
  • the present invention is not limited to fluorescent assays, e.g., fluorescent microscopy or imaging.
  • the methods herein comprise colorimetric assays.
  • the methods may comprise a colorimetric ELISA.
  • the methods herein comprise imaging without a microscope.
  • the methods herein comprise using an image analysis system, which may provide images from surfaces such as a slide or a plate (e.g., microplate well), etc.
  • the methods herein may be used for methods of detecting biological changes in CNS tissue.
  • the methods may be performed in lieu of obtaining imaging of the subject or obtaining a biopsy.
  • FIG. 1 shows the results of GFAP fluorescent imaging and cell counts.
  • Three image planes GFAP1, GFAP2, GFAP3 from one sample were imaged at 5x and fluorescent cells (DAPI[350]/CD14[568]/GFAP[488]) were counted using ImageJ software. Cell counts were fairly high (low thousands). This sample contained on average 10.8% CD14+ cells and of those cells roughly 1.7% were GFAP+ (0.19% of total). There were a few cells which were GFAP+/CD14-.
  • FIG. 2 shows the results of Tau fluorescent imaging and cell counts.
  • Three image planes Tau1, Tau2, Tau3 from one sample were imaged at 5x and fluorescent cells (DAPI[350]/CD14[568]/TAU[488]) were counted using ImageJ software. Cell counts were somewhat lower (due to division of sample between several slides for troubleshooting). This sample contained on average 8.4% CD14+ cells and of those cells roughly 8.7% were TAU+ (0.7% of total). There were a few cells which were TAU+/CD14-.
  • the present invention describes methods for detecting neural-derived debris inside or on the cell surface of circulating neutrophils, and methods for detecting and/or monitoring various states of brain health based on the neural-derived debris in said neutrophils.
  • the methods of the present invention may comprise producing a preparation comprising CNS-derived molecules by introducing to a whole blood sample obtained from outside central nervous system (CNS) tissue of a subject a first detectable binding moiety specific for circulating neutrophils and a second detectable binding moiety specific for a CNS-derived molecule, the first detectable binding moiety being differentially detectable from the second detectable binding moiety; subjecting the preparation to single-cell analysis for detecting the first detectable binding moiety and second detectable binding moiety; and analyzing the CNS-derived molecules in the preparation.
  • CNS central nervous system
  • the single-cell analysis is flow cytometry.
  • the single-cell analysis is a single cell enzyme linked immunosorbent assay (ELISA).
  • the single-cell analysis is a microscopy-based assay.
  • the single-cell analysis comprises placing the preparation on a solid surface, using said surface as a wave guide for illumination, and imaging by direct charge-coupled device (CCD).
  • the first detectable binding moiety, the second detectable binding moiety, or both comprise a fluorescent label, a fluorescent antibody, a nanoparticle, a quantum dot, or a tag.
  • the present invention also features methods for measuring a biomarker inside of or displayed on a cell surface of neutrophils in a sample from a subject.
  • the method comprises staining neutrophils in the sample from the subject with a labeled first binding moiety specific for neutrophils, and staining at least one target biomarker molecule with a labeled second binding moiety specific for the target biomarker, the first binding moiety can be differentiated from the second binding moiety, the target biomarker being a compound derived from central nervous system tissue that is within or displayed on the cell surface of the neutrophils; and detecting the first binding moiety and the second binding moiety, wherein the relationship between the amount or distribution of the first binding moiety and second binding moiety is indicative of an amount of target biomarker molecules inside or displayed on the cell surface of said neutrophils.
  • the sample is whole blood.
  • detecting the binding moieties comprises subjecting the sample to flow cytometry or ELISA.
  • the neutrophils are immobilized prior to staining.
  • the labels of the binding moieties are fluorescent labels.
  • the target biomarker molecule is associated with neurologic disease or neurotrauma.
  • the target biomarker may be one or a combination of: Tau, phosphorylated Tau, hippocalcin-1 , 14-3-3 protein, MBP, UCH-L1, TDP-43, superoxide dismutase (SOD), neuromelanin, glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL), neurofilament heavy chain (NFH), neurofilament medium chain (NFM), phosphorylated NFL, phosphorylated NFH, phosphorylated NFM, internexin (Int), peripherin, UCH-L1, amyloid beta, alpha-synuclein, apo A-l, Apo E, Apo J, a viral antigen, a JC viral antigen, TGF-beta, VEGF, dopamine-beta-hydroxylase (DBH), vitamin D binding protein, histidine-rich glycoprotein, cDNA FLJ78071, apolipoprotein C
  • the present invention is not limited to fluorescence microscopy or fluorescently labeled binding agents (e.g., antibodies). Any appropriate imaging system may be considered (e.g., colorimetric, etc.).
  • the sample is a blood sample.
  • the present invention is not limited to blood samples and may include other fluids such as but not limited to cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • a subject includes a mammal.
  • a mammal may include but is not limited to a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a goat, a horse, or a cow.
  • the present invention is not limited to the aforementioned subjects or patients.
  • the blood sample is one or a few drops of blood (e.g., from a blood draw, a finger prick, etc.) ⁇
  • the blood sample may be 1-2 drops of blood, 1-4 drops of blood, 4-10 drops of blood, etc.
  • the present invention is not limited to the aforementioned volumes of samples.
  • the detection methods herein are advantageous.
  • the methods herein do not require a large amount of sample (e.g., a few drops of blood may be obtained from a finger prick).
  • the methods herein may further comprise introducing to the sample a molecule for inhibiting degradation (or further degradation) of the neural-derived compound (e.g., CNS-derived compound, CNS-derived debris, etc.) in or on the phagocytes.
  • a molecule for inhibiting degradation (or further degradation) of the neural-derived compound e.g., CNS-derived compound, CNS-derived debris, etc.
  • the neural-derived compound e.g., CNS-derived compound, CNS-derived debris, etc.
  • any component that increases the pH of the phagolysosomes, which would inhibit the enzymes in the phagolysosomes may 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 phagolysosomal protease inhibitors.
  • the protease 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., chloroquin, carbonate/bicarbonate buffer, buffers of pH 9.2 or above, weak base buffers, quinine, etc.
  • both a phagolysosomal inhibitor and alkaline buffer are added.
  • a protease 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 sample is obtained.
  • the phagocytes may be obtained, collected, concentrated, etc. via a variety of means.
  • methods may feature a cell-affinity chromatography system herein the phagocytes interact with and/or bind a ligand immobilized on a system such as a filter, a membrane, a slide, a column, etc.
  • the phagocytes may then be eluted after being captured by the chromatography system.
  • the ligand is an antibody that is specific for the cell type of interest, e.g. the phagocyte.
  • the chromatography system may feature a spin column with a resin displaying a phagocyte-specific antibody, wherein the sample (e.g., blood) is introduced to the spin column.
  • the system features a slide displaying a phagocyte-specific antibody, wherein the sample (e.g., blood) is introduced to the slide.
  • the system features a syringe with a membrane displaying a phagocyte-specific antibody, wherein the sample (e.g., blood) is introduced to the syringe.
  • the method comprises introducing magnetic beads to the sample, whereupon phagocytes engulf the magnetic beads, yielding magnetic phagocytes.
  • the method may further comprise separating the magnetic phagocytes using a magnetic separation mechanism.
  • the method comprises introducing to the sample a stimulator to stimulate phagocytosis of the magnetic beads by the phagocytes.
  • the magnetic beads are conjugated with an acid hydrolase inhibitor.
  • the magnetic beads are conjugated with an antibody or antibody component to stimulate phagocytosis.
  • the magnetic beads are introduced to the first fluid sample 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 magnetic beads/particles are coated with a phagolysosomal inhibitor (e.g., leupeptin).
  • the magnetic beads/particles are coated with a mix of compounds, e.g., a phagolysosomal inhibitor (e.g., leupeptin), an antibody (e.g., IgG, IgG(Fc), etc.).
  • a phagolysosomal inhibitor e.g., leupeptin
  • an antibody e.g., IgG, IgG(Fc), etc.
  • the magnetic separation mechanism comprises a magnetic column or magnetic rack.
  • the container (for the blood sample) comprises Ficoll.
  • the container (for the blood sample) does not comprise Ficoll or is free of Ficoll.
  • the magnetic phagocytes are separated using the magnetic separation mechanism 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 sample. In some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism within 48 hours of harvesting of the first fluid sample. In some embodiments, the magnetic phagocytes are separated using the magnetic separation mechanism after the sample has been stored for a period of time.
  • the methods herein may comprise subjecting the sample fluorescence-activated cell sorting (FACS).
  • FACS Fluorescence-activated cell sorting
  • FACS Fluorescence-activated cell sorting
  • FACS is a type of flow cytometry that sorts a mixture of biological cells, one at a time, into separate containers based upon the specific light scattering and fluorescent characteristics of each cell. It provides quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
  • a current of a rapidly flowing 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 stream of cells to break into individual droplets, and the system is adjusted so that there is a low probability of more than one cell 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 is not limited to fluorescent assays, e.g., fluorescent microscopy or imaging.
  • the methods herein comprise colorimetric assays.
  • the methods may comprise a colorimetric ELISA.
  • the methods herein comprise imaging without a microscope.
  • the methods herein comprise using an image analysis system, wherein images may be obtained from surfaces such as a slide or a plate (e.g., microplate well), etc.
  • the methods herein comprise sorting the phagocytes using a magnetic mechanism, e.g., magnetic extraction.
  • the phagocytes are stained with a labeled phagocyte-specific binding moiety.
  • a target biomarker e.g., a CNS-derived biomarker inside or on the surface of phagocytes
  • the methods may further comprise measuring a ratio of the first color to the second color, wherein the ratio of colors is indicative of an amount of target biomarker molecules inside or displayed on the cell surface of said phagocytes.
  • the circulating phagocytes have a specific immunotype. In some embodiments, the circulating phagocytes are concentrated. In some embodiments, the circulating phagocytes are concentrated based on immunotype.
  • the methods herein may also comprise introducing a factor or combination of factors to the sample and/or the phagocytes and/or the fraction, wherein the fraction helps prevent apoptosis of the phagocytes.
  • factors that may be introduced includes epidermal growth factor (EGF), fetal bovine serum (FBS), other growth factors, a nutrient-rich medium, etc.
  • the present invention also features methods for preservation of samples for preserving the amount and/or structure and/or location of the CNS-derived 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 preservation 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 is not limited to the aforementioned methods or compositions.
  • the phagocytes obtained from the sample are permeabilized.
  • the phagocytes are lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
  • Various neural-derived debris antigens or biomarkers may be found inside or on the surface of recirculating phagocytes in the peripheral blood.
  • Such debris antigens may be used to detect (or monitor) neurodegenerative or neuroinflammatory diseases (e.g., diseases as described herein). Debris from other diseases, such as relapse-remitting diseases, may be retrieved specifically from circulating phagocytes as well.
  • the biomarker may be present inside or on the surface of a circulating phagocyte, e.g., peripheral phagocyte.
  • a peripheral phagocyte may be obtained from cerebrospinal fluid (CSF).
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • Such circulating phagocytes may be found in tissues, cells, and/or fluids in the body, for example in blood, peripheral blood mononuclear cells (PBMCs), synovial fluid, cerebrospinal fluid (CSF), central nervous system tissues, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreal fluid, urine the like, or a combination thereof.
  • the biomarker is an intracellular component.
  • the biomarker may be obtained from within a macrophage.
  • the macrophage sample is permeabilized.
  • 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.
  • the biomarkers or antigens or neural-derived biomarkers (or neurodegenerative disease-associated proteins or biomarkers), or fragments thereof include but are not limited to neuromelanin, a Tau protein (or fragment thereof), a Tau protein or fragment thereof comprising a phosphorylated residue (e.g., a phosphorylated serine reside, a phosphorylated threonine reside; e.g., serine 214, serine 235, serine 262, serine 356, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531, serine 552, serine 610, serine 622, serine 641, serine 713, serine 721, serine 726, serine 730, serine 739, threonine 181, threonine 205, threonine 470, threonine 492, threonine 498, threon
  • neuromelanin may be detected in the debris of degenerated dopaminergic neurons in recirculating phagocytes. Abnormal neuromelanin levels may be associated with Parkinson’s disease.
  • the term Tau biomarker may refer to full-length Tau, a fragment thereof, a particular epitope of Tau, e.g., an epitope within a particular region of amino acids.
  • the epitope is in a region from aa 240-441. In some embodiments, the epitope is in a region from aa 243-441. In some embodiments, the epitope is in a region from aa 244-274. In some embodiments, the epitope is in a region from aa 275-305. In some embodiments, the epitope is in a region from aa 306-336. In some embodiments, the epitope is in a region from aa 337-368.
  • the epitope is in a region from aa 388-411. The present invention is not limited to these regions. Further, the epitope may be in shorter regions of amino acids, e.g., aa 244-260, aa 270-280, aa 290-310, aa 330-360, etc.
  • 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 method comprises detecting an epitope of Tau in the region from aa 244-274.
  • the method comprises detecting an epitope of Tau in the region from aa 275-305. In some embodiments, the method comprises detecting an epitope of Tau in the region from aa 306-336. In some embodiments, the method comprises detecting 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.
  • phosphorylation of Tau can decrease its solubility.
  • the method comprises detecting a level of insoluble Tau protein in the sample.
  • an increased level of insoluble Tau protein as compared to a control level of insoluble Tau protein is indicative of the disease or condition of interest or a risk thereof.
  • a combination e.g., a pair
  • biomarker-specific antibodies is used for isolating and detecting the biomarker of interest.
  • the present invention includes a combination of any of the antibodies disclosed herein or antibodies specific to the biomarker of interest not necessarily listed herein, e.g., those that may be produced in the future, those that are commercially available, etc.
  • the biomarkers of interest are being cleaved or modified in a way that makes detection more difficult than isolating or detecting recombinant versions of the biomarker.
  • a commercial antibody that is able to detect a recombinant biomarker may not work well to detect the biomarker.
  • 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.
  • biomarker(s) is a neural-derived biomarker.
  • the biomarker(s) is not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
  • the biomarker may be of various lengths. For example, in some embodiments, the biomarker is from 5 to 20 amino acids. In some embodiments, the biomarker is from 20 to 40 amino acids. In some embodiments, the biomarker is from 40 to 80 amino acids.
  • the biomarker is from 80 to 150 amino acids. In some embodiments, the biomarker is from 150 to 200 amino acids. In some embodiments, the biomarker is from 200 to 300 amino acids. In some embodiments, the biomarker is from 300 to 400 amino acids. In some embodiments, the biomarker is from 400 to 500 amino acids. In some embodiments, the biomarker is from 500 to 600 amino acids.
  • the biomarker may comprise various regions of the full-length protein.
  • the biomarker comprises the amino-terminus (e.g., N-terminus, NH2-terminus, N-terminal end, amino-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.
  • the biomarker comprises about the first 25 amino acids.
  • the biomarker comprises about the first 50 amino acids.
  • the biomarker comprises about the first 75 amino acids.
  • the biomarker comprises about the first 100 amino acids.
  • the biomarker comprises about the first 125 amino acids.
  • the biomarker or fragment thereof comprises the carboxy-terminus (e.g., C-terminus, COOH-terminus, C-terminal 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).
  • 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. In some embodiments, the step of detecting the biomarker further comprises contacting the sample with a secondary antibody that binds to the primary antibody and detecting an antibody-biomarker complex. In some embodiments, detecting the antibody-biomarker complex indicates the presence of the particular disease or condition of investigation or a risk of the particular disease or condition of investigation.
  • 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.
  • a therapy e.g., a steroid or other drug or other therapeutic intervention
  • the sample is obtained from the mammal before a relapse.
  • the sample is obtained during the course of the therapy treatment.
  • 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 mellitus, autoimmune Addison’s disease, autoimmune hepatitis, celiac disease, pemphigous, chronic inflammatory demyelinating polyneuropathy, acute disseminated encephalomyelopathy, sarcoidosis, dermatomyositis and behcet’s disease; neurological diseases or neurotrauma, 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 mild cognitive impairment, traumatic brain injury, Huntington’s disease, amyotrophic lateral sclerosis, cerebral hypoxia, hydrocephalus, progressive supranuclear atrophy, corticobasal degeneration, multiple 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.
  • 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 neuromelanin for Parkinson’s disease.
  • kits for performing the methods described herein e.g., detecting neural-derived biomarkers inside or on the surface of circulating phagocytes (from a sample derived from a subject, e.g., a mammal such as a human).
  • the kit comprises one or more tools for processing samples (e.g., blood samples) and/or for collecting PBMCs, storing PBMCs, processing PBMCs, etc.
  • the kit comprises one or more tools for obtaining phagocytes from the PBMCs.
  • the kit comprises one or more tools for identifying phagocytes.
  • the kit comprises one or more tools for concentrating phagocytes.
  • the kit may comprise one or more binding moieties that bind to a specified appropriate biomarker and one or more binding moieties that bind to a phagocyte.
  • the kit further comprises a means for visualizing the binding of the binding moieties to the biomarker/antigen in the sample (e.g., an antibody-antigen complex), e.g., secondary antibodies.
  • the binding moiety is a monoclonal or a polyclonal antibody.
  • the antibody is derived from a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a goat, a horse, or a cow.
  • the antibody is humanized.
  • the antibody is a chimera.
  • the binding moiety is an aptamer.
  • kits may be purified, e.g., affinity purified for the antigen of interest.
  • the kits further comprise reagents for detecting additional biomarkers, e.g., additional biomarkers in the circulating phagocytes, serum biomarkers, plasma biomarkers, etc.
  • the kits further comprise reagents for capture and/or 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 slides coated with macrophage-specific binding moieties and reagents for automated assays.
  • the kit comprises reagents for multiplex assays.
  • the present invention features methods of detecting and/or monitoring and/or treating diseases described herein, or detecting a risk thereof.
  • the present invention features methods for detecting and/or monitoring multiple sclerosis or detecting a risk of multiple sclerosis, methods of determining drug efficacy, methods for treating multiple sclerosis, etc.
  • the present invention also features methods for detecting and/or monitoring Alzheimer’s disease or detecting a risk of Alzheimer’s disease, or methods for determining drug efficacy, etc.
  • the present invention also features methods for treating Alzheimer’s disease.
  • the present invention also features methods for detecting and/or monitoring Parkinson’s disease or detecting a risk of Parkinson’s disease, or methods for determining drug efficacy, etc.
  • the present invention also features methods for treating Parkinson’s disease.
  • the present invention also features methods for detecting and/or monitoring concussion or detecting a risk of concussion, methods of determining drug efficacy, etc.
  • the present invention also features methods for treating concussion.
  • the present invention also features methods for detecting and/or monitoring central nervous system tissue damage or detecting a risk of central nervous system tissue damage, or methods determining drug efficacy, etc.
  • the present invention also features methods for treating central nervous system tissue damage.
  • the present invention also features methods for detecting and/or monitoring central nervous system repair, methods for determining drug efficacy, etc.
  • the present invention also features methods for detecting and/or monitoring a relapse-remitting disease or detecting a risk of a relapse-remitting disease, methods for determining drug efficacy, etc.
  • the present invention also features methods for treating a relapse-remitting disease.
  • the relapse-remitting disease may be, for example, multiple sclerosis (MS), Lyme disease, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Chron’s disease, etc.
  • the present invention also features a method of detecting and/or monitoring an 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.
  • 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.
  • the biomarkers are detected in the recirculating phagocytes in the collected fluid sample.
  • two or more biomarkers are detected in a sample, e.g., a pattern of biomarkers may be detected in the sample.
  • the biomarker(s) is/are a neural-derived biomarker(s).
  • the biomarker(s) is/are not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
  • the method may feature comparing the amount of biomarker in the sample to a predetermined threshold or control for determining whether or not the level of biomarker in the sample is abnormal.
  • the predetermined threshold or control may be an industry standard (e.g., derived from non-disease controls) a laboratory standard, a patient standard, etc.
  • the predetermined threshold or control may be, for example, a sample (a second sample) obtained from the patient at a time prior to obtaining the first sample.
  • analyzing the level of the biomarker e.g., an abnormal level of the biomarker
  • the predetermined threshold or control level of the biomarker indicates the presence of the neurological condition, the disease, central nervous system tissue damage, CNS repair, etc., or a risk thereof.
  • compositions, treatments, drugs, or therapeutic interventions may include but are not limited to: crenezumab, mastinab, daclizumab, zinbryta, ER-Beta agonist, cyclophosphamide, rHlgM22, ponesimod, alpha-4 Integrin, AMP-110, an antisense oligonucleotide, Immune tolerizing agent, MultiStem, Kv1.3 Blocker, Alemtuzumab, IFNb-1b, BHT-3009-01, IFNb-1a, dimethyl fumarate, IFNb-1a (PEGylated), Natalizumab, MT1301, Abatacept, RCP1063, a compound related to lamitrigene, CNM-Au8, Mesenchymal stem cell transplant, apolipoprotein E-based, modified peptidomimetic, Aimspro, Anti-BAFF Human Ab, NDC-1308, GNbACI, Vatelizumab
  • the present invention also features methods of selecting a patient for a clinical trial for testing a drug for an effect on central nervous system (CNS) tissue damage, CNS repair, or neurodegeneration.
  • the method comprises detecting a level of a biomarker in the phagocytes, the biomarker being associated with CNS tissue damage, CNS system repair, or neurodegeneration, wherein if the level of the biomarker in the phagocytes is abnormal, then the patient has active central nervous system disease.
  • the method may comprise selecting the patient for the clinical trial if the patient has active CNS disease.
  • these cohorts may be used to reevaluate drugs that may have previously failed in clinical 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.
  • the biomarker is one that helps define subgroups of a particular disease state or disease process.
  • the biomarker is TDP-43, which may be used to help define subgroups of ALS.
  • Table 1 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
  • T elevated compared to controls
  • 1 decreased compared to controls
  • — no change compared to controls
  • Eq. equivocal
  • C cerebrospinal fluid
  • S serum
  • P plasma
  • E exosomes
  • R recirculating phagocytes.
  • FIG. 1 shows the results of GFAP fluorescent imaging and cell counts.
  • Three image planes GFAP1, GFAP2, GFAP3 from one sample were imaged at 5x and fluorescent cells (DAPI[350]/CD14[568]/GFAP[488]) were counted using ImageJ software. Cell counts were fairly high (low thousands). This sample contained on average 10.8% CD14+ cells and of those cells roughly 1.7% were GFAP+ (0.19% of total). There were a few cells which were GFAP+/CD14-.
  • FIG. 2 shows Tau the results of fluorescent imaging and cell counts.
  • Three image planes Tau1, Tau2, Tau3 from one sample were imaged at 5x and fluorescent cells (DAPI[350]/CD14[568]/TAU[488]) were counted using ImageJ software. Cell counts were somewhat lower (due to division of sample between several slides for troubleshooting). This sample contained on average 8.4% CD14+ cells and of those cells roughly 8.7% were TAU+ (0.7% of total). There were a few cells which were TAU+/CD14-.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Psychiatry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Psychology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)

Abstract

L'invention concerne des méthodes de détection de débris dérivés de neurones à l'intérieur de la surface cellulaire de neutrophiles circulants ou sur cette dernière. Les méthodes consistent à pratiquer une analyse cellulaire unique de neutrophiles et de biomarqueurs dérivés de neurones associés, à l'aide de dosages tels que la cytométrie en flux et l'ELISA à cellule unique. La présente invention concerne également des méthodes de détection et/ou de surveillance de divers états de santé cérébrale sur la base des débris dérivés de neurones dans lesdits neutrophiles.
PCT/US2022/024482 2007-11-30 2022-04-12 Détection de débris dans des phagocytes recirculants WO2022221322A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/379,431 US20240036065A1 (en) 2007-11-30 2023-10-12 Detection of debris in recirculating phagocytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17/228,416 2021-04-12
US17/228,416 US20220236288A1 (en) 2007-11-30 2021-04-12 Detection of neural-derived debris in recirculating phagocytes

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US17/228,416 Continuation-In-Part US20220236288A1 (en) 2007-11-30 2021-04-12 Detection of neural-derived debris in recirculating phagocytes

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/379,431 Continuation-In-Part US20240036065A1 (en) 2007-11-30 2023-10-12 Detection of debris in recirculating phagocytes

Publications (1)

Publication Number Publication Date
WO2022221322A1 true WO2022221322A1 (fr) 2022-10-20

Family

ID=83640985

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2022/024457 WO2022221303A1 (fr) 2007-11-30 2022-04-12 Détection de débris dérivés de neurones dans des phagocytes en recirculation
PCT/US2022/024482 WO2022221322A1 (fr) 2007-11-30 2022-04-12 Détection de débris dans des phagocytes recirculants

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2022/024457 WO2022221303A1 (fr) 2007-11-30 2022-04-12 Détection de débris dérivés de neurones dans des phagocytes en recirculation

Country Status (1)

Country Link
WO (2) WO2022221303A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120293797A1 (en) * 2009-12-17 2012-11-22 Universiteit Gent Methods and systems for optical characterisation
US20140273000A1 (en) * 2013-03-15 2014-09-18 Peter STYS Methods for analyzing blood to detect diseases associated with abnormal protein aggregation
US20200355684A1 (en) * 2017-07-17 2020-11-12 UNIVERSITé LAVAL Asprv1 as a neutrophil-specific marker and therapeutic target for inflammatory diseases

Family Cites Families (4)

* 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
EP2935628B1 (fr) * 2012-12-19 2018-03-21 Caris Life Sciences Switzerland Holdings GmbH Compositions et procédés pour le criblage d'aptamères
WO2020163794A1 (fr) * 2019-02-08 2020-08-13 Msdx, Inc. Détection de débris dérivés du cerveau dans des phagocytes en recirculation
GB2585252A (en) * 2019-07-05 2021-01-06 Gen2 Neuroscience Ltd Tau epitope and binding molecules

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120293797A1 (en) * 2009-12-17 2012-11-22 Universiteit Gent Methods and systems for optical characterisation
US20140273000A1 (en) * 2013-03-15 2014-09-18 Peter STYS Methods for analyzing blood to detect diseases associated with abnormal protein aggregation
US20200355684A1 (en) * 2017-07-17 2020-11-12 UNIVERSITé LAVAL Asprv1 as a neutrophil-specific marker and therapeutic target for inflammatory diseases

Also Published As

Publication number Publication date
WO2022221303A1 (fr) 2022-10-20

Similar Documents

Publication Publication Date Title
KR102323688B1 (ko) 알파-시뉴클레인 검출 분석 및 알파-시뉴클레인병증의 진단 방법
JP5281397B2 (ja) 脳損傷関連障害の診断法
TWI708058B (zh) 阿茲海默症及其他神經退化性疾病之生物標記及診斷方法
US9625459B2 (en) Method for diagnosing alzheimer's disease (AD)
EP3710031A1 (fr) Dosage à base de complément d'exosomes d'astrocytes à la recherche d'une neuro-inflammation
KR20220106957A (ko) 비드를 사용한 알파-시누클레인 검출
US20220236292A1 (en) Detection of debris in recirculating phagocytes
US20240036065A1 (en) Detection of debris in recirculating phagocytes
WO2022221322A1 (fr) Détection de débris dans des phagocytes recirculants
WO2012015050A1 (fr) Procédé de détection de produit de clivage de protéine précurseur d'amyloïde-β 770β soluble pour le diagnostic de maladies associées à l'accumulation de peptide amyloïde-β
US20120178177A1 (en) Biological Components Within the Cerebrospinal Fluid
WO2020163794A1 (fr) Détection de débris dérivés du cerveau dans des phagocytes en recirculation
EP3404415B1 (fr) Mobilisation de cellules souches pluripotentes pour l'infarctus cérébral ischémique
Gutknecht et al. Microparticle immunocapture assay for quantitation of protein multimer amount and size
TW202136297A (zh) Tau蛋白病變及失智症相關疾病之判定藥及判定方法
US9964547B2 (en) Method for detecting aSyn-specific antibodies in a biological sample
US20220236291A1 (en) Detection of neural-derived debris in recirculating phagocytes
US20220236288A1 (en) Detection of neural-derived debris in recirculating phagocytes
US10066020B2 (en) Methods of detecting cancer
WO2014071359A1 (fr) Détection de maladies neurologiques par mesure de la neuromélanine dans des phagocytes en recirculation
CN117007819A (zh) 用于辅助诊断阿尔茨海默病的试剂盒

Legal Events

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

Ref document number: 22788798

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE