WO2014193999A2 - Méthodes et compositions d'identification de biomarqueurs - Google Patents

Méthodes et compositions d'identification de biomarqueurs Download PDF

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WO2014193999A2
WO2014193999A2 PCT/US2014/039858 US2014039858W WO2014193999A2 WO 2014193999 A2 WO2014193999 A2 WO 2014193999A2 US 2014039858 W US2014039858 W US 2014039858W WO 2014193999 A2 WO2014193999 A2 WO 2014193999A2
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cadherin
cancer
isoform
mucin
precursor
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PCT/US2014/039858
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English (en)
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WO2014193999A3 (fr
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Robert T MANEY
Tassilo HORNUNG
Daniel A. Holterman
David Spetzler
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Caris Science, Inc.
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Publication of WO2014193999A2 publication Critical patent/WO2014193999A2/fr
Publication of WO2014193999A3 publication Critical patent/WO2014193999A3/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4725—Mucins, e.g. human intestinal mucin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • Biomarkers for conditions and diseases such as cancer include biological molecules such as proteins, peptides, lipids, RNAs, DNA and variations and modifications thereof.
  • Biomarkers can provide biosignatures that are used for the diagnosis, prognosis, or theranosis of conditions or diseases.
  • Biomarkers can be detected in bodily fluids, including circulating DNA, RNA, proteins, and vesicles. Circulating biomarkers include proteins such as PSA and CA125, and nucleic acids such as SEPT9 DNA and PCA3 messenger RNA (mRNA). Circulating biomarkers can be associated with circulating vesicles. Vesicles are membrane encapsulated structures that are shed from cells and have been found in a number of bodily fluids, including blood, plasma, serum, breast milk, ascites, bronchoalveolar lavage fluid and urine.
  • Vesicles can take part in the communication between cells as transport vehicles for proteins, RNAs, DNAs, viruses, and prions.
  • MicroRNAs are short RNAs that regulate the transcription and degradation of messenger RNAs. MicroRNAs have been found in bodily fluids and have been observed as a component within vesicles shed from tumor cells.
  • the analysis of circulating biomarkers associated with diseases, including vesicles and/or microRNA, can aid in detection of disease or severity thereof, determining predisposition to a disease, as well as making treatment decisions.
  • Vesicles present in a biological sample provide a source of biomarkers, e.g., the markers are present within a vesicle (vesicle payload), or are present on the surface of a vesicle. Characteristics of vesicles (e.g., size, surface antigens, determination of cell-of-origin, payload) can also provide a diagnostic, prognostic or theranostic readout. There remains a need to identify biomarkers that can be used to detect and treat disease. microRNA, proteins and other biomarkers associated with vesicles as well as the characteristics of a vesicle can provide a diagnosis, prognosis, or theranosis.
  • biomarkers e.g., the markers are present within a vesicle (vesicle payload), or are present on the surface of a vesicle.
  • Characteristics of vesicles e.g., size, surface antigens, determination of cell
  • the present invention provides methods and systems for characterizing a phenotype by detecting biomarkers that are indicative of disease or disease progress.
  • the biomarkers can be circulating biomarkers including without limitation vesicle markers, protein, nucleic acids, mRNA, or and microRNA.
  • the biomarkers can be nucleic acid-protein complexes.
  • Characterizing a phenotype for a subject or individual may include, but is not limited to, the diagnosis of a disease or condition, the prognosis of a disease or condition, the determination of a disease stage or a condition stage, a drug efficacy, a physiological condition, organ distress or organ rejection, disease or condition progression, therapy-related association to a disease or condition, or a specific physiological or biological state.
  • the invention provides a method for detecting a biomarker in a biological sample, comprising: contacting the biological sample with at least one binding agent specific to the biomarker, wherein the biomarker is selected from the group consisting of a mucin protein, TROP2, E-Cadherin, an antibody, a target protein in Table 43, a target protein in Table 46, a target of an antibody in Table 47, and a combination thereof; and detecting the biomarker that forms a complex with the at least one binding agent, thereby detecting the biomarker.
  • the method may further comprise determining an amount of the biomarker that formed a complex with the at least one binding agent.
  • the invention provides a method for detecting a microvesicle population from a biological sample, comprising: contacting the biological sample with at least one binding agent specific to an microvesicle antigen selected from the group consisting of a mucin protein, TROP2, E-Cadherin, an antibody, a target protein in Table 43, a target protein in Table 46, a target of an antibody in Table 47, and a combination thereof; and detecting microvesicles that form a complex with the at least one binding agent, thereby detecting the microvesicle population.
  • the method may further comprise determining an amount of microvesicles that formed a complex with the at least one binding agent.
  • the mucin protein may comprise MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, an isoform of any thereof, or a combination of any thereof.
  • the mucin protein comprises MUC 1 or a derivative or isoform thereof.
  • the isoform can be selected from the group consisting of mucin- 1 isoform 2 precursor or mature form (NP_001018016.1), mucin-1 isoform 3 precursor or mature form (NP_001018017.1), mucin-1 isoform 5 precursor or mature form (NP_001037855.1), mucin-1 isoform 6 precursor or mature form (NP_001037856.1), mucin-1 isoform 7 precursor or mature form (NP_001037857.1), mucin-1 isoform 8 precursor or mature form
  • mucin-1 isoform 9 precursor or mature form NP_001191214.1
  • mucin-1 isoform 10 precursor or mature form NP_001191215.1
  • mucin-1 isoform 11 precursor or mature form NP_001191216.1
  • mucin-1 isoform 12 precursor or mature form NP_001191217.1
  • mucin-1 isoform 14 precursor or mature form NP_001191219.1
  • mucin-1 isoform 15 precursor or mature form NP_001191220.1
  • mucin-1 isoform 16 precursor or mature form NP_001191221.1
  • mucin-1 isoform 17 precursor or mature form NP_001191222.1
  • NP_001191223.1 mucin-1 isoform 19 precursor or mature form (NP_001191224.1), mucin-1 isoform 20 precursor or mature form (NP_001191225.1), mucin-1 isoform 21 precursor or mature form (NP_001191226.1), mucin-1 isoform 1 precursor or mature form (NP_002447.4), ENSP00000357380, ENSP00000357377, ENSP00000389098, ENSP00000357374, ENSP00000357381, ENSP00000339690, ENSP00000342814, ENSP00000357383,
  • the antibody comprises an IgM antibody, IgD antibody, IgG antibody, IgA antibody, IgE antibody or fragment of any thereof.
  • the antibody can be an autoantibody, including without limitation an autoantibody to a tumor antigen.
  • the tumor antigen can be any desired antigen, e.g., at least one of AIBl-coactivator protein, BRCA1, BRCA2, Carbonic anhydrase IX, CD24, CEA, COX- 2, Creatine kinase-BB, DAP -kinase, Endoglin (CD105), ErbB2, Estrogen receptor beta, Etsl, EZH2, GCDFP-15, IGFBP-2, KAI-1 (CD82), KLK15, MAGE-A, Mammaglobin A, Mammaglobin B, MCM2, MUC1 (CA15-3), Progesterone Receptor, SRC-1- coactivator protein, STC-l(Stanniocalcin 1), TEM-8, THRSP, TIMP-1.
  • the autoantibody may be an autoantibody to a microvesicle surface antigen.
  • the biological sample comprises a bodily fluid.
  • the bodily fluid may comprise any useful bodily fluid, e.g., peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, umbilical cord blood,
  • CSF cerebrospinal fluid
  • the methods of the invention may comprise selectively depleting at least one abundant protein from the biological sample prior to the contacting step. In addition or in the alternative, the methods may comprise selectively depleting at least one abundant protein from the biological sample prior to the detecting step. If the biological sample comprises peripheral blood, serum or plasma, the abundant protein can be an abundant blood protein.
  • the at least one abundant protein can be, e.g., at least one of albumin, IgG, transferrin, fibrinogen, fibrin, IgA, a2- Marcroglobulin, IgM, al -Antitrypsin, complement C3, haptoglobulin, apolipoprotein Al, A3 and B; al-Acid Glycoprotein, ceruloplasmin, complement C4, Clq, IgD, prealbumin (transthyretin), plasminogen, a derivative of any thereof, and a combination thereof.
  • the at least one abundant protein in blood may comprise at least one of Albumin, Immunoglobulins, Fibrinogen, Prealbumin, Alpha 1 antitrypsin, Alpha 1 acid glycoprotein, Alpha 1 fetoprotein, Haptoglobin, Alpha 2 macroglobulin, Ceruloplasmin, Transferrin, complement proteins C3 and C4, Beta 2 microglobulin, Beta lipoprotein, Gamma globulin proteins, C-reactive protein (CRP), Lipoproteins (chylomicrons, VLDL, LDL, HDL), other globulins (types alpha, beta and gamma), Prothrombin, Mannose-binding lectin (MBL), a derivative of any thereof, and a combination thereof.
  • Albumin Immunoglobulins
  • Fibrinogen Prealbumin
  • Prealbumin Alpha 1 antitrypsin
  • Alpha 1 acid glycoprotein Alpha 1 fetoprotein
  • Haptoglobin Haptoglobin
  • Alpha 2 macroglobulin Ceruloplasmin
  • the at least one abundant protein may be depleted by various techniques, including without limitation chromatography, immunoaffinity, precipitation, or a combination thereof.
  • the biological sample is contacted with thromboplastin to precipitate fibrinogen.
  • Selectively depleting the at least one abundant protein from the biological sample may include depleting at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%o of the at least one abundant protein.
  • tissue sample a tumor sample, a biopsy, or a cell culture sample.
  • the biological sample used in the methods of the invention may be subjected to various processing steps. These include, e.g., size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, affinity capture, immunoassay, microfluidic separation, flow cytometry or combinations thereof.
  • the processing steps can be performed prior to the contacting and/or prior to the detecting as desired.
  • the biological sample is filtered using a selection membrane having a pore size of from 0.01 ⁇ to about 10 ⁇ , or a molecular weight cut off (MWCO) from about 1 kDa to 10000 kDa.
  • the filtering can be performed prior to the contacting and/or prior to the detecting as desired.
  • the biological sample can be filtered to retain microvesicles prior to the contacting step.
  • the biomarker can be detected using any number of available detection techniques disclosed herein or known in the art.
  • the biomarker is detected using immunoabsorbent capture, affinity purification, affinity capture, immunoassay, Western Blot, flow cytometry, ELISA, antibody array, or combinations thereof.
  • the methods of the invention may further comprise contacting the biological sample with at least one additional binding agent specific to an additional biomarker.
  • the additional biomarker comprises a microvesicle antigen.
  • the additional biomarker can be a biomarker selected from Table 3, Table 4, and/or Table 5 herein.
  • the additional biomarker can be a protein selected from the group consisting of a tetraspanin, CD9, CD63, CD81, MMP7, EpCAM, CD81, MFG-E8, STAT3, EZH2, p53, MACC1, SPDEF, RUNX2, YB-1, AURKA, AURKB, PCSA, AdamlO, BCNP, CD9, EGFR, EpCam, ILIB, KLK2, MMP7, p53, SERPINB3, SPDEF, SSX2, SSX4, EGFR, EpCAM, KLK2, SPDEF, SSX2, SSX4, MUC2, TROP2, a target protein in Table 43, E- Cadherin, an antibody, a target protein in Table 46, a target of an antibody in Table 47, and a combination thereof and a combination thereof.
  • the at least one binding agent and the at least one additional binding agent are selected from the pairs of capture and detector agents in any of Table 44, Tables 49-52, or any capture agent in Table 46 used with any detector agent in Table 47.
  • the pairs of capture and detector agents can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 pairs selected from E-cadherin - EGFR, E- cadherin - TOMM22, E-cadherin - NDRG1, E-cadherin - VDAC2, E-cadherin - KLK6, E-cadherin - MMP7, E- cadherin - EDIL3 (del-1), E-cadherin - CCR5, E-cadherin - BDNF, E-cadherin - HsplO, E-cadherin - GOLPH2, E- cadherin - Hsp40/DNAJB1, E-cadherin - KLK4, E-cadherin - LGALS3BP, E-cadherin - pl30 /RBL2, E-cadherin - SCARB2, E-cadherin - Stanniocalcin 2
  • the pairs of capture and detector agents can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 pairs selected from E-cadherin - CD81, E-cadherin - Anti-EDN-3, E- cadherin - Cytochrome C, E-cadherin - CD 10, E-cadherin - CD151, E-cadherin - seprase/FAP, E-cadherin - HGF, E-cadherin - PAP, E-cadherin - CD41, E-cadherin - IGFBP-2, E-cadherin - TWEAK, E-cadherin - MMP 2, E- cadherin - H3F3A, E-cadherin - DDX1, E-cadherin - 14-3-3 zeta/beta, E-cadherin - IDH2, E-cadherin - CD49d, E- cadherin
  • the pairs of capture and detector agents can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 pairs selected from E-cadherin - CD81, E-cadherin - Anti-EDN-3, E-cadherin - Cytochrome C, E- cadherin - CD 10, E-cadherin - CD151, E-cadherin - seprase/FAP, E-cadherin - HGF, E-cadherin - PAP, E- cadherin - CD41, E-cadherin - IGFBP-2, E-cadherin - TWEAK, E-cadherin - MMP 2, E-cadherin - H3F3A, E- cadherin - DDX1, E-cadherin - 14-3-3 zeta/beta, E-cadherin - IDH2, E-cadherin - CD49d, E-cadherin
  • the at least one binding agent comprises a nucleic acid, DNA molecule, RNA molecule, antibody, antibody fragment, aptamer, peptoid, zDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), lectin, peptide, dendrimer, membrane protein labeling agent, chemical compound, a fragment thereof, a derivative thereof, or a combination thereof.
  • the at least one binding agent can be tethered to a substrate.
  • the at least one binding agent can be labeled.
  • the microvesicle population comprises microvesicles having a diameter between 10 nm and 2000 nm, optionally between 20 nm and 200 nm.
  • the methods of the invention may comprise detecting at least one payload biomarker within the microvesicle population.
  • the at least one payload biomarker may comprise at least one nucleic acid, peptide, protein, lipid, antigen, carbohydrate, and/or proteoglycan.
  • the nucleic acid can be at least one DNA, mRNA, microRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, or shRNA.
  • at least one payload biomarker comprises microRNA or mRNA.
  • the detection of biomarkers by the methods of the invention can be used to characterize a cancer or assist in characterizing such cancer.
  • the presence or the level of the detected biomarker is compared to a reference in order to characterize the cancer.
  • the presence or the level of the detected microvesicle population can be compared to a reference in order to characterize the cancer.
  • the characterizing may comprise providing a prognostic, diagnostic or theranostic determination for the cancer, identifying the presence or risk of the cancer in a subject, or identifying the cancer in a subject as metastatic or aggressive.
  • the characterizing may provide improved sensitivity, specificity, accuracy, or area under a received operating characteristic curve (AUC) compared to alternate methods that detect such biomarkers (e.g., mucin proteins) or microvesicles.
  • the characterizing is performed with improved sensitivity, specificity, accuracy, or AUC compared to alternate methods that characterize the cancer.
  • the characterizing can be performed with a sensitivity, specificity, and/or accuracy of at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.
  • the characterizing can performed with an AUC of at least 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.971, 0.972, 0.973, 0.974, 0.975, 0.976, 0.977, 0.978, 0.978, 0.979, 0.980, 0.981, 0.982, 0.983, 0.984, 0.985, 0.986, 0.987, 0.988, 0.989, 0.99, 0.991, 0.992, 0.993, 0.994, 0.995, 0.996, 0.997, 0.9,
  • the cancer characterized by the methods of the invention can be an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer
  • endometrial cancer ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
  • GIST gestational trophoblastic tumor
  • glioma hairy cell leukemia; head and neck cancer
  • heart cancer Hodgkin lymphoma
  • hypopharyngeal cancer intraocular melanoma
  • islet cell tumors Kaposi sarcoma; kidney cancer;
  • Langerhans cell histiocytosis laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma;
  • mesothelioma metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-
  • Hodgkin lymphoma nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer;
  • oropharyngeal cancer osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma;
  • pleuropulmonary blastoma pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer;
  • T-cell lymphoma T-cell lymphoma
  • testicular cancer throat cancer
  • thymic carcinoma thymoma
  • thyroid cancer transitional cell cancer
  • transitional cell cancer of the renal pelvis and ureter trophoblastic tumor
  • ureter cancer urethral cancer
  • uterine cancer uterine sarcoma
  • vaginal cancer vulvar cancer
  • the cancer can be a prostate cancer.
  • the cancer can be a pancreatic cancer, including without limitation an exocrine pancreas cancer, a pancreatic carcinoma, a ductal adenocarcinoma, a pancreatic intraepithelial neoplasm, an intraductal papillary mucinous neoplasm, an intraductal papillary mucinous neoplasm, a mucinous pancreatic cancer, a pancreatic cystic neoplasm, a pancreatic neuroendocrine tumor, an islet cell tumor, an pancreas endocrine tumor,a pancreatic neuroendocrine carcinoma (PNEC), or a combination thereof.
  • PNEC pancreatic neuroendocrine carcinoma
  • the invention provides use of the at least one reagent to carry out the methods of invention.
  • the invention provides a kit comprising at least one reagent to carry out the methods of invention.
  • the at least one reagent can be selected from the group consisting of at least one reagent capable of binding to a mucin protein, at least one reagent capable of binding to a target protein in Table 43, at least one reagent capable of binding to TROP2 or E-Cadherin, at least one reagent capable of binding to an antibody, at least one reagent capable of binding to a target protein in Table 46 or a target of an antibody in Table 47; at least one reagent capable of binding to a microvesicle surface antigen; at least one lipophilic dye; at least one population of microvesicles; a substrate; a filter; a microfluidic device; a microtiter plate; and a combination thereof.
  • the at least one reagent comprises a binding
  • the invention provides an aptamer that binds to a mucin protein.
  • the mucin protein can be MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, an isoform of any thereof, or a combination of any thereof.
  • the mucin protein comprises MUC 1 or a derivative or isoform thereof.
  • Useful MUC 1 isoforms include without limitation mucin-1 isoform 2 precursor or mature form (NP_001018016.1), mucin-1 isoform 3 precursor or mature form (NP_001018017.1), mucin-1 isoform 5 precursor or mature form (NP_001037855.1), mucin-1 isoform 6 precursor or mature form (NP_001037856.1), mucin-1 isoform 7 precursor or mature form (NP_001037857.1), mucin-1 isoform 8 precursor or mature form (NP_001037858.1), mucin-1 isoform 9 precursor or mature form (NP_001191214.1), mucin-1 isoform 10 precursor or mature form (NP_001191215.1), mucin-1 isoform 11 precursor or mature form (NP_001191216.1), mucin-1 isoform 12 precursor or mature form
  • NP_001191217.1 mucin-1 isoform 13 precursor or mature form (NP_001191218.1), mucin-1 isoform 14 precursor or mature form (NP_001191219.1), mucin-1 isoform 15 precursor or mature form (NP_001191220.1), mucin-1 isoform 16 precursor or mature form (NP_001191221.1), mucin-1 isoform 17 precursor or mature form
  • NP_001191222.1 mucin-1 isoform 18 precursor or mature form (NP_001191223.1), mucin-1 isoform 19 precursor or mature form (NP_001191224.1), mucin-1 isoform 20 precursor or mature form (NP_001191225.1), mucin-1 isoform 21 precursor or mature form (NP_001191226.1), mucin-1 isoform 1 precursor or mature form
  • ENSP00000357381 ENSP00000339690, ENSP00000342814, ENSP00000357383, ENSP00000357375,
  • the aptamer can be capable of interfering with MUC1 interaction with at least one of ABL1, SRC, CTNND1, ERBB2, GSK3B, JUP, PRKCD, APC, GALNT1, GALNT10, GALNT12, JUN, LCK, OSGEP, ZAP70, CTNNB1, EGFR, SOS1, ERBB3, ERBB4, GRB2, ESR1, GALNT2, GALNT4, LYN, TP53, C1GALT1, C1GALT1C1, GALNT3, GALNT6, GCNT1, GCNT4, MUC 12, MUC13, MUC15, MUC 17, MUC 19, MUC2, MUC20, MUC3A, MUC4, MUC5B, MUC6, MUC7, MUCL1, ST3GAL1, ST3GAL3, ST3GAL4, ST6GALNAC2, B3GNT2, B3GNT3, B3GNT4, B3GNT5, B3GNT7, B4GALT5, GALNT11,
  • ST6GALNAC4 GALNT15, MYOD1, SIGLECl, IKBKB, TNFRSF1A, IKBKG, and MUC1.
  • the aptamers of the invention may be identified herein in the form of DNA or RNA. Unless otherwise specified, one of skill in the art will appreciate that an aptamer may generally be synthesized in various forms of nucleic acid. The aptamers may also carry various chemical modifications and remain within the scope of the invention. The aptamers may also comprise any number of modified, synthetic or unnatural nucleic acids other than the primary nucleobases such as cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U, respectively.
  • cytosine DNA and RNA
  • guanine DNA and RNA
  • DNA and RNA adenine
  • DNA and RNA thymine
  • uracil RNA
  • an aptamer of the invention is modified to comprise at least one chemical modification.
  • the modification may include without limitation a chemical substitution at a sugar position; a chemical substitution at a phosphate position; and a chemical substitution at a base position of the nucleic acid.
  • the modification is selected from the group consisting of: biotinylation, incorporation of a fluorescent label, incorporation of a modified nucleotide, a 2'-modified pyrimidine, 3' capping, conjugation to an amine linker, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, conjugation to a drug, conjugation to a cytotoxic moiety, and labeling with a radioisotope, or other modification as disclosed herein.
  • the position of the modification can be varied as desired.
  • the biotinylation, fluorescent label, or cytotoxic moiety can be conjugated to the 5' end of the aptamer.
  • the biotinylation, fluorescent label, or cytotoxic moiety can also be conjugated to the 3' end of the aptamer.
  • the cytotoxic moiety is encapsulated in a nanoparticle.
  • the nanoparticle can be selected from the group consisting of: liposomes, dendrimers, and comb polymers.
  • the cytotoxic moiety comprises a small molecule cytotoxic moiety.
  • the small molecule cytotoxic moiety can include without limtation vinblastine hydrazide, calicheamicin, vinca alkaloid, a cryptophycin, a tubulysin, dolastatin-10, dolastatin-15, auristatin E, rhizoxin, epothilone B, epithilone D, taxoids, maytansinoids and any variants and derivatives thereof.
  • the cytotoxic moiety comprises a protein toxin.
  • the protein toxin can be selected from the group consisting of diphtheria toxin, ricin, abrin, gelonin, and Pseudomonas exotoxin A.
  • Non-immunogenic, high molecular weight compounds for use with the invention include polyalkylene glycols, e.g., polyethylene glycol.
  • radioisotopes include yttrium-90, indium-I l l, iodine-131, lutetium- 177, copper-67, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211, and actinium-225.
  • the aptamer may be labeled with a gamma-emitting radioisotope.
  • an active agent is conjugated to the aptamer.
  • the active agent may be a therapeutic agent or a diagnostic agent.
  • the therapeutic agent may be selected from the group consisting of tyrosine kinase inhibitors, kinase inhibitors, biologically active agents, biological molecules, radionuclides, adriamycin, ansamycin antibiotics, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecotabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, epothilones, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin
  • the invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the aptamer described above or a salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aptamer or a salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • the invention provides a method of treating or ameliorating a disease or disorder, comprising administering the pharmaceutical composition to a subject in need thereof.
  • Administering a therapeutically effective amount of the composition to the subject may result in: (a) an enhancement of the delivery of the active agent to a disease site relative to delivery of the active agent alone; or (b) an enhancement of microvesicles clearance resulting in a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in a blood level of microvesicles targeted by the aptamer; or (c) an decrease in biological activity of microvesicles targeted by the aptamer of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
  • the biological activity of microvesicles comprises immune suppression or transfer of genetic information.
  • the disease or disorder can include without limitation those disclosed herein.
  • the disease or disorder may comprise a neoplastic, proliferative, or inflammatory, metabolic, cardiovascular, or neurological disease or disorder. See section "Phenotypes.”
  • the invention further provides a kit comprising an aptamer disclosed herein, or a pharmaceutical composition thereof.
  • FIG. 1A depicts a method of identifying a biosignature comprising nucleic acid to characterize a phenotype.
  • FIG. IB depicts a method of identifying a biosignature of a vesicle or vesicle population to characterize a phenotype.
  • FIGs. 2A-2H illustrate methods of assessing biomarkers such as microvesicle surface antigens.
  • FIG. 2A is a schematic of a planar substrate coated with a capture agent, such as an aptamer or antibody, which captures vesicles expressing the target antigen of the capture agent.
  • the capture agent may bind a protein expressed on the surface of vesicles shed from diseased cells ("disease vesicle").
  • the detection agent which may also be an aptamer or antibody, carries a detectable label, here a fluorescent signal. The detection agent binds to the captured vesicle and provides a detectable signal via its fluorescent label.
  • the detection agent can detect an antigen that is generally associated with vesicles, or is associated with a cell-of-origin or a disease, e.g., a cancer.
  • FIG. 2B is a schematic of a particle bead conjugated with a capture agent, which captures vesicles expressing the target antigen of the capture agent.
  • the capture agent may bind a protein expressed on the surface of vesicles shed from diseased cells ("disease vesicle").
  • the detection agent which may also be an aptamer or antibody, carries a detectable label, here a fluorescent signal. The detection agent binds to the captured vesicle and provides a detectable signal via its fluorescent label.
  • the detection agent can detect an antigen that is generally associated with vesicles, or is associated with a cell-of-origin or a disease, e.g., a cancer.
  • FIG. 2C is an example of a screening scheme that can be performed by using different combinations of capture and detection agents to the indicated biomarkers. The biomarker combinations can be detected using assays as shown in FIGs. 2A-2B.
  • FIGs. 2D-2E present illustrative schemes for capturing and detecting vesicles to characterize a phenotype.
  • FIG. 2F presents illustrative schemes for assessing vesicle payload to characterize a phenotype.
  • FIG. 2G presents illustrative schemes for capturing and detecting vesicles and optionally assessing payload to characterize a phenotype.
  • FIG. 2H presents illustrative schemes for using a lipid dye to detect vesicles and characterize a phenotype.
  • FIG. 3 illustrates a computer system that can be used in some exemplary embodiments of the invention.
  • FIG. 4 illustrates a method of depicting results using a bead based method of detecting vesicles from a subject.
  • the number of beads captured at a given intensity is an indication of how frequently a vesicle expresses the detection protein at that intensity. The more intense the signal for a given bead, the greater the expression of the detection protein.
  • the figure shows a normalized graph obtained by combining normal patients into one curve and cancer patients into another, and using bio-statistical analysis to differentiate the curves. Data from each individual is normalized to account for variation in the number of beads read by the detection machine, added together, and then normalized again to account for the different number of samples in each population.
  • FIG. 5 illustrates the capture of prostate cancer cells-derived vesicles from plasma with EpCam by assessing TMPRSS2-ERG expression.
  • VCaP purified vesicles were spiked into normal plasma and then incubated with Dynal magnetic beads coated with either the EpCam or isotype control antibody.
  • RNA was isolated directly from the Dynal beads. Equal volumes of RNA from each sample were used for RT-PCR and subsequent Taqman assays.
  • FIG. 6 depicts a bar graph of miR-21 or miR-141 expression with CD9 bead capture.
  • 1 ml of plasma from prostate cancer patients, 250 ng/ml of LNCaP, or normal purified vesicles were incubated with CD9 coated Dynal beads.
  • the RNA was isolated from the beads and the bead supernatant.
  • One sample (#6) was also uncaptured for comparison.
  • microRNA expression was measured with qRT-PCR and the mean CT values for each sample compared.
  • CD9 capture improves the detection of miR-21 and miR-141 in prostate cancer samples.
  • FIGs. 7A-C illustrate detecting vesicles using flow cytometry.
  • FIG. 7A illustrates separation and identification of vesicles using the MoFlo XDP.
  • FIG. 7B illustrates FACS analysis of VCaP cells and exosomes stained with antibodies to CD9, B7H3, PCSA and PSMA.
  • FIG. 7C illustrates different patterns of miR expression were obtained in flow sorted B7H3+ or PSMA+ vesicle populations as compared to overall vesicle population.
  • FIGs. 8A-H illustrate detecting vesicles in a sample wherein the presence or level of the desired vesicles are assessed.
  • FIG. 8A represents a schematic of isolating vesicles from plasma using a column based filtering method, wherein the isolated vesicles are subsequently assessed.
  • FIG. 8B represents a schematic of compression of a membrane of a vesicle due to high-speed centrifugation, such as ultracentrifugation.
  • FIG. 8C represents a schematic of detecting vesicles bound to microspheres using laser detection.
  • FIG. 8D represents an example of detecting prostate derived vesicles bound to a substrate.
  • the microvesicles are captured with capture agents specific to PCSA, PSMA or B7H3 tethered to the substrate.
  • the so-captured vesicles are labeled with fluorescently labeled detection agents specific to CD9, CD63 and CD81.
  • FIG. 8E illustrates correlation of CD9 positive vesicles detected using a microsphere platform (Y-axis) or flow cytometry (X-axis). To calculate median fluorescence intensity (MFIs), vesicles were captured with anti-CD9 antibodies tethered to microspheres and detected using fluorescently labeled detection antibodies specific to CD9, CD63 and CD81.
  • MFIs median fluorescence intensity
  • FIG. 8F illustrates correlation of PSMA, PCSA or B7H3 positive vesicles detected using a microsphere platform (Y-axis) or BCA protein assay (X-axis).
  • Y-axis Y-axis
  • X-axis BCA protein assay
  • vesicles were captured with antibodies to B7H3, PSMA or PCSA tethered to microspheres and detected using fluorescently labeled detection antibodies specific to CD9, CD63 and CD81.
  • FIG. 8G illustrates similar performance for detecting CD81 positive vesicles using a microsphere assay in a single -plex or multi-plex fashion. Vesicles were captured with anti-CD81 antibodies tethered to microspheres and detected using fluorescently labeled detection antibodies specific to CD9, CD63 and CD81.
  • 8H illustrates similar performance for detecting B7H3, CD63, CD9 or EpCam positive vesicles using a microsphere assay in a single -plex or multi-plex fashion.
  • Vesicles were captured with antibodies to B7H3, CD63, CD9 or EpCam tethered to microspheres and detected using fluorescently labeled detection antibodies specific to CD9, CD63 and CD81.
  • FIG. 9A illustrates the ability of a vesicle bio-signature to discriminate between normal prostate and PCa samples.
  • Cancer markers included EpCam and B7H3.
  • General vesicle markers included CD9, CD81 and CD63.
  • Prostate specific markers included PCSA. PSMA can be used as well as PCSA. The test was found to be 98% sensitive and 95% specific for PCa vs normal samples.
  • FIG. 9B illustrates mean fluorescence intensity (MFI) on the MFI.
  • FIG. 10 is a schematic for a decision tree for a vesicle prostate cancer assay for determining whether a sample is positive for prostate cancer.
  • FIG. 11 shows the results of a vesicle detection assay for prostate cancer following the decision tree versus detection using elevated PSA levels.
  • FIG. 12 illustrates levels of miR-145 in vesicles isolated from control and PCa samples.
  • FIGs. 13A-13E illustrate the use of microRNA to identify false negatives from a vesicle -based diagnostic assay for prostate cancer.
  • FIG. 13A illustrates a scheme for using miR analysis within vesicles to convert false negatives into true positives, thereby improving sensitivity.
  • FIG. 13B illustrates a scheme for using miR analysis within vesicles to convert false positives into true negatives, thereby improving specificity.
  • Normalized levels of miR- 107 FIG. 13C
  • miR- 141 FIG.
  • FIG. 13D shows Taqman qRT-PCR verification of increased miR- 107 in plasma cMVs of prostate cancer patients compared to patients without prostate cancer using a different sample cohort.
  • FIGs. 14A-D illustrate KRAS sequencing in a colorectal cancer (CRC) cell line and patient sample.
  • CRC colorectal cancer
  • Samples comprise genomic DNA obtained from the cell line (FIG. 14B) or from a tissue sample from the patient (FIG. 14D), or cDNA obtained from RNA payload within vesicles shed from the cell line (FIG. 14A) or from a plasma sample from the patient (FIG. 14C).
  • FIGs. 15A-B illustrate immunoprecipitation of microRNA from human plasma.
  • FIG. 15A shows the mean quantity of miR- 16 detected in various fractions of human plasma.
  • Beads are the amount of miR- 16 that co- immunoprecipitated using antibodies to Argonaute2 (Ago2), Apolipoprotein Al (ApoAl), GW182, and an IgG control.
  • Dyna refers to immunoprecipitation using Dynabead Protein G
  • Magna refers to Magnabind Protein G beads.
  • Supernt are the amount of miR-16 detected in the supernatant of the immunoprecipitation reactions. See Examples for details.
  • FIG. 15B is the same as FIG. 15A except that miR-92a was detected.
  • FIG. 16 illustrates flow sorting of complexes stained with PE labeled anti-PCSA antibodies and FITC labeled anti-Ago2 antibodies.
  • FIGs. 17A-D illustrate detection of microRNA in PCSA/Ago2 positive complexes in human plasma samples.
  • the plasma samples were from subjects with prostate cancer (PrC) or normal controls (normal).
  • FIG. 17A shows miR-22 copy number in total circulating microvesicle population from human plasma.
  • FIG. 17B shows plasma-derived complexes were sorted using antibodies against PCSA and Argonaute 2 (Ago2). RNA was isolated and the copy number of miR-22 was determined in the population of PCSA/Ago2 double positive events.
  • FIG. 17C shows the number of PCSA/Ago2 double positive events counted by flow cytometry for each plasma sample.
  • FIG. 17D shows copy number of miR-22 divided by the total number of PCSA/Ago2 positive events for each plasma sample. This yields the copy number of miR-22 per PCSA/Ago2 double positive complex.
  • FIGs. 18A-F illustrate dot plots of raw background subtracted fluorescence values of selected mRNAs from microarray profiling of vesicle mRNA payload levels.
  • the Y axis shows raw background subtracted fluorescence values (Raw BGsub Florescence).
  • the X axis shows dot plots for four normal control plasmas and four plasmas from prostate cancer patients.
  • the mRNAs shown are A2ML1 (FIG. 18A), GABARAPL2 (FIG. 18B), PTMA (FIG. 18C), RABAC1 (FIG. 18D), SOX1 (FIG. 18E), and ETFB (FIG. 18F).
  • FIGs. 20A-C illustrate the performance of a three marker panel consisting of the following markers: 1) Epcam detector - MMP7 capture; 2) PCSA detector - MMP7 capture; 3) Epcam detector - BCNP capture.
  • An ROC curve generated using a diagonal linear discriminant analysis in this setting is shown in FIG. 20A.
  • the arrow indicates the threshold point along the curve where sensitivity equals 90% and specificity equals 80%.
  • FIG. 20B shows the distribution of PCA+ and PC A- samples falling on either side of the indicated threshold line.
  • the individual contribution of the Epcam detector - MMP7 capture marker is shown in FIG. 20C.
  • PCA, Current Biopsy refers to men who had a first positive biopsy
  • PCA, Previous Biopsy refers to the watchful waiting cohort.
  • FIGs. 21A-B show ROC curves demonstrating the ability of different vesicle capture and detection agents to distinguish prostate cancer.
  • the performance of a 5-marker panel was determined in two settings using a linear discriminant analysis and 10-fold cross-validation or re-substitution methodology.
  • ROC curves for the Model A setting i.e., all PCa versus all other patient samples
  • the marker panel in this setting consisted of: 1) Epcam detector - MMP7 capture; 2) PCSA detector - MMP7 capture; 3) Epcam detector - BCNP capture; 4) PCSA detector - AdamlO capture; and 5) PCSA detector - KLK2 capture.
  • the marker panel in this setting consisted of: 1) Epcam detector - MMP7 capture; 2) PCSA detector - MMP7 capture; 3) Epcam detector - BCNP capture; 4) PCSA detector - AdamlO capture; and 5) PCSA detector - KLK2 capture.
  • the upper more jagged line corresponds to the re-substitution method.
  • the AUC was 0.90.
  • the calculated AUC was 0.87.
  • the model using cross-validation achieved 92% sensitivity and 50% specificity.
  • the model using cross-validation achieved 82% sensitivity and 80% specificity.
  • ROC curves for the Model C setting i.e., restricted sample set as described below for Table 27 are shown in FIG. 21B.
  • the marker panel in this setting consisted of: 1) Epcam detector - MMP7 capture; 2) PCSA detector - MMP7 capture; 3) Epcam detector - BCNP capture; 4) PCSA detector - AdamlO capture; and 5) CD81 detector - MMP7 capture.
  • the upper more jagged line corresponds to the resubstitution method.
  • the AUC was 0.91.
  • the calculated AUC was 0.89.
  • the cross-validation model achieved 95% sensitivity and 60% specificity.
  • FIGs. 22A-D shows levels of microRNA species in PCSA+ circulating microvesicles from the plasma of men with prostate cancer and benign prostate disorders.
  • the Ct from the Exiqon cards for miR-1974 (which overlaps a mitochondrial tRNA) is shown in the various pools.
  • the prostate cancer samples had higher levels of this miR than other samples.
  • FIG. 22B shows the copy number of the miR in the pools as measured by taqman analysis using an ABI 7900.
  • FIG. 22C the Ct from the Exiqon cards for miR-320b is shown in the various pools.
  • the prostate cancer samples had lower levels of this miR than other samples.
  • FIG. 22A shows the Ct from the Exiqon cards for miR-1974 (which overlaps a mitochondrial tRNA) is shown in the various pools.
  • the prostate cancer samples had higher levels of this miR than other samples.
  • FIG. 22B shows the copy number of the miR in the pools as measured by taqman analysis using
  • FIG. 22D shows the copy number of miR-320b in the pools as measured by taqman analysis using an ABI 7900.
  • FIG. 23 shows detection of a standard curve for a synthetic miR16 standard (10 ⁇ 6 - 10 ⁇ 1) and detection of miR16 in triplicate from a human plasma sample. As indicated by the legend, the data was taken from a Fluidigm
  • ArrayTM IFCs or with an ABI 7900HT Taqman assay (Applied Biosystems, Foster City, CA). All levels were determined under multiplex conditions.
  • FIG. 24A illustrates a protein gel demonstrating removal of HSA and antibody heavy and light chains in the indicated samples.
  • the columns in the gel are as follows: “Raw” (Plasma without any treatment); “Cone” (Plasma concentrated via nanomembrane filtration); “FTp” (Plasma flow through from treatment with Pierce Albumin and IgG Removal Kit, Thermo Fisher Scientific Inc., Rockford, IL USA); “FTv” (Plasma flow through from treatment with Vivapure® Anti-HSA/IgG Kit from Sartorius Stedim North America Inc., Edgewood, NY USA); “IgG” (IgG control); “M” (molecular weight marker).
  • FIG. 24B shows an example using the protocol to detect microvesicles.
  • the cMVs were detected using Anti-MMP7-FITC antibody conjugate (Millipore anti-MMP7 monoclonal antibody 7B2).
  • the plot shows the frequency of events detected versus concentration of the detection antibody.
  • FIG. 24C shows EpCam expression in human serum albumin (HSA) depleted plasma sample.
  • the x-axis refers to concentration of EpCam+ vesicles in various aliquots.
  • the Y axis illustrates median fluorescent intensity (MFI) detected in a microbead assay using PE labeled anti-EpCAM antibodies to detect the vesicles.
  • "Isotype” refers to detection using PE anti-IgG antibodies as a control.
  • FIG. 24D is similar to FIG. 24C except that PE-labeled anti- MMP7 antibodies were used to detect the microvesicles. Shown are samples that were pre-treated to remove HSA ("HSA depleted") or not ("HSA non-depleted"), "iso” refers to the anti-IgG antibody controls.
  • FIG. 60E illustrates detection of vesicles in plasma after treatment with thromboplastin to precipitate fibrinogen.
  • the Y axis illustrates median fluorescent intensity (MFI) detected in a microbead assay using bead-conjugated anti-KLK2 to capture the vesicles and a PE labeled anti-EpCAM aptamer to detect the vesicles.
  • MFI median fluorescent intensity
  • the X-axis groups 4 plasma samples (cancer sample CI, cancer sample C2, benign sample Bl, benign sample B2) into 6 experimental conditions, VI -V6. As indicated by the thromboplastin incubation time and concentration below the plot, the thromboplastin treatment stringency increased from VI -V6.
  • FIGs. 25A-D illustrate the use of an anti-EpCAM aptamer (Aptamer 4; SEQ ID NO. 1) to detect a microvesicle population. Vesicles in patient plasma samples were captured using bead-conjugated antibodies to the indicated microvesicle surface antigens (FIG. 25A: EGFR; FIG. 25B: PBP; FIG. 25C: EpCAM; FIG. 25D: KLK2).
  • Fluorescently labeled Aptamer 4 was used as a detector in the microbead assay.
  • the figure shows average median fluorescence values (MFI values) for three prostate cancer (C1-C3) and three normal samples (N1-N3) in each plot. In each plot, the samples from left to right are ordered as: CI, C2, C3, Nl, N2, N3.
  • FIGs. 26A-F illustrate detection of microvesicles using lipid dyes and anti -protein antibodies.
  • FIG. 26A and FIG. 26B illustrate staining of VCap derived vesicles.
  • the vesicles were concentrated using ultrafiltration then stained simultaneously with an anti-tetraspanin-FITC cocktail (consisting antibodies to CD9, CD63, CD81), anti- EGFR-PE-Cy7 and the lipid dye Dil for 20 minutes at 37°C while shaking.
  • the solution was diluted with 500 ⁇ PBS-BN, vortexed and analyzed on a MoFlo flow cytometer (Beckman Coulter, Inc., Indianapolis IN).
  • FIG. 26A the vesicles were first gated for Dil+ events then EGFR+/tetraspanin+ events were counted. As indicated, 0% double negative events corresponding to cellular debris were observed.
  • FIG. 26B the vesicles were first gated for tetraspanin+ events then EGFR+/DiI+ events were counted. As indicated, 29% double negative events corresponding to cellular debris were observed.
  • FIG. 26C and FIG. 26D illustrate staining of vesicles concentrated from plasma of cancer-positive patients. Experimental conditions were otherwise identical to FIG. 26A and FIG. 26B, respectively.
  • FIG. 26E and FIG. 26F illustrate staining of vesicles concentrated from plasma of cancer-negative patients.
  • FIGs. 27A-E illustrate analysis of carboxyfluorescein diacetate succinimidyl ester (CFSE) stained microvesicles.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • FIG. 27A shows serial dilution of vesicles stained with 40 ⁇ of CFSE according to vendor instructions.
  • CFSE fluorescence was determined at several time -points (0, 15, 30 and 45 min post incubation, as indicated in the figure) to evaluate enzymatic activity over time. The CFSE fluorescent signal was consistent after 15 min of incubation and fluorescence values correleated to microvesicle concentration.
  • FIG. 27B shows a standard curve generated using CFSE stained microvesicles. 50 x 10 6 microvesicles as determined using flow cytometry were stained with 40 ⁇ in 400 ⁇ to create the standard curve. The curve was generated by detecting fluorescence in a series of dilutions using a Viaa7 RT-PCR machine. See Examples for details.
  • FIG. 27C shows the effects of CFSE concentration ( ⁇ ) on microvesicle staining.
  • FIG. 27D and FIG. 27E illustrate determination of microvesicle concentration in a test sample using a standard curve.
  • the protocol is outlined in detail in the Examples herein. Briefly, the standard curve and test samples were stained with 370 ⁇ CFSE then incubated at room temperature before they were loaded on 96-well (MicroAmp) plate.
  • fluorescence relative units Y-axis, Viia-7 system readings
  • X-axis microvesicle concentration
  • a phenotype of a biological sample e.g., a sample from a cell culture, an organism, or a subject.
  • the phenotype can be characterized by assessing at least one biomarkers.
  • the biomarkers can be associated with a vesicle or vesicle population, either presented vesicle surface antigens or vesicle payload.
  • vesicle payload comprises entities encapsulated within a vesicle.
  • Vesicle associated biomarkers can comprise both membrane bound and soluble biomarkers.
  • the biomarkers can also be circulating biomarkers, such as nucleic acids (e.g., microRNA) or protein/polypeptide, or functional fragments thereof, assessed in a bodily fluid.
  • biomarkers such as nucleic acids (e.g., microRNA) or protein/polypeptide, or functional fragments thereof, assessed in a bodily fluid.
  • nucleic acids e.g., microRNA
  • protein/polypeptide e.g., protein/polypeptide, or functional fragments thereof, assessed in a bodily fluid.
  • the terms "purified” or “isolated” as used herein in reference to vesicles or biomarker components mean partial or complete purification or isolation of such components from a cell or organism.
  • reference to vesicle isolation using a binding agent includes binding a vesicle with the binding agent whether or not such binding results in complete isolation of the vesicle apart from other biological entities in the starting material.
  • a method of characterizing a phenotype by analyzing a circulating biomarker e.g., a nucleic acid biomarker
  • a biological sample is obtained, e.g., a bodily fluid, tissue sample or cell culture.
  • Nucleic acids are isolated from the sample 103.
  • the nucleic acid can be DNA or RNA, e.g., microRNA. Assessment of such nucleic acids can provide a biosignature for a phenotype.
  • At least one nucleic acid markers that are indicative of the phenotype can be determined.
  • Various aspects of the present invention are directed to biosignatures determined by assessing at least one nucleic acid molecules (e.g., microRNA) present in the sample 105, where the biosignature corresponds to a predetermined phenotype 107.
  • FIG. IB illustrates a scheme 100B of using vesicles to determine a biosignature and/or characterize a phenotype.
  • a biological sample is obtained 102, and at least one vesicles of interest, e.g., all vesicles, or vesicles from a particular cell-of-origin and/or vesicles associated with a particular disease state, are isolated from the sample 104.
  • the vesicles can be analyzed 106 by characterizing surface antigens associated with the vesicles and/or determining the presence or levels of components present within the vesicles ("payload").
  • the term "antigen" as used herein refers generally to a biomarker that can be bound by a binding agent, whether the binding agent is an antibody, aptamer, lectin, or other binding agent for the biomarker and regardless of whether such biomarker illicits an immune response in a host.
  • Vesicle payload including without limitation protein, including peptides and polypeptides, nucleic acids such as DNA and RNAs, lipids and/or carbohydrates.
  • RNA payload includes messenger RNA (mRNA) and microRNA (also referred to herein as miRNA or miR).
  • mRNA messenger RNA
  • miRNA miRNA
  • a phenotype is characterized based on the biosignature of the vesicles 108.
  • schemes 100A and 100B are performed together to characterize a phenotype.
  • vesicles and nucleic acids e.g., microRNA
  • multiple biomarkers can be assessed sequentially or concurrently to characterize a phenotype.
  • a subpopulation of vesicles can be assessed by concurrently detecting two vesicle surface antigens, e.g., using binding agents to both capture and detect vesicles.
  • a subpopulation of vesicles can be assessed by sequentially detecting a vesicle surface antigen, e.g., to capture vesicles, and then the captured vesicles can be assessed for payload such as mRNA, microRNA or soluble protein.
  • characterizing a phenotype comprises both the concurrent assessment of at least one biomarker and sequential assessment of at least one other biomarker.
  • a vesicle subpopulation that is detecting using binding agents to more than one surface antigen can be sorted, and then payload can be assessed, e.g., at least one miRs.
  • payload can be assessed, e.g., at least one miRs.
  • biomarkers comprising assessing vesicle surface markers or payload markers in one sample and comparing the markers to another sample.
  • Markers that distinguish between the samples can be used as biomarkers according to the invention.
  • Such samples can be from a subject or group of subjects.
  • the groups can be, e.g., diseased versus normal (e.g., non- diseased), known responders and non-responders to a given treatment for a given disease or disorder.
  • Biomarkers discovered to distinguish the known responders and non-responders provide a biosignature of whether a subject is likely to respond to a treatment such as a therapeutic agent, e.g., a drug or biologic.
  • the term "about,” e.g., when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods.
  • "about” refers to ⁇ 10%.
  • phenotype can mean any trait or characteristic that is attributed to a biomarker profile that is identified using in part or in whole the compositions and/or methods of the invention.
  • a phenotype can be a diagnostic, prognostic or theranostic determination based on a characterized biomarker profile for a sample obtained from a subject.
  • a phenotype can be any observable characteristic or trait of, such as a disease or condition, a stage of a disease or condition, susceptibility to a disease or condition, prognosis of a disease stage or condition, a physiological state, or response / potential response to therapeutics.
  • a phenotype can result from a subject's genetic makeup as well as the influence of environmental factors and the interactions between the two, as well as from epigenetic modifications to nucleic acid sequences.
  • a phenotype in a subject can be characterized by obtaining a biological sample from a subject and analyzing the sample.
  • characterizing a phenotype for a subject or individual may include detecting a disease or condition (including pre-symptomatic early stage detecting), determining a prognosis, diagnosis, or theranosis of a disease or condition, or determining the stage or progression of a disease or condition.
  • Characterizing a phenotype can include identifying appropriate treatments or treatment efficacy for specific diseases, conditions, disease stages and condition stages, predictions and likelihood analysis of disease progression, particularly disease recurrence, metastatic spread or disease relapse.
  • a phenotype can also be a clinically distinct type or subtype of a condition or disease, such as a cancer or tumor.
  • Phenotype determination can also be a determination of a physiological condition, or an assessment of organ distress or organ rejection, such as post-transplantation.
  • the products and processes described herein allow assessment of a subject on an individual basis, which can provide benefits of more efficient and economical decisions in treatment.
  • the invention relates to the analysis of a biological sample to identify a biosignature to predict whether a subject is likely to respond to a treatment for a disease or disorder. Characterizating a phenotype includes predicting the responder / non-responder status of the subject, wherein a responder responds to a treatment for a disease and a non-responder does not respond to the treatment. Vesicles can be analyzed in the subject and compared to vesicle analysis of previous subjects that were known to respond or not to a treatment. If the vesicle biosignature in a subject more closely aligns with that of previous subjects that were known to respond to the treatment, the subject can be characterized, or predicted, as a responder to the treatment.
  • the subject can be characterized, or predicted as a non-responder to the treatment.
  • the treatment can be for any appropriate disease, disorder or other condition.
  • the method can be used in any disease setting where a vesicle biosignature that correlates with responder / non-responder status is known.
  • phenotype can mean any trait or characteristic that is attributed to a vesicle biosignature that is identified using methods of the invention.
  • a phenotype can be the identification of a subject as likely to respond to a treatment, or more broadly, it can be a diagnostic, prognostic or theranostic determination based on a characterized biosignature for a sample obtained from a subject.
  • the phenotype comprises a disease or condition such as those listed in Table 1.
  • the phenotype can comprise the presence of or likelihood of developing a tumor, neoplasm, or cancer.
  • a cancer detected or assessed by products or processes described herein includes, but is not limited to, breast cancer, ovarian cancer, lung cancer, colon cancer, hyperplastic polyp, adenoma, colorectal cancer, high grade dysplasia, low grade dysplasia, prostatic hyperplasia, prostate cancer, melanoma, pancreatic cancer, brain cancer (such as a glioblastoma), hematological malignancy, hepatocellular carcinoma, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cell carcinoma (RCC) or gastric cancer.
  • GIST gastrointestinal stromal tumor
  • RRCC renal cell carcinoma
  • the colorectal cancer can be CRC Dukes B or Dukes C-D.
  • the hematological malignancy can be B-Cell Chronic Lymphocytic Leukemia, B-Cell Lymphoma-DLBCL, B-Cell Lymphoma-DLBCL-germinal center-like, B-Cell Lymphoma-DLBCL-activated B-cell-like, and Burkitt's lymphoma.
  • the phenotype can be a premalignant condition, such as actinic keratosis, atrophic gastritis, leukoplakia, erythroplasia, Lymphomatoid Granulomatosis, preleukemia, fibrosis, cervical dysplasia, uterine cervical dysplasia, xeroderma pigmentosum, Barrett's Esophagus, colorectal polyp, or other abnormal tissue growth or lesion that is likely to develop into a malignant tumor.
  • Transformative viral infections such as FHV and HPV also present phenotypes that can be assessed according to the invention.
  • the cancer characterized by the methods of the invention can comprise, without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, a germ cell tumor, a blastoma, or other cancers.
  • Carcinomas include without limitation epithelial neoplasms, squamous cell neoplasms squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multiple endocrine
  • Sarcoma includes without limitation Askin's tumor, botryodies, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and
  • Lymphoma and leukemia include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leuk
  • Germ cell tumors include without limitation germinoma, dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus turmor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma.
  • Blastoma includes without limitation nephroblastoma, meduUoblastoma, and retinoblastoma.
  • cancers include without limitation labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, meduUoblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma
  • the cancer under analysis may be a lung cancer including non-small cell lung cancer and small cell lung cancer (including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma), colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor.
  • non-small cell lung cancer and small cell lung cancer including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma
  • colon cancer breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblast
  • the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia;
  • adrenocortical carcinoma AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer;
  • astrocytomas atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, meduUoblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic my
  • endometrial cancer endometrial cancer
  • ependymoblastoma ependymoma
  • ependymoma ependymoma
  • esophageal cancer esthesioneuroblastoma
  • Ewing sarcoma extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor
  • GIST gestational trophoblastic tumor
  • glioma hairy cell leukemia; head and neck cancer
  • heart cancer Hodgkin lymphoma
  • hypopharyngeal cancer intraocular melanoma
  • islet cell tumors Kaposi sarcoma; kidney cancer;
  • Langerhans cell histiocytosis laryngeal cancer; lip cancer; liver cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma;
  • mesothelioma metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non- Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer;
  • oropharyngeal cancer osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma;
  • pleuropulmonary blastoma pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer;
  • T-cell lymphoma testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm's tumor.
  • the methods of the invention can be used to characterize these and other cancers.
  • characterizing a phenotype can be providing a diagnosis, prognosis or theranosis of one of the cancers disclosed herein.
  • the cancer comprises an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), lung non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non epithelial ovarian cancer
  • AML acute myeloid
  • the phenotype can also be an inflammatory disease, immune disease, or autoimmune disease.
  • the disease may be inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes, autoimmune hepatitis, Multiple Sclerosis, Myasthenia Gravis, Type I diabetes, Rheumatoid Arthritis, Psoriasis, Systemic Lupus Erythematosis (SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing Spondylitis Sjogrens Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organ rejection, Primary Sclerosing Cholangitis, or sepsis.
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • pelvic inflammation vasculitis
  • psoriasis psoriasis
  • diabetes autoimmune hepatitis
  • the phenotype can also comprise a cardiovascular disease, such as atherosclerosis, congestive heart failure, vulnerable plaque, stroke, or ischemia.
  • the cardiovascular disease or condition can be high blood pressure, stenosis, vessel occlusion or a thrombotic event.
  • the phenotype can also comprise a neurological disease, such as Multiple Sclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD), schizophrenia, bipolar disorder, depression, autism, Prion Disease, Pick's disease, dementia, Huntington disease (HD), Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophic lateral sclerosis,
  • a neurological disease such as Multiple Sclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD), schizophrenia, bipolar disorder, depression, autism, Prion Disease, Pick's disease, dementia, Huntington disease (HD), Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophic lateral sclerosis,
  • MS Multiple Sclerosis
  • PD Parkinson's
  • the phenotype may also be a condition such as fibromyalgia, chronic neuropathic pain, or peripheral neuropathic pain.
  • the phenotype may also comprise an infectious disease, such as a bacterial, viral or yeast infection.
  • the disease or condition may be Whipple's Disease, Prion Disease, cirrhosis, methicillin-resistant staphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria, tuberculosis, or influenza.
  • Viral proteins, such as HIV or HCV-like particles can be assessed in a vesicle, to characterize a viral condition.
  • the phenotype can also comprise a perinatal or pregnancy related condition (e.g. preeclampsia or preterm birth), metabolic disease or condition, such as a metabolic disease or condition associated with iron metabolism.
  • a perinatal or pregnancy related condition e.g. preeclampsia or preterm birth
  • metabolic disease or condition such as a metabolic disease or condition associated with iron metabolism.
  • hepcidin can be assayed in a vesicle to characterize an iron deficiency.
  • the metabolic disease or condition can also be diabetes, inflammation, or a perinatal condition.
  • the methods of the invention can be used to characterize these and other diseases and disorders that can be assessed via a candidate biosignature comprising one or a plurality of biomarkers.
  • characterizing a phenotype can be providing a diagnosis, prognosis or theranosis of one of the diseases and disorders disclosed herein.
  • a biosignature for any of the conditions or diseases disclosed herein can comprise one or more biomarkers in one of several different categories of markers, wherein the categories include one or more of: 1) disease specific biomarkers; 2) cell- or tissue-specific biomarkers; 3) vesicle-specific markers (e.g., general vesicle biomarkers); 4. angiogenesis-specific biomarkers; and 5) immunomodulatory biomarkers. Examples of such markers for use in methods and compositions of the invention are disclosed herein. Furthermore, a biomarker known in the art that is characterized to have a role in a particular disease or condition can be adapted for use as a target in compositions and methods of the invention.
  • biomarkers can be all vesicle surface markers, or a combination of vesicle surface markers and vesicle payload markers (i.e., molecules enclosed by a vesicle).
  • the biological sample assessed can be any biological fluid, or can comprise individual components present within such biological fluid (e.g., vesicles, nucleic acids, proteins, or complexes thereof).
  • One or more phenotypes of a subject can be determined by analyzing one or more vesicles, such as vesicles, in a biological sample obtained from the subject.
  • a subject or patient can include, but is not limited to, mammals such as bovine, avian, canine, equine, feline, ovine, porcine, or primate animals (including humans and non-human primates).
  • a subject can also include a mammal of importance due to being endangered, such as a Siberian tiger; or economic importance, such as an animal raised on a farm for consumption by humans, or an animal of social importance to humans, such as an animal kept as a pet or in a zoo.
  • Examples of such animals include, but are not limited to, carnivores such as cats and dogs; swine including pigs, hogs and wild boars; ruminants or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, camels or horses. Also included are birds that are endangered or kept in zoos, as well as fowl and more particularly domesticated fowl, i.e. poultry, such as turkeys and chickens, ducks, geese, guinea fowl. Also included are domesticated swine and horses (including race horses). In addition, any animal species connected to commercial activities are also included such as those animals connected to agriculture and aquaculture and other activities in which disease monitoring, diagnosis, and therapy selection are routine practice in husbandry for economic productivity and/or safety of the food chain.
  • the subject can have a pre-existing disease or condition, such as cancer.
  • the subject may not have any known pre-existing condition.
  • the subject may also be non-responsive to an existing or past treatment, such as a treatment for cancer.
  • a sample used and/or assessed via the compositions and methods of the invention includes any relevant biological sample that can be used for biomarker assessment, including without limitation sections of tissues such as biopsy or tissue removed during surgical or other procedures, bodily fluids, autopsy samples, frozen sections taken for histological purposes, and cell cultures.
  • samples include blood and blood fractions or products (e.g., serum, buffy coat, plasma, platelets, red blood cells, and the like), sputum, malignant effusion, cheek cells tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, other biological or bodily fluids (e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, and the like), etc.
  • blood and blood fractions or products e.g., serum, buffy coat, plasma, platelets, red blood cells, and the like
  • sputum e.g., malignant effusion
  • cheek cells tissue e.g., cultured cells (e.
  • the sample can comprise biological material that is a fresh frozen & formalin fixed paraffin embedded (FFPE) block, formalin- fixed paraffin embedded, or is within an RNA preservative + formalin fixative. More than one sample of more than one type can be used for each patient.
  • FFPE fresh frozen & formalin fixed paraffin embedded
  • the sample used in the methods described herein can be a formalin fixed paraffin embedded (FFPE) sample.
  • the FFPE sample can be one or more of fixed tissue, unstained slides, bone marrow core or clot, core needle biopsy, malignant fluids and fine needle aspirate (FNA).
  • the fixed tissue comprises a tumor containing formalin fixed paraffin embedded (FFPE) block from a surgery or biopsy.
  • the unstained slides comprise unstained, charged, unbaked slides from a paraffin block.
  • bone marrow core or clot comprises a decalcified core.
  • a formalin fixed core and/or clot can be paraffin-embedded.
  • the core needle biopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 3-4, paraffin embedded biopsy samples.
  • An 18 gauge needle biopsy can be used.
  • the malignant fluid can comprise a sufficient volume of fresh pleural/ascitic fluid to produce a 5x5x2mm cell pellet.
  • the fluid can be formalin fixed in a paraffin block.
  • the core needle biopsy comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g., 4-6, paraffin embedded aspirates.
  • a sample may be processed according to techniques understood by those in the art.
  • a sample can be without limitation fresh, frozen or fixed cells or tissue.
  • a sample comprises formalin- fixed paraffin-embedded (FFPE) tissue, fresh tissue or fresh frozen (FF) tissue.
  • FFPE formalin- fixed paraffin-embedded
  • a sample can comprise cultured cells, including primary or immortalized cell lines derived from a subject sample.
  • a sample can also refer to an extract from a sample from a subject.
  • a sample can comprise DNA, RNA or protein extracted from a tissue or a bodily fluid. Many techniques and commercial kits are available for such purposes.
  • the fresh sample from the individual can be treated with an agent to preserve RNA prior to further processing, e.g., cell lysis and extraction.
  • Samples can include frozen samples collected for other purposes. Samples can be associated with relevant information such as age, gender, and clinical symptoms present in the subject; source of the sample; and methods of collection and storage of the sample.
  • a sample is typically obtained from
  • a biopsy comprises the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself.
  • Any biopsy technique known in the art can be applied to the molecular profiling methods of the present invention.
  • the biopsy technique applied can depend on the tissue type to be evaluated (e.g., colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, lung, breast, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors.
  • Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy.
  • An "excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • Molecular profiling can use a "core -needle biopsy” of the tumor mass, or a “fine -needle aspiration biopsy” which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.
  • PCR Polymerase chain reaction
  • the biological sample assessed using the compositions and methods of the invention can be any useful bodily or biological fluid, including but not limited to peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, other lavage fluids, cells, cell culture, or a cell culture supernatant.
  • CSF cerebro
  • a biological sample may also include the blastocyl cavity, umbilical cord blood, or maternal circulation which may be of fetal or maternal origin.
  • the biological sample may also be a cell culture, tissue sample or biopsy from which vesicles and other circulating biomarkers may be obtained. For example, cells of interest can be cultured and vesicles isolated from the culture.
  • biomarkers or more particularly biosignatures disclosed herein can be assessed directly from such biological samples (e.g., identification of presence or levels of nucleic acid or polypeptide biomarkers or functional fragments thereof) using various methods, such as extraction of nucleic acid molecules from blood, plasma, serum or any of the foregoing biological samples, use of protein or antibody arrays to identify polypeptide (or functional fragment) biomarker(s), as well as other array, sequencing, PCR and proteomic techniques known in the art for identification and assessment of nucleic acid and polypeptide molecules.
  • one or more components present in such samples can be first isolated or enriched and further processed to assess the presence or levels of selected biomarkers, to assess a given biosignature (e.g., isolated microvesicles prior to profiling for protein and/or nucleic acid biomarkers).
  • a given biosignature e.g., isolated microvesicles prior to profiling for protein and/or nucleic acid biomarkers.
  • Table 1 presents a non-limiting listing of diseases, conditions, or biological states and corresponding biological samples that may be used for analysis according to the methods of the invention.
  • Table 1 Examples of Biological Samples for Vesicle Analysis for Various
  • Blood derivatives include plasma and serum.
  • Blood plasma is the liquid component of whole blood, and makes up approximately 55% of the total blood volume. It is composed primarily of water with small amounts of minerals, salts, ions, nutrients, and proteins in solution. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma.
  • Blood serum refers to blood plasma without fibrinogen or other clotting factors (i.e., whole blood minus both the cells and the clotting factors).
  • the biological sample may be obtained through a third party, such as a party not performing the analysis of the biomarkers, whether direct assessment of a biological sample or by profiling one or more vesicles obtained from the biological sample.
  • a third party such as a party not performing the analysis of the biomarkers, whether direct assessment of a biological sample or by profiling one or more vesicles obtained from the biological sample.
  • the sample may be obtained through a clinician, physician, or other health care manager of a subject from which the sample is derived.
  • the biological sample may obtained by the same party analyzing the vesicle.
  • biological samples be assayed, are archived (e.g., frozen) or ortherwise stored in under preservative conditions.
  • the volume of the biological sample used for biomarker analysis can be in the range of between 0.1-20 mL, such as less than about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.1 mL.
  • a sample of bodily fluid can be used as a sample for characterizing a phenotype.
  • biomarkers in the sample can be assessed to provide a diagnosis, prognosis and/or theranosis of a disease.
  • the biomarkers can be circulating biomarkers, such as circulating proteins or nucleic acids.
  • the biomarkers can also be associated with a vesicle or vesicle population.
  • Methods of the invention can be applied to assess one or more vesicles, as well as one or more different vesicle populations that may be present in a biological sample or in a subject.
  • Analysis of one or more biomarkers in a biological sample can be used to determine whether an additional biological sample should be obtained for analysis.
  • analysis of one or more vesicles in a sample of bodily fluid can aid in determining whether a tissue biopsy should be obtained.
  • a sample from a patient can be collected under conditions that preserve the circulating biomarkers and other entities of interest contained therein for subsequent analysis.
  • the samples are processed using one or more of CellSave Preservative Tubes (Veridex, North Raritan, NJ), PAXgene Blood DNA Tubes (QIAGEN GmbH, Germany), and RNAlater (QIAGEN GmbH, Germany).
  • CellSave Preservative Tubes are sterile evacuated blood collection tubes. Each tube contains a solution that contains Na2EDTA and a cell preservative. The EDTA absorbs calcium ions, which can reduce or eliminate blood clotting. The preservative preserves the morphology and cell surface antigen expression of epithelial and other cells. The collection and processing can be performed as described in a protocol provided by the manufacturer. Each tube is evacuated to withdraw venous whole blood following standard phlebotomy procedures as known to those of skill in the art. CellSave tubes are disclosed in US Patent Numbers 5,466,574; 5,512,332;
  • PAXgene Blood DNA Tube is a plastic, evacuated tube for the collection of whole blood for the isolation of nucleic acids.
  • the tubes can be used for blood collection, transport and storage of whole blood specimens and isolation of nucleic acids contained therein, e.g., DNA or RNA.
  • Blood is collected under a standard phlebotomy protocol into an evacuated tube that contains an additive. The collection and processing can be performed as described in a protocol provided by the manufacturer.
  • PAXgene tubes are disclosed in US Patent Nos. 5,906,744; 4,741,446; 4,991, 104, each of which is incorporated by reference in its entirety herein.
  • RNAlater RNA Stabilization Reagent
  • RNA can be unstable in harvested samples.
  • the aqueous RNAlater reagent permeates tissues and other biological samples, thereby stabilizing and protecting the RNA contained therein. Such protection helps ensure that downstream analyses reflect the expression profile of the RNA in the tissue or other sample.
  • the samples are submerged in an appropriate volume of RNAlater reagent immediately after harvesting. The collection and processing can be performed as described in a protocol provided by the manufacturer.
  • the reagent preserves RNA for up to 1 day at 37°C, 7 days at 18-25°C, or 4 weeks at 2-8°C, allowing processing, transportation, storage, and shipping of samples without liquid nitrogen or dry ice.
  • the samples can also be placed at -20°C or -80°C, e.g., for archival storage.
  • the preserved samples can be used to analyze any type of
  • RNA including without limitation total RNA, mRNA, and microRNA.
  • RNAlater can also be useful for collecting samples for DNA, RNA and protein analysis. RNAlater is disclosed in US Patent Nos. 5,346,994, each of which is incorporated by reference in its entirety herein.
  • the biological sample of the invention is understood to comprise a sample containing a separated, depleted, enriched, isolated, or otherwise processed derivative of another biological sample.
  • a component of a patient sample or a cell culture can be isolated from the patient sample or the cell culture and resuspended in a buffer for further analysis.
  • the derivative component suspended in the buffer is a biological sample that can be assessed according to the methods of the invention.
  • the component can be any useful biological entity as disclosed herein or known in the art, including without limitation circulating biomarkers, vesicles, proteins, nucleic acids, lipids or carbohydrates.
  • the biological sample can be the biological entity, including without limitation circulating biomarkers, vesicles, proteins, nucleic acids, lipids or carbohydrates.
  • Methods of the invention can include assessing one or more vesicles, including assessing vesicle populations.
  • a vesicle as used herein, is a membrane vesicle that is shed from cells. Vesicles or membrane vesicles include without limitation: circulating microvesicles (cMVs), microvesicle, exosome, nanovesicle, dexosome, bleb, blebby, prostasome, microparticle, intralumenal vesicle, membrane fragment, intralumenal endosomal vesicle, endosomal-like vesicle, exocytosis vehicle, endosome vesicle, endosomal vesicle, apoptotic body, multivesicular body, secretory vesicle, phospholipid vesicle, liposomal vesicle, argosome, texasome, secresome, tolerosome, melanosome, onco
  • Vesicles may be produced by different cellular processes, the methods of the invention are not limited to or reliant on any one mechanism, insofar as such vesicles are present in a biological sample and are capable of being characterized by the methods disclosed herein. Unless otherwise specified, methods that make use of a species of vesicle can be applied to other types of vesicles. Vesicles comprise spherical structures with a lipid bilayer similar to cell membranes which surrounds an inner compartment which can contain soluble components, sometimes referred to as the payload. In some embodiments, the methods of the invention make use of exosomes, which are small secreted vesicles of about 40-100 nm in diameter. For a review of membrane vesicles, including types and characterizations, see Thery et al, Nat Rev Immunol. 2009 Aug;9(8):581-93. Some properties of different types of vesicles include those in Table 2:
  • PPS phosphatidylserine
  • EM electron microscopy
  • Vesicles include shed membrane bound particles, or "microparticles," that are derived from either the plasma membrane or an internal membrane. Vesicles can be released into the extracellular environment from cells.
  • Cells releasing vesicles include without limitation cells that originate from, or are derived from, the ectoderm, endoderm, or mesoderm. The cells may have undergone genetic, environmental, and/or any other variations or alterations.
  • the cell can be tumor cells.
  • a vesicle can reflect any changes in the source cell, and thereby reflect changes in the originating cells, e.g., cells having various genetic mutations.
  • a vesicle is generated intracellularly when a segment of the cell membrane spontaneously invaginates and is ultimately exocytosed (see for example, Keller et al, Immunol. Lett. 107 (2): 102-8 (2006)).
  • Vesicles also include cell-derived structures bounded by a lipid bilayer membrane arising from both herniated evagination (blebbing) separation and sealing of portions of the plasma membrane or from the export of any intracellular membrane-bounded vesicular structure containing various membrane-associated proteins of tumor origin, including surface-bound molecules derived from the host circulation that bind selectively to the tumor-derived proteins together with molecules contained in the vesicle lumen, including but not limited to tumor-derived microRNAs or intracellular proteins.
  • a vesicle shed into circulation or bodily fluids from tumor cells may be referred to as a "circulating tumor-derived vesicle.”
  • a vesicle shed into circulation or bodily fluids from tumor cells
  • a circulating tumor-derived vesicle When such vesicle is an exosome, it may be referred to as a circulating-tumor derived exosome (CTE).
  • CTE circulating-tumor derived exosome
  • a vesicle can be derived from a specific cell of origin.
  • CTE as with a cell-of-origin specific vesicle, typically have one or more unique biomarkers that permit isolation of the CTE or cell- of-origin specific vesicle, e.g., from a bodily fluid and sometimes in a specific manner.
  • a cell or tissue specific markers are used to identify the cell of origin. Examples of such cell or tissue specific markers are disclosed herein and can further be accessed in the Tissue-specific Gene Expression and Regulation (TiGER) Database, available at bioinfo.wilmer.jhu.edu/tiger/; Liu et al. (2008) TiGER: a database for tissue-specific gene expression and regulation.
  • TiGER Tissue-specific Gene Expression and Regulation
  • a vesicle can have a diameter of greater than about 10 nm, 20 nm, or 30 nm.
  • a vesicle can have a diameter of greater than 40 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1500 nm, 2000 nm or greater than 10,000 nm.
  • a vesicle can have a diameter of about 20-2000 nm, about 20-1500 nm, about 30-1000 nm, about 30-800 nm, about 30-200 nm, or about 30-100 nm.
  • the vesicle has a diameter of less than 10,000 nm, 2000 nm, 1500 nm, 1000 nm, 800 nm, 500 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm or less than 10 nm.
  • the term "about" in reference to a numerical value means that variations of 10% above or below the numerical value are within the range ascribed to the specified value. Typical sizes for various types of vesicles are shown in Table 2. Vesicles can be assessed to measure the diameter of a single vesicle or any number of vesicles.
  • the range of diameters of a vesicle population or an average diameter of a vesicle population can be determined.
  • Vesicle diameter can be assessed using methods known in the art, e.g., imaging technologies such as electron microscopy.
  • a diameter of one or more vesicles is determined using optical particle detection. See, e.g., U.S. Patent 7,751,053, entitled “Optical Detection and Analysis of Particles" and issued July 6, 2010; and U.S. Patent 7,399,600, entitled “Optical Detection and Analysis of Particles” and issued July 15, 2010.
  • the methods of the invention comprise assessing vesicles directly assayed from a biological sample without prior isolation, purification, or concentration from the biological sample.
  • the amount of vesicles in the sample can by itself provide a biosignature that provides a diagnostic, prognostic or theranostic determination.
  • the vesicle in the sample may be isolated, captured, purified, or concentrated from a sample prior to analysis.
  • isolation, capture or purification as used herein comprises partial isolation, partial capture or partial purification apart from other components in the sample.
  • Vesicle isolation can be performed using various techniques as described herein, e.g., chromatography, filtration, centrifugation, flow cytometry, affinity capture (e.g., to a planar surface or bead), and/or using microfluidics.
  • Vesicles such as exosomes can be assessed to provide a phenotypic characterization by comparing vesicle characteristics to a reference.
  • surface antigens on a vesicle are assessed.
  • the surface antigens can provide an indication of the anatomical origin and/or cellular of the vesicles and other phenotypic information, e.g., tumor status.
  • a patient sample e.g., a bodily fluid such as blood, serum or plasma
  • a bodily fluid such as blood, serum or plasma
  • the surface antigens may comprise any informative biological entity that can be detected on the vesicle membrane surface, including without limitation surface proteins, lipids, carbohydrates, and other membrane components.
  • positive detection of colon derived vesicles expressing tumor antigens can indicate that the patient has colorectal cancer.
  • methods of the invention can be used to characterize any disease or condition associated with an anatomical or cellular origin, by assessing, for example, disease-specific and cell-specific biomarkers of one or more vesicles obtained from a subject.
  • one or more vesicle payloads are assessed to provide a phenotypic characterization.
  • the payload with a vesicle comprises any informative biological entity that can be detected as encapsulated within the vesicle, including without limitation proteins and nucleic acids, e.g., genomic or cDNA, mRNA, or functional fragments thereof, as well as microRNAs (miRs).
  • methods of the invention are directed to detecting vesicle surface antigens (in addition or exclusive to vesicle payload) to provide a phenotypic characterization.
  • vesicles can be characterized by using binding agents (e.g., antibodies or aptamers) that are specific to vesicle surface antigens, and the bound vesicles can be further assessed to identify one or more payload components disclosed therein.
  • the levels of vesicles with surface antigens of interest or with payload of interest can be compared to a reference to characterize a phenotype.
  • overexpression in a sample of cancer-related surface antigens or vesicle payload e.g., a tumor associated mRNA or microRNA, as compared to a reference, can indicate the presence of cancer in the sample.
  • the biomarkers assessed can be present or absent, increased or reduced based on the selection of the desired target sample and comparison of the target sample to the desired reference sample.
  • target samples include: disease; treated/not-treated; different time points, such as a in a longitudinal study; and non-limiting examples of reference sample: non-disease; normal; different time points; and sensitive or resistant to candidate treatment(s).
  • MicroRNAs comprise one class biomarkers assessed via methods of the invention.
  • MicroRNAs also referred to herein as miRNAs or miRs, are short RNA strands approximately 21-23 nucleotides in length.
  • MiRNAs are encoded by genes that are transcribed from DNA but are not translated into protein and thus comprise non-coding RNA.
  • the miRs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to the resulting single strand miRNA.
  • the pre -miRNA typically forms a structure that folds back on itself in self-complementary regions.
  • Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and can function to regulate translation of proteins. Identified sequences of miRNA can be accessed at publicly available databases, such as www.microRNA.org, www.mirbase.org, or www.mirz.unibas.ch/cgi/miRNA.cgi.
  • miRNAs are generally assigned a number according to the naming convention " mir- [number]." The number of a miRNA is assigned according to its order of discovery relative to previously identified miRNA species. For example, if the last published miRNA was mir-121, the next discovered miRNA will be named mir- 122, etc. When a miRNA is discovered that is homologous to a known miRNA from a different organism, the name can be given an optional organism identifier, of the form [organism identifier] - mir- [number]. Identifiers include hsa for Homo sapiens and mmu for Mus Musculus.
  • a human homolog to mir-121 might be referred to as hsa- mir-121 whereas the mouse homolog can be referred to as mmu-mir-121 and the rat homolog can be referred to as rno-mir-121, etc.
  • Mature microRNA is commonly designated with the prefix “miR” whereas the gene or precursor miRNA is designated with the prefix “mir.”
  • mir- 121 is a precursor for miR- 121.
  • the genes/precursors can be delineated by a numbered suffix.
  • mir-121-1 and mir- 121-2 can refer to distinct genes or precursors that are processed into miR- 121.
  • Lettered suffixes are used to indicate closely related mature sequences.
  • mir- 12 la and mir- 12 lb can be processed to closely related miRNAs miR- 12 la and miR- 12 lb, respectively.
  • any microRNA (miRNA or miR) designated herein with the prefix mir-* or miR-* is understood to encompass both the precursor and/or mature species, unless otherwise explicitly stated otherwise.
  • miR-121 would be the predominant product whereas miR-121 * is the less common variant found on the opposite arm of the precursor.
  • the miRs can be distinguished by the suffix "5p" for the variant from the 5' arm of the precursor and the suffix "3p" for the variant from the 3 ' arm.
  • miR-121-5p originates from the 5' arm of the precursor whereas miR-121-3p originates from the 3 ' arm.
  • miR-121-5p may be referred to as miR-121-s whereas miR- 121 -3p may be referred to as miR-121-as.
  • Plant miRNAs follow a different naming convention as described in Meyers et al., Plant Cell. 2008 20(12):3186-3190.
  • miRNAs are involved in gene regulation, and miRNAs are part of a growing class of non- coding RNAs that is now recognized as a major tier of gene control.
  • miRNAs can interrupt translation by binding to regulatory sites embedded in the 3 '-UTRs of their target mRNAs, leading to the repression of translation.
  • Target recognition involves complementary base pairing of the target site with the miRNA's seed region (positions 2-8 at the miRNA's 5' end), although the exact extent of seed complementarity is not precisely determined and can be modified by 3' pairing.
  • miRNAs function like small interfering RNAs (siRNA) and bind to perfectly complementary mRNA sequences to destroy the target transcript.
  • miRNAs Characterization of a number of miRNAs indicates that they influence a variety of processes, including early development, cell proliferation and cell death, apoptosis and fat metabolism. For example, some miRNAs, such as lin-4, let-7, mir-14, mir-23, and bantam, have been shown to play critical roles in cell differentiation and tissue development. Others are believed to have similarly important roles because of their differential spatial and temporal expression patterns.
  • the miRNA database available at miRBase comprises a searchable database of published miRNA sequences and annotation. Further information about miRBase can be found in the following articles, each of which is incorporated by reference in its entirety herein: Griffiths- Jones et al., miRBase: tools for microRNA genomics. NAR 2008 36(Database Issue):D154-D158; Griffiths- Jones et al., miRBase: microRNA sequences, targets and gene nomenclature. NAR 2006 34(Database Issue):D140-D144; and Griffiths- Jones, S. The microRNA Registry. NAR 2004 32(Database Issue):D109-Dl l 1. Representative miRNAs contained in Release 16 of miRBase, made available September 2010.
  • microRNAs are known to be involved in cancer and other diseases and can be assessed in order to characterize a phenotype in a sample. See, e.g., Ferracin et al., Micromarkers: miRNAs in cancer diagnosis and prognosis, Exp Rev Mol Diag, Apr 2010, Vol. 10, No. 3, Pages 297-308; Fabbri, miRNAs as molecular biomarkers of cancer, Exp Rev Mol Diag, May 2010, Vol. 10, No. 4, Pages 435-444. Techniques to isolate and characterize vesicles and miRs are disclosed herein and/or known to those of skill in the art. In addition to the methodology presented herein, additional methods can be found in U.S. Patent No.
  • Circulating biomarkers include biomarkers that are detectable in body fluids, such as blood, plasma, serum.
  • body fluids such as blood, plasma, serum.
  • circulating cancer biomarkers include cardiac troponin T (cTnT), prostate specific antigen (PSA) for prostate cancer and CA125 for ovarian cancer.
  • Circulating biomarkers according to the invention include any appropriate biomarker that can be detected in bodily fluid, including without limitation protein, nucleic acids, e.g., DNA, mRNA and microRNA, lipids, carbohydrates and metabolites.
  • Circulating biomarkers can include biomarkers that are not associated with cells, such as biomarkers that are membrane associated, embedded in membrane fragments, part of a biological complex, or free in solution.
  • circulating biomarkers are biomarkers that are associated with one or more vesicles present in the biological fluid of a subject.
  • Circulating biomarkers have been identified for use in characterization of various phenotypes. See, e.g., Ahmed N, et al., Proteomic -based identification of haptoglobin- 1 precursor as a novel circulating biomarker of ovarian cancer. Br. J. Cancer 2004; Mathelin et al., Circulating proteinic biomarkers and breast cancer, Gynecol Obstet Fertil. 2006 Jul-Aug;34(7-8):638-46. Epub 2006 Jul 28; Ye et al., Recent technical strategies to identify diagnostic biomarkers for ovarian cancer. Expert Rev Proteomics.
  • a vesicle or a population of vesicles may be isolated, purified, concentrated or otherwise enriched prior to and/or during analysis.
  • the terms "purified,” “isolated,” or similar as used herein in reference to vesicles or biomarker components are intended to include partial or complete purification or isolation of such components from a cell or organism.
  • Analysis of a vesicle can include quantitiating the amount one or more vesicle populations of a biological sample.
  • a heterogeneous population of vesicles can be quantitated, or a homogeneous population of vesicles, such as a population of vesicles with a particular biomarker profile, a particular biosignature, or derived from a particular cell type can be isolated from a heterogeneous population of vesicles and quantitated.
  • Analysis of a vesicle can also include detecting, quantitatively or qualitatively, one or more particular biomarker profile or biosignature of a vesicle, as described herein.
  • a vesicle can be stored and archived, such as in a bio-fluid bank and retrieved for analysis as necessary.
  • a vesicle may also be isolated from a biological sample that has been previously harvested and stored from a living or deceased subject.
  • a vesicle may be isolated from a biological sample which has been collected as described in King et al, Breast Cancer Res 7(5): 198-204 (2005).
  • a vesicle can be isolated from an archived or stored sample.
  • a vesicle may be isolated from a biological sample and analyzed without storing or archiving of the sample.
  • a third party may obtain or store the biological sample, or obtain or store the vesicle for analysis.
  • An enriched population of vesicles can be obtained from a biological sample.
  • vesicles may be concentrated or isolated from a biological sample using size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • Size exclusion chromatography such as gel permeation columns, centrifugation or density gradient centrifugation, and filtration methods can be used.
  • a vesicle can be isolated by differential centrifugation, anion exchange and/or gel permeation chromatography (for example, as described in US Patent Nos. 6,899,863 and 6,812,023), sucrose density gradients, organelle electrophoresis (for example, as described in U.S. Patent No. 7, 198,923), magnetic activated cell sorting (MACS), or with a nanomembrane ultrafiltration concentrator.
  • anion exchange and/or gel permeation chromatography for example, as described in US Patent Nos. 6,899,863 and 6,812,023
  • sucrose density gradients sucrose density gradients
  • organelle electrophoresis for example, as described in U.S. Patent No. 7, 198,923
  • MCS magnetic activated cell sorting
  • vesicle can be isolated from a biological sample using a system that uses multiple antibodies that are specific to the most abundant proteins found in a biological sample, such as blood. Such a system can remove up to several proteins at once, thus unveiling the lower abundance species such as cell-of-origin specific vesicles.
  • This type of system can be used for isolation of vesicles from biological samples such as blood, cerebrospinal fluid or urine.
  • the isolation of vesicles from a biological sample may also be enhanced by high abundant protein removal methods as described in Chromy et al.
  • vesicles from a biological sample may also be enhanced by removing serum proteins using glycopeptide capture as described in Zhang et al, Mol Cell Proteomics 2005;4:144-155.
  • vesicles from a biological sample such as urine may be isolated by differential centrifugation followed by contact with antibodies directed to cytoplasmic or anti-cytoplasmic epitopes as described in Pisitkun et al, Proc Natl Acad Sci USA, 2004;101:13368-13373.
  • Plasma contains a large variety of proteins including albumin, immunoglobulins, and clotting proteins such as fibrinogen.
  • About 60% of plasma protein comprises the protein albumin (e.g., human serum albumin or HSA), which contributes to osmotic pressure of plasma to assist in the transport of lipids and steroid hormones.
  • Globulins make up about 35% of plasma proteins and are used in the transport of ions, hormones and lipids assisting in immune function.
  • About 4% of plasma protein comprises fibrinogen which is essential in the clotting of blood and can be converted into the insoluble protein fibrin.
  • blood proteins include: Prealbumin, Alpha 1 antitrypsin, Alpha 1 acid glycoprotein, Alpha 1 fetoprotein, Haptoglobin, Alpha 2 macroglobulin, Ceruloplasmin, Transferrin, complement proteins C3 and C4, Beta 2 microglobulin, Beta lipoprotein, Gamma globulin proteins, C- reactive protein (CRP), Lipoproteins (chylomicrons, VLDL, LDL, HDL), other globulins (types alpha, beta and gamma), Prothrombin and Mannose -binding lectin (MBL).
  • CRP C- reactive protein
  • Lipoproteins chylomicrons, VLDL, LDL, HDL
  • other globulins types alpha, beta and gamma
  • Prothrombin and Mannose -binding lectin MBL
  • any of these proteins including classes of proteins, or derivatives thereof (such as fibrin which is derived from the cleavage of fibrinogen) can be selectively depleted from a biological sample prior to further analysis performed on the sample. Without being bound by theory, removal of such background proteins may facilitate more sensitive, accurate, or precise detection of the biomarkers of interest in the sample.
  • Abundant proteins in blood or blood derivatives include without limitation albumin, IgG, transferrin, fibrinogen, IgA, a 2 -Macroglobulin, IgM, ai -Antitrypsin, complement C3, haptoglobulin, apolipoprotein Al, apolipoprotein A3, apolipoprotein B, ⁇ -Acid Glycoprotein, ceruloplasmin, complement C4, C 1 q, IgD, prealbumin (transthyretin), and plasminogen.
  • Such proteins can be depleted using commercially available columns and kits. Examples of such columns comprise the Multiple Affinity Removal System from Agilent Technologies (Santa Clara, CA).
  • This system include various cartridges designed to deplete different protein profiles, including the following cartridges with performance characteristics according to the manufacturer: Human 14, which eliminates approximately 94% of total protein (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2 -macroglobulin, alphal-acid glycoprotein (orosomucoid), IgM, apolipoprotein AI, apolipoprotein All, complement C3 and transthyretin); Human 7, which eliminates approximately 85 - 90% of total protein (albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, and fibrinogen); Human 6, which eliminates approximately 85 - 90% of total protein (albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin); Human Albumin/IgG, which eliminates approximately 69% of total protein (albumin and IgG); and Human Albumin, which eliminates approximately 50-55% of
  • the ProteoPrep® 20 Plasma Immunodepletion Kit from Sigma- Aldrich is intended to specifically remove the 20 most abundant proteins from human plasma or serum, which is about remove 97-98% of the total protein mass in plasma or serum (Sigma- Aldrich, St. Louis, MO). According to the manufacturer, the ProteoPrep® 20 removes: albumin, IgG, transferrin, fibrinogen, IgA, a 2 - Macroglobulin, IgM, a r Antitrypsin, complement C3, haptoglobulin, apolipoprotem Al, A3 and B; a Acid Glycoprotein, ceruloplasmin, complement C4, Clq; IgD, prealbumin, and plasminogen.
  • ProteoPrep® columns to remove albumin (HSA) and immunoglobulins (IgG).
  • the ProteomeLab IgY-12 High Capacity Proteome Partitioning kits from Beckman Coulter (Fullerton, CA) are specifically designed to remove twelve highly abundant proteins (Albumin, IgG, Transferrin, Fibrinogen, IgA, a2 -macroglobulin, IgM, al -Antitrypsin, Haptoglobin, Orosomucoid, Apolipoprotem A-I, Apolipoprotem A-II) from the human biological fluids such as serum and plasma.
  • the Pure ProteomeTM Human Albumin/Immunoglobulin Depletion Kit from Millipore is a magnetic bead based kit that enables high depletion efficiency (typically >99%) of Albumin and all Immunoglobulins (i.e., IgG, IgA, IgM, IgE and IgD) from human serum or plasma samples.
  • the ProteoExtract ® Albumin/IgG Removal Kit also from Millipore, is designed to deplete >80% of albumin and IgG from body fluid samples.
  • Other similar protein depletion products include without limitation the following: AurumTM Affi-Gels Blue mini kit (Bio-Rad, Hercules, CA, USA); Vivapure® anti-HSA/IgG kit (Sartorius Stedim Biotech, Goettingen, Germany), Qproteome albumin/IgG depletion kit (Qiagen, Hilden, Germany); Seppro® MIXED 12-LC20 column (GenWay Biotech, San Diego, CA, USA); Abundant Serum Protein Depletion Kit (Norgen Biotek Corp., Ontario, Canada); GBC Human Albumin/IgG/Transferrin 3 in 1 Depletion Column/Kit (Good Biotech Corp., Taiwan). These systems and similar systems can be used to remove abundant proteins from a biological sample, thereby improving the ability to detect low abundance circulating biomarkers such as proteins and vesicles.
  • Thromboplastin is a plasma protein aiding blood coagulation through conversion of prothrombin to thrombin. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.
  • thromboplastin is a protein that can be used to facilitate precipitation of fibrinogen/fibrin (blood clotting factors) out of plasma.
  • a blood sample can be treated with thromboplastin to deplete fibrinogen/fibrin.
  • Thromboplastin removal can be performed in addition to or as an alternative to immunoaffinity protein removal as described above using methods known in the art. Precipitation of other proteins and/or other sample particulate can also improve detection of circulating biomarkers such as vesicles in a sample. For example, ammonium sulfate treatment as known in the art can be used to precipitate immunoglobulins and other highly abundant proteins.
  • the invention provides a method of detecting a presence or level of one or more circulating biomarker such as a microvesicle in a biological sample, comprising: (a) providing a biological sample comprising or suspected to comprise the one or more circulating biomarker; (b) selectively depleting one or more abundant protein from the biological sample provided in step (a); (c) performing affinity selection of the one or more circulating biomarker from the sample depleted in step (b), thereby detecting the presence or level of one or more circulating biomarker.
  • the biological sample may comprise a bodily fluid, e.g., peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, umbilical cord blood, or a derivative of any thereof.
  • the biological sample comprises peripheral blood, serum or plasma. See Example
  • An abundant protein may comprise a protein in the sample that is present in the sample at a high enough concentration to potentially interfere with downstream processing or analysis. Typically, an abundant protein is not the target of any further analysis of the sample.
  • the abundant protein may constitute at least 10 "5 , 10 "4 , 10 "3 , 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or at least 99% of the total protein mass in the sample.
  • the abundant protein is present at less than 10 "5 % of the total protein mass in the sample, e.g., in the case of a rare target of interest.
  • the one or more abundant protein may comprise one or more of albumin, IgG, transferrin, fibrinogen, fibrin, IgA, a2-Marcroglobulin, IgM, al -Antitrypsin, complement C3, haptoglobulin, apolipoprotein Al, A3 and B; al-Acid Glycoprotein, ceruloplasmin, complement C4, Clq, IgD, prealbumin (transthyretin), plasminogen, a derivative of any thereof, and a combination thereof.
  • the one or more abundant protein in blood or a blood derivative may also comprise one or more of Albumin, Immunoglobulins, Fibrinogen, Prealbumin, Alpha 1 antitrypsin, Alpha 1 acid glycoprotein, Alpha 1 fetoprotein, Haptoglobin, Alpha 2 macroglobulin, Ceruloplasmin, Transferrin, complement proteins C3 and C4, Beta 2 microglobulin, Beta lipoprotein, Gamma globulin proteins, C- reactive protein (CRP), Lipoproteins (chylomicrons, VLDL, LDL, HDL), other globulins (types alpha, beta and gamma), Prothrombin, Mannose -binding lectin (MBL), a derivative of any thereof, and a combination thereof.
  • Albumin Immunoglobulins
  • Fibrinogen Prealbumin
  • Prealbumin Alpha 1 antitrypsin
  • Alpha 1 acid glycoprotein Alpha 1 fetoprotein
  • Haptoglobin Haptoglobin
  • selectively depleting the one or more abundant protein comprises contacting the biological sample with thromboplastin to initiate precipitation of fibrin.
  • the one or more abundant protein may also be depleted by immunoaffinity, precipitation, or a combination thereof.
  • the sample can be treated with thromboplastin to precipitate fibrin, and then the sample may be passed through a column to remove HSA, IgG, and other abundant proteins as desired.
  • "Selectively depleting" the one or more abundant protein comprises depleting the abundant protein from the sample at a higher percentage than depletion another entity in the sample, such as another protein or microvesicle, including a target of interest for downstream processing or analysis.
  • Selectively depleting the one or more abundant protein may comprise depleting the abundant protein at a 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7- fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 10 4 -fold, 10 5 -fold, 10 6 -fold, 10 7 -fold, 10 8 -fold, 10 9 -fold, 10 10 -fold, 10 n -
  • selectively depleting the one or more abundant protein from the biological sample comprises depleting at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the one or more abundant protein.
  • Removal of highly abundant proteins and other non-desired entities can further be facilitated with a non- stringent size exclusion step.
  • the sample can be processed using a high molecular weight cutoff size exclusion step to preferentially enrich high molecular weight vesicles apart from lower molecular weight proteins and other entities.
  • a sample is processed with a column (e.g., a gel filtration column) or filter having a molecular weight cutoff (MWCO) of 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or greater than 10000 kiloDaltons (kDa).
  • a column e.g., a gel filtration column
  • kDa kiloDaltons
  • a 700 kDa filtration column is used.
  • the vesicles will be retained or flow more slowly than the column or filter than the lower molecular weight entities.
  • Such columns and filters are known in the art.
  • Isolation or enrichment of a vesicle from a biological sample can also be enhanced by use of sonication (for example, by applying ultrasound), detergents, other membrane-activating agents, or any combination thereof.
  • sonication for example, by applying ultrasound
  • detergents for example, by applying detergents
  • other membrane-activating agents for example, detergents, other membrane-activating agents, or any combination thereof.
  • ultrasonic energy can be applied to a potential tumor site, and without being bound by theory, release of vesicles from a tissue can be increased, allowing an enriched population of vesicles that can be analyzed or assessed from a biological sample using one or more methods disclosed herein.
  • the consistency of the results can be optimized as necessary using various concentration or isolation procedures.
  • Such steps can include agitation such as shaking or vortexing, different isolation techniques such as polymer based isolation, e.g., with PEG, and concentration to different levels during filtration or other steps.
  • agitation such as shaking or vortexing
  • different isolation techniques such as polymer based isolation, e.g., with PEG
  • concentration to different levels during filtration or other steps.
  • concentration can be applied at various stages of testing the vesicle containing sample.
  • the sample itself, e.g., a bodily fluid such as plasma or serum
  • the sample is vortexed after one or more sample treatment step, e.g., vesicle isolation, has occurred. Agitation can occur at some or all appropriate sample treatment steps as desired.
  • Additives can be introduced at the various steps to improve the process, e.g., to control aggregation or degradation of the biomarkers of interest.
  • the results can also be optimized as desireable by treating the sample with various agents.
  • agents include additives to control aggregation and/or additives to adjust pH or ionic strength.
  • Additives that control aggregation include blocking agents such as bovine serum albumin (BSA), milk or StabilGuard® (a BSA-free blocking agent; Product code SG02, Surmodics, Eden Prairie, MN), chaotropic agents such as guanidium hydro chloride, and detergents or surfactants.
  • BSA bovine serum albumin
  • StabilGuard® a BSA-free blocking agent
  • Product code SG02 Surmodics, Eden Prairie, MN
  • chaotropic agents such as guanidium hydro chloride
  • detergents or surfactants such as guanidium hydro chloride, and detergents or surfactants.
  • Useful ionic detergents include sodium dodecyl sulfate (SDS, sodium lauryl sulfate (SLS)), sodium laureth sulfate (SLS, sodium lauryl ether sulfate (SLES)), ammonium lauryl sulfate (ALS), cetrimonium bromide, cetrimonium chloride, cetrimonium stearate, and the like.
  • SDS sodium dodecyl sulfate
  • SLS sodium lauryl sulfate
  • SLES sodium laureth sulfate
  • ALS ammonium lauryl sulfate
  • cetrimonium bromide cetrimonium chloride
  • cetrimonium stearate and the like.
  • Non-ionic (zwitterionic) detergents include polyoxyethylene glycols, polysorbate 20 (also known as Tween 20), other polysorbates (e.g., 40, 60, 65, 80, etc), Triton-X (e.g., X100, XI 14), 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), CHAPSO, deoxycholic acid, sodium deoxycholate, NP-40, glycosides, octyl-thio-glucosides, maltosides, and the like.
  • polyoxyethylene glycols polysorbate 20 (also known as Tween 20), other polysorbates (e.g., 40, 60, 65, 80, etc), Triton-X (e.g., X100, XI 14), 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), CHAPSO,
  • Pluronic F-68 a surfactant shown to reduce platelet aggregation, is used to treat samples containing vesicles during isolation and/or detection.
  • F68 can be used from a 0.1% to 10% concentration, e.g., a 1%, 2.5% or 5% concentration.
  • the pH and/or ionic strength of the solution can be adjusted with various acids, bases, buffers or salts, including without limitation sodium chloride (NaCl), phosphate -buffered saline (PBS), tris-buffered saline (TBS), sodium phosphate, potassium chloride, potassium phosphate, sodium citrate and saline- sodium citrate (SSC) buffer.
  • NaCl sodium chloride
  • PBS phosphate -buffered saline
  • TBS tris-buffered saline
  • SSC saline- sodium citrate
  • NaCl is added at a concentration of 0. P/o to 10%, e.g., P/o, 2.5% or 5% final concentration.
  • Tween 20 is added to 0.005 to 2% concentration, e.g., 0.05%, 0.25% or 0.5 % final concentration.
  • Blocking agents for use with the invention comprise inert proteins, e.g., milk proteins, non-fat dry milk protein, albumin, BSA, casein, or serum such as newborn calf serum (NBCS), goat serum, rabbit serum or salmon serum.
  • inert proteins e.g., milk proteins, non-fat dry milk protein, albumin, BSA, casein, or serum such as newborn calf serum (NBCS), goat serum, rabbit serum or salmon serum.
  • the proteins can be added at a 0.1% to 10% concentration, e.g., P/o, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9% or 10% concentration.
  • BSA is added to 0.1% to 10%
  • concentration e.g., P/o, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9% or 10% concentration.
  • the sample is treated according to the methodology presented in U.S. Patent Application 11/632946, filed July 13, 2005, which application is incorporated herein by reference in its entirety.
  • Commercially available blockers may be used, such as SuperBlock, StartingBlock, Protein-Free from Pierce (a division of Thermo Fisher Scientific, Rockford, IL).
  • SSC/detergent e.g., 20X SSC with 0.5% Tween 20 or 0.1% Triton-X 100
  • SSC/detergent is added to 0.1% to 10% concentration, e.g., at 1.0% or 5.0% concentration.
  • the methods of detecting vesicles and other circulating biomarkers can be optimized as desired with various combinations of protocols and treatments as described herein.
  • a detection protocol can be optimized by various combinations of agitation, isolation methods, and additives.
  • the patient sample is vortexed before and after isolation steps, and the sample is treated with blocking agents including BSA and/or F68. Such treatments may reduce the formation of large aggregates or protein or other biological debris and thus provide a more consistent detection reading.
  • a vesicle can be isolated from a biological sample by filtering a biological sample from a subject through a filtration module and collecting from the filtration module a retentate comprising the vesicle, thereby isolating the vesicle from the biological sample.
  • the method can comprise filtering a biological sample from a subject through a filtration module comprising a filter (also referred to herein as a selection membrane); and collecting from the filtration module a retentate comprising the vesicle, thereby isolating the vesicle from the biological sample.
  • the filter retains molecules greater than about 100 kiloDaltons. In such cases, microvesicles are generally found within the retentate of the filtration process whereas smaller entities such as proteins, protein complexes, nucleic acids, etc, pass through into the filtrate.
  • the method can be used when determining a biosignature of one or more microvesicle.
  • the method can also further comprise contacting the retentate from the filtration to a plurality of substrates, wherein each substrate is coupled to one or more capture agents, and each subset of the plurality of substrates comprises a different capture agent or combination of capture agents than another subset of the plurality of substrates.
  • Also provided herein is a method of determining a biosignature of a vesicle in a sample comprising:
  • the filtration module comprises a filter that retains molecules greater than about 100 or 150 kiloDaltons.
  • the method disclosed herein can further comprise characterizing a phenotype in a subject by filtering a biological sample from a subject through a filtration module, collecting from the filtration module a retentate comprising one or more vesicles; detecting a biosignature of the one or more vesicles; and characterizing a phenotype in the subject based on the biosignature, wherein characterizing is with at least 70% sensitivity.
  • characterizing comprises determining an amount of one or more vesicle having the biosignature.
  • the characterizing can be from about 80% to 100% sensitivity.
  • the method comprises filtering a biological sample from a subject through a filtration module; collecting from the filtration module a retentate comprising the plurality of vesicles, applying the plurality of vesicles to a plurality of capture agents, wherein the plurality of capture agents is coupled to a plurality of substrates, and each subset of the plurality of substrates is differentially labeled from another subset of the plurality of substrates; capturing at least a subset of the plurality of vesicles; and determining a biosignature for at least a subset of the captured vesicles.
  • each substrate is coupled to one or more capture agents, and each subset of the plurality of substrates comprises a different capture agent or combination of capture agents as compared to another subset of the plurality of substrates.
  • at least a subset of the plurality of substrates is intrinsically labeled, such as comprising one or more labels.
  • the substrate can be a particle or bead, or any combination thereof.
  • the filter retains molecules greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or greater than 10000 kiloDaltons (kDa).
  • the filtration module comprises a filter that retains molecules greater than about 100 or 150 kiloDaltons.
  • the filtration module comprises a filter that retains molecules greater than about 9, 20, 100 or 150 kiloDaltons.
  • the filtration module comprises a filter that retains molecules greater than about 7000 kDa.
  • the method for multiplex analysis of a plurality of vesicles comprises filtering a biological sample from a subject through a filtration module, wherein the filtration module comprises a filter that retains molecules greater than about 100 kiloDaltons; collecting from the filtration module a retentate comprising the plurality of vesicles; applying the plurality of vesicles to a plurality of capture agents, wherein the plurality of capture agents is coupled to a microarray; capturing at least a subset of the plurality of vesicles on the microarray; and determining a biosignature for at least a subset of the captured vesicles.
  • the filter retains molecules greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or greater than 10000 kiloDaltons (kDa).
  • the filtration module comprises a filter that retains molecules greater than about 100 or 150 kiloDaltons.
  • the filtration module comprises a filter that retains molecules greater than about 9, 20, 100 or 150 kiloDaltons.
  • the filtration module comprises a filter that retains molecules greater than about 7000 kDa.
  • the biological sample can be clarified prior to isolation by filtration. Clarification comprises selective removal of cellular debris and other undesirable materials. For example, cellular debris and other components that may interfere with detection of the circulating biomarkers can be removed.
  • the clarification can be by low-speed centrifugation, such as at about 5,000x g, 4,000x g, 3,000x g, 2,000x g, l,000x g, or less.
  • the supernatant, or clarified biological sample, containing the vesicle can then be collected and filtered to isolate the vesicle from the clarified biological sample.
  • the biological sample is not clarified prior to isolation of a vesicle by filtration.
  • isolation of a vesicle from a sample does not use high-speed centrifugation, such as ultracentrifugation.
  • isolation may not require the use of centrifugal speeds, such as about 100,000x g or more.
  • isolation of a vesicle from a sample uses speeds of less than 50,000 x g, 40,000 x g, 30,000 x g, 20,000 x g, 15,000 x g, 12,000 x g, or 10,000 x g.
  • the filtration module used to isolate the circulating biomarkers from the biological sample is a fiber- based filtration cartridge.
  • the fiber can be a hollow polymeric fiber, such as a polypropylene hollow fiber.
  • a biological sample can be introduced into the filtration module by pumping the sample fluid, such as a biological fluid as disclosed herein, into the module with a pump device, such as a peristaltic pump.
  • the pump flow rate can vary, such as at about 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mL/minute.
  • the flow rate can be adjusted given the configuration, e.g., size and throughput, of the filtration module.
  • the filtration module can be a membrane filtration module.
  • the membrane filtration module can comprise a filter disc membrane, such as a hydrophilic polyvinylidene difluoride (PVDF) filter disc membrane housed in a stirred cell apparatus (e.g., comprising a magnetic stirrer).
  • PVDF polyvinylidene difluoride
  • the sample moves through the filter as a result of a pressure gradient established on either side of the filter membrane.
  • the filter can comprise a material having low hydrophobic absorptivity and/or high hydrophilic properties.
  • the filter can have an average pore size for vesicle retention and permeation of most proteins as well as a surface that is hydrophilic, thereby limiting protein adsorption.
  • the filter can comprise a material selected from the group consisting of polypropylene, PVDF, polyethylene, polyfluoroethylene, cellulose, secondary cellulose acetate, polyvinylalcohol, and ethylenevinyl alcohol (EVAL®, Kuraray Co., Okayama, Japan). Additional materials that can be used in a filter include, but are not limited to, polysulfone and polyethersulfone.
  • the filtration module can have a filter that retains molecules greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, or 900 kiloDaltons (kDa), such as a filter that has a MWCO (molecular weight cut off) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, or 900 kDa, respectively.
  • a filter that retains molecules greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250
  • the filtration module has a MWCO of 1000 kDa, 1500 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa, 6000 kDa, 6500 kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa, 9500 kDa, 10000 kDa, or greater than 10000 kDa.
  • Ultrafiltration membranes with a range of MWCO of 9 kDa, 20 kDa and/or 150 kDa can be used.
  • the filter within the filtration module has an average pore diameter of about 0.01 ⁇ to about 0.15 ⁇ , and in some embodiments from about 0.05 ⁇ to about 0.12 ⁇ . In some embodiments, the filter has an average pore diameter of about 0.06 ⁇ , 0.07 ⁇ , 0.08 ⁇ , 0.09 ⁇ , 0.1 ⁇ , 0.11 ⁇ or 0.2 ⁇ .
  • the filtration module can be a commerically available column, such as a column typically used for concentrating proteins or for isolating proteins (e.g., ultrafiltration). Examples include, but are not limited to, columns from Millpore (Billerica, MA), such as Amicon® centrifugal filters, or from Pierce® (Rockford, IL), such as Pierce Concentrator filter devices. Useful columns from Pierce include disposable ultrafiltration centrifugal devices with a MWCO of 9 kDa, 20 kDa and/or 150 kDa. These concentrators consist of a high-performance regenerated cellulose membrane welded to a conical device.
  • the filters can be as described in U.S. Patents 6,269,957 or 6,357,601, both of which applications are incorporated by reference in their entirety herein.
  • the retentate comprising the isolated vesicle can be collected from the filtration module.
  • the retentate can be collected by flushing the retentate from the filter.
  • Selection of a filter composition having hydrophilic surface properties, thereby limiting protein adsorption, can be used, without being bound by theory, for easier collection of the retentate and minimize use of harsh or time-consuming collection techniques.
  • the collected retentate can then be used subsequent analysis, such as assessing a biosignature of one or more vesicles in the retentate, as further described herein.
  • the analysis can be directly performed on the collected retentate.
  • the collected retentate can be further concentrated or purified, prior to analysis of one or more vesicles.
  • the retentate can be further concentrated or vesicles further isolated from the retentate using size exclusion chromatography, density gradient centrifugation, differential centrifugation, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof, such as described herein.
  • the retentate can undergo another step of filtration.
  • the vesicle prior to isolation of a vesicle using a filter, the vesicle is concentrated or isolated using techniques including without limitation size exclusion chromatography, density gradient centrifugation, differential centrifugation, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • Combinations of filters can be used for concentrating and isolating biomarkers.
  • the biological sample may first be filtered through a filter having a porosity or pore size of between about 0.01 ⁇ to about 10 ⁇ , e.g., 0.01 ⁇ to about 2 ⁇ or about 0.05 ⁇ to about 1.5 ⁇ , and then the sample is filtered.
  • a filter that retains molecules greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or greater than 10000 kiloDaltons (kDa), such as a filter that has a MWCO (molecular weight cut off) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900,
  • MWCO molecular weight cut off
  • the filter has a pore size of about O.Ol, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 ⁇ .
  • the filter may be a syringe filter.
  • the method comprises filtering the biological sample through a filter, such as a syringe filter, wherein the syringe filter has a porosity of greater than about 1 ⁇ , prior to filtering the sample through a filtration module comprising a filter that retains molecules greater than about 100 or 150 kiloDaltons.
  • the filter is 1.2 ⁇ filter and the filtration is followed by passage of the sample through a 7 ml or 20 ml concentrator column with a 150 kDa cutoff. Multiple concentrator columns may be used, e.g., in series. For example, a 7000 MWCO filtration unit can be used before a 150 MWCO unit.
  • the filtration module can be a component of a microfluidic device.
  • Microfluidic devices which may also be referred to as "lab-on-a-chip” systems, biomedical micro-electro-mechanical systems (bioMEMs), or multicomponent integrated systems, can be used for isolating, and analyzing, vesicles.
  • bioMEMs biomedical micro-electro-mechanical systems
  • Such systems miniaturize and compartmentalize processes that allow for binding of vesicles, detection of biomarkers, and other processes, such as further described herein.
  • the filtration module and assessment can be as described in Grant, R., et al., A filtration-based protocol to isolate human Plasma Membrane-derived Vesicles and exosomes from blood plasma, J Immunol Methods (2011) 371: 143-51 (Epub 2011 Jun 30), which reference is incorporated herein by reference in its entirety.
  • a microfluidic device can also be used for isolation of a vesicle by comprising a filtration module.
  • a microfluidic device can use one more channels for isolating a vesicle from a biological sample based on size from a biological sample.
  • a biological sample can be introduced into one or more microfluidic channels, which selectively allows the passage of vesicles.
  • the microfluidic device can further comprise binding agents, or more than one filtration module to select vesicles based on a property of the vesicles, for example, size, shape, deformability, biomarker profile, or biosignature.
  • the retentate from a filtration step can be further processed before assessment of microvesicles or other biomarkers therein.
  • the retentate is diluted prior to biomarker assessment, e.g., with an appropriate diluent such as a biologically compatible buffer.
  • the retentate is serially diluted.
  • the invention provides a method for detecting a microvesicle population from a biological sample comprising: a) concentrating the biological sample using a selection membrane having a pore size of from 0.01 ⁇ to about 10 ⁇ , or a molecular weight cut off (MWCO) from about 1 kDa to 10000 kDa; b) diluting a retentate from the concentration step into one or more aliquots; and c) contacting each of the one or more aliquots of retentate with one or more binding agent specific to a molecule of at least one microvesicle in the microvesicle population.
  • MWCO molecular weight cut off
  • the invention provides a method for detecting a microvesicle population from a biological sample comprising: a) concentrating the biological sample using a selection membrane having a pore size of from 0.01 ⁇ to about 10 ⁇ , or a molecular weight cut off (MWCO) from about 1 kDa to 10000 kDa; and b) contacting one or more aliquots of the retentate from the concentrating step with one or more binding agent specific to a molecule of at least one microvesicle in the microvesicle population.
  • a selection membrane having a pore size of from 0.01 ⁇ to about 10 ⁇ , or a molecular weight cut off (MWCO) from about 1 kDa to 10000 kDa
  • MWCO molecular weight cut off
  • the selection membrane can be sized to retain the desired biomarkers in the retentate or to allow the desired biomarkers to pass through the filter into the filtrate.
  • the filter membrane can be chosen to have a certain pore size or MWCO value.
  • the selection membrane can have a pore size of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 ⁇ .
  • the selection membrane can also have a MWCO of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 kDa.
  • the retentate can be separated and/or diluted into any number of desired aliquots. For example, multiple aliquots without any dilution or the same dilution can be used to determine reproducibility. In another example, multiple aliquots at different dilutions can be used to construct a concentration curve. In an embodiment, the retentate is separated and/or diluted into at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400 aliquots. The aliquots can be at a same dilution or at different dilutions.
  • a dilution factor is the ratio of the final volume of a mixture (the mixture of the diluents and aliquot) divided by the initial volume of the aliquot.
  • the retentate can be diluted into one or more aliquots at a dilution factor of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 and/or 100000.
  • the retentate can be diluted into one or more aliquot at a dilution factor of about
  • the retentate can be diluted into multiple aliquots.
  • the retentate is diluted into one or more aliquots at a dilution factor of about 100, 250, 500, 1000, 10000 and 100000.
  • the method can further comprise detecting an amount of microvesicles in each aliquot of retentate, e.g., that formed a complex with the one or more binding agent.
  • the curve can be used to determine a linear range of the amount of microvesicles in each aliquot detected versus dilution factor.
  • a concentration of the detected microvesicles for the biological sample can be determined using the amount of microvesicles determined in one or more aliquot within the linear range.
  • the concentration can be compared to a reference concentration, e.g., in order to characterize a phenotype as described herein.
  • the invention also provides a related method comprising filtering a biological sample from a subject through a filtration module and collecting a filtrate comprising the vesicle, thereby isolating the vesicle from the biological sample.
  • a biological sample from a subject through a filtration module and collecting a filtrate comprising the vesicle, thereby isolating the vesicle from the biological sample.
  • cells and other large entities can be retained in the retentate while microvesicles pass through into the filtrate.
  • strategies to retain and filter microvesicles can be used in concert.
  • a sample can be filtered with a selection membrane that allows microvesicles to pass through, thereby isolating the microvesicles from large particles (cells, complexes, etc).
  • the filtrate comprising the microvesicle can then be filtered using a selection membrane that retains microvesicles, thereby isolating the microvesicles from smaller particles (proteins, nucleic acids, etc).
  • the isolated microvesicles can be further assessed according to the methods of the invention, e.g., to characterize a phenotype.
  • Vesicles can be isolated using a polymeric precipitation method. The method can be in combination with or in place of the other isolation methods described herein.
  • the sample containing the vesicles is contacted with a formulation of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the polymeric formulation is incubated with the vesicle containing sample then precipitated by centrifugation.
  • the PEG can bind to the vesicles and can be treated to specifically capture vesicles by addition of a capture moiety, e.g., a pegylated-binding protein such as an antibody.
  • PEG derivatives including methoxypolyethylene glycols, poly (ethylene oxide), and various polymers of formula HO-CH 2 -(CH 2 -0-CH 2 -)n- CH 2 -OH having different molecular weights.
  • the vesicles are concentrated from a sample using the polymer precipitation method and the isolated vesicles are further separated using another approach.
  • the second step can be used to identify a subpopulation of vesicles, e.g., that display certain biomarkers.
  • the second separation step can comprise size exclusion, a binding agent, an antibody capture step, microbeads, as described herein.
  • vesicles are isolated according to the ExoQuickTM and ExoQuick-TCTM kits from System Biosciences, Mountain View, CA USA. These kits use a polymer-based precipitation method to pellet vesicles. Similarly, the vesicles can be isolated using the Total Exosome Isolation (from Serum) or Total Exosome Isolation (from Cell Culture Media) kits from Invitrogen / Life Technologies (Carlsbad, CA USA). The Total Exosome Isolation reagent forces less-soluble components such as vesicles out of solution, allowing them to be collected by a short, low-speed centrifugation. The reagent is added to the biological sample, and the solution is incubated overnight at 2 °C to 8 °C. The precipitated vesicles are recovered by standard centrifugation.
  • ExoQuickTM and ExoQuick-TCTM kits from System Biosciences, Mountain View, CA USA. These kits use a polymer-based precipitation method to pellet vesic
  • Binding agents include agents that are capable of binding a target biomarker.
  • a binding agent can be specific for the target biomarker, meaning the agent is capable of binding a target biomarker.
  • the target can be any useful biomarker disclosed herein, such as a biomarker on the vesicle surface.
  • the target is a single molecule, such as a single protein, so that the binding agent is specific to the single protein.
  • the target can be a group of molecules, such as a family or proteins having a similar epitope or moiety, so that the binding agent is specific to the family or group of proteins.
  • the group of molecules can also be a class of molecules, such as protein, DNA or RNA.
  • the binding agent can be a capture agent used to capture a vesicle by binding a component or biomarker of a vesicle.
  • a capture agent comprises an antibody or fragment thereof, or an aptamer, that binds to an antigen on a vesicle.
  • the capture agent can be optionally coupled to a substrate and used to isolate a vesicle, as further described herein.
  • a binding agent is an agent that binds to a circulating biomarker, such as a vesicle or a component of a vesicle.
  • the binding agent can be used as a capture agent and/or a detection agent.
  • a capture agent can bind and capture a circulating biomarker, such as by binding a component or biomarker of a vesicle.
  • the capture agent can be a capture antibody or capture antigen that binds to an antigen on a vesicle.
  • a detection agent can bind to a circulating biomarker thereby facilitating detection of the biomarker.
  • a capture agent comprising an antibody or aptamer that is sequestered to a substrate can be used to capture a vesicle in a sample
  • a detection agent comprising an antibody or aptamer that carries a label can be used to detect the captured vesicle via detection of the detection agent's label.
  • a vesicle is assessed using capture and detection agents that recognize the same vesicle biomarkers.
  • a vesicle population can be captured using a tetraspanin such as by using an anti-CD9 antibody bound to a substrate, and the captured vesicles can be detected using a fluorescently labeled anti-CD9 antibody to label the captured vesicles.
  • a vesicle is assessed using capture and detection agents that recognize different vesicle biomarkers.
  • a vesicle population can be captured using a cell-specific marker such as by using an anti-PCSA antibody bound to a substrate, and the captured vesicles can be detected using a fluorescently labeled anti-CD9 antibody to label the captured vesicles.
  • the vesicle population can be captured using a general vesicle marker such as by using an anti-CD9 antibody bound to a substate, and the captured vesicles can be detected using a fluorescently labeled antibody to a cell-specific or disease specific marker to label the captured vesicles.
  • antigen as used herein is meant to encompass any entity that is capable of being bound by a binding agent, regardless of the type of binding agent or the immunogenicity of the biomarker.
  • the antigen further encompasses a functional fragment thereof.
  • an antigen can encompass a protein biomarker capable of being bound by a binding agent, including a fragment of the protein that is capable of being bound by a binding agent.
  • a vesicle is captured using a capture agent that binds to a biomarker on a vesicle.
  • the capture agent can be coupled to a substrate and used to isolate a vesicle, as further described herein.
  • a capture agent is used for affinity capture or isolation of a vesicle present in a substance or sample.
  • a binding agent can be used after a vesicle is concentrated or isolated from a biological sample.
  • a vesicle can first be isolated from a biological sample before a vesicle with a specific biosignature is isolated or detected.
  • the vesicle with a specific biosignature can be isolated or detected using a binding agent for the biomarker.
  • a vesicle with the specific biomarker can be isolated or detected from a heterogeneous population of vesicles.
  • a binding agent may be used on a biological sample comprising vesicles without a prior isolation or concentration step.
  • a binding agent is used to isolate or detect a vesicle with a specific biosignature directly from a biological sample.
  • a binding agent can be a nucleic acid, protein, or other molecule that can bind to a component of a vesicle.
  • the binding agent can comprise DNA, RNA, monoclonal antibodies, polyclonal antibodies, Fabs, Fab', single chain antibodies, synthetic antibodies, aptamers (DNA/RNA), peptoids, zDNA, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), lectins, synthetic or naturally occurring chemical compounds (including but not limited to drugs, labeling reagents), dendrimers, or a combination thereof.
  • the binding agent can be a capture antibody.
  • the binding agent comprises a membrane protein labeling agent.
  • vesicles are isolated or captured as described herein, and one or more membrane protein labeling agent is used to detect the vesicles.
  • a single binding agent can be employed to isolate or detect a vesicle.
  • a combination of different binding agents may be employed to isolate or detect a vesicle.
  • at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75 or 100 different binding agents may be used to isolate or detect a vesicle from a biological sample.
  • the one or more different binding agents for a vesicle can form a biosignature of a vesicle, as further described below.
  • Different binding agents can also be used for multiplexing. For example, isolation or detection of more than one population of vesicles can be performed by isolating or detecting each vesicle population with a different binding agent. Different binding agents can be bound to different particles, wherein the different particles are labeled. In another embodiment, an array comprising different binding agents can be used for multiplex analysis, wherein the different binding agents are differentially labeled or can be ascertained based on the location of the binding agent on the array. Multiplexing can be accomplished up to the resolution capability of the labels or detection method, such as described below. The binding agents can be used to detect the vesicles, such as for detecting cell-of-origin specific vesicles.
  • a binding agent or multiple binding agents can themselves form a binding agent profile that provides a biosignature for a vesicle.
  • One or more binding agents can be selected from Fig. 2 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein. For example, if a vesicle population is detected or isolated using two, three, four or more binding agents in a differential detection or isolation of a vesicle from a heterogeneous population of vesicles, the particular binding agent profile for the vesicle population provides a biosignature for the particular vesicle population.
  • the vesicle can be detected using any number of binding agents in a multiplex fashion.
  • the binding agent can also be used to form a biosignature for a vesicle.
  • the biosignature can be used to characterize a phenotype.
  • the binding agent can be a lectin.
  • Lectins are proteins that bind selectively to polysaccharides and glycoproteins and are widely distributed in plants and animals.
  • lectins such as those derived from Galanthus nivalis in the form of Galanthus nivalis agglutinin ("GNA”), Narcissus pseudonarcissus in the form of Narcissus pseudonarcissus agglutinin ("NPA”) and the blue green algae Nostoc ellipsosporum called "cyanovirin” (Boyd et al. Antimicrob Agents Chemother 41(7): 1521 1530, 1997; Hammar et al.
  • High mannose glycoprotein refers to glycoproteins having mannose -mannose linkages in the form of a-l ⁇ 3 or a- 1 ⁇ 6 mannose -mannose linkages.
  • the binding agent can be an agent that binds one or more lectins.
  • Lectin capture can be applied to the isolation of the biomarker cathepsin D since it is a glycosylated protein capable of binding the lectins Galanthus nivalis agglutinin (GNA) and concanavalin A (ConA).
  • GAA Galanthus nivalis agglutinin
  • ConA concanavalin A
  • the binding agent can be an antibody.
  • a vesicle may be isolated using one or more antibodies specific for one or more antigens present on the vesicle.
  • a vesicle can have CD63 on its surface, and an antibody, or capture antibody, for CD63 can be used to isolate the vesicle.
  • a vesicle derived from a tumor cell can express EpCam, the vesicle can be isolated using an antibody for EpCam and CD63.
  • antibodies for isolating vesicles can include an antibody, or capture antibody, to CD9, PSCA, TNFR, CD63, B7H3, MFG-E8, EpCam, Rab, CD81, STEAP, PCSA, PSMA, or 5T4.
  • Other antibodies for isolating vesicles can include an antibody, or capture antibody, to DR3, STEAP, epha2, TMEM211, MFG-E8, Tissue Factor (TF), unc93A, A33, CD24, NGAL, EpCam, MUC17, TROP2, or TETS.
  • the capture agent is an antibody to CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP, or EGFR.
  • the capture agent can also be used to identify a biomarker of a vesicle.
  • a capture agent such as an antibody to CD9 would identify CD9 as a biomarker of the vesicle.
  • a plurality of capture agents can be used, such as in multiplex analysis.
  • the plurality of captures agents can comprise binding agents to one or more of: CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP, and EGFR.
  • the plurality of capture agents comprise binding agents to CD9, CD63, CD81, PSMA, PCSA, B7H3, MFG-E8, and/or EpCam. In yet other embodiments, the plurality of capture agents comprises binding agents to CD9, CD63, CD81, PSMA, PCSA, B7H3, EpCam, PSCA, ICAM, STEAP, and/or EGFR.
  • the plurality of capture agents comprises binding agents to TMEM211, MFG-E8, Tissue Factor (TF), and/or CD24.
  • the antibodies referenced herein can be immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen and synthetic antibodies.
  • the immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule.
  • Antibodies include, but are not limited to, polyclonal, monoclonal, bispecific, synthetic, humanized and chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, Fv or Fv' portions, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of any of the above.
  • An antibody, or generally any molecule "binds specifically" to an antigen (or other molecule) if the antibody binds preferentially to the antigen, and, e.g., has less than about 30%, 20%, 10%, 5% or 1% cross-reactivity with another molecule.
  • the binding agent can also be a polypeptide or peptide.
  • Polypeptide is used in its broadest sense and may include a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds.
  • the polypeptides may be naturally occurring, processed forms of naturally occurring polypeptides (such as by enzymatic digestion), chemically synthesized or recombinantly expressed.
  • the polypeptides for use in the methods of the present invention may be chemically synthesized using standard techniques.
  • the polypeptides may comprise D-amino acids (which are resistant to L- amino acid-specific proteases), a combination of D- and L- amino acids, ⁇ amino acids, or various other designer or non-naturally occurring amino acids (e.g., ⁇ -methyl amino acids, Ca- methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids may include ornithine for lysine, and norleucine for leucine or isoleucine.
  • the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare polypeptides with novel properties.
  • a polypeptide may be generated that incorporates a reduced peptide bond, i.e., R i-CH 2 -NH-R 2 , where R i and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a polypeptide would be resistant to protease activity, and would possess an extended half- live in vivo.
  • Polypeptides can also include peptoids (N-substituted glycines), in which the side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the a-carbons, as in amino acids.
  • Polypeptides and peptides are intended to be used interchangeably throughout this application, i.e.
  • peptide may also include polypeptides and where the term polypeptides is used, it may also include peptides.
  • protein is also intended to be used interchangeably throughout this application with the terms “polypeptides” and “peptides” unless otherwise specified.
  • a vesicle may be isolated, captured or detected using a binding agent.
  • the binding agent can be an agent that binds a vesicle "housekeeping protein," or general vesicle biomarker.
  • the biomarker can be CD63, CD9, CD81, CD82, CD37, CD53, Rab-5b, Annexin V or MFG-E8.
  • Tetraspanins a family of membrane proteins with four transmembrane domains, can be used as general vesicle markers.
  • the tetraspanins include CD151, CD53, CD37, CD82, CD81, CD9 and CD63.
  • TSPAN1 TSPAN1
  • TSPAN2 TSPAN-2
  • TSPAN3 TSPAN-3
  • TSPAN4 TSPAN-4, NAG-2
  • TSPAN5 TSPAN-5
  • TSPAN6 TSPAN-6
  • TSPAN7 CD231, TALLA-1, A15
  • TSPAN8 CO-029
  • TSPAN9 NET-5
  • TSPAN10 Oculospanin
  • TSPAN11 CD151-like
  • T SPAN 12 NET -2
  • TSPAN13 NET-6
  • TSPAN14 TSPAN15
  • TSPAN16 TSPAN16
  • TSPAN17 TSPAN18
  • TSPAN19 TSPAN20
  • UPK1B UPK1B
  • TSPAN21 UPla, UPK1A
  • TSPAN22 RS, PRPH2
  • TSPAN23 ROM1
  • TSPAN24 CD151
  • TSPAN25 CD53
  • TSPAN26 TSPAN26
  • vesicle markers include those listed in Table 3. Any of these proteins can be used as vesicle markers. Furthermore, any of the markers disclosed herein or in Table 3 can be selected in identifying a candidate biosignature for a disease or condition, where the one or more selected biomarkers have a direct or indirect role or function in mechanisms involved in the disease or condition.
  • the binding agent can also be an agent that binds to a vesicle derived from a specific cell type, such as a tumor cell (e.g. binding agent for Tissue factor, EpCam, B7H3, RAGE or CD24) or a specific cell-of-origin.
  • a tumor cell e.g. binding agent for Tissue factor, EpCam, B7H3, RAGE or CD24
  • the binding agent used to isolate or detect a vesicle can be a binding agent for an antigen selected from Fig. 1 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • the binding agent for a vesicle can also be selected from those listed in Fig. 2 of International Patent Application Serial No.
  • the binding agent can be for an antigen such as a tetraspanin, MFG-E8, Annexin V, 5T4, B7H3, caveolin, CD63, CD9, E-Cadherin, Tissue factor, MFG-E8, TMEM211, CD24, PSCA, PCSA, PSMA, Rab- 5B, STEAP, TNFR1, CD81, EpCam, CD59, CD81, ICAM, EGFR, or CD66.
  • a binding agent for a platelet can be a glycoprotein such as GpIa-IIa, GpIIb-IIIa, GpIIIb, Gplb, or GpIX.
  • a binding agent can be for an antigen comprisine one or more of CD9, Erb2, Erb4, CD81, Erb3, MUC16, CD63, DLL4, HLA-Drpe, B7H3, IFNAR, 5T4, PCSA, MICB, PSMA, MFG-E8, Mucl, PSA, Muc2, Unc93a, VEGFR2, EpCAM, VEGF A, TMPRSS2, RAGE, PSCA, CD40, Mucl7, IL-17-RA, and CD80.
  • the binding agent can be one or more of CD9, CD63, CD81, B7H3, PCSA, MFG-E8, MUC2, EpCam, RAGE and Mucl 7.
  • One or more binding agents can be used for isolating or detecting a vesicle.
  • the binding agent used can be selected based on the desire of isolating or detecting a vesicle derived from a particular cell type or cell- of-origin specific vesicle.
  • a binding agent can also be linked directly or indirectly to a solid surface or substrate.
  • a solid surface or substrate can be any physically separable solid to which a binding agent can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, particles such as beads, columns, optical fibers, wipes, glass and modified or functionalized glass, quartz, mica, diazotized membranes (paper or nylon), poly formaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (PTFE, Teflon®), etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals
  • the substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Such coatings can facilitate the use of the array with a biological sample.
  • an antibody used to isolate a vesicle can be bound to a solid substrate such as a well, such as commercially available plates (e.g. from Nunc, Milan Italy). Each well can be coated with the antibody.
  • the antibody used to isolate a vesicle is bound to a solid substrate such as an array.
  • the array can have a predetermined spatial arrangement of molecule interactions, binding islands, biomolecules, zones, domains or spatial arrangements of binding islands or binding agents deposited within discrete boundaries.
  • the term array may be used herein to refer to multiple arrays arranged on a surface, such as would be the case where a surface bore multiple copies of an array. Such surfaces bearing multiple arrays may also be referred to as multiple arrays or repeating arrays.
  • Arrays typically contain addressable moieties that can detect the presense of an entity, e.g., a vesicle in the sample via a binding event.
  • An array may be referred to as a microarray.
  • Arrays or microarrays include without limitation DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays and SNP microarrays, microRNA arrays, protein microarrays, antibody microarrays, tissue microarrays, cellular microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays).
  • DNA arrays typically comprise addressable nucleotide sequences that can bind to sequences present in a sample.
  • MicroRNA arrays e.g., the MMChips array from the University of Louisville or commercial systems from Agilent, can be used to detect microRNAs.
  • Protein microarrays can be used to identify protein-protein interactions, including without limitation identifying substrates of protein kinases, transcription factor protein-activation, or to identify the targets of biologically active small molecules. Protein arrays may comprise an array of different protein molecules, commonly antibodies, or nucleotide sequences that bind to proteins of interest. In a non-limiting example, a protein array can be used to detect vesicles having certain proteins on their surface.
  • Antibody arrays comprise antibodies spotted onto the protein chip that are used as capture molecules to detect proteins or other biological materials from a sample, e.g., from cell or tissue lysate solutions.
  • antibody arrays can be used to detect vesicle-associated biomarkers from bodily fluids, e.g., serum or urine.
  • Tissue microarrays comprise separate tissue cores assembled in array fashion to allow multiplex histological analysis.
  • Cellular microarrays, also called transfection microarrays comprise various capture agents, such as antibodies, proteins, or lipids, which can interact with cells to facilitate their capture on addressable locations. Cellular arrays can also be used to capture vesicles due to the similarity between a vesicle and cellular membrane.
  • Chemical compound microarrays comprise arrays of chemical compounds and can be used to detect protein or other biological materials that bind the compounds.
  • Carbohydrate arrays (glycoarrays) comprise arrays of carbohydrates and can detect, e.g., protein that bind sugar moieties.
  • a binding agent can also be bound to particles such as beads or microspheres.
  • particles such as beads or microspheres.
  • an antibody specific for a component of a vesicle can be bound to a particle, and the antibody-bound particle is used to isolate a vesicle from a biological sample.
  • the microspheres may be magnetic or fluorescently labeled.
  • a binding agent for isolating vesicles can be a solid substrate itself.
  • latex beads such as aldehyde/sulfate beads (Interfacial Dynamics, Portland, OR) can be used.
  • a binding agent bound to a magnetic bead can also be used to isolate a vesicle.
  • a biological sample such as serum from a patient can be collected for colon cancer screening.
  • the sample can be incubated with anti-CCSA-3 (Colon Cancer-Specific Antigen) coupled to magnetic microbeads.
  • a low-density microcolumn can be placed in the magnetic field of a MACS Separator and the column is then washed with a buffer solution such as Tris- buffered saline.
  • the magnetic immune complexes can then be applied to the column and unbound, non-specific material can be discarded.
  • the CCSA-3 selected vesicle can be recovered by removing the column from the separator and placing it on a collection tube.
  • a buffer can be added to the column and the magnetically labeled vesicle can be released by applying the plunger supplied with the column.
  • the isolated vesicle can be diluted in IgG elution buffer and the complex can then be centrifuged to separate the microbeads from the vesicle.
  • the pelleted isolated cell-of-origin specific vesicle can be resuspended in buffer such as phosphate -buffered saline and quantitated.
  • a proteolytic enzyme such as trypsin can be used for the release of captured vesicles without the need for centrifugation.
  • the proteolytic enzyme can be incubated with the antibody captured cell-of-origin specific vesicles for at least a time sufficient to release the vesicles.
  • a binding agent such as an antibody, for isolating vesicles is preferably contacted with the biological sample comprising the vesicles of interest for at least a time sufficient for the binding agent to bind to a component of the vesicle.
  • an antibody may be contacted with a biological sample for various intervals ranging from seconds days, including but not limited to, about 10 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 7 hours, 10 hours, 15 hours, 1 day, 3 days, 7 days or 10 days.
  • a binding agent such as an antibody specific to an antigen listed in Fig. 1 of International Patent
  • labels include without limitation a magnetic label, a fluorescent moiety, an enzyme, a chemiluminescent probe, a metal particle, a non-metal colloidal particle, a polymeric dye particle, a pigment molecule, a pigment particle, an electrochemically active species, semiconductor nanocrystal or other nanoparticles including quantum dots or gold particles, fluorophores, quantum dots, or radioactive labels.
  • Protein labels include green fluorescent protein (GFP) and variants thereof (e.g., cyan fluorescent protein and yellow fluorescent protein); and luminescent proteins such as luciferase, as described below.
  • Radioactive labels include without limitation radioisotopes (radionuclides), such as 3 ⁇ 4 n C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, m In, 123 1, 124 1, 125 1, 131 1, 133 Xe, 177 Lu, 211 At, or 213 Bi.
  • radioisotopes radioisotopes (radionuclides), such as 3 ⁇ 4 n C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, m In, 123 1, 124 1, 125 1, 131 1, 133 Xe, 177 Lu, 211 At, or 213 Bi.
  • Fluorescent labels include without limitation a rare earth chelate (e.g., europium chelate), rhodamine; fluorescein types including without limitation FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; a rhodamine type including without limitation TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; Cy3, Cy5, dapoxyl, NBD, Cascade Yellow, dansyl, PyMPO, pyrene, 7-diethylaminocoumarin-3-carboxylic acid and other coumarin derivatives, Marina BlueTM, Pacific BlueTM, Cascade BlueTM, 2-anthracenesulfonyl, PyMPO, 3,4,9, 10-perylene-tetracarboxylic acid, 2,7- difluorofluorescein (Oregon GreenTM 488-X), 5-carboxyfluorescein, Texas RedTM-X, Alexa Fluor 430, 5- carboxy
  • the fluorescent label can be one or more of FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ, Gold540 and LIZ.
  • a binding agent can be directly or indirectly labeled, e.g., the label is attached to the antibody through biotin-streptavidin.
  • an antibody is not labeled, but is later contacted with a second antibody that is labeled after the first antibody is bound to an antigen of interest.
  • various enzyme-substrate labels are available or disclosed (see for example, U.S. Pat. No. 4,275,149).
  • the enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques.
  • the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically.
  • the enzyme may alter the fluorescence or chemiluminescence of the substrate.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • glucose oxidase galactose oxidase
  • enzyme- substrate combinations include, but are not limited to, horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- ⁇ -D- galactosidase) or fluorogenic substrate 4-methylumbelliferyl ⁇ -D-galactosidase.
  • HRP horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethylbenzidine hydrochloride
  • AP alkaline phosphata
  • the binding agent may be linked to a solid surface or substrate, such as arrays, particles, wells and other substrates described above.
  • Methods for direct chemical coupling of antibodies, to the cell surface are known in the art, and may include, for example, coupling using glutaraldehyde or maleimide activated antibodies.
  • Methods for chemical coupling using multiple step procedures include biotinylation, coupling of trinitrophenol (TNP) or digoxigenin using for example succinimide esters of these compounds. Biotinylation can be accomplished by, for example, the use of D-biotinyl-N-hydroxysuccinimide.
  • Succinimide groups react effectively with amino groups at pH values above 7, and preferentially between about pH 8.0 and about pH 8.5.
  • Biotinylation can be accomplished by, for example, treating the cells with dithiothreitol followed by the addition of biotin maleimide.
  • assays using particles or microspheres are capable of use with a binding agent.
  • antibodies or aptamers are easily conjugated with commercially available beads. See, e.g., Fan et al , Illumina universal bead arrays. Methods Enzymol. 2006 410:57-73; Srinivas et al. Anal. Chem. 2011 Oct. 21, Aptamer functionalized Microgel Particles for Protein Detection; See also, review article on aptamers as therapeutic and diagnostic agents, Brody and Gold, Rev. Mol. Biotech. 2000, 74:5-13.
  • Multiparametric assays or other high throughput detection assays using bead coatings with cognate ligands and reporter molecules with specific activities consistent with high sensitivity automation can be used.
  • a binding agent for a biomarker or vesicle such as a capture agent (e.g. capture antibody)
  • a capture agent e.g. capture antibody
  • Each binding agent for each individual binding assay can be coupled to a distinct type of microsphere (i.e., microbead) and the assay reaction takes place on the surface of the microsphere, such as depicted in FIG. 2B.
  • a binding agent for a vesicle can be a capture antibody or aptamer coupled to a bead.
  • Dyed microspheres with discrete fluorescence intensities are loaded separately with their appropriate binding agent or capture probes.
  • the different bead sets carrying different binding agents can be pooled as necessary to generate custom bead arrays. Bead arrays are then incubated with the sample in a single reaction vessel to perform the assay. See FIGs. 8C-D for illustrative methods of detecting microvesicles using microbeads with antibody binding agents.
  • a bead substrate can provide a platform for attaching one or more binding agents, including aptamer(s) or antibodies.
  • binding agents including aptamer(s) or antibodies.
  • One of skill will appreciate that the illustrative schemes shown in FIGs. 8C-D can employ aptamers along with or instead of antibodies.
  • multiple different bead sets e.g., those commercially available from Illumina, Inc., San Diego, CA, USA, or Luminex Corporation, Austin, TX, USA
  • Beads can also be used for different purposes, e.g., detection and/or isolation.
  • a bead can be conjugated to an aptamer used to detect the presence (quantitatively or qualitatively) of a given biomarker, or it can also be used to isolate a component present in a selected biological sample (e.g., cell, cell-fragment or vesicle comprising the target molecule to which the binding agent is configured to bind or associate).
  • a component present in a selected biological sample e.g., cell, cell-fragment or vesicle comprising the target molecule to which the binding agent is configured to bind or associate.
  • Various molecules of organic origin can be conjugated to a microbeads, e.g., polysterene beads, through use of commercially available kits.
  • an assay can use multiple types of binding agents.
  • a bead may be conjugated to an aptamer which serves to bind and capture a biomarker, and a labeled antibody can be used to further detect the captured biomarker.
  • a bead may be conjugated to an antibody which serves to bind and capture a biomarker, and a labeled aptamer can be used to further detect the captured biomarker. Any such useful combinations of binding agents are contemplated by the invention.
  • One or more binding agent can be used with any bead based substrate, including but not limited to magnetic capture method, fluorescence activated cell sorting (FACS) or laser cytometry.
  • Magnetic capture methods can include, but are not limited to, the use of magnetically activated cell sorter (MACS) microbeads or magnetic columns.
  • MCS magnetically activated cell sorter
  • bead or particle based methods that can be used in the methods of the invention include the bead systems described in any of U.S. Patent Nos.
  • Isolation or detection of circulating biomarkers e.g., protein antigens, from a biological sample, or of the biomarker- comprising cells, cell fragments or vesicles may also be achieved using a cytometry process.
  • biomarkers e.g., protein antigens
  • aptamers or antibodies can be used in an assay comprising using a particle such as a bead or microsphere.
  • Flow cytometry can be used for sorting microscopic particles suspended in a stream of fluid. As particles pass through they can be selectively charged and on their exit can be deflected into separate paths of flow. It is therefore possible to separate populations from an original mix, such as a biological sample, with a high degree of accuracy and speed.
  • Flow cytometry allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus.
  • a beam of light, usually laser light, of a single frequency (color) is directed onto a hydrodynamically focused stream of fluid.
  • a number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescent detectors.
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source.
  • This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is possible to deduce various facts about the physical and chemical structure of each individual particle.
  • FSC correlates with the cell size and SSC depends on the inner complexity of the particle, such as shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness.
  • Flow cytometers can analyze several thousand particles every second in "real time” and can actively separate out and isolate particles having specified properties. They offer high-throughput automated quantification, and separation, of the set parameters for a high number of single cells during each analysis session.
  • Flow cytomers can have multiple lasers and fluorescence detectors, allowing multiple labels to be used to more precisely specify a target population by their phenotype.
  • a flow cytometer such as a multicolor flow cytometer, can be used to detect one or more vesicles with multiple fluorescent labels or colors.
  • the flow cytometer can also sort or isolate different vesicle populations, such as by size or by different markers.
  • the flow cytometer may have one or more lasers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more lasers.
  • the flow cytometer can detect more than one color or fluorescent label, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different colors or fluorescent labels.
  • the flow cytometer can have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fluorescence detectors.
  • Examples of commercially available flow cytometers that can be used to detect or analyze one or more vesicles, to sort or separate different populations of vesicles, include, but are not limited to the MoFloTM XDP Cell Sorter (Beckman Coulter, Brea, CA), MoFloTM Legacy Cell Sorter (Beckman Coulter, Brea, CA), BD FACSAriaTM Cell Sorter (BD Biosciences, San Jose, CA), BDTM LSRII (BD Biosciences, San Jose, CA), and BD FACSCaliburTM (BD Biosciences, San Jose, CA).
  • MoFloTM XDP Cell Sorter Beckman Coulter, Brea, CA
  • MoFloTM Legacy Cell Sorter Beckman Coulter, Brea, CA
  • BD FACSAriaTM Cell Sorter BD Biosciences, San Jose, CA
  • BDTM LSRII BD Biosciences, San Jose, CA
  • BD FACSCaliburTM BD Biosciences, San Jose, CA
  • the flow cytometer can sort, and thereby collect or sort more than one population of vesicles based one or more characteristics. For example, two populations of vesicles differ in size, such that the vesicles within each population have a similar size range and can be differentially detected or sorted. In another embodiment, two different populations of vesicles are differentially labeled.
  • the data resulting from flow-cytometers can be plotted in 1 dimension to produce histograms or seen in 2 dimensions as dot plots or in 3 dimensions with newer software.
  • the regions on these plots can be sequentially separated by a series of subset extractions which are termed gates.
  • Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology.
  • the plots are often made on logarithmic scales. Because different fluorescent dye's emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally.
  • Fluorophores for labeling biomarkers may include those described in Ormerod, Flow Cytometry 2nd ed., Springer-Verlag, New York (1999), and in Nida et al, Gynecologic Oncology 2005 ;4 889-894 which is incorporated herein by reference.
  • a multiplexed assay including but not limited to a flow cytometry assay, one or more different target molecules can be assessed.
  • at least one of the target molecule is a biomarker, e.g., a microvesicle surface antigen.
  • flow cytometry is used to assess a microvesicle population in a biological sample.
  • the microvesicle population can be sorted from other particles (e.g., cell debris, protein aggregates, etc) in a sample by labeling the vesicles using one or more general vesicle marker.
  • the general vesicle marker can be a marker in Table 3.
  • Commonly used vesicle markers include tetraspanins such as CD9, CD63 and/or CD81. Vesicles comprising one or more tetraspanin are sometimes refered to as "Tet+" herein to indicate that the vesicles are tetraspanin-positive.
  • the sorted microvesicles can be further assessed using methodology described herein. E.g., surface antigens on the sorted microvesicles can be detected using flow or other methods.
  • payload within the sorted microvesicles is assessed.
  • a population of microvesicles is contacted with a labeled binding agent to a surface antigen of interest, the contacted microvesicles are sorted using flow cytometry, and payload with the microvesicles is assessed.
  • the payload may be polypeptides, nucleic acids (e.g., mRNA or microRNA) or other biological entities as desired.
  • Such assessment is used to characterize a phenotype as described herein, e.g., to diagnose, prognose or theranose a cancer.
  • flow sorting is used to distinguish microvesicle populations from other biological complexes.
  • Ago2+/Tet+ and Ago2+/Tet- particles are detected using flow methodology to separate Ago2+ vesicles from vesicle-free Ago2+ complexes, respectively.
  • Multiplex experiments comprise experiments that can simultaneously measure multiple analytes in a single assay. Vesicles and associated biomarkers can be assessed in a multiplex fashion. Different binding agents can be used for multiplexing different circulating biomarkers, e.g., microRNA, protein, or vesicle populations. Different biomarkers, e.g., different vesicle populations, can be isolated or detected using different binding agents. Each population in a biological sample can be labeled with a different signaling label, such as a fluorophore, quantum dot, or radioactive label, such as described above. The label can be directly conjugated to a binding agent or indirectly used to detect a binding agent that binds a vesicle. The number of populations detected in a multiplexing assay is dependent on the resolution capability of the labels and the summation of signals, as more than two differentially labeled vesicle populations that bind two or more affinity elements can produce summed signals.
  • Different binding agents can be used for multiplexing
  • Multiplexing of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75 or 100 different circulating biomarkers may be performed.
  • one population of vesicles specific to a cell-of- origin can be assayed along with a second population of vesicles specific to a different cell-of-origin, where each population is labeled with a different label.
  • a population of vesicles with a particular biomarker or biosignature can be assayed along with a second population of vesicles with a different biomarker or biosignature.
  • hundreds or thousands of vesicles are assessed in a single assay.
  • multiplex analysis is performed by applying a plurality of vesicles comprising more than one population of vesicles to a plurality of substrates, such as beads.
  • Each bead is coupled to one or more capture agents.
  • the plurality of beads is divided into subsets, where beads with the same capture agent or combination of capture agents form a subset of beads, such that each subset of beads has a different capture agent or combination of capture agents than another subset of beads.
  • the beads can then be used to capture vesicles that comprise a component that binds to the capture agent.
  • the different subsets can be used to capture different populations of vesicles.
  • the captured vesicles can then be analyzed by detecting one or more biomarkers.
  • Flow cytometry can be used in combination with a particle -based or bead based assay.
  • Multiparametric immunoassays or other high throughput detection assays using bead coatings with cognate ligands and reporter molecules with specific activities consistent with high sensitivity automation can be used.
  • beads in each subset can be differentially labeled from another subset.
  • a binding agent or capture agent for a vesicle, such as a capture antibody can be immobilized on addressable beads or microspheres.
  • Each binding agent for each individual binding assay can be coupled to a distinct type of microsphere (i.e., microbead) and the binding assay reaction takes place on the surface of the microspheres.
  • Microspheres can be distinguished by different labels, for example, a microsphere with a specific capture agent would have a different signaling label as compared to another microsphere with a different capture agent.
  • microspheres can be dyed with discrete fluorescence intensities such that the fluorescence intensity of a microsphere with a specific binding agent is different than that of another microsphere with a different binding agent. Biomarkers bound by different capture agents can be differentially detected using different labels.
  • a microsphere can be labeled or dyed with at least 2 different labels or dyes.
  • the microsphere is labeled with at least 3, 4, 5, 6, 7, 8, 9, or 10 different labels.
  • Different microspheres in a plurality of microspheres can have more than one label or dye, wherein various subsets of the microspheres have various ratios and combinations of the labels or dyes permitting detection of different microspheres with different binding agents.
  • the various ratios and combinations of labels and dyes can permit different fluorescent intensities.
  • the various ratios and combinations maybe used to generate different detection patters to identify the binding agent.
  • the microspheres can be labeled or dyed externally or may have intrinsic fluorescence or signaling labels. Beads can be loaded separately with their appropriate binding agents and thus, different vesicle populations can be isolated based on the different binding agents on the differentially labeled microspheres to which the different binding agents are coupled.
  • multiplex analysis can be performed using a planar substrate, wherein the substrate comprises a plurality of capture agents.
  • the plurality of capture agents can capture one or more populations of vesicles, and one or more biomarkers of the captured vesicles detected.
  • the planar substrate can be a microarray or other substrate as further described herein.
  • a vesicle may be isolated or detected using a binding agent for a novel component of a vesicle, such as an antibody for a novel antigen specific to a vesicle of interest.
  • Novel antigens that are specific to a vesicle of interest may be isolated or identified using different test compounds of known composition bound to a substrate, such as an array or a plurality of particles, which can allow a large amount of chemical/structural space to be adequately sampled using only a small fraction of the space.
  • the novel antigen identified can also serve as a biomarker for the vesicle.
  • a novel antigen identified for a cell-of-origin specific vesicle can be a useful biomarker.
  • agent or "reagent” as used in respect to contacting a sample can mean any entity designed to bind, hybridize, associate with or otherwise detect or facilitate detection of a target molecule, including target polypeptides, peptides, nucleic acid molecules, leptins, lipids, or any other biological entity that can be detected as described herein or as known in the art.
  • agents/reagents are well known in the art, and include but are not limited to universal or specific nucleic acid primers, nucleic acid probes, antibodies, aptamers, peptoid, peptide nucleic acid, locked nucleic acid, lectin, dendrimer, chemical compound, or other entities described herein or known in the art.
  • a binding agent can be identified by screening either a homogeneous or heterogeneous vesicle population against test compounds. Since the composition of each test compound on the substrate surface is known, this constitutes a screen for affinity elements.
  • a test compound array comprises test compounds at specific locations on the substrate addressable locations, and can be used to identify one or more binding agents for a vesicle.
  • the test compounds can all be unrelated or related based on minor variations of a core sequence or structure.
  • the different test compounds may include variants of a given test compound (such as polypeptide isoforms), test compounds that are structurally or compositionally unrelated, or a combination thereof.
  • a test compound can be a peptoid, polysaccharide, organic compound, inorganic compound, polymer, lipids, nucleic acid, polypeptide, antibody, protein, polysaccharide, or other compound.
  • the test compound can be natural or synthetic.
  • the test compound can comprise or consist of linear or branched heteropolymeric compounds based on any of a number of linkages or combinations of linkages (e.g., amide, ester, ether, thiol, radical additions, metal coordination, etc.), dendritic structures, circular structures, cavity structures or other structures with multiple nearby sites of attachment that serve as scaffolds upon which specific additions are made.
  • Thes test compound can be spotted on a substrate or synthesized in situ, using standard methods in the art.
  • the test compound can be spotted or synthesized in situ in combinations in order to detect useful interactions, such as cooperative binding.
  • the test compound can be a polypeptide with known amino acid sequence, thus, detection of a test compound binding with a vesicle can lead to identification of a polypeptide of known amino sequence that can be used as a binding agent.
  • a homogenous population of vesicles can be applied to a spotted array on a slide containing between a few and 1,000,000 test polypeptides having a length of variable amino acids.
  • the polypeptides can be attached to the surface through the C-terminus.
  • the sequence of the polypeptides can be generated randomly from 19 amino acids, excluding cysteine.
  • the binding reaction can include a non-specific competitor, such as excess bacterial proteins labeled with another dye such that the specificity ratio for each polypeptide binding target can be determined.
  • the polypeptides with the highest specificity and binding can be selected. The identity of the polypeptide on each spot is known, and thus can be readily identified.
  • An array can also be used for identifying an antibody as a binding agent for a vesicle.
  • Test antibodies can be attached to an array and screened against a heterogeneous population of vesicles to identify antibodies that can be used to isolate or identify a vesicle.
  • a homogeneous population of vesicles such as cell-of-origin specific vesicles can also be screened with an antibody array.
  • Other than identifying antibodies to isolate or detect a homogeneous population of vesicles, one or more protein biomarkers specific to the homogenous population can be identified.
  • Commercially available platforms with test antibodies pre-selected or custom selection of test antibodies attached to the array can be used. For example, an antibody array from Full Moon Biosystems can be screened using prostate cancer cell derived vesicles identifying antibodies to Bcl-XL, ERCC1, Keratin 15, CD81/TAPA-1, CD9, Epithelial
  • ESA Specific Antigen
  • Mast Cell Chymase as binding agents, and the proteins identified can be used as biomarkers for the vesicles.
  • the biomarker can be present or absent, underexpressed or overexpressed, mutated, or modified in or on a vesicle and used in characterizing a condition.
  • An antibody or synthetic antibody to be used as a binding agent can also be identified through a peptide array.
  • Another method is the use of synthetic antibody generation through antibody phage display.
  • Ml 3 bacteriophage libraries of antibodies e.g. Fabs
  • Fabs antibodies
  • Each phage particle displays a unique antibody and also encapsulates a vector that contains the encoding DNA.
  • Highly diverse libraries can be constructed and represented as phage pools, which can be used in antibody selection for binding to immobilized antigens. Antigen-binding phages are retained by the immobilized antigen, and the nonbinding phages are removed by washing.
  • the retained phage pool can be amplified by infection of an Escherichia coli host and the amplified pool can be used for additional rounds of selection to eventually obtain a population that is dominated by antigen-binding clones.
  • individual phase clones can be isolated and subjected to DNA sequencing to decode the sequences of the displayed antibodies.
  • phase display and other methods known in the art high affinity designer antibodies for vesicles can be generated.
  • Bead-based assays can also be used to identify novel binding agents to isolate or detect a vesicle.
  • a test antibody or peptide can be conjugated to a particle.
  • a bead can be conjugated to an antibody or peptide and used to detect and quantify the proteins expressed on the surface of a population of vesicles in order to discover and specifically select for novel antibodies that can target vesicles from specific tissue or tumor types.
  • Any molecule of organic origin can be successfully conjugated to a polystyrene bead through use of a commercially available kit according to manufacturer's instructions.
  • Each bead set can be colored a certain detectable wavelength and each can be linked to a known antibody or peptide which can be used to specifically measure which beads are linked to exosomal proteins matching the epitope of previously conjugated antibodies or peptides.
  • the beads can be dyed with discrete fluorescence intensities such that each bead with a different intensity has a different binding agent as described above.
  • a purified vesicle preparation can be diluted in assay buffer to an appropriate concentration according to empirically determined dynamic range of assay.
  • a sufficient volume of coupled beads can be prepared and approximately 1 ⁇ of the antibody-coupled beads can be aliqouted into a well and adjusted to a final volume of approximately 50 ⁇ .
  • the beads can be washed to ensure proper binding conditions.
  • An appropriate volume of vesicle preparation can then be added to each well being tested and the mixture incubated, such as for 15-18 hours.
  • a sufficient volume of detection antibodies using detection antibody diluent solution can be prepared and incubated with the mixture for 1 hour or for as long as necessary.
  • the beads can then be washed before the addition of detection antibody (biotin expressing) mixture composed of streptavidin phycoereythin.
  • detection antibody biotin expressing
  • the beads can then be washed and vacuum aspirated several times before analysis on a suspension array system using software provided with an instrument.
  • the identity of antigens that can be used to selectively extract the vesicles can then be elucidated from the analysis.
  • Assays using imaging systems can be used to detect and quantify proteins expressed on the surface of a vesicle in order to discover and specifically select for and enrich vesicles from specific tissue, cell or tumor types.
  • Antibodies, peptides or cells conjugated to multiple well multiplex carbon coated plates can be used. Simultaneous measurement of many analytes in a well can be achieved through the use of capture antibodies arrayed on the patterned carbon working surface. Analytes can then be detected with antibodies labeled with reagents in electrode wells with an enhanced electro-chemiluminescent plate. Any molecule of organic origin can be successfully conjugated to the carbon coated plate. Proteins expressed on the surface of vesicles can be identified from this assay and can be used as targets to specifically select for and enrich vesicles from specific tissue or tumor types.
  • the binding agent can also be an aptamer, which refers to nucleic acids that can bond molecules other than their complementary sequence.
  • An aptamer typically contains 30-80 nucleic acids and can have a high affinity towards a certain target molecule (K d 's reported are between 10 "n -10 " 6 mole/l).
  • An aptamer for a target can be identified using systematic evolution of ligands by exponential enrichment (SELEX) ⁇ Tuerk & Gold, Science 249:505-510, 1990; Ellington & Szostak, Nature 346:818-822, 1990), such as described in U.S. Pat. Nos.
  • a library of nucleic acids can be contacted with a target vesicle, and those nucleic acids specifically bound to the target are partitioned from the remainder of nucleic acids in the library which do not specifically bind the target.
  • the partitioned nucleic acids are amplified to yield a ligand- enriched pool. Multiple cycles of binding, partitioning, and amplifying (i.e., selection) result in identification of one or more aptamers with the desired activity.
  • Another method for identifying an aptamer to isolate vesicles is described in U.S. Pat. No.
  • binding agent can mean that an agent has a greater affinity for its target than other targets, typically with a much great affinity, but does not require that the binding agent is absolutely specific for its target.
  • the methods for isolating or identifying vesicles can be used in combination with microfluidic devices.
  • the methods of isolating or detecting a vesicle, such as described herien, can be performed using a microfluidic device.
  • Microfluidic devices which may also be referred to as "lab-on-a-chip” systems, biomedical micro-electromechanical systems (bioMEMs), or multicomponent integrated systems, can be used for isolating and analyzing a vesicle.
  • Such systems miniaturize and compartmentalize processes that allow for binding of vesicles, detection of biosignatures, and other processes.
  • a microfluidic device can also be used for isolation of a vesicle through size differential or affinity selection.
  • a microfluidic device can use one more channels for isolating a vesicle from a biological sample based on size or by using one or more binding agents for isolating a vesicle from a biological sample.
  • a biological sample can be introduced into one or more microfluidic channels, which selectively allows the passage of a vesicle. The selection can be based on a property of the vesicle, such as the size, shape, deformability, or biosignature of the vesicle.
  • a heterogeneous population of vesicles can be introduced into a microfluidic device, and one or more different homogeneous populations of vesicles can be obtained.
  • different channels can have different size selections or binding agents to select for different vesicle populations.
  • a microfluidic device can isolate a plurality of vesicles wherein at least a subset of the plurality of vesicles comprises a different biosignature from another subset of the plurality of vesicles.
  • the microfluidic device can isolate at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 different subsets of vesicles, wherein each subset of vesicles comprises a different biosignature.
  • the microfluidic device can comprise one or more channels that permit further enrichment or selection of a vesicle.
  • a population of vesicles that has been enriched after passage through a first channel can be introduced into a second channel, which allows the passage of the desired vesicle or vesicle population to be further enriched, such as through one or more binding agents present in the second channel.
  • Array-based assays and bead-based assays can be used with microfluidic device.
  • the binding agent can be coupled to beads and the binding reaction between the beads and vesicle can be performed in a microfluidic device. Multiplexing can also be performed using a microfluidic device.
  • Different compartments can comprise different binding agents for different populations of vesicles, where each population is of a different cell- of-origin specific vesicle population. In one embodiment, each population has a different biosignature.
  • the hybridization reaction between the microsphere and vesicle can be performed in a microfluidic device and the reaction mixture can be delivered to a detection device.
  • the detection device such as a dual or multiple laser detection system can be part of the microfluidic system and can use a laser to identify each bead or microsphere by its color-coding, and another laser can detect the hybridization signal associated with each bead.
  • microfluidic device can be used in the methods of the invention.
  • microfluidic devices that may be used, or adapted for use with vesicles, include but are not limited to those described in U.S. Pat.
  • microfluidic devices for use with the invention include devices comprising elastomeric layers, valves and pumps, including without limitation those disclosed in U.S. Patent Nos. 5,376,252, 6,408,878, 6,645,432, 6,719,868, 6,793,753, 6,899,137, 6,929,030, 7,040,338, 7, 118,910, 7,144,616, 7,216,671, 7,250,128, 7,494,555, 7,501,245, 7,601,270, 7,691,333, 7,754,010, 7,837,946; U.S. Patent Application Nos.
  • the devices are composed of elastomeric material.
  • Certain devices are designed to conduct thermal cycling reactions (e.g., PCR) with devices that include one or more elastomeric valves to regulate solution flow through the device.
  • the devices can comprise arrays of reaction sites thereby allowing a plurality of reactions to be performed.
  • the devices can be used to assess circulating microRNAs in a multiplex fashion, including microRNAs isolated from vesicles.
  • the microfluidic device comprises (a) a first plurality of flow channels formed in an elastomeric substrate; (b) a second plurality of flow channels formed in the elastomeric substrate that intersect the first plurality of flow channels to define an array of reaction sites, each reaction site located at an intersection of one of the first and second flow channels; (c) a plurality of isolation valves disposed along the first and second plurality of flow channels and spaced between the reaction sites that can be actuated to isolate a solution within each of the reaction sites from solutions at other reaction sites, wherein the isolation valves comprise one or more control channels that each overlay and intersect one or more of the flow channels; and (d) means for simultaneously actuating the valves for isolating the reaction sites from each other.
  • MicroRNAs can be detected in each of the reaction sites by using PCR methods.
  • the method can comprise the steps of the steps of: (i) providing a microfluidic device, the microfluidic device comprising: a first fluidic channel having a first end and a second end in fluid communication with each other through the channel; a plurality of flow channels, each flow channel terminating at a terminal wall; wherein each flow channel branches from and is in fluid communication with the first fluidic channel, wherein an aqueous fluid that enters one of the flow channels from the first fluidic channel can flow out of the flow channel only through the first fluidic channel; and, an inlet in fluid communication with the first fluidic channel, the inlet for introducing a sample fluid; wherein each flow channel is associated with a valve that when closed isolates one end of the flow channel from the first fluidic channel, whereby an isolated reaction site is formed between the valve and the terminal wall; a control channel; wherein each the valve
  • the PCR used to detect microRNA is digital PCR, which is described by Brown, et al., U.S. Pat. No. 6, 143,496, titled “Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized chambers and methods of filling chambers", and by Vogelstein, et al, U.S. Pat. No. 6,446,706, titled “Digital PCR", both of which are hereby incorporated by reference in their entirety.
  • digital PCR a sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions, such as the reaction sites of the micro fluidic device described above.
  • the partitioning of the sample allows one to count the molecules by estimating according to Poisson. As a result, each part will contain "0" or “1” molecules, or a negative or positive reaction, respectively.
  • nucleic acids may be quantified by counting the regions that contain PCR end-product, positive reactions.
  • starting copy number is proportional to the number of PCR amplification cycles.
  • Digital PCR is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification.
  • the method can provide a sensitive approach to detecting microRNAs in a sample.
  • a microfluidic device for isolating or detecting a vesicle comprises a channel of less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, of 60 mm in width, or between about 2-60, 3-50, 3-40, 3-30, 3-20, or 4-20 mm in width.
  • the microchannel can have a depth of less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65 or 70 ⁇ , or between about 10-70, 10-40, 15-35, or 20-30 ⁇ . Furthermore, the microchannel can have a length of less than about 1, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cm.
  • the microfluidic device can have grooves on its ceiling that are less than about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6, 65, 70, 75, or 80 ⁇ wide, or between about 40-80, 40-70, 40-60 or 45-55 ⁇ wide.
  • the grooves can be less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 ⁇ deep, such as between about 1-50, 5-40, 5-30, 3-20 or 5-15 ⁇ .
  • the microfluidic device can have one or more binding agents attached to a surface in a channel, or present in a channel.
  • the microchannel can have one or more capture agents, such as a capture agent for one or more general microvesicle antigen in Table 3 or a cell-of-origin or cancer related antigen in Table 4 or Table 5, including without limitation EpCam, CD9, PCSA, CD63, CD81, PSMA, B7H3, PSCA, ICAM, STEAP, KLK2, SSX2, SSX4, PBP, SPDEF, and/or EGFR.
  • a microchannel surface is treated with avidin and a capture agent, such as an antibody, that is biotinylated can be injected into the channel to bind the avidin.
  • a capture agent such as an antibody
  • the capture agents are present in chambers or other components of a microfluidic device.
  • the capture agents can also be attached to beads that can be manipulated to move through the microfluidic channels.
  • the capture agents are attached to magnetic beads. The beads can be manipulated using magnets.
  • a biological sample can be flowed into the microfluidic device, or a microchannel, at rates such as at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 ⁇ per minute, such as between about 1-50, 5-40, 5-30, 3-20 or 5-15 ⁇ per minute.
  • One or more vesicles can be captured and directly detected in the microfluidic device. Alternatively, the captured vesicle may be released and exit the microfluidic device prior to analysis. In another embodiment, one or more captured vesicles are lysed in the microchannel and the lysate can be analyzed, e.g., to examine payload within the vesicles.
  • Lysis buffer can be flowed through the channel and lyse the captured vesicles.
  • the lysis buffer can be flowed into the device or microchannel at rates such as at least about a, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 ⁇ per minute, such as between about 1-50, 5-40, 10-30, 5-30 or 10-35 ⁇ per minute.
  • the lysate can be collected and analyzed, such as performing RT-PCR, PCR, mass spectrometry, Western blotting, or other assays, to detect one or more biomarkers of the vesicle.
  • the various isolation and detection systems described herein can be used to isolate or detect circulating biomarkers such as vesicles that are informative for diagnosis, prognosis, disease stratification, theranosis, prediction of responder / non-responder status, disease monitoring, treatment monitoring and the like as related to such diseases and disorders. Combinations of the isolation techniques are within the scope of the invention.
  • a sample can be run through a chromatography column to isolate vesicles based on a property such as size of electrophoretic motility, and the vesicles can then be passed through a microfluidic device. Binding agents can be used before, during or after these steps.
  • biomarkers such as vesicles can be combined as desired.
  • a biological sample can be treated to prevent aggregation, remove undesired particulate and/or deplete highly abundant proteins.
  • the steps used can be chosen to optimize downstream analysis steps.
  • biomarkers such as vesicles can be isolated, e.g., by chromotography, centrifugation, density gradient, filtration, precipitation, or affinity techniques.
  • any number of the later steps can be combined, e.g., a sample could be subjected to one or more of chromotography, centrifugation, density gradient, filtration and precipitation in order to isolate or concentrate all or most microvesicles.
  • affinity techniques e.g., using binding agents to one or more target of interest, can be used to isolate or identify microvesicles carrying desired biomarker profiles.
  • Microfluidic systems can be employed to perform some or all of these steps.
  • An exemplary isolation scheme for isolating and analysis of microvesicles includes the following: Plasma or serum collection -> highly abundant protein removal -> ultrafiltration -> nanomembrane concentration -> flow cytometry or particle-based assay.
  • circulating biomarkers such as vesicles can be isolated or concentrated by at least about 2-fold, 3-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 125-fold, 130-fold, 140-fold, 150-fold, 160- fold, 170-fold, 175-fold, 180-fold, 190-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, 500-fold, 5
  • the vesicles are isolated or concentrated concentrated by at least 1 order of magnitude, 2 orders of magnitude, 3 orders of magnitude, 4 orders of magnitude, 5 orders of magnitude, 6 orders of magnitude, 7 orders of magnitude, 8 orders of magnitude, 9 orders of magnitude, or 10 orders of magnitude or more.
  • the circulating biomarkers can be assessed, e.g., in order to characterize a phenotype as described herein.
  • the concentration or isolation steps themselves shed light on the phenotype of interest.
  • affinity methods can detect the presence or level of specific biomarkers of interest.
  • the bindings agent disclosed herein can be used to isolate or detect a vesicle, such as a cell-of-origin vesicle or vesicle with a specific biosignature.
  • the binding agent can be used to isolate or detect a heterogeneous population of vesicles from a sample or can be used to isolate or detect a homogeneous population of vesicles, such as cell-of-origin specific vesicles with specific biosignatures, from a heterogeneous population of vesicles.
  • a homogeneous population of vesicles can be analyzed and used to characterize a phenotype for a subject.
  • Cell-of-origin specific vesicles are esicles derived from specific cell types, which can include, but are not limited to, cells of a specific tissue, cells from a specific tumor of interest or a diseased tissue of interest, circulating tumor cells, or cells of maternal or fetal origin.
  • the vesicles may be derived from tumor cells or lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, or fetal cells.
  • the isolated vesicle can also be from a particular sample type, such as urinary vesicle.
  • a cell-of-origin specific vesicle from a biological sample can be isolated using one or more binding agents that are specific to a cell-of-origin.
  • Vesicles for analysis of a disease or condition can be isolated using one or more binding agent specific for biomarkers for that disease or condition.
  • a vesicle can be concentrated prior to isolation or detection of a cell-of-origin specific vesicle, such as through centrifugation, chromatography, or filtration, as described above, to produce a heterogeneous population of vesicles prior to isolation of cell-of-origin specific vesicles.
  • the vesicle is not concentrated, or the biological sample is not enriched for a vesicle, prior to isolation of a cell-of-origin vesicle.
  • FIG. IB illustrates a flowchart which depicts one method 100B for isolating or identifying a cell-of-origin specific vesicle.
  • a biological sample is obtained from a subject in step 102.
  • the sample can be obtained from a third party or from the same party performing the analysis.
  • cell-of-origin specific vesicles are isolated from the biological sample in step 104.
  • the isolated cell-of-origin specific vesicles are then analyzed in step 106 and a biomarker or biosignature for a particular phenotype is identified in step 108.
  • the method may be used for a number of phenotypes.
  • vesicles are concentrated or isolated from a biological sample to produce a homogeneous population of vesicles.
  • a heterogeneous population of vesicles may be isolated using centrifugation, chromatography, filtration, or other methods as described above, prior to use of one or more binding agents specific for isolating or identifying vesicles derived from specific cell types.
  • a cell-of-origin specific vesicle can be isolated from a biological sample of a subject by employing one or more binding agents that bind with high specificity to the cell-of-origin specific vesicle.
  • a single binding agent can be employed to isolate a cell-of-origin specific vesicle.
  • a combination of binding agents may be employed to isolate a cell-of-origin specific vesicle. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, or 100 different binding agents may be used to isolate a cell-of- origin vesicle. Therefore, a vesicle population (e.g., vesicles having the same binding agent profile) can be identified by using a single or a plurality of binding agents.
  • One or more binding agents can be selected based on their specificity for a target antigen(s) that is specific to a cell-of-origin, e.g., a cell-of-origin that is related to a tumor, autoimmune disease, cardiovascular disease, neurological disease, infection or other disease or disorder.
  • the cell-of-origin can be from a cell that is informative for a diagnosis, prognosis, disease stratification, theranosis, prediction of responder / non-responder status, disease monitoring, treatment monitoring and the like as related to such diseases and disorders.
  • the cell-of-origin can also be from a cell useful to discover biomarkers for use thereto.
  • Non-limiting examples of antigens which may be used singularly, or in combination, to isolate a cell-of-origin specific vesicle, disease specific vesicle, or tumor specific vesicle, are shown in Fig. 1 of International Patent Application Serial No. PCT US2011/031479, entitled
  • the antigen can comprise membrane bound antigens which are accessible to binding agents.
  • the antigen can be a biomarker related to characterizing a phenotype.
  • binding agents e.g., antibodies, aptamers and lectins
  • the binding agents can recognize antigens specific to the desired cell type or location and/or recognize biomarkers associated with the desired cells.
  • the cells can be, e.g., tumor cells, other diseased cells, cells that serve as markers of disease such as activated immune cells, etc.
  • binding agents for any cells of interest can be useful for isolating vesicles associated with those cells.
  • binding agents disclosed herein can be used for detecting vesicles of interest.
  • a binding agent to a vesicle biomarker can be labeled directly or indirectly in order to detect vesicles bound by one of more of the same or different binding agents.
  • a number of targets for binding agents useful for binding to vesicles associated with cancer, autoimmune diseases, cardiovascular diseases, neurological diseases, infection or other disease or disorders are presented in Table 4.
  • a vesicle derived from a cell associated with one of the listed disorders can be characterized using one of the antigens in the table.
  • the binding agent e.g., an antibody or aptamer, can recognize an epitope of the listed antigens, a fragment thereof, or binding agents can be used against any appropriate combination.
  • Other antigens associated with the disease or disorder can be recognized as well in order to characterize the vesicle.
  • any applicable antigen that can be used to assess an informative vesicle is contemplated by the invention for isolation, capture or detection in order to characterize a vesicle.
  • biomarkers disclosed here are illustrative, and Applicants contemplate incorporating various biomarkers disclosed across different disease states or conditions.
  • method of the invention may use various biomarkers across different diseases or conditions, where the biomarkers are useful for providing a diagnostic, prognostic or theranostic signature.
  • angiogenic, inflammatory or immune-associated antigens (or biomarkers) disclosed herein or know in the art can be used in methods of the invention to screen a biological sample in identification of a biosignature.
  • the flexibility of Applicants' multiplex approach to assessing microvesicle populations facilitates assessing various markers (and in some instances overlapping markers) for different conditions or diseases whose etiology necessarily may share certain cellular and biological mechanisms, e.g., different cancers implicating biomarkers for angiogenesis, or immune response regulation or modulation.
  • the combination of such overlapping biomarkers with tissue or cell- specific biomarkers, along with microvesicle-associated biomarkers provides a powerful series of tools for practicing the methods and compositions of the invention.
  • a cell-of-origin specific vesicle may be isolated using novel binding agents, using methods as described herein. Furthermore, a cell-of-origin specific vesicle can also be isolated from a biological sample using isolation methods based on cellular binding partners or binding agents of such vesicles. Such cellular binding partners can include but are not limited to peptides, proteins, RNA, DNA, apatmers, cells or serum-associated proteins that only bind to such vesicles when one or more specific biomarkers are present.
  • Isolation or deteciton of a cell-of-origin specific vesicle can be carried out with a single binding partner or binding agent, or a combination of binding partners or binding agents whose singular application or combined application results in cell-of-origin specific isolation or detection.
  • binding agents are provided in Fig. 2 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • a vesicle for characterizing breast cancer can be isolated with one or more binding agents including, but not limited to, estrogen, progesterone, trastuzumab, CCNDl, MYC PNA, IGF-1 PNA, MYC PNA, SC4 aptamer (Ku), AII-7 aptamer (ERB2), Galectin -3, mucin-type O-glycans, L-PHA, Galectin-9, or any combination thereof.
  • binding agents including, but not limited to, estrogen, progesterone, trastuzumab, CCNDl, MYC PNA, IGF-1 PNA, MYC PNA, SC4 aptamer (Ku), AII-7 aptamer (ERB2), Galectin -3, mucin-type O-glycans, L-PHA, Galectin-9, or any combination thereof.
  • a binding agent may also be used for isolating or detecting a cell-of-origin specific vesicle based on: i) the presence of antigens specific for cell-of-origin specific vesicles; ii) the absence of markers specific for cell-of-origin specific vesicles; or iii) expression levels of biomarkers specific for cell-of-origin specific vesicles.
  • a heterogeneous population of vesicles can be applied to a surface coated with specific binding agents designed to rule out or identify the cell-of-origin characteristics of the vesicles.
  • binding agents such as antibodies
  • Various binding agents can be arrayed on a solid surface or substrate and the heterogeneous population of vesicles is allowed to contact the solid surface or substrate for a sufficient time to allow interactions to take place.
  • Specific binding or non-binding to given antibody locations on the array surface or substrate can then serve to identify antigen specific characteristics of the vesicle population that are specific to a given cell-of-origin. That is, binding events can signal the presence of a vesicle having an antigen recognized by the bound antibody. Conversely, lack of binding events can signal the absence of vesicles having an antigen recognized by the bound antibody.
  • a cell-of-origin specific vesicle can be enriched or isolated using one or more binding agents using a magnetic capture method, fluorescence activated cell sorting (FACS) or laser cytometry as described above.
  • FACS fluorescence activated cell sorting
  • Magnetic capture methods can include, but are not limited to, the use of magnetically activated cell sorter (MACS) microbeads or magnetic columns.
  • MCS magnetically activated cell sorter
  • immunoaffinity and magnetic particle methods that can be used are described in U.S. Patent Nos. 4,551,435, 4,795,698, 4,925,788, 5, 108,933, 5,186,827, 5,200,084 or 5,158,871.
  • a cell-of-origin specific vesicle can also be isolated following the general methods described in U.S. Patent No. 7,399,632, by using combination of antigens specific to a vesicle.
  • any other appropriate method for isolating or otherwise enriching the cell-of-origin specific vesicles with respect to a biological sample may also be used in combination with the present invention.
  • size exclusion chromatography such as gel permeation columns, centrifugation or density gradient centrifugation, and filtration methods can be used in combination with the antigen selection methods described herein.
  • the cell-of-origin specific vesicles may also be isolated following the methods described in Koga et al., Anticancer Research, 25:3703-3708 (2005), Taylor et al., Gynecologic Oncology, 110:13-21 (2008), Nanjee et al, Clin Chem,
  • Vesicles can be isolated and/or detected to provide diagnosis, prognosis, disease stratification, theranosis, prediction of responder / non-responder status, disease monitoring, treatment monitoring and the like.
  • vesicles are isolated from cells having a disease or disorder, e.g., cells derived from a tumor or malignant growth, a site of autoimmune disease, cardiovascular disease, neurological disease, or infection.
  • the isolated vesicles are derived from cells related to such diseases and disorders, e.g., immune cells that play a role in the etiology of the disease and whose analysis is informative for a diagnosis, prognosis, disease stratification, theranosis, prediction of responder / non-responder status, disease monitoring, treatment monitoring and the like as relates to such diseases and disorders.
  • the vesicles are further useful to discover novel biomarkers. By identifying biomarkers associated with vesicles, isolated vesicles can be assessed for characterizing a phenotype as described herein.
  • methods of the invention are directed to characterizing presence of a cancer or likelihood of a cancer occurring in an individual by assessing one or more microvesicle population present in a biological sample from an individual.
  • Microvesicles can be isolated using one or more processes disclosed herein or practiced in the art.
  • microvesicles populations can each separately or collectively provide a disease phenotype characterization for the individual by comparing the biomarker profile, or biosignature, for the microvesicle population(s) with a reference sample to provide a diagnostic, prognostic or theranostic characterization for the test sample.
  • the vesicle population(s) can be assessed from various biological samples and bodily fluids such as disclosed herein.
  • a phenotype of a subject is characterized by analyzing a biological sample and determining the presence, level, amount, or concentration of one or more populations of circulating biomarkers in the sample, e.g., circulating vesicles, proteins or nucleic acids.
  • characterization includes determining whether the circulating biomarkers in the sample are altered as compared to a reference, which can also be referred to a standard or a control.
  • An alteration can include any measurable difference between the sample and the reference, including without limitation an absolute presence or absence, a quantitative level, a relative level compared to a reference, e.g., the level of all vesicles present, the level of a housekeeping marker, and/or the level of a spiked-in marker, an elevated level, a decreased level, overexpression, underexpression, differential expression, a mutation or other altered sequence, a modification (glycosylation, phosphorylation, epigenetic change) and the like.
  • circulating biomarkers are purified or concentrated from a sample prior to determining their amount. Unless otherwise specified, "purified” or “isolated” as used herein refer to partial or complete purification or isolation.
  • Circulating biomarkers are directly assessed from a sample, without prior purification or concentration.
  • Circulating vesicles can be cell-of-origin specific vesicles or vesicles with a specific biosignature.
  • a biosignature includes specific pattern of biomarkers, e.g., patterns of biomarkers indicative of a phenotype that is desireable to detect, such as a disease phenotype.
  • the biosignature can comprise one or more circulating biomarkers.
  • a biosignature can be used when characterizing a phenotype, such as a diagnosis, prognosis, theranosis, or prediction of responder / non-responder status.
  • the biosignature is used to determine a physiological or biological state, such as pregnancy or the stage of pregnancy.
  • the biosignature can also be used to determine treatment efficacy, stage of a disease or condition, or progression of a disease or condition.
  • the amount of one or more vesicles can be proportional or inversely proportional to an increase in disease stage or progression.
  • the detected amount of vesicles can also be used to monitor progression of a disease or condition or to monitor a subject's response to a treatment.
  • the circulating biomarkers can be evaluated by comparing the level of circulating biomarkers with a reference level or value.
  • the reference value can be particular to physical or temporal endpoint.
  • the reference value can be from the same subject from whom a sample is assessed, or the reference value can be from a representative population of samples (e.g., samples from normal subjects not exhibiting a symptom of disease). Therefore, a reference value can provide a threshold measurement which is compared to a subject sample's readout for a biosignature assayed in a given sample.
  • Such reference values may be set according to data pooled from groups of sample corresponding to a particular cohort, including but not limited to age (e.g., newborns, infants, adolescents, young, middle-aged adults, seniors and adults of varied ages), racial/ethnic groups, normal versus diseased subjects, smoker v. non-smoker, subject receiving therapy versus untreated subject, different time points of treatment for a particular individual or group of subjects similarly diagnosed or treated or combinations thereof. Furthermore, by determining a biosignature at different timepoints of treatment for a particular individual, the individual's response to the treatment or progression of a disease or condition for which the individual is being treated for, can be monitored.
  • a reference value may be based on samples assessed from the same subject so to provide individualized tracking.
  • frequent testing of a biosignature in samples from a subject provides better comparisons to the reference values previously established for that subject.
  • Such time course measurements are used to allow a physician to more accurately assess the subject's disease stage or progression and therefore inform a better decision for treatment.
  • the variance of a biosignature is reduced when comparing a subject's own biosignature over time, thus allowing an individualized threshold to be defined for the subject, e.g., a threshold at which a diagnosis is made.
  • Temporal intrasubject variation allows each individual to serve as their own longitudinal control for optimum analysis of disease or physiological state.
  • the level of vesicles derived from prostate cells is measured in a subject's blood over time.
  • a spike in the level of prostate- derived vesicles in the subject's blood can indicate hyperproliferation of prostate cells, e.g., due to prostate cancer.
  • Reference values can be established for unaffected individuals (of varying ages, ethnic backgrounds and sexes) without a particular phenotype by determining the biosignature of interest in an unaffected individual.
  • a reference value for a reference population can be used as a baseline for detection of one or more circulating biomarker populations in a test subject. If a sample from a subject has a level or value that is similar to the reference, the subject can be identified to not have the disease, or of having a low likelihood of developing a disease.
  • reference values or levels can be established for individuals with a particular phenotype by determining the amount of one or more populations of vesicles in an individual with the phenotype.
  • an index of values can be generated for a particular phenotype. For example, different disease stages can have different values, such as obtained from individuals with the different disease stages. A subject's value can be compared to the index and a diagnosis or prognosis of the disease can be determined, such as the disease stage or progression wherein the subject's levels most closely correlate with the index.
  • an index of values is generated for therapeutic efficacies. For example, the level of vesicles of individuals with a particular disease can be generated and noted what treatments were effective for the individual.
  • the levels can be used to generate values of which is a subject's value is compared, and a treatment or therapy can be selected for the individual, e.g., by predicting from the levels whether the subject is likely to be a responder or non-responder for a treatment.
  • a reference value is determined for individuals unaffected with a particular cancer, by isolating or detecting circulating biomarkers with an antigen that specifically targets biomarkers for the particular cancer.
  • individuals with varying stages of colorectal cancer and noncancerous polyps can be surveyed using the same techniques described for unaffected individuals and the levels of circulating vesicles for each group can be determined.
  • the levels are defined as means ⁇ standard deviations from at least two separate experiments, performed in at least duplicate or triplicate. Comparisons between these groups can be made using statistical tests to determine statistical significance of distinguishing biomarkers observed. In some embodiments, statistical significance is determined using a parametric statistical test.
  • the parametric statistical test can comprise, without limitation, a fractional factorial design, analysis of variance (ANOVA), a t-test, least squares, a Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression.
  • the parametric statistical test can comprise a one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance.
  • statistical significance is determined using a nonparametric statistical test. Examples include, but are not limited to, a Wilcoxon signed-rank test, a Mann- Whitney test, a Kruskal-Wallis test, a Friedman test, a Spearman ranked order correlation coefficient, a Kendall Tau analysis, and a nonparametric regression test.
  • statistical significance is determined at a p-value of less than 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001.
  • the p-values can also be corrected for multiple comparisons, e.g., using a Bonferroni correction, a modification thereof, or other technique known to those in the art, e.g., the Hochberg correction, Holm-Bonferroni correction, Sidak correction, Dunnett's correction or Tukey's multiple comparisons.
  • an ANOVA is followed by Tukey's correction for post-test comparing of the biomarkers from each population.
  • a biosignature comprising more than one marker can be evaluated using multivariate modeling techniques to build a classifier using techniques described herein or known in the art.
  • Reference values can also be established for disease recurrence monitoring (or exacerbation phase in MS), for therapeutic response monitoring, or for predicting responder / non-responder status.
  • a reference value for vesicles is determined using an artificial vesicle, also referred to herein as a synthetic vesicle.
  • an artificial vesicle also referred to herein as a synthetic vesicle.
  • Methods for manufacturing artificial vesicles are known to those of skill in the art, e.g., using liposomes.
  • Artificial vesicles can be manufactured using methods disclosed in US20060222654 and US4448765, which are incorporated herein by reference in its entirety. Artificial vesicles can be constructed with known markers to facilitate capture and/or detection.
  • artificial vesicles are spiked into a bodily sample prior to processing.
  • the level of intact synthetic vesicle can be tracked during processing, e.g., using filtration or other isolation methods disclosed herein, to provide a control for the amount of vesicles in the initial versus processed sample.
  • artificial vesicles can be spiked into a sample before or after any processing steps.
  • artificial vesicles are used to calibrate equipment used for isolation and detection of vesicles.
  • Artificial vesicles can be produced and used a control to test the viability of an assay, such as a bead-based assay.
  • the artificial vesicle can bind to both the beads and to the detection antibodies.
  • the artificial vesicle contains the amino acid sequence/conformation that each of the antibodies binds.
  • the artificial vesicle can comprise a purified protein or a synthetic peptide sequence to which the antibody binds.
  • the artificial vesicle could be a bead, e.g., a polystyrene bead, that is capable of having biological molecules attached thereto. If the bead has an available carboxyl group, then the protein or peptide could be attached to the bead via an available amine group, such as using carbodiimide coupling.
  • the artificial vesicle can be a polystyrene bead coated with avidin and a biotin is placed on the protein or peptide of choice either at the time of synthesis or via a biotin-maleimide chemistry.
  • the proteins/peptides to be on the bead can be mixed together in ratio specific to the application the artificial vesicle is being used for, and then conjugated to the bead.
  • These artificial vesicles can then serve as a link between the capture beads and the detection antibodies, thereby providing a control to show that the components of the assay are working properly.
  • the value can be a quantitative or qualitative value.
  • the value can be a direct measurement of the level of vesicles (example, mass per volume), or an indirect measure, such as the amount of a specific biomarker.
  • the value can be a quantitative, such as a numerical value. In other embodiments, the value is qualitiative, such as no vesicles, low level of vesicles, medium level, high level of vesicles, or variations thereof.
  • the reference value can be stored in a database and used as a reference for the diagnosis, prognosis, theranosis, disease stratification, disease monitoring, treatment monitoring or prediction of non-responder / responder status of a disease or condition based on the level or amount of circulating biomarkers, such as total amount of vesicles or microRNA, or the amount of a specific population of vesicles or microRNA, such as cell-of- origin specific vesicles or microRNA or microRNA from vesicles with a specific biosignature.
  • a method of determining a diagnosis for a cancer consider a method of determining a diagnosis for a cancer.
  • Vesicles or other circulating biomarkers from reference subjects with and without the cancer are assessed and stored in the database.
  • the reference subjects provide biosignature indicative of the cancer or of another state, e.g., a healthy state.
  • a sample from a test subject is then assayed and the microRNA biosignature is compared against those in the database. If the subject's biosignature correlates more closely with reference values indicative of cancer, a diagnosis of cancer may be made. Conversely, if the subject's biosignature correlates more closely with reference values indicative of a healthy state, the subject may be determined to not have the disease.
  • this example is non-limiting and can be expanded for assessing other phenotypes, e.g., other diseases, prognosis, theranosis, disease stratification, disease monitoring, treatment monitoring or prediction of non-responder / responder status, and the like.
  • a biosignature for characterizing a phenotype can be determined by detecting circulating biomarkers such as vesicles, including biomarkers associate with vesicles such as surface antigens or payload.
  • the payload e.g., protein or species of RNA such as mRNA or microRNA, can be assessed within a vesicle. Alternately, the payload in a sample is analyzed to characterize the phenotype without isolating the payload from the vesicles. Many analytical techniques are available to assess vesicles. In some embodiments, vesicle levels are characterized using mass spectrometry, flow cytometry, immunocytochemical staining, Western blotting, electrophoresis,
  • vesicles can be characterized and quantitatively measured using flow cytometry as described in Clayton et al, Journal of Immunological Methods 2001; 163-174, which is herein incorporated by reference in its entirety.
  • Vesicle levels may be determined using binding agents as described above.
  • a binding agent to vesicles can be labeled and the label detected and used to determine the amount of vesicles in a sample.
  • the binding agent can be bound to a substrate, such as arrays or particles, such as described above.
  • the vesicles may be labeled directly.
  • Electrophoretic tags or eTags can be used to determine the amount of vesicles.
  • eTags are small fluorescent molecules linked to nucleic acids or antibodies and are designed to bind one specific nucleic acid sequence or protein, respectively. After the eTag binds its target, an enzyme is used to cleave the bound eTag from the target. The signal generated from the released eTag, called a "reporter,” is proportional to the amount of target nucleic acid or protein in the sample.
  • the eTag reporters can be identified by capillary electrophoresis.
  • each eTag reporter that is, its electrical charge divided by its molecular weight— makes it show up as a specific peak on the capillary electrophoresis readout.
  • the amount or level of vesicles can be determined.
  • the vesicle level can determined from a heterogeneous population of vesicles, such as the total population of vesicles in a sample.
  • the vesicles level is determined from a homogenous population, or substantially homogenous population of vesicles, such as the level of specific cell-of-origin vesicles, such as vesicles from prostate cancer cells.
  • the level is determined for vesicles with a particular biomarker or combination of biomarkers, such as a biomarker specific for prostate cancer. Determining the level vesicles can be performed in conjunction with determining the biomarker or combination of biomarkers of a vesicle.
  • determining the amount of vesicle may be performed prior to or subsequent to determining the biomarker or combination of biomarkers of the vesicles.
  • Determining the amount of vesicles can be assayed in a multiplexed manner. For example, determining the amount of more than one population of vesicles, such as different cell-of-origin specific vesicles with different biomarkers or combination of biomarkers, can be performed, such as those disclosed herein.
  • Performance of a diagnostic or related test is typically assessed using statistical measures.
  • the performance of the characterization can be assessed by measuring sensitivity, specificity and related measures. For example, a level of circulating biomarkers of interest can be assayed to characterize a phenotype, such as detecting a disease. The sensitivity and specificity of the assay to detect the disease is determined.
  • a true positive is a subject with a characteristic, e.g., a disease or disorder, correctly identified as having the characteristic.
  • a false positive is a subject without the characteristic that the test improperly identifies as having the characteristic.
  • a true negative is a subject without the characteristic that the test correctly identifies as not having the characteristic.
  • a false negative is a person with the characteristic that the test improperly identifies as not having the characteristic. The ability of the test to distinguish between these classes provides a measure of test performance.
  • the specificity of a test is defined as the number of true negatives divided by the number of actual negatives (i.e., sum of true negatives and false positives). Specificity is a measure of how many subjects are correctly identified as negatives. A specificity of 100% means that the test recognizes all actual negatives - for example, all healthy people will be recognized as healthy. A lower specificity indicates that more negatives will be determined as positive.
  • the sensitivity of a test is defined as the number of true positives divided by the number of actual positives (i.e., sum of true positives and false negatives). Sensitivity is a measure of how many subjects are correctly identified as positives. A sensitivity of 100% means that the test recognizes all actual positives - for example, all sick people will be recognized as sick. A lower sensitivity indicates that more positives will be missed by being determined as negative.
  • the accuracy of a test is defined as the number of true positives and true negatives divided by the sum of all true and false positives and all true and false negatives. It provides one number that combines sensitivity and specificity measurements.
  • Sensitivity, specificity and accuracy are determined at a particular discrimination threshold value.
  • a common threshold for prostate cancer (PCa) detection is 4 ng/niL of prostate specific antigen (PSA) in serum.
  • PSA prostate specific antigen
  • a level of PSA equal to or above the threshold is considered positive for PCa and any level below is considered negative.
  • the threshold is varied, the sensitivity and specificity will also vary. For example, as the threshold for detecting cancer is increased, the specificity will increase because it is harder to call a subject positive, resulting in fewer false positives. At the same time, the sensitivity will decrease.
  • a receiver operating characteristic curve is a graphical plot of the true positive rate (i.e., sensitivity) versus the false positive rate (i.e., 1 - specificity) for a binary classifier system as its discrimination threshold is varied.
  • the ROC curve shows how sensitivity and specificity change as the threshold is varied.
  • the Area Under the Curve (AUC) of an ROC curve provides a summary value indicative of a test's performance over the entire range of thresholds.
  • the AUC is equal to the probability that a classifier will rank a randomly chosen positive sample higher than a randomly chosen negative sample.
  • An AUC of 0.5 indicates that the test has a 50% chance of proper ranking, which is equivalent to no discriminatory power (a coin flip also has a 50% chance of proper ranking).
  • An AUC of 1.0 means that the test properly ranks (classifies) all subjects.
  • the AUC is equivalent to the Wilcoxon test of ranks.
  • a biosignature according to the invention can be used to characterize a phenotype with at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70% sensitivity, such as with at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87% sensitivity.
  • the phenotype is characterized with at least 87.1, 87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88.0, or 89% sensitivity, such as at least
  • the phenotype can be characterized with at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sensitivity.
  • a biosignature according to the invention can be used to characterize a phenotype of a subject with at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97% specificity, such as with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99
  • a biosignature according to the invention can be used to characterize a phenotype of a subject, e.g., based on a level of a circulating biomarker or other characteristic, with at least 50% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% specificity; at least 55% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%o specificity; at least 60% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% specificity; at least 65% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% specificity; at least 70% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% specificity; at least 75% sensitivity and at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% specificity; at least 80% sensitivity and at least 60
  • a biosignature according to the invention can be used to characterize a phenotype of a subject with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97% accuracy, such as with at least 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%o
  • a biosignature according to the invention is used to characterize a phenotype of a subject with an AUC of at least 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, or 0.97, such as with at least 0.971, 0.972, 0.973, 0.974, 0.975, 0.976, 0.977, 0.978, 0.978, 0.979, 0.980, 0.981, 0.982, 0.983, 0.984, 0.985, 0.986, 0.987, 0.988, 0.989, 0.99, 0.991, 0.992, 0.993,
  • the confidence level for determining the specificity, sensitivity, accuracy or AUC may be determined with at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% confidence.
  • Biosignature according to the invention can be used to classify a sample.
  • Techniques for discriminate analysis are known to those of skill in the art. For example, a sample can be classified as, or predicted to be, a responder or non-responder to a given treatment for a given disease or disorder. Many statistical classification techniques are known to those of skill in the art.
  • supervised learning approaches a group of samples from two or more groups are analyzed with a statistical classification method.
  • One or more biomarkers e.g., a panel of biomarkers that forms a biosignature, can be discovered that can be used to build a classifier that differentiates between the two or more groups. A new sample can then be analyzed so that the classifier can associate the new with one of the two or more groups.
  • Commonly used supervised classifiers include without limitation the neural network (multi-layer perceptron), support vector machines, k-nearest neighbors, Gaussian mixture model, Gaussian, naive Bayes, decision tree and radial basis function (RBF) classifiers.
  • Linear classification methods include Fisher's linear discriminant, logistic regression, naive Bayes classifier, perceptron, and support vector machines (SVMs).
  • Other classifiers for use with the invention include quadratic classifiers, k-nearest neighbor, boosting, decision trees, random forests, neural networks, pattern recognition, Bayesian networks and Hidden Markov models.
  • Multivariate models that can be used to evaluate a biosignature comprising a presence or level of one or more biomarker include the following:
  • LDA is a well understood classification method that performs well for cases where predictors follow a generally normal distribution. The method can serve as a benchmark for more complex methods.
  • DLDA is version of discriminant analysis which assumes that predictors are independent, an assumption that may not hold true. However, when training data sets are too small to properly estimate covariances between predictors, well-fit DLDA model may consistently outperform more complex models.
  • This method is commonly known within the mRNA micorarray community as "PAM" (prediction analysis for microarrays). The method is similar to other for discriminate analysis methods but uses more robust (stabilized) estimates of variance.
  • SVMs are a popular variety of machine learning. SVMs often outperforming traditional statistical methods when predictors are not easily transformed to a multivariate normal distribution. The final SVM model can be expressed in much the same way as an LDA model.
  • This method generates binary decision trees, using "boosting" to combine weakly performing trees in a weighted fashion to form a stronger ensemble.
  • Lasso (Lasso) [00303] This approach fits a logistic regression model using "lasso" penalized maximum likelihood method. This approach tends to pick one representative marker from a set of highly correlated markers, returning zero values for coefficients of the remaining markers.
  • a classifier's performance can be estimated using a "training" set of sample to build a classifier and an independent "test" set of samples to test the model.
  • Other techniques can be used in the art to estimate predictive performance, such as cross-validation methods.
  • One round of cross-validation involves partitioning a sample of data into complementary subsets, performing the analysis on one subset (the training set), and validating the analysis on the other subset (the validation set or testing set). To reduce variability, multiple rounds of cross-validation can be performed using different partitions, and the validation results are averaged over the rounds.
  • Common types of cross- validation include the following:
  • the sample group is partitioned into k-partitions. One partition is used as the test set and the remainder are used as the training set. The process is repeated k times (or k folds) using each of the partitions once as the test set. The performance of the classifier model is averaged over the iterations. 10-fold cross validation is common though other numbers can be selected depending on sample size, computational resources, and the like.
  • Classification using supervised methods is generally performed by the following methodology:
  • [00315] Gather a training set. These can include, for example, samples that are from a subject with or without a disease or disorder, subjects that are known to respond or not respond to a treatment, subjects whose disease progresses or does not progress, etc. The training samples are used to "train" the classifier.
  • [00316] Determine the input "feature" representation of the learned function.
  • the accuracy of the learned function depends on how the input object is represented.
  • the input object is transformed into a feature vector, which contains a number of features that are descriptive of the object.
  • the number of features should not be too large, because of the curse of dimensionality; but should be large enough to accurately predict the output.
  • the features might include a set of biomarkers such as those described herein.
  • [00317] Determine the structure of the learned function and corresponding learning algorithm.
  • a learning algorithm is chosen, e.g., artificial neural networks, decision trees, Bayes classifiers or support vector machines. The learning algorithm is used to build the classifier.
  • the learning algorithm is run the gathered training set. Parameters of the learning algorithm may be adjusted by optimizing performance on a subset (called a validation set) of the training set, or via cross-validation. After parameter adjustment and learning, the performance of the algorithm may be measured on a test set of naive samples that is separate from the training set.
  • the classifier can be used to classify a sample, e.g., that of a subject who is being analyzed by the methods of the invention.
  • a classifier can be built using data for levels of circulating biomarkers of interest in reference subjects with and without a disease as the training and test sets. Circulating biomarker levels found in a sample from a test subject are assessed and the classifier is used to classify the subject as with or without the disease.
  • a classifier can be built using data for levels of vesicle biomarkers of interest in reference subjects that have been found to respond or not respond to certain diseases as the training and test sets. The vesicle biomarker levels found in a sample from a test subject are assessed and the classifier is used to classify the subject as with or without the disease.
  • Unsupervised learning approaches can also be used with the invention.
  • Clustering is an unsupervised learning approach wherein a clustering algorithm correlates a series of samples without the use the labels. The most similar samples are sorted into "clusters.” A new sample could be sorted into a cluster and thereby classified with other members that it most closely associates.
  • Many clustering algorithms well known to those of skill in the art can be used with the invention, such as hierarchical clustering.
  • a biosignature can be obtained according to the invention by assessing a vesicle population, including surface and payload vesicle associated biomarkers, and/or circulating biomarkers including microRNA and protein.
  • a biosignature derived from a subject can be used to characterize a phenotype of the subject.
  • a biosignature can further include the level of one or more additional biomarkers, e.g., circulating biomarkers or biomarkers associated with a vesicle of interest.
  • a biosignature of a vesicle of interest can include particular antigens or biomarkers that are present on the vesicle.
  • the biosignature can also include one or more antigens or biomarkers that are carried as payload within the vesicle, including the microRNA under examination.
  • the biosignature can comprise a combination of one or more antigens or biomarkers that are present on the vesicle with one or more biomarkers that are detected in the vesicle.
  • the biosignature can further comprise other information about a vesicle aside from its biomarkers. Such information can include vesicle size, circulating half-life, metabolic half-life, and specific activity in vivo or in vitro.
  • the biosignature can comprise the biomarkers or other characteristics used to build a classifier.
  • the microRNA is detected directly in a biological sample.
  • RNA in a bodily fluid can be isolated using commercially available kits such as mirVana kits (Applied Biosystems/ Ambion, Austin, TX), MagMAXTM RNA Isolation Kit (Applied Biosystems/ Ambion, Austin, TX), and QIAzol Lysis Reagent and RNeasy Midi Kit (Qiagen Inc., Valencia CA).
  • mirVana kits Applied Biosystems/ Ambion, Austin, TX
  • MagMAXTM RNA Isolation Kit Applied Biosystems/ Ambion, Austin, TX
  • QIAzol Lysis Reagent and RNeasy Midi Kit Qiagen Inc., Valencia CA.
  • Particular species of microRNAs can be determined using array or PCR techniques as described below.
  • the microRNA payload with vesicles is assessed in order to characterize a phenotype.
  • the vesicles can be purified or concentrated prior to determining the biosignature. For example, a cell- of-origin specific vesicle can be isolated and its biosignature determined. Alternatively, the biosignature of the vesicle can be directly assayed from a sample, without prior purification or concentration.
  • the biosignature of the invention can be used to determine a diagnosis, prognosis, or theranosis of a disease or condition or similar measures described herein.
  • a biosignature can also be used to determine treatment efficacy, stage of a disease or condition, or progression of a disease or condition, or responder / non-responder status. Furthermore, a biosignature may be used to determine a physiological state, such as pregnancy.
  • a characteristic of a vesicle in and of itself can be assessed to determine a biosignature.
  • the characteristic can be used to diagnose, detect or determine a disease stage or progression, the therapeutic implications of a disease or condition, or characterize a physiological state.
  • Such characteristics include without limitation the level or amount of vesicles, vesicle size, temporal evaluation of the variation in vesicle half-life, circulating vesicle half- life, metabolic half- life of a vesicle, or activity of a vesicle.
  • Biomarkers that can be included in a biosignature include one or more proteins or peptides (e.g., providing a protein signature), nucleic acids (e.g. RNA signature as described, or a DNA signature), lipids (e.g. lipid signature), or combinations thereof.
  • the biosignature can also comprise the type or amount of drug or drug metabolite present in a vesicle, (e.g., providing a drug signature), as such drug may be taken by a subject from which the biological sample is obtained, resulting in a vesicle carrying the drug or metabolites of the drug.
  • a biosignature can also include an expression level, presence, absence, mutation, variant, copy number variation, truncation, duplication, modification, or molecular association of one or more biomarkers.
  • a genetic variant, or nucleotide variant refers to changes or alterations to a gene or cDNA sequence at a particular locus, including, but not limited to, nucleotide base deletions, insertions, inversions, and substitutions in the coding and non-coding regions. Deletions may be of a single nucleotide base, a portion or a region of the nucleotide sequence of the gene, or of the entire gene sequence. Insertions may be of one or more nucleotide bases.
  • the genetic variant may occur in transcriptional regulatory regions, untranslated regions of mRNA, exons, introns, or exon/intron junctions.
  • the genetic variant may or may not result in stop codons, frame shifts, deletions of amino acids, altered gene transcript splice forms or altered amino acid sequence.
  • nucleic acid biomarkers including nucleic acid payload within a vesicle, is assessed for nucleotide variants.
  • the nucleic acid biomarker may comprise one or more RNA species, e.g., mRNA, miRNA, snoRNA, snRNA, rRNAs, tRNAs, siRNA, hnRNA, shRNA, enhancer RNA (eRNA), or a combination thereof.
  • DNA payload can be assessed to form a DNA signature.
  • RNA signature or DNA signature can also include a mutational, epigenetic modification, or genetic variant analysis of the RNA or DNA present in the vesicle.
  • Epigenetic modifications include patterns of DNA methylation. See, e.g., Lesche R. and Eckhardt F., DNA methylation markers: a versatile diagnostic tool for routine clinical use. Curr Opin Mol Ther. 2007 Jun;9(3):222-30, which is incorporated herein by reference in its entirety.
  • a biomarker can be the methylation status of a segment of DNA.
  • a biosignature can comprise one or more miRNA signatures combined with one or more additional signatures including, but not limited to, an mRNA signature, DNA signature, protein signature, peptide signature, antigen signature, or any combination thereof.
  • the biosignature can comprise one or more miRNA biomarkers with one or more DNA biomarkers, one or more mRNA biomarkers, one or more snoRNA biomarkers, one or more protein biomarkers, one or more peptide biomarkers, one or more antigen biomarkers, one or more antigen biomarkers, one or more lipid biomarkers, or any combination thereof.
  • a biosignature can comprise a combination of one or more antigens or binding agents (such as ability to bind one or more binding agents), such as listed in Figs. 1 and 2, respectively, of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein, or those described elsewhere herein.
  • the biosignature can further comprise one or more other biomarkers, such as, but not limited to, miRNA, DNA (e.g. single stranded DNA, complementary DNA, or noncoding DNA), or mRNA.
  • the biosignature of a vesicle can comprise a combination of one or more antigens, such as shown in Fig. 1 of International Patent Application Serial No.
  • the biosignature can comprise one or more biomarkers, for example miRNA, with one or more antigens specific for a cancer cell (for example, as shown in Fig. 1 of International Patent Application Serial No. PCT/US2011/031479).
  • a vesicle used in the subject methods has a biosignature that is specific to the cell-of- origin and is used to derive disease-specific or biological state specific diagnostic, prognostic or therapy-related biosignatures representative of the cell-of-origin.
  • a vesicle has a biosignature that is specific to a given disease or physiological condition that is different from the biosignature of the cell-of-origin for use in the diagnosis, prognosis, staging, therapy-related determinations or physiological state characterization.
  • Biosignatures can also comprise a combination of cell-of-origin specific and non-specific vesicles.
  • Biosignatures can be used to evaluate diagnostic criteria such as presence of disease, disease staging, disease monitoring, disease stratification, or surveillance for detection, metastasis or recurrence or progression of disease.
  • a biosignature can also be used clinically in making decisions concerning treatment modalities including therapeutic intervention.
  • a biosignature can further be used clinically to make treatment decisions, including whether to perform surgery or what treatment standards should be used along with surgery (e.g., either pre-surgery or post-surgery).
  • a biosignature of circulating biomarkers that indicates an aggressive form of cancer may call for a more aggressive surgical procedure and/or more aggressive therapeutic regimen to treat the patient.
  • a biosignature can be used in therapy related diagnostics to provide tests useful to diagnose a disease or choose the correct treatment regimen, such as provide a theranosis.
  • Theranostics includes diagnostic testing that provides the ability to affect therapy or treatment of a diseased state.
  • Theranostics testing provides a theranosis in a similar manner that diagnostics or prognostic testing provides a diagnosis or prognosis, respectively.
  • theranostics encompasses any desired form of therapy related testing, including predictive medicine, personalized medicine, integrated medicine, pharmacodiagnostics and Dx/Rx partnering. Therapy related tests can be used to predict and assess drug response in individual subjects, i.e., to provide personalized medicine.
  • Predicting a drug response can be determining whether a subject is a likely responder or a likely non-responder to a candidate therapeutic agent, e.g., before the subject has been exposed or otherwise treated with the treatment. Assessing a drug response can be monitoring a response to a drug, e.g., monitoring the subject's improvement or lack thereof over a time course after initiating the treatment. Therapy related tests are useful to select a subject for treatment who is particularly likely to benefit from the treatment or to provide an early and objective indication of treatment efficacy in an individual subject. Thus, a biosignature as disclosed herein may indicate that treatment should be altered to select a more promising treatment, thereby avoiding the great expense of delaying beneficial treatment and avoiding the financial and morbidity costs of administering an ineffective drug(s).
  • Therapy related diagnostics are also useful in clinical diagnosis and management of a variety of diseases and disorders, which include, but are not limited to cardiovascular disease, cancer, infectious diseases, sepsis, neurological diseases, central nervous system related diseases, endovascular related diseases, and autoimmune related diseases. Therapy related diagnostics also aid in the prediction of drug toxicity, drug resistance or drug response. Therapy related tests may be developed in any suitable diagnostic testing format, which include, but are not limited to, e.g., immunohistochemical tests, clinical chemistry, immunoassay, cell-based technologies, nucleic acid tests or body imaging methods. Therapy related tests can further include but are not limited to, testing that aids in the determination of therapy, testing that monitors for therapeutic toxicity, or response to therapy testing.
  • a biosignature can be used to predict or monitor a subject's response to a treatment.
  • a biosignature can be determined at different time points for a subject after initiating, removing, or altering a particular treatment.
  • a determination or prediction as to whether a subject is responding to a treatment is made based on a change in the amount of one or more components of a biosignature (i.e., the microRNA, vesicles and/or biomarkers of interest), an amount of one or more components of a particular biosignature, or the biosignature detected for the components.
  • a subject's condition is monitored by determining a biosignature at different time points. The progression, regression, or recurrence of a condition is determined.
  • the invention provides a method of monitoring a status of a disease or other medical condition in a subject, comprising isolating or detecting a biosignature from a biological sample from the subject, detecting the overall amount of the components of a particular biosignature, or detecting the biosignature of one or more components (such as the presence, absence, or expression level of a biomarker).
  • the biosignatures are used to monitor the status of the disease or condition.
  • One or more novel biosignatures of a vesicle can also be identified.
  • one or more vesicles can be isolated from a subject that responds to a drug treatment or treatment regimen and compared to a reference, such as another subject that does not respond to the drug treatment or treatment regimen. Differences between the biosignatures can be determined and used to identify other subjects as responders or non-re sponders to a particular drug or treatment regimen.
  • a biosignature is used to determine whether a particular disease or condition is resistant to a drug. If a subject is drug resistant, a physician need not waste valuable time with such drug treatment. To obtain early validation of a drug choice or treatment regimen, a biosignature is determined for a sample obtained from a subject. The biosignature is used to assess whether the particular subject's disease has the biomarker associated with drug resistance. Such a determination enables doctors to devote critical time as well as the patient's financial resources to effective treatments.
  • biosignature may be used to assess whether a subject is afflicted with disease, is at risk for developing disease or to assess the stage or progression of the disease.
  • a biosignature can be used to assess whether a subject has prostate cancer, colon cancer, or other cancer as described herein. Futhermore, a biosignature can be used to determine a stage of a disease or condition, such as colon cancer.
  • determining the amount of vesicles, such a heterogeneous population of vesicles, and the amount of one or more homogeneous population of vesicles, such as a population of vesicles with the same biosignature can be used to characterize a phenotype. For example, determination of the total amount of vesicles in a sample (i.e. not cell-type specific) and determining the presence of one or more different cell-of-origin specific vesicles can be used to characterize a phenotype.
  • Threshold values, or reference values or amounts can be determined based on comparisons of normal subjects and subjects with the phenotype of interest, as further described below, and criteria based on the threshold or reference values determined. The different criteria can be used to characterize a phenotype.
  • One criterion can be based on the amount of a heterogeneous population of vesicles in a sample.
  • general vesicle markers such as CD9, CD81, and CD63 can be used to determine the amount of vesicles in a sample.
  • the expression level of CD9, CD81, CD63, or a combination thereof can be detected and if the level is greater than a threshold level, the criterion is met.
  • the criterion is met if if level of CD9, CD81, CD63, or a combination thereof is lower than a threshold value or reference value.
  • the criterion can be based on whether the amount of vesicles is higher than a threshold or reference value. Another criterion can be based on the amount of vesicles with a specific biosignature. If the amount of vesicles with the specific biosignature is lower than a threshold or reference value, the criterion is met. In another embodiment, if the amount of vesicles with the specific biosignature is higher than a threshold or reference value, the criterion is met. A criterion can also be based on the amount of vesicles derived from a particular cell type. If the amount is lower than a threshold or reference value, the criterion is met. In another embodiment, if the amount is higher than a threshold value, the criterion is met.
  • vesicles from prostate cells are determined by detecting the biomarker PCSA or PSCA, and that a criterion is met if the level of detected PCSA or PSCA is greater than a threshold level.
  • the threshold can be the level of the same markers in a sample from a control cell line or control subject.
  • Another criterion can be based on whether the amount of vesicles derived from a cancer cell or comprising one or more cancer specific biomarkers. For example, the biomarkers B7H3, EpCam, or both, can be determined and a criterion met if the level of detected B7H3 and/or EpCam is greater than a threshold level or within a predetermined range.
  • a criterion can also be the reliability of the result, such as meeting a quality control measure or value.
  • a detected amount of B7H3 and/or EpCam in a test sample that is above the amount of these markers in a control sample may indicate the presence of a cancer in the test sample.
  • a biosignature is used to assess whether a subject has prostate cancer by detecting one or more of the general vesicle markers CD9, CD63 and CD81 ; one or more prostate epithelial markers including PCSA or PSMA; and one or more cancer markers such as B7H3 and/or EpCam. Higher levels of the markers in a sample from a subject than in a control individual without prostate cancer indicates the presence of the prostate cancer in the subject. In some embodiments, the multiple markers are assessed in a multiplex fashion.
  • the criterion can be applied to vesicle characteristics such as amount of vesicles present, amount of vesicles with a particular biosignature present, amount of vesicle payload biomarkers present, amount of microRNA or other circulating biomarkers present, and the like. The ratios of appropriate biomarkers can be determined.
  • the criterion could be a ratio of an vesicle surface protein to another vesicle surface protein, a ratio of an vesicle surface protein to a microRNA, a ratio of one vesicle population to another vesicle population, a ratio of one circulating biomarker to another circulating biomarker, etc.
  • a phenotype for a subject can be characterized based on meeting any number of useful criteria.
  • at least one criterion is used for each biomarker.
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100 criteria are used.
  • a number of different criteria can be used when the subject is diagnosed with a cancer: 1) if the amount of microRNA in a sample from a subject is higher than a reference value; 2) if the amount of a microRNA within cell type specific vesicles (i.e.
  • the method can further include a quality control measure, such that the results are provided for the subject if the samples meet the quality control measure. In some embodiments, if the criteria are met but the quality control is questionable, the subject is reassessed.
  • a single measure is determined for assessment of multiple biomarkers, and the measure is compared to a reference.
  • a test for prostate cancer might comprise multiplying the level of PSA against the level of miR-141 in a blood sample. The criterion is met if the product of the levels is above a threshold, indicating the presense of the cancer.
  • a number of binding agents to general vesicle markers can carry the same label, e.g., the same fluorophore. The level of the detected label can be compared to a threshold.
  • Criterion can be applied to multiple types of biomarkers in addition to multiple biomarkers of the same type.
  • the levels of one or more circulating biomarkers e.g., RNA, DNA, peptides), vesicles, mutations, etc.
  • a biosignature can have different criteria.
  • a biosignature used to diagnose a cancer can include overexpression of one miR species as compared to a reference and underexpression of a vesicle surface antigen as compared to another reference.
  • a biosignature can be determined by comparing the amount of vesicles, the structure of a vesicle, or any other informative characteristic of a vesicle.
  • Vesicle structure can be assessed using transmission electron microscopy, see for example, Hansen et ah, Journal of Biomechanics 31, Supplement 1: 134-134(1) (1998), or scanning electron microscopy.
  • Various combinations of methods and techniques or analyzing one or more vesicles can be used to determine a phenotype for a subject.
  • a biosignature can include without limitation the presence or absence, copy number, expression level, or activity level of a biomarker.
  • Other useful components of a biosignature include the presence of a mutation (e.g., mutations which affect activity of a transcription or translation product, such as substitution, deletion, or insertion mutations), variant, or post-translation modification of a biomarker.
  • Post-translational modification of a protein biomarker include without limitation acylation, acetylation, phosphorylation, ubiquitination, deacetylation, alkylation, methylation, amidation, biotinylation, gamma-carboxylation, glutamylation, glycosylation, glycyation, hydroxylation, covalent attachment of heme moiety, iodination, isoprenylation, lipoylation, prenylation, GPI anchor formation, myristoylation, farnesylation, geranylgeranylation, covalent attachment of nucleotides or derivatives thereof, ADP-ribosylation, flavin attachment, oxidation, palmitoylation, pegylation, covalent attachment of phosphatidylinositol, phosphopantetheinylation, polysialylation, pyroglutamate formation, racemization of proline by prolyl isomerase, tRNA-mediation addition of amino acids such
  • the methods described herein can be used to identify a biosignature that is associated with a disease, condition or physiological state.
  • the biosignature can also be used to determine if a subject is afflicted with cancer or is at risk for developing cancer.
  • a subject at risk of developing cancer can include those who may be predisposed or who have pre -symptomatic early stage disease.
  • a biosignature can also be used to provide a diagnostic or theranostic determination for other diseases including but not limited to autoimmune diseases, inflammatory bowel diseases, cardiovascular disease, neurological disorders such as Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, sepsis or pancreatitis or any disease, conditions or symptoms listed in Figs. 3-58 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • the biosignature can also be used to identify a given pregnancy state from the peripheral blood, umbilical cord blood, or amniotic fluid (e.g. miRNA signature specific to Downs Syndrome) or adverse pregnancy outcome such as pre-eclampsia, pre-term birth, premature rupture of membranes, intrauterine growth restriction or recurrent pregnancy loss.
  • amniotic fluid e.g. miRNA signature specific to Downs Syndrome
  • adverse pregnancy outcome such as pre-eclampsia, pre-term birth, premature rupture of membranes, intrauterine growth restriction or recurrent pregnancy loss.
  • the biosignature can also be used to indicate the health of the mother, the fetus at all developmental stages, the pre -implantation embryo or a newborn.
  • a biosignature can be used for pre-symptomatic diagnosis. Furthermore, the biosignature can be used to detect disease, determine disease stage or progression, determine the recurrence of disease, identify treatment protocols, determine efficacy of treatment protocols or evaluate the physiological status of individuals related to age and environmental exposure.
  • Monitoring a biosignature of a vesicle can also be used to identify toxic exposures in a subject including, but not limited to, situations of early exposure or exposure to an unknown or unidentified toxic agent.
  • vesicles can shed from damaged cells and in the process compartmentalize specific contents of the cell including both membrane components and engulfed cytoplasmic contents. Cells exposed to toxic agents/chemicals may increase vesicle shedding to expel toxic agents or metabolites thereof, thus resulting in increased vesicle levels.
  • monitoring vesicle levels, vesicle biosignature, or both allows assessment of an individual's response to potential toxic agent(s).
  • a vesicle and/or other biomarkers of the invention can be used to identify states of drug-induced toxicity or the organ injured, by detecting one or more specific antigen, binding agent, biomarker, or any combination thereof.
  • the level of vesicles, changes in the biosignature of a vesicle, or both can be used to monitor an individual for acute, chronic, or occupational exposures to any number of toxic agents including, but not limited to, drugs, antibiotics, industrial chemicals, toxic antibiotic metabolites, herbs, household chemicals, and chemicals produced by other organisms, either naturally occurring or synthetic in nature.
  • a biosignature can be used to identify conditions or diseases, including cancers of unknown origin, also known as cancers of unknown primary (CUP).
  • a vesicle may be isolated from a biological sample as previously described to arrive at a heterogeneous population of vesicles.
  • the heterogeneous population of vesicles can then be contacted with substrates coated with specific binding agents designed to rule out or identify antigen specific characteristics of the vesicle population that are specific to a given cell-of-origin.
  • the biosignature of a vesicle can correlate with the cancerous state of cells.
  • Compounds that inhibit cancer in a subject may cause a change, e.g., a change in biosignature of a vesicle, which can be monitored by serial isolation of vesicles over time and treatment course.
  • the level of vesicles or changes in the level of vesicles with a specific biosignature can be monitored.
  • characterizing a phenotype of a subject comprises a method of determining whether the subject is likely to respond or not respond to a therapy.
  • the methods of the invention also include determining new biosignatures useful in predicting whether the subject is likely to respond or not.
  • One or more subjects that respond to a therapy (responders) and one or more subjects that do not respond to the same therapy (non-responders) can have their vesicles interrogated. Interrogation can be performed to identify vesicle biosignatures that classify a subject as a responder or non-responder to the treatment of interest.
  • the presence, quantity, and payload of a vesicle are assayed.
  • the payload of a vesicle includes, for example, internal proteins, nucleic acids such as miRNA, lipids or carbohydrates.
  • a sample from responders may be analyzed for one or more of the following: amount of vesicles, amount of a unique subset or species of vesicles, biomarkers in such vesicles, biosignature of such vesicles, etc.
  • vesicles such as microvesicles or exosomes from responders and non-responders are analyzed for the presence and/or quantity of one or more miRNAs, such as miRNA 122, miR-548c-5p, miR-362-3p, miR-422a, miR- 597, miR-429, miR-200a, and/or miR-200b.
  • miRNAs such as miRNA 122, miR-548c-5p, miR-362-3p, miR-422a, miR- 597, miR-429, miR-200a, and/or miR-200b.
  • miRNAs such as miRNA 122, miR-548c-5p, miR-362-3p, miR-422a, miR- 597, miR-429, miR-200a, and/or miR-200b.
  • a difference in biosignatures between responders and non-responders can be used for theranosis.
  • vesicles are obtained from subjects having
  • the vesicles from both groups of subjects are assayed for unique biosignatures that are associated with all subjects in that group but not in subjects from the other group.
  • biosignatures or biomarkers can then used as a diagnostic for the presence or absence of the condition or disease, or to classify the subject as belonging on one of the groups (those with/without disease, aggressive/non-aggressive disease, responder/non-responder, etc).
  • characterizing a phenotype of a subject comprises a method of staging a disease.
  • the methods of the invention also include determining new biosignatures useful in staging.
  • vesicles are assayed from patients having a stage I cancer and patients having stage II or stage III of the same cancer.
  • vesicles are assayed in patients with metastatic disease.
  • a difference in biosignatures or biomarkers between vesicles from each group of patient is identified (e.g., vesicles from stage III cancer may have an increased expression of one or more genes or miRNA's), thereby identifying a biosignature or biomarker that distinguishes different stages of a disease.
  • biosignature can then be used to stage patients having the disease.
  • a biosignature is determined by assaying vesicles from a subject over a period of time, e.g., daily, semiweekly, weekly, biweekly, semimonthly, monthly, bimonthly, semiquarterly, quarterly, semiyearly, biyearly or yearly.
  • the biosignatures in patients on a given therapy can be monitored over time to detect signatures indicative of responders or non-responders for the therapy.
  • patients with differing stages of disease or in differing stages of a clinical trial have a biosignature interrogated over time. The payload or physical attributes of the vesicles in each point in time can be compared.
  • a temporal pattern can thus form a biosignature that can then be used for theranosis, diagnosis, prognosis, disease stratification, treatment monitoring, disease monitoring or making a prediction of responder / non-responder status.
  • a biomarker e.g., miR 122
  • an increasing amount of a biomarker in vesicles over a time course is associated with metastatic cancer, as opposed to a stagnant amounts of the biomarker in vesicles over the time course that are associated with non-metastatic cancer.
  • a time course may last over at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 6 weeks, 8 weeks, 2 months, 10 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, one year, 18 months, 2 years, or at least 3 years.
  • the level of vesicles, level of vesicles with a specific biosignature, or a biosignature of a vesicle can also be used to assess the efficacy of a therapy for a condition.
  • the level of vesicles, level of vesicles with a specific biosignature, or a biosignature of a vesicle can be used to assess the efficacy of a cancer treatment, e.g., chemotherapy, radiation therapy, surgery, or any other therapeutic approach useful for inhibiting cancer in a subject.
  • a biosignature can be used in a screening assay to identify candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) that have a modulatory effect on the biosignature of a vesicle.
  • candidate or test compounds or agents e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs
  • Compounds identified via such screening assays may be useful, for example, for modulating, e.g., inhibiting, ameliorating, treating, or preventing conditions or diseases.
  • a biosignature for a vesicle can be obtained from a patient who is undergoing successful treatment for a particular cancer.
  • Cells from a cancer patient not being treated with the same drug can be cultured and vesicles from the cultures obtained for determining biosignatures.
  • the cells can be treated with test compounds and the biosignature of the vesicles from the cultures can be compared to the biosignature of the vesicles obtained from the patient undergoing successful treatment.
  • the test compounds that results in biosignatures that are similar to those of the patient undergoing successful treatment can be selected for further studies.
  • the biosignature of a vesicle can also be used to monitor the influence of an agent (e.g., drug compounds) on the biosignature in clinical trials. Monitoring the level of vesicles, changes in the biosignature of a vesicle, or both, can also be used in a method of assessing the efficacy of a test compound, such as a test compound for inhibiting cancer cells.
  • an agent e.g., drug compounds
  • the methods and compositions disclosed herein also provide a system for optimizing the treatment of a subject having such a disease, condition or syndrome.
  • the level of vesicles, the biosignature of a vesicle, or both, can also be used to determine the effectiveness of a particular therapeutic intervention (pharmaceutical or non- pharmaceutical) and to alter the intervention to 1) reduce the risk of developing adverse outcomes, 2) enhance the effectiveness of the intervention or 3) identify resistant states.
  • the methods and compositions disclosed herein also provide a system for optimizing the treatment of a subject having such a disease, condition or syndrome.
  • a therapy-related approach to treating a disease, condition or syndrome by integrating diagnostics and therapeutics to improve the real-time treatment of a subject can be determined by identifying the biosignature of a vesicle.
  • Tests that identify the level of vesicles, the biosignature of a vesicle, or both, can be used to identify which patients are most suited to a particular therapy, and provide feedback on how well a drug is working, so as to optimize treatment regimens. For example, in pregnancy-induced hypertension and associated conditions, therapy- related diagnostics can flexibly monitor changes in important parameters (e.g., cytokine and/or growth factor levels) over time, to optimize treatment.
  • important parameters e.g., cytokine and/or growth factor levels
  • therapy-related diagnostics as determined by a biosignature disclosed herein, can provide key information to optimize trial design, monitor efficacy, and enhance drug safety.
  • therapy-related diagnostics can be used for patient stratification, determination of patient eligibility (inclusion/exclusion), creation of homogeneous treatment groups, and selection of patient samples that are optimized to a matched case control cohort.
  • Such therapy-related diagnostic can therefore provide the means for patient efficacy enrichment, thereby minimizing the number of individuals needed for trial recruitment.
  • therapy-related diagnostics are useful for monitoring therapy and assessing efficacy criteria.
  • therapy-related diagnostics can be used to prevent adverse drug reactions or avoid medication error and monitor compliance with the therapeutic regimen.
  • the invention provides a method of identifying responder and non-re sponders to a treatment undergoing clinical trials, comprising detecting biosignatures comprising circulating biomarkers in subjects enrolled in the clinical trial, and identifying biosignatures that distinguish between responders and non- responders.
  • the biosignatures are measured in a drug naive subject and used to predict whether the subject will be a responder or non-responder. The prediction can be based upon whether the biosignatures of the drug naive subject correlate more closely with the clinical trial subjects identified as responders, thereby predicting that the drug naive subject will be a responder.
  • the methods of the invention can predict that the drug naive subject will be a non-responder.
  • the prediction can therefore be used to stratify potential responders and non-responders to the treatment.
  • the prediction is used to guide a course of treatment, e.g., by helping treating physicians decide whether to administer the drug.
  • the prediction is used to guide selection of patients for enrollment in further clinical trials.
  • biosignatures that predict responder / non-responder status in Phase II trials can be used to select patients for a Phase III trial, thereby increasing the likelihood of response in the Phase III patient population.
  • the method can be adapted to identify biosignatures to stratify subjects on criteria other than responder / non-responder status.
  • the criterion is treatment safety. Therefore the method is followed as above to identify subjects who are likely or not to have adverse events to the treatment.
  • biosignatures that predict safety profile in Phase II trials can be used to select patients for a Phase III trial, thereby increasing the treatment safety profile in the Phase III patient population.
  • the level of vesicles, the biosignature of a vesicle, or both can be used to monitor drug efficacy, determine response or resistance to a given drug, or both, thereby enhancing drug safety.
  • vesicles are typically shed from colon cancer cells and can be isolated from the peripheral blood and used to isolate one or more biomarkers e.g., KRAS mRNA which can then be sequenced to detect KRAS mutations.
  • the mRNA can be reverse transcribed into cDNA and sequenced (e.g., by Sanger sequencing, pyrosequencing, NextGen sequencing, RT-PCR assays) to determine if there are mutations present that confer resistance to a drug (e.g., cetuximab or panitumimab).
  • a drug e.g., cetuximab or panitumimab.
  • vesicles that are specifically shed from lung cancer cells are isolated from a biological sample and used to isolate a lung cancer biomarker, e.g., EGFR mRNA.
  • the EGFR mRNA is processed to cDNA and sequenced to determine if there are EGFR mutations present that show resistance or response to specific drugs or treatments for lung cancer.
  • One or more biosignatures can be grouped so that information obtained about the set of biosignatures in a particular group provides a reasonable basis for making a clinically relevant decision, such as but not limited to a diagnosis, prognosis, or management of treatment, such as treatment selection.
  • samples e.g., serum and tissue biobanks
  • methods and compositions disclosed herein are used for conducting prospective analysis on a sample (e.g., serum and/or tissue collected from individuals in a clinical trial) for the purpose of correlating qualitative and quantitative biosignatures of vesicleswith clinical outcomes in terms of disease state, disease stage, progression, prognosis; therapeutic efficacy or selection; or physiological conditions can also be performed.
  • a biosignature for a vesicle can be used to identify a cell-of-origin specific vesicle. Furthermore, a biosignature can be determined based on a surface marker profile of a vesicle or contents of a vesicle.
  • the biosignatures used to characterize a phenotype according to the invention can comprise multiple components (e.g., microRNA, vesicles or other biomarkers) or characteristics (e.g., vesicle size or morphology).
  • the biosignatures can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100 components or characteristics.
  • a biosignature with more than one component or characteristic, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100 components, may provide higher sensitivity and/or specificity in characterizing a phenotype.
  • assessing a plurality of components or characteristics provides increased sensitivity and/or specificity as compared to assessing fewer components or characteristics.
  • the methods of the invention comprise determining an optimal number of components or characteristics.
  • a biosignature according to the invention can be used to characterize a phenotype with a sensitivity, specificity, accuracy, or similar performance metric as described above.
  • the biosignatures can also be used to build a classifier to classify a sample as belonging to a group, such as belonging to a group having a disease or not, a group having an aggressive disease or not, or a group of responders or non-responders.
  • a classifier is used to determine whether a subject has an aggressive or non-aggressive cancer. In the illustrative case of prostate cancer, this can help a physician to determine whether to watch the cancer, i.e., prescribe "watchful waiting," or perform a prostatectomy.
  • a classifier is used to determine whether a breast cancer patient is likely to respond or not to tamoxifen, thereby helping the physician to determine whether or not to treat the patient with tamoxifen or another drug.
  • the methods and compositions of the invention can be used in assays to detect the presence or level of one or more biomarker of interest.
  • the biomarker can be any useful biomarker disclosed herein or known to those of skill in the art.
  • the biomarker comprises a protein or polypeptide.
  • protein protein
  • polypeptide and “peptide” are used interchangeably unless stated otherwise.
  • the biomarker can be a nucleic acid, including DNA, RNA, and various subspecies of any thereof as disclosed herein or known in the art.
  • the biomarker can comprise a lipid.
  • the biomarker can comprise a carbohydrate.
  • the biomarker can also be a complex, e.g., a complex comprising protein, nucleic acids, lipids and/or carbohydrates.
  • the biomarker comprises a microvesicle.
  • a biosignature comprising more than one biomarker can comprise one type of biomarker or multiple types of biomarkers.
  • a biosignature can comprise multiple proteins, multiple nucleic acids, multiple lipids, multiple carbohydrates, multiple biomarker complexes, multiple microvesicles, or a combination of any thereof.
  • the biosignature may comprise one or more microvesicle, one or more protein, and one or more microRNA, wherein the one or more protein and/or one or more microRNA is optionally in association with the microvesicle as a surface antigen and/or payload, as appropriate.
  • the biosignature can include the presence or absence, expression level, mutational state, genetic variant state, or any modification (such as epigenetic modification, or post-translation modification) of a biomarker disclosed herein (e.g., Tables 3, 4 or 5) or previously disclosed (e.g. any one or more biomarker listed in Figs. 1, 3- 60 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein).
  • a biomarker disclosed herein e.g., Tables 3, 4 or 5
  • previously disclosed e.g. any one or more biomarker listed in Figs. 1, 3- 60 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • methods of the invention can be adapted to assess one or more biomarkers disclosed herein for a disease
  • one or more biomarkers disclosed herein for condition x may readily be utilized in obtaining a biosignature for a different condition y, based on the teachings of the instant disclosure and methods of the invention.
  • the expression level of a biomarker can be compared to a control or reference, to determine the overexpression or underexpression (or upregulation or downregulation) of a biomarker in a sample.
  • the control or reference level comprises the amount of a same biomarker, such as a miRNA, in a control sample from a subject that does not have or exhibit the condition or disease.
  • control of reference levels comprises that of a housekeeping marker whose level is minimally affected, if at all, in different biological settings such as diseased versus non-diseased states.
  • control or reference level comprises that of the level of the same marker in the same subject but in a sample taken at a different time point. Other types of controls are described herein.
  • Nucleic acid biomarkers include various RNA or DNA species.
  • the biomarker can be mRNA, microRNA (miRNA), small nucleolar RNAs (snoRNA), small nuclear RNAs (snRNA), ribosomal RNAs (rRNA), heterogeneous nuclear RNA (hnRNA), ribosomal RNAS (rRNA), siRNA, transfer RNAs (tRNA), or shRNA.
  • the DNA can be double-stranded DNA, single stranded DNA, complementary DNA, or noncoding DNA.
  • miRNAs are short ribonucleic acid (RNA) molecules which average about 22 nucleotides long.
  • miRNAs act as post- transcriptional regulators that bind to complementary sequences in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), which can result in gene silencing.
  • mRNAs target messenger RNA transcripts
  • One miRNA may act upon 1000s of mRNAs. miRNAs play multiple roles in negative regulation, e.g., transcript degradation and sequestering, translational suppression, and may also have a role in positive regulation, e.g., transcriptional and translational activation. By affecting gene regulation, miRNAs can influence many biologic processes. Different sets of expressed miRNAs are found in different cell types and tissues.
  • Biomarkers for use with the invention further include peptides, polypeptides, or proteins, which terms are used interchangeably throughout unless otherwise noted.
  • the protein biomarker comprises its modification state, truncations, mutations, expression level (such as overexpression or underexpression as compared to a reference level), and/or post-translational modifications, such as described above.
  • a biosignature for a disease can include a protein having a certain post-translational modification that is more prevalent in a sample associated with the disease than without.
  • a biosignature may include a number of the same type of biomarkers (e.g., two or more different microRNA or mRNA species) or one or more of different types of biomarkers (e.g. mRNAs, miRNAs, proteins, peptides, ligands, and antigens).
  • biomarkers e.g., two or more different microRNA or mRNA species
  • biomarkers e.g. mRNAs, miRNAs, proteins, peptides, ligands, and antigens.
  • One or more biosignatures can comprise at least one biomarker selected from those listed in Figs. 1, 3-60 of International Patent Application Serial No. PCT/US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • a specific cell-of-origin biosignature may include one or more biomarkers.
  • PCT/US2011/031479 depict tables which lists a number of disease or condition specific biomarkers that can be derived and analyzed from a vesicle.
  • the biomarker can also be CD24, midkine, hepcidin, TMPRSS2-ERG, PCA-3, PSA, EGFR, EGFRvIII, BRAF variant, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c- myc, IGFR, PI3K, Akt, BRCA1, BRCA2, PTEN, VEGFR-2, VEGFR-1, Tie-2, TEM-1, CD276, HER-2, HER-3, or HER-4.
  • the biomarker can also be annexin V, CD63, Rab-5b, or caveolin, or a miRNA, such as let-7a; miR-15b; miR-16; miR-19b; miR-21 ; miR-26a; miR-27a; miR-92; miR-93; miR-320 or miR-20.
  • the biomarker can also be of any gene or fragment thereof as disclosed in PCT Publication No. WO2009/ 100029, such as those listed in Tables 3 - 15 therein.
  • a vesicle comprises a cell fragment or cellular debris derived from a rare cell, such as described in PCT Publication No. WO2006054991.
  • One or more biomarkers such as CD 146, CD 105, CD31, CD 133, CD 106, or a combination thereof, can be assessed for the vesicle.
  • a capture agent for the one or more biomarkers is used to isolate or detect a vesicle.
  • one or more of the biomarkers CD45, cytokeratin (CK) 8, CK18, CK19, CK20, CEA, EGFR, GUC, EpCAM, VEGF, TS, Muc-1, or a combination thereof is assessed for a vesicle.
  • a tumor-derived vesicle is CD45-, CK+ and comprises a nucleic acid, wherein the membrane vesicle has an absence of, or low expression or detection of CD45, has detectable expression of a cytokeratin (such as CK8, CK18, CK19, or CK20), and detectable expression of a nucleic acid.
  • any number of useful biomarkers that can be assessed as part of a vesicle biosignature are disclosed throughout the application, including without limitation CD9, EphA2, EGFR, B7H3, PSM, PCSA, CD63, STEAP, CD81, ICAM1, A33, DR3, CD66e, MFG-E8, TROP-2, Mammaglobin, Hepsin, NPGP/NPFF2, PSCA, 5T4, NGAL, EpCam, neurokinin receptor-1 (NK-1 or NK-1R), NK-2, Pai-1, CD45, CD10, HER2/ERBB2, AGTR1, NPY1R, MUC1, ESA, CD133, GPR30, BCA225, CD24, CA15.3 (MUC1 secreted), CA27.29 (MUC1 secreted), NMDAR1,
  • NMDAR2 MAGEA, CTAG1B, Y-ESO-1, SPB, SPC, NSE, PGP9.5, P2RX7, NDUFB7, NSE, GAL3, osteopontin, CHI3L1, IC3b, mesothelin, SPA, AQP5, GPCR, hCEA-CAM, PTP IA-2, CABYR, TMEM211, ADAM28, UNC93A, MUC17, MUC2, ILlOR-beta, BCMA, HVEM/TNFRSF14, Trappin-2 Elafm, ST2/IL1 R4, TNFRF14, CEACAM1, TPA1, LAMP, WF, WH1000, PECAM, BSA, TNFR, or a combination thereof.
  • biomarkers useful for assessment in methods and compositions disclosed herein include those associated with conditions or physiological states as disclosed in U.S. Patent No. 6329179 and 7,625,573; U.S. Patent Publication Nos. 2002/106684, 2004/005596, 2005/0159378, 2005/0064470, 2006/116321, 2007/0161004, 2007/0077553, 2007/104738, 2007/0298118, 2007/0172900, 2008/0268429, 2010/0062450, 2007/0298118, 2009/0220944 and 2010/0196426; U.S. Patent Application Nos. 12/524,432, 12/524,398, 12/524,462; Canadian Patent CA 2453198; and International PCT Patent Publication Nos. WO1994022018, WO2001036601,
  • WO2003063690 WO2003044166, WO2003076603, WO2005121369, WO2005118806, WO/2005/078124, WO2007126386, WO2007088537, WO2007103572, WO2009019215, WO2009021322, WO2009036236, WO2009100029, WO2009015357, WO2009155505, WO 2010/065968 and WO 2010/070276; each of which patent or application is incorporated herein by reference in their entirety.
  • biomarkers disclosed in these patents and applications can be assessed as part of a signature for characterizing a phenotype, such as providing a diagnosis, prognosis or theranosis of a cancer or other disease.
  • methods and techniques disclosed therein can be used to assess biomarkers, including vesicle biomarkers and microRNAs.
  • Another group of useful biomarkers for assessment in methods and compositions disclosed herein include those associated with cancer diagnostics, prognostics and theranostics as disclosed in US Patents 6,692,916, 6,960,439, 6,964,850, 7,074,586; U.S. Patent Application Nos. 11/159,376, 11/804,175, 12/594, 128, 12/514,686, 12/514,775, 12/594,675, 12/594,911, 12/594,679, 12/741,787, 12/312,390; and International PCT Patent Application Nos.
  • Biomarkers further include those described in U.S. Patent Application Nos., 10/703, 143 and US 10/701,391 for inflammatory disease; 11/529,010 for rheumatoid arthritis; 11/454,553 and 11/827,892 for multiple sclerosis; 11/897,160 for transplant rejection; 12/524,677 for lupus;
  • biomarkers disclosed in these patents and applications can be assessed as part of a signature for characterizing a phenotype, such as providing a diagnosis, prognosis or theranosis of a cancer or other disease.
  • methods and techniques disclosed therein can be used to assess biomarkers, including vesicle biomarkers and microRNAs.
  • Still other biomarkers useful for assessment in methods and compositions disclosed herein include those associated with conditions or physiological states as disclosed in Wieczorek et al., Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans, Proc Natl Acad Sci U S A. 1985 May;82(10):3455-9; Wieczorek et al , Diagnostic and prognostic value of RNA-proteolipid in sera of patients with malignant disorders following therapy: first clinical evaluation of a novel tumor marker, Cancer Res. 1987 Dec 1 ;47(23):6407-12; Escola et al.
  • B cell-derived exosomes can present allergen peptides and activate allergen-specific T cells to proliferate and produce TH2-like cytokines J Allergy Clin Immunol (2007) 120: 1418-1424; Aoki et al. Identification and characterization of microvesicles secreted by 3T3-L adipocytes: redox- and hormone dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles Endocrinol (2007) 148:3850-3862; Baj-Krzyworzeka et al.
  • Tumour- derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes Cencer Immunol Immunother (2006) 55:808-18; Skog et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers Nature Cell Biol (2008) 10: 1470-76; El-Hefnawy et al.
  • Decay-accelerating factor (CD 55) and membrane inhibitor of reactive lysis (CD 59) are released within exosomes during In vitro maturation of reticulocytes. Blood 91:2573-2580 (1998); Lamparski et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 270:211-226 (2002); Keller et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int'l 72: 1095-1102 (2007); Runz et al.
  • CD24 is an independent prognostic marker of survival in nonsmall cell lung cancer patients, Brit J Cancer 88:231- 236 (2003); Lim and Oh, The Role of CD24 in Various Human Epithelial Neoplasias, Pathol Res Pract 201:479-86 (2005); Matutes et al., The Immunophenotype of Splenic Lymphoma with Villous Lymphocytes and its Relevance to the Differential Diagnosis With Other B-Cell Disorders, Blood 83: 1558-1562 (1994); Pirruccello and Lang, Differential Expression of CD24- Related Epitopes in Mycosis Fungoides/Sezary Syndrome: A Potential Marker for Circulating Sezary Cells, Blood 76:2343-2347 (1990).
  • biomarkers disclosed in these publications can be assessed as part of a signature for characterizing a phenotype, such as providing a diagnosis, prognosis or theranosis of a cancer or other disease.
  • methods and techniques disclosed therein can be used to assess biomarkers, including vesicle biomarkers and microRNAs.
  • biomarkers useful for assessment in methods and compositions disclosed herein include those associated with conditions or physiological states as disclosed in Rajendran et al, Proc Natl Acad Sci USA 2006;103:11172-11177, Taylor et al, Gynecol Oncol 2008;110:13-21, Zhou et al, Kidney Int 2008;74:613-621, Buning et al, Immunology 2008, Prado et al. J Immunol 2008;181:1519-1525, Vella et al. (2008) Vet Immunol Immunopathol 124(3-4): 385-93, Gould et al. (2003). Proc Natl Acad Sci USA 100(19): 10592-7, Fang et al.
  • biomarkers disclosed in these publications can be assessed as part of a signature for characterizing a phenotype, such as providing a diagnosis, prognosis or theranosis of a cancer or other disease.
  • the methods and techniques disclosed therein can be used to assess biomarkers, including vesicle biomarkers and microRNAs.
  • the invention provides a method of assessing a cancer comprising detecting a level of one or more circulating biomarkers in a sample from a subject selected from the group consisting of CD9, HSP70, Gal3, MIS, EGFR, ER, ICB3, CD63, B7H4, MUC1, DLL4, CD81, ERB3, VEGF, BCA225, BRCA, CA125, CD174, CD24, ERB2, NGAL, GPR30, CYFRA21, CD31, cMET, MUC2 or ERB4.
  • CD9 HSP70, Gal3, MIS, EGFR, ER, ICB3, CD63, B7H4, MUC1, DLL4, CD81, ERB3, VEGF, BCA225, BRCA, BCA200, CA125, CD174, CD24, ERB2, NGAL, GPR30, CYFRA21, CD31, cMET, MUC2 or ERB4.
  • the one or more circulating biomarkers are selected from the group consisting of CD9, EphA2, EGFR, B7H3, PSMA, PCSA, CD63, STEAP, STEAP, CD81, B7H3, STEAP1, ICAM1 (CD54), PSMA, A33, DR3, CD66e, MFG-8e, EphA2, Hepsin, TMEM211, EphA2, TROP-2, EGFR, Mammoglobin, Hepsin, NPGP/NPFF2, PSCA, 5T4, NGAL, NK-2, EpCam, NGAL, NK-IR, PSMA, 5T4, PAI-1, and CD45.
  • the one or more circulating biomarkers are selected from the group consisting of CD9, MIS Rii, ER, CD63, MUC1, HER3, STAT3, VEGFA, BCA, CA125, CD24, EPCAM, and ERB B4. Any number of useful biomarkers can be assessed from these groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the one or more biomarkers are one or more of Gal3, BCA200, OPN and NCAM, e.g., Gal3 and BCA200, OPN and NCAM, or all four. Assessing the cancer may comprise diagnosing, prognosing or theranosing the cancer.
  • the cancer can be a breast cancer.
  • the markers can be associated with a vesicle or vesicle population.
  • the one or more circulating biomarker can be a vesicle surface antigen or vesicle payload.
  • Vesicle surface antigens can further be used as capture antigens, detector antigens, or both.
  • the invention further provides a method for predicting a response to a therapeutic agent comprising detecting a level of one or more circulating biomarkers in a sample from a subject selected from the group consisting of CD9, HSP70, Gal3, MIS, EGFR, ER, ICB3, CD63, B7H4, MUC1, DLL4, CD81, ERB3, VEGF, BCA225,
  • Biomarkers can also be selected from the group consisting of CD9, EphA2, EGFR, B7H3, PSMA, PCSA, CD63, STEAP, STEAP, CD81, B7H3, STEAP1, ICAM1 (CD54), PSMA, A33, DR3, CD66e, MFG-8e, EphA2, Hepsin, TMEM211, EphA2, TROP-2, EGFR, Mammoglobin, Hepsin, NPGP/NPFF2, PSCA, 5T4, NGAL, NK-2, EpCam, NGAL, NK-1R, PSMA, 5T4, PAI-1, and CD45.
  • the one or more circulating biomarkers are selected from the group consisting of CD9, MIS Rii, ER, CD63, MUC1, HER3, STAT3, VEGF A, BCA, CA125, CD24, EPCAM, and ERB B4. Any number of useful biomarkers can be assessed from these groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the one or more biomarkers are one or more of Gal3, BCA200, OPN and NCAM, e.g., Gal3 and BCA200, OPN and NCAM, or all four.
  • the therapeutic agent can be a therapeutic agent for treating cancer.
  • the cancer can be a breast cancer.
  • the markers can be associated with a vesicle or vesicle population.
  • the one or more circulating biomarker can be a vesicle surface antigen or vesicle payload.
  • Vesicle surface antigens can further be used as capture antigens, detector antigens, or both.
  • Various methods or platforms can be used to assess or detect biomarkers identified herein. Examples of such methods or platforms include but are not limited to using an antibody array, microbeads, or other method disclosed herein or known in the art. For example, a capture antibody or aptamer to the one or more biomarkers can be bound to the array or bead. The captured vesicles can then be detected using a detectable agent. In some embodiments, captured vesicles are detected using an agent, e.g., an antibody or aptamer, that recognizes general vesicle biomarkers that detect the overall population of vesicles, such as a tetraspanin or MFG-E8.
  • an agent e.g., an antibody or aptamer, that recognizes general vesicle biomarkers that detect the overall population of vesicles, such as a tetraspanin or MFG-E8.
  • the captured vesicles are detected using markers specific for vesicle origin, e.g., a type of tissue or organ.
  • the captured vesicles are detected using CD31, a marker for cells or vesicles of endothelial origin.
  • the biomarkers used for capture can also be used for detection, and vice versa.
  • Methods of the invention can be used to assess various diseases or conditions, where biomarkers correspond to various such diseases or conditions.
  • methods of the invention are applied to assess one or more cancers, such as those disclosed herein, wherein a method comprises detecting a level of one or more circulating biomarker in a sample from a subject selected from the group consisting of 5T4 (trophoblast), ADAM10,
  • the methods can comprise detecting protein, RNA or DNA of the specified target biomarker.
  • the one or more marker can be assessed directly from a biological fluid, such as those fluids disclosed herein, or can be assessed for its association with a vesicle, e.g., as a vesicle surface antigen or as vesicle payload (e.g., soluble protein, mRNA or DNA).
  • a particular biosignature determined using methods and compositions of the invention can comprise any number of useful biomarkers, e.g., a biosignature can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different biomarkers (or in some cases different molecules of the same biomarkers, such protein and nucleic acid).
  • Vesicle surface antigens can also be used as capture antigens, detector antigens, or both, as disclosed herein or in applications incorporated by reference.
  • Methods and compositions of the invention are applied to assess various aspects of a cancer, including identifying different informative aspects of a cancer, e.g., identifying a biosignature that is indicative of metastasis, angiogenesis, or classifying different stages, classes or subclasses of the same tumor or tumor lineage.
  • methods of the invention comprise determining if a disease or condition affects
  • the one or more circulating biomarker for immunomodulation can be one or more of CD45, FasL, CTLA4, CD80 and CD83.
  • the one or more circulating biomarker for metastatis can be one or more of Mucl, CD147, TIMP1, TIMP2, MMP7, and MMP9.
  • the one or more circulating biomarker for angiogenesis can be one or more of HIF2a, Tie2, Angl, DLL4 and VEGFR2. Any number of useful biomarkers can be assessed from the groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • the cancer can be a breast cancer.
  • the markers can be associated with a vesicle or vesicle population.
  • the one or more circulating biomarker can be a vesicle surface antigen or vesicle payload. Vesicle surface antigens can further be used as capture antigens, detector antigens, or both.
  • a biosignature can comprise DLL4 or cMET.
  • Delta-like 4 (DLL4) is a Notch-ligand and is up-regulated during angiogenesis.
  • cMET (also referred to as c-Met, MET, or MNNG HOS Transforming gene) is a proto- oncogene that encodes a membrane receptor tyrosine kinase whose ligand is hepatocyte growth factor (HGF).
  • HGF hepatocyte growth factor
  • the MET protein is sometimes referred to as the hepatocyte growth factor receptor (HGFR).
  • HGFR hepatocyte growth factor receptor
  • MET is normally expressed on epithelial cells, and improper activation can trigger tumor growth, angiogenesis and metastasis.
  • DLL4 and cMET can be used as biomarkers to detect a vesicle population.
  • Biomarkers that can be derived and analyzed from a vesicle include miRNA (miR), miRNA*nonsense (miR*), and other RNAs (including, but not limited to, mRNA, preRNA, priRNA, hnRNA, snRNA, siRNA, shRNA).
  • miRNA biomarker can include not only its miRNA and microRNA* nonsense, but its precursor molecules: pri-microRNAs (pri-miRs) and pre-microRNAs (pre-miRs).
  • the sequence of a miRNA can be obtained from publicly available databases such as http://www.mirbase.org/, http://www.microrna.org/, or any others available.
  • the methods of the invention comprise isolating vesicles, and assessing the miRNA payload within the isolated vesicles.
  • the biomarker can also be a nucleic acid molecule (e.g. DNA), protein, or peptide.
  • the presence or absence, expression level, mutations (for example genetic mutations, such as deletions, translocations, duplications, nucleotide or amino acid substitutions, and the like) can be determined for the biomarker. Any epigenetic modulation or copy number variation of a biomarker can also be analyzed.
  • the one or more biomarkers analyzed can be indicative of a particular tissue or cell of origin, disease, or physiological state. Furthermore, the presence, absence or expression level of one or more of the biomarkers described herein can be correlated to a phenotype of a subject, including a disease, condition, prognosis or drug efficacy.
  • the specific biomarker and biosignature set forth below constitute non-inclusive examples for each of the diseases, condition comparisons, conditions, and/or physiological states.
  • the one or more biomarker assessed for a phenotype can be a cell-of-origin specific vesicle.
  • the one or more miRNAs used to characterize a phenotype may be selected from those disclosed in PCT Publication No. WO2009/036236.
  • one or more miRNAs listed in Tables I-VI ( Figures 6-11) therein can be used to characterize colon adenocarcinoma, colorectal cancer, prostate cancer, lung cancer, breast cancer, b- cell lymphoma, pancreatic cancer, diffuse large BCL cancer, CLL, bladder cancer, renal cancer, hypoxia-tumor, uterine leiomyomas, ovarian cancer, hepatitis C virus-associated hepatocellular carcinoma, ALL, Alzheimer's disease, myelofibrosis, myelofibrosis, polycythemia vera, thrombocythemia, HIV, or HIV-I latency, as further described herein.
  • the one or more miRNAs can be detected in a vesicle.
  • the one or more miRNAs can be miR-223, miR- 484, miR-191, miR-146a, miR-016, miR-026a, miR-222, miR-024, miR-126, and miR-32.
  • One or more miRNAs can also be detected in PBMC.
  • the one or more miRNAs can be miR-223, miR-150, miR-146b, miR-016, miR-484, miR-146a, miR-191, miR-026a, miR-019b, or miR-020a.
  • the one or more miRNAs can be used to characterize a particular disease or condition.
  • one or more miRNAs can be detected, such as miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, miR-205 or any combination thereof.
  • the one or more miRNAs may be upregulated or overexpressed.
  • the one or more miRNAs is used to characterize hypoxia-tumor.
  • the one or more miRNA may be miR-23, miR-24, miR-26, miR-27, miR-103, miR-107, miR-181, miR-210, or miR-213, and may be upregulated.
  • One or more miRNAs can also be used to characterize uterine leiomyomas.
  • the one or more miRNAs used to characterize a uterine leiomyoma may be a let-7 family member, miR-21, miR-23b, miR-29b, or miR-197. The miRNA can be upregulated.
  • Myelofibrosis can also be characterized by one or more miRNAs, such as miR-190, which can be upregulated; miR-31, miR-150 and miR-95, which can be downregulated, or any combination thereof.
  • miRNAs such as miR-190, which can be upregulated; miR-31, miR-150 and miR-95, which can be downregulated, or any combination thereof.
  • myelofibrosis, polycythemia vera or thrombocythemia can also be characterized by detecting one or more miRNAs, such as, but not limited to, miR-34a, miR-342, miR-326, miR- 105, miR-149, miR- 147, or any combination thereof.
  • the one or more miRNAs may be downregulated.
  • phenotypes that can be characterized by assessing a vesicle for one or more biomarkers are futher described herein.
  • the one or more biomarkers can be detected using a probe.
  • a probe can comprise an oligonucleotide, such as DNA or RNA, an aptamer, monoclonal antibody, polyclonal antibody, Fabs, Fab', single chain antibody, synthetic antibody, peptoid, zDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), lectin, synthetic or naturally occurring chemical compound (including but not limited to a drug or labeling reagent), dendrimer, or a combination thereof.
  • the probe can be directly detected, for example by being directly labeled, or be indirectly detected, such as through a labeling reagent.
  • the probe can selectively recognize a biomarker.
  • a probe that is an oligonucleotide can selectively hybridize to a miRNA biomarker.
  • the invention provides for the diagnosis, theranosis, prognosis, disease stratification, disease staging, treatment monitoring or predicting responder / non-responder status of a disease or disorder in a subject.
  • the invention comprises assessing vesicles from a subject, including assessing biomarkers present on the vesicles and/or assessing payload within the vesicles, such as protein, nucleic acid or other biological molecules. Any appropriate biomarker that can be assessed using a vesicle and that relates to a disease or disorder can be used the carry out the methods of the invention. Furthermore, any appropriate technique to assess a vesicle as described herein can be used.
  • biomarkers for specific diseases that can be assessed according to the methods of the invention include the biomarkers described in International Patent Application Serial No. PCT US2011/031479, entitled “Circulating Biomarkers for Disease” and filed April 6, 2011, which application is incorporated by reference in its entirety herein.
  • biomarkers or specific biomarkers described herein can be assessed to identify a biosignature or to identify a candidate biosignature.
  • exemplary biomarkers include without limitation those in Table 3, Table 4 or Table 5. The markers in the table can be used for capture and/or detection of vesicles for
  • the markers can be detected as protein or as mRNA, which can be circulating freely or in a complex with other biological molecules.
  • the markers can be detected as vesicle surface antigens or and vesicle payload.
  • the "Illustrative Class" indicates indications for which the markers are known markers. Those of skill will appreciate that the markers can also be used in alternate settings in certain instances. For example, a marker which can be used to characterize one type disease may also be used to characterize another disease as appropriate.
  • Ensembl www.ensembl.org
  • gene symbols and names below correspond to those approved by HUGO
  • protein names are those recommended by UniProtKB/Swiss-Prot. Common alternatives are provided as well.
  • biomarkers are referred to by Ensembl reference numbers, which are of the form "ENSG” followed by a number, e.g., ENSG00000005893 which corresponds to LAMP2.
  • HNF 1A JAK3, KDR, MLH1, PTPN11, RBI, RET, c-Kit, EGFR, PIK3CA, NRAS, GNA11, GNAQ, KRAS, BRAF
  • ERBB2 (HER2), ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HNF 1A, HRAS, IDH1, JAK2, JAK3, KDR (VEGFR2), KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RBI, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL
  • KRAS KRAS, MGMT-Me, MSH2, MSI, NRAS, PGP (MDR-1), PIK3CA, PR, PTEN, ROS1 translocation, RRM1, SPARC, TLE3, TOPOl, TOP02A, TS, TUBB3, VEGFR2
  • Colon cancer AREG, BRAF, EGFR, EML4-ALK, ERCCl, EREG, KRAS, MSI, NRAS, PIK3CA, PTEN, treatment TS, VEGFR2
  • Colon cancer AREG, BRAF, EGFR, EML4-ALK, ERCCl, EREG, KRAS, MSI, NRAS, PIK3CA, PTEN, treatment TS, VEGFR2
  • NSCLC cancer BRAF, BRCAl, cMET, EGFR, EGFR w/T790M, EML4-ALK, ERCCl, Her2 Exon 20 treatment insert, KRAS, MSH2, PIK3CA, PTEN, ROS1 (trans), RRM1, TLE3, TS, VEGFR2 associated markers
  • FGFRl FGFR2, FGFR3, ERBB4, SMO, DDR2, GRB1, PTCH, SHH, PD1, UGT1A1, BIM, ESR1, MLL, AR, CDK4, SMAD4
  • AREG ARFRP1, ARID 1 A, ARID2, ASNS, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXL, BAP1, BARD1, BCL2, BCL2L2, BCL6, BCOR, BCORL1, BCR, BIRC5 (survivin), BLM, BRAF, BRCA1, BRCA2, BRIP1, BTK, CA2, CARD11, CAV, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD33, CD52 (CDW52), CD79A, CD79B, CDC73, CDH1, CDK12, CDK2, CDK4, CDK6, CDK8, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CES2, CHEK1, CHEK2, CIC, CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, CTNNB1, DAXX, DCK, DDR2,
  • Cytohesions cytohesin-1 cytohesin-1 (CYTHl), cytohesin-2 (CYTH2; ARNO), cytohesin-3 (CYTH3; Grpl ; ARN03), cytohesin-4 (CYTH4)
  • Tissue (Breast) BIG H3, GCDFP-15, PR(B), GPR 30, CYFRA 21, BRCA 1, BRCA 2, ESR 1, ESR2
  • IFNAR Inllammation/Imm MFG-E8, IFNAR, CD40, CD80, MICB, HLA-DRb, IL-17-Ra
  • HSPA8 Common vesicle HSPA8, CD63, Actb, GAPDH, CD9, CD81, ANXA2, HSP90AA1, ENOl, YWHAZ, markers PDCD6IP, CFL1, SDCBP, PKN2, MSN, MFGE8, EZR, YWHAG, PGK1, EEF1A1, PPIA,
  • GLC1F GK, ANXA6, ANXAl, ALDOA, ACTG1, TPI1, LAMP2, HSP90AB1, DPP4, YWHAB, TSG101, PFN1, LDHB, HSPA1B, HSPA1A, GSTP1, GNAI2, GDI2, CLTC, ANXA5, YWHAQ, TUBA 1 A, THBS1, PRDX1, LDHA, LAMP1, CLU, CD86
  • Metastatic Prostate hsa-miR-100, hsa-miR-1236, hsa-miR-1296, hsa-miR-141, hsa-miR-146b-5p, hsa-miR-17*, Cancer hsa-miR-181a, hsa-miR-200b, hsa-miR-20a*, hsa-miR-23a*, hsa-miR-331-3p, hsa-miR-375, hsa-miR-452, hsa-miR-572, hsa-miR-574-3p, hsa-miR-577, hsa-miR-582-3p, hsa-miR-937, miR-lOa, miR-134, miR-141, miR-200b, miR-30a, miR-32, miR-375
  • Prostate Cancer FLNA DCRN, HER 3 (ErbB3), VCAN, CD9, GAL3, CDADC1, GM-CSF, EGFR, RANK, Vesicle Markers CSA, PSMA, ChickenlgY, B7H3, PCSA, CD63, CD3, MUC1, TGM2, CD81, S100-A4,
  • Prostate Cancer NT5E CD73
  • A33 ABL2
  • ADAM 10 AFP
  • ALA ALIX
  • ALPL ALPL
  • AMACR AMACR
  • Apo J/CLU Vesicle Markers ASCA, ASPH (A-10), ASPH (D01P), AURKB, B7H3, B7H4, BCNP, BDNF, CA125
  • PSIP1/LEDGF PSMA
  • RAGE RAGE
  • RANK RAGE
  • RUNX2 RUNX2
  • S 100-A4 seprase/FAP
  • SERPINB3, SIM2 C-15
  • SPARC SPC
  • SPDEF SPP1, SSX2, SSX4, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB (poly), Trop2, Tsg 101, TWEAK, UNC93A, VCAN, VEGF A, wnt-5a(C-16), XAGE, XAGE-1
  • GGPS1 Farnesyltransferase/geranylgeranyl diphosphate synthase 1
  • Fatty acid synthase (FASN) Fatty acid synthase (FASN), FTL (light and heavy)
  • Prostate PCSA+ miR-182, miR-663, miR-155, mirR-125a-5p, miR-548a-5p, miR-628-5p, miR-517*, miR- cMVs
  • Prostate Cancer miR-183 -96- 182 cluster (miRs-183, 96 and 182), metal ion transporter such as hZIPl,
  • Prostate Cancer RAD23B FBP1, TNFRSF1A, CCNG2, NOTCH3, ETV1, BID, SIM2, LETMD1, ANXAl, miR-519d, miR-647
  • Prostate Cancer RAD23B FBP1, TNFRSF1A, NOTCH3, ETV1, BID, SIM2, ANXA1, BCL2
  • HIST1H3B HLA-A, HSP90AA1, NRGN, PRDX5, PTMA, RABAC1, RABAGAP1L, RPL22, SAP 18, SEPW1, SOX1
  • CD66eCEA CD81, CD81, CD9, CD9, CDA, CDADCl, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3(ErbB3), HSP70, HSPB1, hVEGFR2, iC3b, IL-1B, IL6R, IL6Unc, IL7Ralpha/CD127, IL8, INSIG-2, Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUCl, Mucl, MUCl 7, MUC2, Ncam, NDUFB7, NGAL, NK-2R(C-21), NT5E
  • miR-588 Inflammatory miR-588, miR-1258, miR-16-2*, miR-938, miR-526b, miR-92b*, let-7d, miR-378*, miR- Disease 124, miR-376c, miR-26b, miR-1204, miR-574-3p, miR-195, miR-499-3p, miR-2110, miR- 888

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Abstract

La présente invention concerne des biomarqueurs pouvant être évalués pour des procédés de diagnostic, de thérapie ou de pronostic, afin d'identifier des phénotypes, tels qu'une pathologie ou une maladie, ou le stade ou la progression d'une maladie, sélectionner des schémas thérapeutiques candidats pour des maladies, des états, des stades de maladie et des stades d'un état, et déterminer l'efficacité d'un traitement. Des biomarqueurs circulants qui proviennent d'un liquide organique peuvent être utilisés dans le profilage d'états physiologiques ou la détermination de phénotypes. Ceux-ci incluent des acides nucléiques, des protéines et des structures circulantes, telles que des microvésicules, ainsi que des complexes acides nucléiques/protéines.
PCT/US2014/039858 2013-05-28 2014-05-28 Méthodes et compositions d'identification de biomarqueurs WO2014193999A2 (fr)

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