[go: up one dir, main page]

WO2025090771A1 - Profilage de microvésicules de cellules immunitaires pour la surveillance et le traitement d'un rejet de greffe de donneur - Google Patents

Profilage de microvésicules de cellules immunitaires pour la surveillance et le traitement d'un rejet de greffe de donneur Download PDF

Info

Publication number
WO2025090771A1
WO2025090771A1 PCT/US2024/052821 US2024052821W WO2025090771A1 WO 2025090771 A1 WO2025090771 A1 WO 2025090771A1 US 2024052821 W US2024052821 W US 2024052821W WO 2025090771 A1 WO2025090771 A1 WO 2025090771A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
donor
biomarker
transplant
immune cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/052821
Other languages
English (en)
Inventor
Prashanth VALLABHAJOSYULA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Original Assignee
Yale University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yale University filed Critical Yale University
Publication of WO2025090771A1 publication Critical patent/WO2025090771A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • 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
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • the invention provides a method for assessing conditional status of a 2 53258724.1 Attorney Docket No.047162-7412WO1(02347) donor transplant in a subject, the method comprising: (i) obtaining and/or having obtained at least one biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; and (iv) comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; wherein the amount and/or
  • the immune cell microvesicles are T cell microvesicles and/or B cell microvesicles.
  • obtaining and/or having obtained at least one biological sample from the subject comprises obtaining and/or having obtained biological samples from the subject at multiple timepoints post-transplant.
  • the baseline value is determined by quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker from a reference biological sample obtained from the subject pre-transplant, on transplant day, or within about 1 day to within about 5 days post-transplant.
  • the quantified amount of the immune cell microvesicle biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the immune cell microvesicle biomarker.
  • the at least one biological sample comprises plasma.
  • the conditional status indicates rejection of the donor transplant, tolerance of the donor transplant, or recovery from rejection of the donor transplant (e.g., after treatment of a rejection event).
  • 3 53258724.1 Attorney Docket No.047162-7412WO1(02347) (a) the immune cell is a T cell and the rejection is acute cellular rejection (ACR); or (b) the immune cell is a B cell and the rejection is antibody-mediated rejection (AMR).
  • the ACR is at least grade 2 ACR.
  • the immune cell is a T cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD3 antibody; and/or (b) the immune cell is a B cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD19 antibody and/or an anti-CD38 antibody.
  • the method further comprises: (i) detecting and/or isolating donor cell microvesicles and/or at least one donor cell microvesicle biomarker from the at least one biological sample; (ii) quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; and (iii) comparing the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample to a baseline value of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; wherein the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample compared to the baseline value indicates the conditional status of the donor transplant.
  • the baseline value is determined by quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker from a reference biological sample obtained from the subject pre-transplant, on transplant day, or within about 1 day to within about 5 days post-transplant.
  • the quantified amount of the donor cell biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the donor cell biomarker.
  • the at least one immune cell microvesicle biomarker and/or the at least one donor cell microvesicle biomarker is a protein, a messenger RNA (mRNA), and/or a 4 53258724.1 Attorney Docket No.047162-7412WO1(02347) micro RNA.
  • the immune cell microvesicles and/or the donor cell microvesicles are characterized by a diameter of about 30 nm to about 400 nm, preferably wherein the diameter is 100 nm or less.
  • the donor transplant is an organ, tissue, or cells selected from the group consisting of heart, lung(s), kidney(s), liver, pancreas, pancreatic islet tissue(s), and pancreatic beta cells.
  • the donor transplant is heart or lung(s).
  • the method comprises quantifying the amount and/or relative level of the immune cell microvesicles and comparing the amount and/or relative level of the immune cell microvesicles to the baseline value, wherein an increase of at least about 40% in the amount and/or relative level of the immune cell microvesicles compared to the baseline value indicates rejection of the donor transplant in the subject.
  • the at least one immune cell microvesicle biomarker is selected from the group consisting of miR let7i, miR 21a, miR 101b, CD4 protein, CD4 mRNA, CD8 protein, CD8 mRNA, donor-specific T cell receptor (TCR) protein, interferon gamma (IFN ⁇ ) mRNA, CD19 mRNA, CD38 mRNA, CD38 protein, HLA-DR mRNA, HLA-DR protein, ICOSL mRNA, CD20 mRNA, IL6-R mRNA, and BAFF-R mRNA, or any combination thereof.
  • TCR T cell receptor
  • IFN ⁇ interferon gamma
  • the at least one immune cell microvesicle biomarker is miR let7i, miR 21a, and/or miR 101b, and the control biomarker is hsa miR 26; and/or (b) the at least one immune cell microvesicle biomarker is CD4 protein, CD8 protein, and/or donor-specific TCR protein, and the control biomarker is TSG101 protein.
  • the at least one immune cell microvesicle biomarker is miR let7i and/or miR 21a, and the conditional status of the donor transplant is rejection when the relative level of the miR let7i and/or the miR 21a is at least about 4-fold greater than the baseline value;
  • the at least one immune cell microvesicle biomarker is miR 101b, and the conditional status of the donor transplant is rejection when the relative level of the miR 101b is at least about 3-fold greater than the baseline value; 5 53258724.1 Attorney Docket No.047162-7412WO1(02347)
  • the at least one immune cell microvesicle biomarker is CD4 protein, and the conditional status of the donor transplant is rejection when the relative level of the CD4 protein is at least about 2.2-fold greater than the baseline value;
  • the at least one immune cell microvesicle biomarker is CD8 protein, and the conditional status of the donor transplant is rejection when the relative level of the CD8
  • the donor transplant is heart
  • the at least one donor cell microvesicle biomarker is selected from the group consisting of Troponin T protein, Troponin T mRNA, Troponin I protein, and Troponin I mRNA, or any combination thereof.
  • the at least one donor cell microvesicle biomarker is Troponin T protein and the control biomarker is TSG101 protein; and/or (b) the at least one donor cell microvesicle biomarker is Troponin T mRNA and the control biomarker is GAPDH mRNA.
  • the at least one donor cell microvesicle biomarker is Troponin T protein, and the conditional status of the donor transplant is rejection when the relative level of the Troponin T protein is at least about 40% less than the baseline value; and/or (b) the at least one donor cell microvesicle biomarker is Troponin T mRNA, and the conditional status of the donor transplant is rejection when the relative level of the Troponin T mRNA is at least about 60% less than the baseline value.
  • the donor transplant is lung(s), and: (a) the immune cell is a T cell and the at least one immune cell microvesicle biomarker is CD8 mRNA, IFN ⁇ mRNA, HLA-DR mRNA, or any combination thereof; and/or (b) the immune cell is a B cell and the at least one immune cell microvesicle biomarker is CD19 mRNA, CD20 mRNA, CD38 mRNA, IL6-R mRNA, BAFF-R 6 53258724.1 Attorney Docket No.047162-7412WO1(02347) mRNA, IFN ⁇ mRNA, HLA-DR mRNA, ICOSL mRNA, or any combination thereof.
  • the invention provides a method of treating rejection of a donor transplant in a subject, the method comprising: (i) obtaining and/or having obtained at least one biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; and (iv) determining the conditional status of the donor transplant, wherein the determining comprises comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; wherein the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell
  • the immune cell microvesicles are T cell microvesicles and/or B cell microvesicles.
  • obtaining and/or having obtained at least one biological sample from the subject comprises obtaining and/or having obtained biological samples from the subject at multiple timepoints post-transplant.
  • the baseline value is determined by quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker from a reference biological sample obtained from the subject pre-transplant, on 7 53258724.1 Attorney Docket No.047162-7412WO1(02347) transplant day, or within about 1 day to within about 5 days post-transplant.
  • the quantified amount of the immune cell microvesicle biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the immune cell microvesicle biomarker.
  • the at least one biological sample comprises plasma.
  • the immune cell is a T cell and the rejection is acute cellular rejection (ACR); and/or (b) the immune cell is a B cell and the rejection is antibody-mediated rejection (AMR).
  • ACR is at least grade 2 ACR.
  • the rejection is ACR and the rejection therapy comprises any one or more of bolus intravenous steroids, mycophenolate mofetil, and thymoglobulin therapy; and/or (b) the rejection is AMR and the rejection therapy comprises an anti-CD20 antibody (e.g., rituximab or ofatumumab).
  • the immune cell is a T cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD3 antibody; and/or (b) the immune cell is a B cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD19 antibody and/or an anti-CD38 antibody.
  • the method further comprises: (i) detecting and/or isolating donor cell microvesicles and/or at least one donor cell microvesicle biomarker from the at least one biological sample; (ii) quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; and (iii) comparing the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample to a baseline value of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; wherein the amount and/or relative level of 8 53258724.1 Attorney Docket No.047162-7412WO1(02347) the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample compared to the baseline value indicates the conditional status of the donor transplant.
  • the baseline value is determined by quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker from a reference biological sample obtained from the subject pre-transplant, on transplant day, or within about 1 day to within about 5 days post-transplant.
  • the quantified amount of the donor cell biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the donor cell biomarker.
  • the at least one immune cell microvesicle biomarker and/or the at least one donor cell microvesicle biomarker is a protein, a messenger RNA (mRNA), and/or a micro RNA.
  • the immune cell microvesicles and/or the donor cell microvesicles are characterized by a diameter of about 30 nm to about 400 nm, preferably wherein the diameter is 100 nm or less.
  • the donor transplant is selected from the group consisting of heart, lung(s), kidney(s), liver, pancreas, pancreatic islet tissue(s), and pancreatic beta cells. In some embodiments, the donor transplant is heart or lung(s).
  • the method comprises quantifying the amount and/or relative level of the immune cell microvesicles and comparing the amount and/or relative level of the immune cell microvesicles to the baseline value, wherein an increase of at least about 40% in the amount and/or relative level of the immune cell microvesicles compared to the baseline value indicates rejection of the donor transplant in the subject.
  • the at least one immune cell microvesicle biomarker is selected from the group consisting of miR let7i, miR 21a, miR 101b, CD4 protein, CD4 mRNA, CD8 protein, CD8 mRNA, donor-specific T cell receptor (TCR) protein, interferon gamma (IFN ⁇ ) mRNA, CD19 mRNA, CD38 mRNA, CD38 protein, HLA-DR mRNA, HLA-DR protein, ICOSL mRNA, CD20 mRNA, IL6-R mRNA, BAFF-R mRNA, and any combination thereof.
  • TCR T cell receptor
  • IFN ⁇ interferon gamma
  • the at least one immune cell microvesicle biomarker is miR let7i, miR 21a, and/or 9 53258724.1 Attorney Docket No.047162-7412WO1(02347) miR 101b, and the control biomarker is hsa miR 26; and/or (b) the at least one immune cell microvesicle biomarker is CD4 protein, CD8 protein, and/or donor-specific TCR protein, and the control biomarker is TSG101 protein.
  • the at least one immune cell microvesicle biomarker is miR let7i and/or miR 21a, and the conditional status of the donor transplant is rejection when the relative level of the miR let7i and/or the miR 21a is at least about 4-fold greater than the baseline value;
  • the at least one immune cell microvesicle biomarker is miR 101b, and the conditional status of the donor transplant is rejection when the relative level of the miR 101b is at least about 3-fold greater than the baseline value;
  • the at least one immune cell microvesicle biomarker is CD4 protein, and the conditional status of the donor transplant is rejection when the relative level of the CD4 protein is at least about 2.2-fold greater than the baseline value;
  • the at least one immune cell microvesicle biomarker is CD8 protein, and the conditional status of the donor transplant is rejection when the relative level of the CD8 protein is at least about 2.5-fold greater than the baseline value; and/or (e) the at least one
  • the donor transplant is heart
  • the at least one donor cell microvesicle biomarker is selected from the group consisting of Troponin T protein, Troponin T mRNA, Troponin I protein, and Troponin I mRNA, or any combination thereof.
  • the at least one donor cell microvesicle biomarker is Troponin T protein and the control biomarker is TSG101 protein
  • the at least one donor cell microvesicle biomarker is Troponin T mRNA and the control biomarker is GAPDH mRNA.
  • the at least one donor cell microvesicle biomarker is Troponin T protein, and the 10 53258724.1 Attorney Docket No.047162-7412WO1(02347) conditional status of the donor transplant is rejection when the relative level of the Troponin T protein is at least about 40% less than the baseline value; and/or (b) the at least one donor cell microvesicle biomarker is Troponin T mRNA, and the conditional status of the donor transplant is rejection when the relative level of the Troponin T mRNA is at least about 60% less than the baseline value.
  • the donor transplant is lung(s), and: (a) the immune cell is a T cell and the at least one immune cell microvesicle biomarker is CD8 mRNA, IFN ⁇ mRNA, HLA-DR mRNA, or any combination thereof; and/or (b) the immune cell is a B cell and the at least one immune cell microvesicle biomarker is CD19 mRNA, CD20 mRNA, CD38 mRNA, IL6-R mRNA, BAFF-R mRNA, IFN ⁇ mRNA, HLA-DR mRNA, ICOSL mRNA, or any combination thereof.
  • FIG.1 is a schematic of the study design and development of T cell sEV platform. Three study arms were designed to ascertain circulating T cell small extracellular microvesicle (sEV) profiles in heterotopic heart transplantation model.
  • sEV small extracellular microvesicle
  • BALB/c heart was transplanted into T cell and B cell immunodeficient C57BL/6 Prkdc SCID recipient to assess for nonspecific CD3 sEV signal and donor passenger T cell sEV signal in recipient blood.
  • BALB/c [H2-k d ] heart was transplanted into wild type C57BL/6 [H2-k b ] recipient across full MHC mismatch, which reliably leads to ACR mediated allograft asystole by day 12 to 14.
  • Control arm syngeneic C57BL/6 into C57BL/6 transplant was performed to assess for changes in circulating T cell sEV signal from isograft transplantation.
  • FIGs.2A – 2C illustrate the finding that Jurkat T cells release sEVs expressing exosome markers and T cell markers into culture supernatant.
  • Jurkat T cells were studied to assess 11 53258724.1 Attorney Docket No.047162-7412WO1(02347) whether T cells release sEVs with specific markers.
  • FIG.2B is a wide field electron microscopy image of Jurkat T cell sEVs.
  • FIG.2C is a Western blot of Jurkat sEVs showing expression of exosome markers TSG101, CD63, flotillin-1, and T cell markers CD4, CD8, and TCR.
  • Jurkat sEVs were also checked for absence of expression of calnexin and cytochrome c per MISEV guidelines.
  • FIGs.3A – 3F illustrate the finding that ACR leads to time specific changes in circulating T cell sEVs.
  • FIG.3A NTA image plots of isolated plasma sEVs from post-transplant time points in all 3 study arms, along with mean sEV size, and quantity (# particles/ ⁇ g sEV protein).
  • FIG.3B Electron microscopy of plasma sEVs from day 7 post-transplant in Control, Rejection, and Maintenance arms.
  • FIG.3C Western blot of plasma sEVs was performed for expression of exosome markers CD63, flotillin-1, and TSG101, with absence of cytochrome c, calnexin, and apolipoprotein E per MISEV guidelines.
  • FIG.3D CD3-expressing T cell sEV subset signal (red) compared to the total plasma sEV pool (blue) on the NTA fluorescence mode is shown for animal receiving syngeneic transplant (Control arm). Percentage nanoparticles positive for CD3 expression is also shown.
  • FIG.3E Circulating CD3-expressing T cell sEV subset signal over serial time points in one animal undergoing heart transplantation in the Maintenance arm is shown. Naive wild type C57BL/6 plasma sEVs and IgG isotype control are also shown.
  • FIG.3F CD3 sEV signal in total plasma sEV pool from a single recipient animal over serial follow-up after full MHC mismatch heart transplant is shown (Rejection arm); along with naive, wild type control and IgG isotype control. Anti-CD3 ⁇ antibody conjugated quantum dot was utilized for T cell sEV subset quantitation on the NTA fluorescence mode. Time specific changes in the T cell sEV signal was seen only in the Rejection arm recipient animal.
  • FIGs.4A – 4F illustrates the finding that circulating T cell sEV signal heralds early ACR.
  • FIG.4A Scatter plot of circulating T cell sEV quantitative signal in the total plasma sEV pool is shown for the Maintenance (black) and Rejection (red) study arms. Each data point represents a sacrificed animal for the respective postoperative time point. In the Maintenance arm, slight increase in CD3 sEV signal was seen on PODs 1 and 2, after which the signal remained at baseline, pre-heart transplant (pre-HTP) levels. In Control arm, plasma CD3 sEV signal also unchanged, similar to baseline signal (data not shown).
  • FIG.4B In the Control and Rejection arms, peripheral blood B cells (CD19 + B220+) and CD3+ T cell counts and their subpopulations, CD3+ CD4+ T cells and CD3+ CD8+ T cells, were analyzed by FACS to assess whether the changes in circulating T cell sEV signal in Rejection arm reflected concomitant changes in circulating T cell numbers during ACR. Day 0 versus day 7 samples were analyzed as peak CD3 sEV signal was noted on day 7. Gating strategy for FACS is shown for one of 5 experiments in each study arm. In 3 animals, Sham operation was performed, where the animal’s abdomen was opened and the aorta and inferior vena cava were clamped and then released, and then the abdomen was closed.
  • FIG.4C Representative FACS results for the Control arm. The numbers of peripheral blood B cell and T cells, and their CD4+ and CD8+ T cell subpopulations were similar in the Control versus Rejection study arms.
  • FIG.4D Representative FACS results for the Rejection arm. The numbers of peripheral blood B cell and T cells, and their CD4+ and CD8+ T cell subpopulations were similar in the Control versus Rejection study arms.
  • FIG.4E Representative FACS results for the Sham Operation.
  • FIGs.5A – 5D illustrate the finding that ACR leads to changes in T cell sEV cargoes reflecting an alloreactivity profile.
  • FIG.5A Schematic for purification of circulating T cell EVs is shown. Given the noted time course of T cell EV signal during ACR, characterization of T cell sEV cargoes from days 4 and 7 after transplantation was performed.
  • FIG.5B Western blot of anti-CD3 antibody bead unbound sEV fraction was performed for confirmation of absence of CD3, CD4, and CD8 proteins as quality control. Jurkat cell positive control is also shown. Representative one of 3 experiments is shown.
  • FIG.5C Anti-CD3 antibody bead-bound sEVs 13 53258724.1 Attorney Docket No.047162-7412WO1(02347) were eluted and analyzed on NTA to confirm that intact nanoparticles in the sEV size range were being enriched using this methodology.
  • the resulting eluted bead-bound sEVs representing the enriched T cell sEV fraction yielded ⁇ 1-1.5 x10 8 sEVs, suggesting that the T cell sEV signal was being enriched between 66 to 100-fold compared to total plasma sEV analysis. Summary data for 5 independent experiments performed for Rejection arm POD 7 is shown.
  • FIG.5D Western blot analysis of T cell sEV protein cargoes revealed upregulation of several T cell activation markers including IFN ⁇ , Serca 2, CD4, and TCR. Upregulation of these markers was noted in day 7 versus day 4 samples in Rejection arm but not Control arm. FoxP3 expression was also noted, indicating a regulatory T cell contribution to circulating T cell sEV subpopulation. Plasma sEVs from naive, wild type C57BL/6 is also shown to confirm that at the baseline, pre-transplant setting, T cell markers are not readily detectable in plasma sEVs. Flotillin-1 is an exosome marker. One of 3 experiments is shown.
  • FIG.5E RT-PCR of T cell sEV mRNA showed upregulation of T cell activation markers in day 7 versus 4 samples in Rejection arm but not Control arm. CD3 ⁇ , TCR, IFN ⁇ , and IL2 expression along with ⁇ -actin control is shown (1 of 3 experiments).
  • FIG.5F T cell sEV cargo expression from syngeneic transplants in Control arm showed low level detection of T cell markers CD4 and TCR, without any changes in expression over serial follow-up. Flotillin-1 is an exosome marker. C57BL/6 wild type plasma control is shown as control, along with Jurkat cells as positive control. One of 3 experiments is shown.
  • FIGs.6A – 6F illustrate the finding that T cell sEV miRNA cargo profiles reflect alloactivation state.
  • FIG.6A Principal component analysis plot of Rejection arm day 4 and day 7 CD3 sEV NGS samples showed distinct miRNA cargo populations.
  • FIG.6B Volcano plot of fold change vs. p-value revealed differentially expressed miRNAs from day 4 to day 7. miRNAs upregulated in day 4 and 7 are highlighted in blue and orange, respectively.
  • FIG.6C Heatmap of top 31 differentially expressed miRNAs (
  • FIG.6D Pathway analysis of differentially regulated miRNA gene targets in days 4 versus 7 is shown.
  • FIG.6E Venn diagrams of enriched apoptosis 14 53258724.1 Attorney Docket No.047162-7412WO1(02347) and immune-specific GO biological processes for day 4 and day 7 upregulated miRNA gene targets is shown. Day 4 showed upregulation miRNAs targeting genes involved in immune response pathways and day 7 showed miRNAs targeting genes associated with apoptosis pathways.
  • FIG.6F Bar plot of genes targeted by multiple differentially expressed miRNAs. Gene targets of miRNAs upregulated in day 4 and day 7 are shaded in blue and orange, respectively. Only gene targets with >1 miRNA hit are shown.
  • FIGs.8A – 8J illustrate the finding that circulating T cell sEVs enriched from Rejection arm animals independently mediate donor cardiomyocyte specific cytotoxicity.
  • FIG.8A Schematic of plasma components analyzed for potential to mediate donor cardiomyocyte cytotoxicity is shown. Total plasma sEVs from POD 7 in Rejection and Control arms was harvested. From this, T cell sEVs were enriched using anti-CD3 antibody beads. The unbound sEVs with the plasma secretome was then ultracentrifuged to obtain the secretome and unbound sEV fraction.
  • donor BALB/c cardiomyocytes or recipient C57BL/6 cardiomyocytes were incubated with the following isolated fractions from either the Rejection arm or Control arm: total plasma sEVs (1x1010 sEVs), CD3 sEVs (bound fraction) (1.5x108 sEVs), bead unbound sEVs, and remnant secretome.
  • FIG.8B Confocal microscopy images of TUNEL assay for BALB/c cells incubated with Control arm total plasma sEVs, CD3 sEVs, unbound sEVs, and secretome failed to show apoptosis.
  • FIG.8C BALB/c cells incubated with Rejection arm total plasma sEVs and CD3 sEVs showed >45% apoptosis, but minimal ( ⁇ 6%) apoptosis was seen with unbound sEVs and secretome.
  • FIG.8D C57BL/6 cells incubated with 15 53258724.1 Attorney Docket No.047162-7412WO1(02347) Control arm plasma components showed ⁇ 5% apoptosis.
  • FIG.8E C57BL/6 cells incubated with Rejection arm plasma sEVs, CD3 sEVs, unbound sEVs, and secretome failed to show any apoptosis.
  • FIG.8F BALB/c cells treated with DNase I (positive control) showed near 100% apoptosis.
  • FIG.8G C57BL/6 cells treated with DNase I showed >97% apoptosis (positive control).
  • FIG.8I CD3 sEVs from C57BL/6 wild type, BALB/c wild type, Control arm day 7, and Rejection arm day 7 were assessed for expression of pro- apoptotic proteins known to be critical for T cell mediated cytotoxicity. All study groups showed expression of Fas ligand (FasL), perforin-1, granzyme B, TCR, CD38, MHC II, but higher levels of these pro-apoptotic markers and activation markers were seen in the Rejection arm. Flotillin-1 and TSG101 are exosome markers. One of two experiments is shown.
  • FIG.8J Relative expression of pro-apoptotic markers and activation markers normalized to expression of exosome marker TSG101 is shown.
  • FIG.9A Electron microscopy of plasma sEVs in Subject 2 is shown.
  • FIG.9B NTA light scatter analysis of sEVs isolated in Subjects 1-3 is shown with mean particle concentration and size.
  • FIG.9C sEV protein markers were assessed by Western blot for expression of exosome markers TSG101, flotillin-1, CD63, Alix-1, and for absence of cytochrome c, calnexin-1, and ApoE, per MISEV guidelines. Representative Western blot for Subject 2 is shown.
  • FIG.9D CD3 sEVs were enriched from plasma sEVs using anti-CD3 antibody conjugated beads, and the bead bound fraction was eluted and analyzed on NTA to confirm enrichment of intact sEVs. Representative NTA for 3 time points (PODs 1, 13, and 98) in Subject 2 is shown with particle number and size distribution.
  • FIG.9E Western blot analysis of CD3 antibody bead bound sEVs was performed to validate expression of T cell markers. Representative analysis for Subject 2, PODs 1, 13, and 98 is shown along with Jurkat cell positive control for expression of CD8 in the T cell sEV fraction. Flotillin-1, exosome marker expression is also shown.
  • FIG.9F Western blot analysis of CD3 antibody unbound sEVs was performed to validate absence of T cell markers. Representative analysis for Subject 2 at 3 postoperative time points is shown for expression of CD3 and flotillin-1. CD3 expression was not seen in the unbound sEV fraction, but flotillin-1 (exosome marker) expression was detected. Jurkat EVs and Jurkat cell positive controls are shown.
  • FIG.9G Western blot analysis of T cell sEVs in Subject 1 for expression of T cell inflammatory and proapoptotic proteins is shown, including perforin-1, granzyme B, IFN ⁇ , Fas L, CD38, and MHC II (HLA-DR). EMB histology data (ACR 0 versus 1 versus 2) is also shown.
  • FIG.9H Western blot analysis of T cell sEVs in Subject 2 for expression of T cell inflammatory and proapoptotic proteins is shown, including perforin-1, granzyme B, IFN ⁇ , Fas L, CD38, and MHC II (HLA-DR). EMB histology data (ACR 0 versus 1 versus 2) is also shown. Grade 2 ACR by EMB correlated with increased expression of these T cell activation marker proteins. Flotillin-1 is an exosome marker protein.
  • FIG.9I Western blot analysis of T cell sEVs in Subject 3 for expression of T cell inflammatory and proapoptotic proteins is shown, including perforin-1, granzyme B, IFN ⁇ , Fas L, CD38, and MHC II (HLA-DR). EMB histology data (ACR 0 versus 1 versus 2) is also shown. Grade 2 ACR by EMB correlated with increased expression of these T cell activation marker proteins. Flotillin-1 is an exosome marker protein.
  • FIG.9J Stem loop RT-qPCR for miR let 7i, miR 21a, and miR 101b expression in T cell sEVs in Subject 1 at EMB-matched postoperative time points is shown.
  • FIG.9K Stem loop RT-qPCR for miR let 7i, miR 21a, and miR 101b expression in T cell sEVs in Subject 2 at EMB-matched postoperative time points is shown. Relative expression of candidate miRNAs compared to POD 1 baseline (set to 1) is shown. Correlation of miRNA expression with EMB histology data showed increased expression of these miRNAs in T cell sEV fraction at time points of grade 2 ACR.
  • FIG.9L Stem loop RT- 17 53258724.1 Attorney Docket No.047162-7412WO1(02347) qPCR for miR let 7i, miR 21a, and miR 101b expression in T cell sEVs in Subject 3 at EMB- matched postoperative time points is shown. Relative expression of candidate miRNAs compared to POD 1 baseline (set to 1) is shown. Correlation of miRNA expression with EMB histology data showed increased expression of these miRNAs in T cell sEV fraction at time points of grade 2 ACR.
  • FIG.10 is a schematic of T cell EV mediated targeted suppression of donor heart cardiomyocytes.
  • Transplanted heart cardiomyocytes immediately release EVs into the blood stream that localize to secondary lymphoid organs, including regional lymph nodes.
  • Donor EVs promote alloantigen recognition and activation of T cell clones via semi-direct pathway, along with direct and indirect pathway-based activation.
  • These EVs localize to the allograft cardiomyocytes.
  • T cell EVs also bind to donor cardiomyocyte EVs in peripheral circulation, potentially facilitating their clearance.
  • T cell EVs bind to donor cardiomyocytes and initiate targeted injury, including suppression of EV release by the donor heart. Both these processes lead to decrease in donor EV signal in peripheral blood early during ACR.
  • T cell EVs carrying pro- inflammatory cytokines and pro-apoptotic markers mediate changes in the local immune environment in the donor heart tissue that contributes to the ACR process, and possibly facilitates migration of alloreactive T cells to the allograft.8)
  • proliferated T cell clones egress out of the regional lymph nodes and migrate to the donor heart tissue to potentiate targeted cytotoxicity.
  • FIG.11 is a table of the antibodies used in the experiments disclosed herein.
  • FIG.12 is a table providing clinical information in the 3 heart transplant patients with grade 2 ACR episodes. Grade >2 ACR episodes were treated with additional immunosuppression as detailed in the table.
  • University of Pennsylvania Surveillance EMB Schedule during study period: EMB weeks 1, 2, 4, 6, 8, and every 2 weeks until week 12-16 based on whether patient has any grade > 2 ACR episodes, and then monthly EMB until 1 st annual visit. After 1 st annual visit, surveillance EMB every 3 months during 2 nd year post-transplant until 2 nd annual visit. No surveillance EMB after 2 nd annual visit, only for-cause EMB.
  • FIGs.13A – 13C are tables showing stem loop RT-qPCR assay results for quantitation of miRNAs let 7i, miR 101b, and miR 21a in circulating T cell EVs in 3 heart transplant patients.
  • 18 53258724.1 Attorney Docket No.047162-7412WO1(02347)
  • POD postoperative day
  • SD standard deviation.
  • FIG.13A stem loop RT-qPCR assay results for Patient 1.
  • FIG.13B stem loop RT-qPCR assay results for Patient 2.
  • FIG.13C stem loop RT-qPCR assay results for Patient 3.
  • FIG.14 shows data demonstrating time specific down regulation of cardiac troponin T protein and mRNA cargoes in donor heart EVs from recipient circulation in the rodent heart transplant ACR model.
  • FIG.14A Troponin T protein results from donor heart EVs.
  • FIG.14B Troponin T mRNA results from donor heart EVs.
  • FIG. 14C CD4 protein results from recipient T cell EVs.
  • FIG.14D CD8 protein results from recipient T cell EVs.
  • FIG.14E TCR protein results from recipient T cell EVs.
  • FIG.14F miR Let 7i results from recipient T cell EVs.
  • FIG.14G miR 101b results from recipient T cell EVs.
  • FIG.14H miR 21a results from recipient T cell EVs.
  • FIG.15A Troponin T protein results from donor heart EVs.
  • FIG.15B Troponin T mRNA results from donor heart EVs.
  • FIG.15A Troponin T protein results from donor heart EVs.
  • FIG.15B Troponin T mRNA results from donor heart EVs.
  • FIG.15A Troponin T protein results from donor heart EVs.
  • FIG.15B Troponin
  • FIG.15C CD4 protein results from recipient T cell EVs.
  • FIG.15D CD8 protein results from recipient T cell EVs.
  • FIG.15E TCR protein results from recipient T cell EVs.
  • FIG.15F miR Let 7i results from recipient T cell EVs.
  • FIG.15G miR 101b results from recipient T cell EVs.
  • FIG.15H miR 21a results from recipient T cell EVs.
  • FIG.17A Troponin T protein results from donor heart EVs.
  • FIG.17B Troponin T mRNA results from donor heart EVs.
  • FIG. 17C CD4 protein results from recipient T cell EVs.
  • FIG.17D CD8 protein results from recipient T cell EVs.
  • FIG.17E TCR protein results from recipient T cell EVs.
  • FIG.17F miR Let 7i results from recipient T cell EVs.
  • FIG.17G miR 101b results from recipient T cell EVs.
  • FIG.17H miR 21a results from recipient T cell EVs.
  • FIG.18A Troponin T protein results from donor heart EVs.
  • FIG.18B Troponin T mRNA results from donor heart EVs.
  • FIG. 18C CD4 protein results from recipient T cell EVs.
  • FIG.18D CD8 protein results from recipient T cell EVs.
  • FIG.18E TCR protein results from recipient T cell EVs.
  • FIG.18F miR Let 7i results from recipient T cell EVs.
  • FIG.18G miR 101b results from recipient T cell EVs.
  • FIG.18H miR 21a results from recipient T cell EVs.
  • FIG.19A Troponin T protein results from donor heart EVs.
  • FIG.19B Troponin T mRNA results from donor heart EVs.
  • FIG. 19C CD4 protein results from recipient T cell EVs.
  • FIG.19D CD8 protein results from recipient T cell EVs.
  • FIG.19E TCR protein results from recipient T cell EVs.
  • FIG.19F miR Let 7i results from recipient T cell EVs.
  • FIG.19G miR 101b results from recipient T cell EVs.
  • FIG.19H miR 21a results from recipient T cell EVs.
  • FIGs.20A – 20H show results of cargo analysis for all eight candidate biomarkers in Patient 7.
  • FIG.20A Troponin T protein results from donor heart EVs.
  • FIG.20B Troponin T mRNA results from donor heart EVs.
  • FIG. 20C CD4 protein results from recipient T cell EVs.
  • FIG.20D CD8 protein results from recipient T cell EVs.
  • FIG.20E TCR protein results from recipient T cell EVs.
  • FIG.20F miR Let 7i results from recipient T cell EVs.
  • FIG.20G miR 101b results from recipient T cell EVs.
  • FIG.20H miR 21a results from recipient T cell EVs.
  • FIG.21A Troponin T protein results from donor heart EVs.
  • FIG.21B Troponin T mRNA results from donor heart EVs.
  • FIG.21A Troponin T protein results from donor heart EVs.
  • FIG.21B Troponin T mRNA results from donor heart EVs.
  • FIG.21A Troponin T protein results from donor heart EVs.
  • FIG.21B Troponin
  • FIG. 21C CD4 protein results from recipient T cell EVs.
  • FIG.21D CD8 protein results from recipient T cell EVs.
  • FIG.21E TCR protein results from recipient T cell EVs.
  • FIG.21F miR Let 7i results from recipient T cell EVs.
  • FIG.21G miR 101b results from recipient T cell EVs.
  • FIG.21H miR 21a results from recipient T cell EVs.
  • FIG.22A Troponin T protein results from donor heart EVs.
  • FIG.22B Troponin T mRNA results from donor heart EVs.
  • FIG. 22C CD4 protein results from recipient T cell EVs.
  • FIG.22D CD8 protein results from recipient T cell EVs.
  • FIG.22E TCR protein results from recipient T cell EVs.
  • FIG.22F miR Let 7i results from recipient T cell EVs.
  • FIG.22G miR 101b results from recipient T cell EVs.
  • FIG.22H miR 21a results from recipient T cell EVs.
  • FIGs.23A – 23H show results of cargo analysis for all eight candidate biomarkers in Patient 10.
  • FIG.23A Troponin T protein results from donor heart EVs.
  • FIG.23B Troponin T mRNA results from donor heart EVs.
  • FIG. 23C CD4 protein results from recipient T cell EVs.
  • FIG.23D CD8 protein results from 21 53258724.1 Attorney Docket No.047162-7412WO1(02347) recipient T cell EVs.
  • FIG.23E TCR protein results from recipient T cell EVs.
  • FIG.23F miR Let 7i results from recipient T cell EVs.
  • FIG.23G miR 101b results from recipient T cell EVs.
  • FIG.23H miR 21a results from recipient T cell EVs.
  • FIGs.24A – 24H show scatter plot results of cargo analysis for the indicated biomarker from all 10 patients. Circles indicate no ACR, triangles indicate grade 1 ACR, and squares indicated grade 2 ACR.
  • FIG.24A is a scatter plot of CD4 levels.
  • FIG.24B is a scatter plot of CD8 levels.
  • FIG.24C is a scatter plot of TCR levels.
  • FIG.24D is a scatter plot of Troponin T protein levels.
  • FIG.24E is a scatter plot of Troponin T mRNA levels.
  • FIG.24F is a scatter plot of miR Let 7i levels.
  • FIG.24G is a scatter plot of miR 101b levels.
  • FIG.24H is a scatter plot of miR 21a levels.
  • FIGs.25A – 25H show box plot results of cargo analysis for the indicated biomarker from all 10 patients.
  • FIG.25A is a box plot of CD4 levels across ACR grades.
  • FIG.25B is a box plot of CD8 levels across ACR grades.
  • FIG.25C is a box plot of TCR levels across ACR grades.
  • FIG.25D is a box plot of Troponin T protein levels across ACR grades.
  • FIG.25E is a box plot of Troponin T mRNA levels across ACR grades.
  • FIG.25F is a box plot of miR Let 7i levels across ACR grades.
  • FIG.25G is a box plot of miR 101b levels across ACR grades.
  • FIG.25H is a box plot of miR 21a levels across ACR grades.
  • FIGs.26A – 26H show Area Under the Receiver Operating Characteristics (AUROC) curves calculated via logistic regression for the indicated biomarker from all 10 patients.
  • FIG. 26A is an AUROC plot for CD4.
  • FIG.26B is an AUROC plot for CD8.
  • FIG.26C is an AUROC plot for TCR.
  • FIG.26D is an AUROC plot for Troponin T protein.
  • FIG.26E is an AUROC plot for Troponin T mRNA.
  • FIG.26F is an AUROC plot for miR Let 7i.
  • FIG.26G is an AUROC plot for miR 101b.
  • FIG.26H is an AUROC plot for miR 21a.
  • FIGs.27A – 27E show Area Under the Receiver Operating Characteristics (AUROC) curves calculated via generalized estimating equations for the indicated biomarker from all 10 patients.
  • FIG.27A is an AUROC plot for CD4.
  • FIG.27B is an AUROC plot for CD8.
  • FIG.27C is an AUROC plot for TCR.
  • FIG.27D is an AUROC plot for Troponin T protein.
  • FIG.27E is an AUROC plot for miR 101b.
  • FIG.28 is a schematic showing changes in circulating tissue/ cell specific sEVs associated with AR.
  • FIGs.29A-29B show data from a rodent heterotopic heart transplant model of ACR.
  • MHC full mismatch model of heart transplantation BALB/c into C57BL/6
  • ACR with T cell infiltration into cardiac allograft occurs by day 5
  • fulminant rejection with allograft asystole is complete by days 11-13.
  • donor heart EVs were purified using anti-H2kd antibody conjugated beads and shown to express cardiomyocyte marker troponin T protein and mRNA.
  • troponin T expression in donor heart EV subset decreased early during ACR.
  • FIG.29A Western blot for troponin T and TSG101 is shown.
  • FIG.29B RT-PCR for troponin T mRNA is shown.
  • FIG.30 shows the methodological design for sEV isolation and enrichment of donor heart sEV, T cell sEV, and B cell sEV subpopulations from peripheral blood. Peripheral blood sample is collected in EDTA tube and processed for plasma.
  • Total plasma sEVs are isolated by size exclusion chromatography and ultracentrifugation, after which quality control is performed by NTA, electron microscopy, and Western blot for expression of exosome markers including CD63, TSG101, alix, flotillin 1; along with absence of cytochrome c, calnexin, and apolipoprotein per MISEV guidelines.
  • Cell/tissue specific sEVs are enriched using magnetic beads conjugated to antibodies specific for surface markers expressed on sEV subpopulation of interest.
  • Donor heart sEVs are enriched using anti-donor HLA I specific antibody beads and the bound fraction is eluted and assessed by NTA for intact sEVs.
  • T cell sEVs are enriched using anti-CD3 antibody beads and B cell sEVs are enriched using anti-CD19 antibody beads and the bound fractions are eluted to check for isolation of intact sEVs by NTA.
  • the unbound fraction is correspondingly checked for absence of CD3 (T cell marker) and CD19 (B cell marker) by Western blot.
  • 23 53258724.1 Attorney Docket No.047162-7412WO1(02347) Downstream analysis of sEV protein and RNA cargoes was performed after these quality control steps.
  • Ab antibody
  • HLA human leukocyte antigen.
  • FIG.31 shows the study design and distribution of acute rejection episodes.
  • FIGs.32A-32D show data for total plasma extracellular vesicle characterization.
  • Per MISEV guidelines Thery, et al., J Extracell Vesicles.2018;7(1)), isolated plasma EVs were quality checked and phenotyped.
  • FIG.32A Electron microscopy showed isolation of EVs ⁇ 200 nm in size, consistent with small EVs (sEVs). Representative electron microscopy for POD 1 timepoints in Patient 1-3 is shown.
  • NTA Nanoparticle tracking analysis
  • FIG.32D Western blot analysis of plasma sEVs was performed for expression of exosome markers CD63, TSG101, flotillin 1, and alix.
  • FIGs.33A-33F show assessment of enriched circulating sEV subpopulations. Bound and unbound sEV fractions for enrichment of T cell sEVs, B cell sEVs, and donor heart sEVs were quality checked.
  • FIG.33A For T cell 24 53258724.1 Attorney Docket No.047162-7412WO1(02347) sEV enrichment, bead bound sEVs were eluted and studied by NTA to confirm purification of intact sEVs. Representative analysis in Patient 7 is shown.
  • FIG.33B Anti-CD3 unbound sEVs were studied for absence of CD3 expression by Western blot. Analysis from Patient 7 is shown.
  • FIG.33C For B cell sEVs, eluted fraction from CD19 antibody bead bound sEVs was analyzed by NTA. Analysis from Patient 7 who had AMR is shown.
  • FIG.33D Anti-CD19 antibody bead unbound sEVs were studied by Western blot for absence of CD19 expression. Analysis from Patient 7 is shown. Human peripheral blood mononuclear cell (PBMC) extract is shown as positive control.
  • FIG.33E For donor heart sEV enrichment, based on HLA I mismatch, sEVs bound to anti-donor HLA I antibody beads were eluted and assessed by NTA. Data from Patient 7 is shown.
  • FIG.33F Anti-donor HLA I antibody bead unbound sEVs were checked for absence of cardiomyocyte markers troponin T (cTnT) and I (cTnI) by Western blot. Analysis in Patient 7 is shown. Heart tissue is shown as positive control.
  • FIGs.34A-34D show donor heart sEV and T cell sEV cargo profiles in Patient 1. The relative expression of sEV cargo compared to POD 1 time point (donor heart sEVs) or pretransplant time point (T cell sEVs) is shown. Results of EMB are delineated for each time- matched sample for ACR grades 0, 1, or 2. ACR episodes treated with escalation of immunosuppression are also shown.
  • FIG.34A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown. Relative expression of cTnT was normalized to TSG101 expression. The normalized expression for POD 1 was set to value of 1, and cTnT expression for all other postoperative time points is shown relative to the POD 1 value.
  • FIG. 34B RT-qPCR for donor heart sEV cTnT to ⁇ -actin mRNA relative expression normalized to POD 1 value set at 1 is shown.
  • FIG.34C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown. Expression of each T cell marker was normalized to TSG101 expression.
  • FIG. 34D Stem loop RT-qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown.
  • Candidate miR biomarker expression was normalized to internal control miR 26 expression.
  • EMB result for each time matched sample is shown, along with time points of escalation of immunosuppression therapy.
  • FIGs.35A-35D show donor heart sEV and T cell sEV cargo profiles in Patient 2.
  • 35A Western blot for donor heart sEV cTnT and TSG101 is shown along with its relative expression.
  • FIG.35B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ -actin mRNA relative expression is shown.
  • FIG.35C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.35D Stem loop RT-qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown.
  • FIGs.36A-36D show donor heart sEV and T cell sEV cargo profiles in Patient 3.
  • FIG. 36A Western blot for donor heart sEV cTnT and TSG101 is shown along with its relative expression.
  • FIG.36B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ -actin mRNA relative expression is shown.
  • FIG.36C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.36D Stem loop RT-qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown, along with time points of escalation of immunosuppression therapy.
  • FIGs.37A-37D show donor heart sEV and T cell sEV cargo profiles in Patient 4.
  • FIG. 37A Western blot for donor heart sEV cTnT and TSG101 is shown along with its relative expression.
  • FIG.37B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ -actin mRNA relative expression is shown.
  • FIG.37C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.37D Stem loop RT-qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown, along with time points of escalation of immunosuppression therapy.
  • FIGs.38A-38D show donor heart sEV and T cell sEV cargo profiles in Patient 5.
  • FIG. 38A Western blot for donor heart sEV cTnT and TSG101 is shown along with its relative expression.
  • FIG.38B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ -actin mRNA relative expression is shown.
  • FIG.38C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.38D Stem loop RT-qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown.
  • FIGs.39A-39F show donor heart sEV and T cell sEV cargo profiles in Patient 6. Availability of more plasma sample enabled analysis for other potential cell-specific markers in sEV subpopulations.
  • FIG.39A Western blot for donor heart sEV cTnT, cTnI, and TSG101 is shown along with its relative expression. cTnI, cardiac isoform troponin I, also showed similar expression pattern as cTnT.
  • FIG.39B RT-PCR for cTnT is shown for the postoperative samples, along with heart tissue as positive control. ⁇ -actin mRNA control is shown.
  • FIG.39C RT-qPCR for donor heart sEV cTnT and cTnI mRNAs to ⁇ -actin mRNA relative expression is shown.
  • FIG.39D Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.39E Stem loop RT- qPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown.
  • FIG.39F Given noted expression of CD4, CD8 protein markers in T cell sEVs, mRNA expression of these markers was also assessed. T cell sEV RT-qPCR for CD4, CD8, and IFN ⁇ mRNAs with expression normalized to ⁇ -actin mRNA is shown. EMB result for each time matched sample is shown, along with time points of escalation of immunosuppression therapy.
  • FIGs.40A-40D show donor heart sEV and T cell sEV cargo profiles in Patient 8.
  • FIG. 40A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown along with its relative expression.
  • FIG.40B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ - actin mRNA relative expression is shown.
  • FIG.40C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.40D Stem loop RTqPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown. There were 8 grade 0 ACR events noted by EMB. Expression profiles of the 8 candidate biomarkers remained stable over follow-up.
  • FIGs.41A-41D show donor heart sEV and T cell sEV cargo profiles in Patient 9.
  • FIG. 41A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown along with its relative expression.
  • FIG.41B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ - actin mRNA relative expression is shown.
  • FIG.41C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.41D Stem loop RTqPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown.
  • FIGs.42A-42D show donor heart sEV and T cell sEV cargo profiles in Patient 10.
  • FIG. 42A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown along with its relative expression.
  • FIG.42B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ - actin mRNA relative expression is shown.
  • FIG.42C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.42D Stem loop RTqPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown. There were 7 grade 0 ACR events noted by EMB. Expression profiles of the 8 candidate biomarkers remained stable over follow-up.
  • FIGs.43A-43D show donor heart sEV and T cell sEV cargo profiles in Patient 11.
  • FIG.43A-43D show donor heart sEV and T cell sEV cargo profiles in Patient 11.
  • FIG.43A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown along with its relative expression.
  • FIG.43B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ - actin mRNA relative expression is shown.
  • FIG.43C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.43D Stem loop RTqPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown.
  • FIGs.44A-44D show donor heart sEV and T cell sEV cargo profiles in Patient 12.
  • FIG. 44A Western blot for donor heart sEV cTnT and TSG101 (canonical exosome marker) is shown along with its relative expression.
  • FIG.44B RT-qPCR for donor heart sEV cTnT mRNA to ⁇ - actin mRNA relative expression is shown.
  • FIG.44C Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101.
  • FIG.44D Stem loop RTqPCR for expression of miR let 7i, miR 101b, and miR 21a in T cell sEVs is shown. EMB result for each time matched sample is shown. There were 5 grade 0 ACR events noted by EMB. Expression profiles of the 8 candidate biomarkers remained stable over follow-up.
  • FIGs.45A-45G show donor heart sEV, T cell sEV, and B cell sEV cargo profiles in Patient 7. This patient developed moderate ACR on POD 7 EMB, with remission on repeat biopsy on POD 15 (mild ACR without myocyte damage) but with development of donor HLA 28 53258724.1 Attorney Docket No.047162-7412WO1(02347) specific antibody.
  • FIG. 45A EMB histology from PODs 57 and 85 is shown. Hematoxylin and eosin staining (4X and 40X magnification) and immunohistochemistry for C4d expression is shown.
  • FIG.45B Western blot for donor heart sEV cTnT, cTnI, and TSG101 is shown along with its relative expression.
  • FIG.45C RT-PCR for cTnT is shown for the postoperative samples, along with heart tissue as positive control. ⁇ -actin mRNA control is shown.
  • FIG.45D RT-qPCR for donor heart sEV cTnT and cTnI mRNAs to ⁇ -actin mRNA relative expression is shown. AMR led to decrease in expression of cTnT and cTnI mRNAs in donor heart sEVs for PODs 71 and 85 time points. After initiation of treatment with rituximab and plasmapheresis on PODs 89 and 90, return in cTnT and cTnI mRNA signals was noted for PODs 92 and 93 timepoints.
  • FIG.45E Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101. Unlike with moderate ACR, CD8 and TCR protein expression in T cell sEVs did not increase with AMR. Increase in CD4 expression was noted for time points of AMR, PODs 57 and 71.
  • FIG.45F B cells sEVs were enriched using anti-CD19 antibody conjugated beads and their protein cargo was tested for expression of immune cell activation protein markers HLA-DR (HLA II) and CD38 by Western blot. Expression relative to TSG101 expression is shown.
  • FIG.45E Western blot for T cell sEV CD4, CD8, TCR, and TSG101 is shown, along with relative expression of each T cell marker normalized to TSG101. Unlike with moderate ACR, CD8 and TCR protein expression in T cell sEVs did not increase with AMR. Increase in CD4 expression was noted for time points of AMR, PODs
  • FIGs.46A-46D show statistical analysis to delineate the diagnostic potential of sEV 29 53258724.1 Attorney Docket No.047162-7412WO1(02347) cargo profiles for detection of grade 2 ACR.
  • Moderate or greater ACR detected on EMB is clinically significant as it mandates escalation in immunosuppression therapy and repeat EMBs until there is resolution of ACR before re-titration of immunosuppression to lower levels. For this reason, the eight candidate biomarkers were analyzed for diagnostic potential to differentiate between grade 2 ACR (moderate) versus grade 0 (none) or grade 1 (mild) ACR episodes – CD4 protein, CD8 protein, TCR protein, cTnT protein, cTnT mRNA, miR let7i, miR 101b, and miR 21a.Wilcoxon rank sum tests were conducted to compare biomarker levels at EMB matched time points of none/ mild versus moderate ACR.
  • FIG.46B Receiver operating characteristic curves for the candidate biomarkers generated by logistic regression are shown.
  • FIG.46C Optimal cut-off points for each biomarker using the Youden method are shown, along with sensitivity, specificity, PPV, and NPV at the optimal cut- off is shown.
  • FIG.46D After treatment initiation for grade > 2 ACR episode, as repeat EMBs are required to assess for resolution of ACR, the extent to which candidate biomarker levels could enable noninvasive detection of resolution of ACR was assessed. Spearman correlations for each biomarker is shown. The results indicate that cTnT mRNA (correlation coefficient - 0.867) and miR 21a (correlation coefficient 0.853) showed the highest correlation.
  • FIGs.47A-47E show receiver operating characteristic curves for the candidate biomarkers generated by generalized estimating equations.
  • FIG.47A ROC curve for CD4.
  • FIG. 47B ROC curve for CD8.
  • FIG.47C ROC curve for TCR.
  • FIG.47D ROC curve for Troponin T protein.
  • FIG.47E ROC curve for Troponin T mRNA.
  • ROC curves for cTnT mRNA, miR let 7i, and miR 21a do not have results because the logistic model for probability has fitted value very close to 1 and so the estimates diverge.
  • FIGs.48A–48C show pertinent clinical data for Patients 1 to 3.
  • FIG.48A Donor specific antibody titers for HLA-DQ, HLA-DP, and HLA-DR in Patients 1 to 3 are shown. Patient 1 showed acute upregulation of donor specific antibodies at the POD 30 time point.
  • FIG. 48B Computer tomography imaging is shown for Patients 1 and 3. Acute bilateral infiltration was noted in Patient 1 for POD 30 time out.
  • FIG.48C Pulmonary function tests for Patients 1 to 3 are shown.
  • FIGs.49A–49E show characterization of plasma sEVs in Patient 1.
  • FIG.49A Western blot analysis of plasma sEVs for exosome markers is shown – CD63, flotillin 1, TSG 101, alix 1, and CD81.
  • FIG.49B NTA of plasma sEVs for PODs 1, 3, and 30 timepoints in Patient 1 are shown. This demonstrated that the phenotype of the majority of the isolated sEVs were in the exosome size range, under 150 nm.
  • FIG.49C Representative NTA screen shot of a video of plasma sEVs for POD 1 in Patient 1.
  • FIG.49D Representative NTA screen shot of a videos of plasma sEVs for POD 3 in Patient 1.
  • FIG.49E Representative NTA screen shot of a video of plasma sEVs for POD 30 in Patient 1.
  • FIGs.50A–50I show data related to circulating T cell sEV and B cell sEV cargoes in a lung transplant patient with mixed ACR/ AMR.
  • FIG.50A Workflow for B cell sEV enrichment and downstream analysis is shown.
  • FIG.50B Anti-CD19 antibody bead-unbound sEVs were analyzed by Western blot for CD19 absence and flotillin-1 (exosome marker) expression. Peripheral blood mononuclear cells (PBMCs) are positive control.
  • FIG.50C Nanoparticle tracking analysis (NTA) of eluted anti-CD19 antibody bead-bound sEVs is shown.
  • FIG.50D Western blot for expression of B cell activation markers HLA-DR, CD20, CD38, IFN ⁇ , IL6-R, BAFF-R, and exosome markers TSG101 and flotillin-1 in B cell sEVs is shown.
  • FIG.50E Western blot of T cell sEVs, enriched from plasma total sEV pool using anti-CD3 antibody beads, is shown for CD3 absence and flotillin-1 expression; along with Jurkat T cell positive control.
  • FIG.50F NTA of eluted T cell sEVs is shown.
  • FIG.50G Western blot for expression of T cell markers, CD4 and CD8 in T cell sEVs is shown.
  • FIG.50H RT-qPCR assays for T cell sEV mRNA cargoes CD8, IFN ⁇ , HLA-DR, and CD4 in Patients 1-3 at POD-1, POD-3, and POD-30 timepoints are shown.
  • FIG.50I RT-qPCR assays for B cell sEV 31 53258724.1 Attorney Docket No.047162-7412WO1(02347) mRNA cargoes, CD19, CD20, IL6-R, BAFF-R, IFN ⁇ , HLA-DR, CD38, and ICOSL, in Patients 1-3 at POD-1, POD-3, and POD-30 timepoints are shown.
  • the present invention relates generally to methods and kits for assessing conditional status, and of treating rejection, of a donor transplant in a subject.
  • the invention includes a method for assessing conditional status of a donor transplant in a subject, the method comprising (i) obtaining and/or having obtained at least one biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; and (iv) comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesic
  • the invention includes a method of treating rejection of a donor transplant in a subject, the method comprising: (i) obtaining and/or having obtained at least one biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; (iv) determining the conditional status of the donor transplant, wherein the determining comprises comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; wherein the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell micro
  • Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. So that the disclosure may be more readily understood, select terms are defined below.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • “About,” as used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, in some instances ⁇ 5%, in some instances ⁇ 1%, and in some instance ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • biological sample refers to a sample of biological material obtained from a subject, e.g., a human subject, including a biological fluid, e.g., blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, biliary fluid and combinations thereof.
  • a biological fluid e.g., blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor
  • the presence of one or more biomarkers is determined in a blood sample obtained from a subject. In certain non-limiting embodiments, the presence of one or more biomarkers is determined in a plasma sample obtained from a subject. In certain non- 34 53258724.1 Attorney Docket No.047162-7412WO1(02347) limiting embodiments, the presence of one or more biomarkers is detected in a urine sample obtained from a subject.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (such as an mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • the non-coding strand used as the template for transcription of a gene or cDNA
  • encoding the protein or other product of that gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.
  • extracellular vesicle small extracellular vesicle
  • small extracellular microvesicle small extracellular microvesicle
  • extracellular microvesicle extracellular microvesicle
  • EV extracellular microvesicle
  • sEV sEV
  • microvesicle are used interchangeably herein, and refer to vesicles that are released from a cell into bodily fluids.
  • sEVs are lipid bilayer vesicles with a size range of about 30 nm to about 2,000 nm in diameter that are released by several types of cells into the extracellular spaces during physiological and pathological conditions. Their membranes contain surface markers of their parent cell type, and their contents reflect the conditional changes imposed on their cells of origin. EVs are found in body fluids, including but not limited to blood, synovial fluids, breast milk, saliva, plasma, and urine. EVs harbor nucleic acids, proteins, lipids and metabolites that reflect their cellular origin.
  • the microvesicle is a vesicle that is released from a cell by exocytosis of intracellular multi vesicular bodies.
  • the microvesicles are exosomes.
  • the microvesicles have an average diameter less than about 400 nm.
  • the microvesicles have an average diameter ranging in size from about 30 nm to about 400 nm.
  • the microvesicles have an average diameter of 100 nm or less. “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules.
  • the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • “Instructional material(s)” as used herein includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of a composition and/or compound of the invention in a kit.
  • the instructional material may describe a method of using the composition and/or compound of the invention in a method of the invention.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.
  • Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
  • isolated means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can 36 53258724.1 Attorney Docket No.047162-7412WO1(02347) exist in a non-native environment such as, for example, a host cell.
  • nucleosides nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • modulating is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as, a human.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject, or individual is a human.
  • non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, fowl, pigs, horses, cows, goats, sheep, etc.
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.”
  • the monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic 37 53258724.1 Attorney Docket No.047162-7412WO1(02347) acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • therapeutic as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.
  • therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
  • the therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • To “treat” a disease or disorder as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • the methods of the present invention are useful for assessing conditional status of a donor transplant in a subject, and for treating the rejection of a donor transplant in a subject when the conditional status indicates that a rejection event of the donor transplant is predicted and/or detected in the subject.
  • the methods disclosed herein provide time-sensitive, accurate biomarker-based platforms for surveillance, diagnosis, and treatment of donor transplant rejection in a patient that do not rely on invasive transplant tissue biopsies which are not practical for frequent repeated testing. Accordingly, the methods of the present invention are ideally suitable for ongoing surveillance and frequent / repeated testing of transplant recipients, thereby providing a significant advantage over current biopsy-based techniques.
  • the methods of the present invention are additionally suitable for detecting and diagnosing transplant rejection soon after the patient has received the donor transplant, i.e., within the first 30 days after transplant, when most acute rejection episodes occur. Furthermore, the methods of the present invention enable noninvasive monitoring for efficacy of treatment of transplant rejection episodes, thereby allowing the practitioner to stop treatment once the patient has recovered from the rejection event. As such, the methods disclosed herein reduce and/or prevent immunosuppression-related complications in donor transplant recipients, such as infections and malignancies.
  • the invention includes a method for assessing conditional status of a donor transplant in a subject, the method comprising (i) obtaining and/or having obtained at least one 39 53258724.1 Attorney Docket No.047162-7412WO1(02347) biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; and (iv) comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; wherein the amount and/or relative level of the immune cell microvesic
  • the invention includes a method of treating rejection of a donor transplant in a subject, the method comprising: (i) obtaining and/or having obtained at least one biological sample from the subject post-transplant; (ii) detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from the at least one biological sample; (iii) quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; (iv) determining the conditional status of the donor transplant, wherein the determining comprises comparing the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker in the at least one biological sample to a baseline value of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker; wherein the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell micro
  • the donor transplant is allogeneic in relation to the subject. In some embodiments of the methods disclosed herein, the donor transplant is xenogeneic in relation to the subject. The methods may be repeated to predict and/or detect additional rejection events in the subject and to treat such rejection events appropriately. In this way, the methods of the invention 40 53258724.1 Attorney Docket No.047162-7412WO1(02347) enable rapid, simple, and safe surveillance and treatment of rejection of a donor transplant as needed, as well as monitoring of the efficacy of the rejection treatment, thereby reducing and/or preventing immunosuppression-related complications. In some embodiments of the methods disclosed herein, the conditional status indicates rejection of the donor transplant.
  • the conditional status indicates tolerance of the donor transplant. In some embodiments, the conditional status indicates recovery from rejection of the donor transplant, for example, after the subject has received treatment for a rejection event. In some embodiments, the method comprises stopping the treatment once the subject has recovered from a rejection event.
  • the immune cell microvesicles are derived from, (e.g., originate from and/or are released from) an immune cell selected from the group consisting of a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a macrophage, a dendritic cell, a natural killer cell, a lymphocyte, a T cell, and a B cell.
  • the immune cell microvesicles are derived from a T cell (i.e., T cell microvesicles). In some embodiments, the immune cell microvesicles are derived from a B cell (i.e., B cell microvesicles).
  • the rejection is acute cellular rejection (ACR). In some embodiments, the ACR is at least grade 2 ACR. It is known in the art that rejection events which are at least grade 2 ACR require immunosuppression therapy. When the rejection is ACR, the immune cell is a T cell.
  • the immune cell is a T cell and the detecting and/or isolating immune cell microvesicles comprises use of an antibody specific for T cells, such as an anti-CD3 antibody.
  • Any T cell specific marker e.g., CD3, CD4, and/or CD8 will allow for detection and/or isolation of immune cell microvesicle (i.e., T cell microvesicles).
  • the rejection is antibody- mediated rejection (AMR).
  • AMR antibody- mediated rejection
  • the immune cell is a B cell.
  • the immune cell is a B cell and the detecting and/or isolating immune cell microvesicles comprises use of an antibody specific for B cells, such as an anti-CD19 antibody and/or an anti-CD38 antibody.
  • Any B cell specific marker e.g., CD5, CD19, CD20, CD22, CD23, CD24, CD27, and/or CD38
  • 41 53258724.1 Attorney Docket No.047162-7412WO1(02347)
  • the rejection is ACR and the rejection therapy targets T cells.
  • the rejection is ACR and the rejection therapy comprises any one or more of steroids (e.g., bolus intravenous steroids), mycophenolate mofetil, and thymoglobulin therapy.
  • the rejection is AMR and the rejection therapy targets B cells.
  • the rejection is AMR and the rejection therapy comprises an anti-CD20 antibody.
  • anti-CD20 antibodies include rituximab and ofatumumab.
  • obtaining and/or having obtained at least one biological sample from the subject comprises obtaining and/or having obtained biological samples from the subject at multiple timepoints post-transplant, that is, after the subject has received the donor transplant.
  • the method comprises obtaining and/or having obtained at least one biological sample from the subject per day post- transplant.
  • the methods comprise obtaining and/or having obtained at least one biological sample from the subject per day post-transplant for at least 30 days.
  • steps of the methods disclosed herein can be performed for each biological sample at the time the biological sample is obtained or shortly thereafter (e.g., on the same day) to enable real-time surveillance, diagnosis, and treatment of donor transplant rejection in a patient.
  • the method further comprises (i) detecting and/or isolating donor cell microvesicles and/or at least one donor cell microvesicle biomarker from the at least one biological sample; (ii) quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; (iii) comparing the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample to a baseline value of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; wherein the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample compared to the baseline value indicates the conditional status of the donor transplant.
  • the biological sample, or a portion or fraction thereof is stored, such as stored at a cold temperature and/or frozen, until it is analyzed to quantify the amount 42 53258724.1 Attorney Docket No.047162-7412WO1(02347) and/or relative level of the immune cell and/or donor cell microvesicles and/or the at least one immune cell and/or donor cell microvesicle biomarker.
  • the method comprises obtaining or having obtained at least one reference biological sample.
  • the reference biological sample is obtained from the subject.
  • the reference biological sample is obtained from a reference individual, i.e., an individual who has not received a donor transplant, or an individual who has received a donor transplant and the donor transplant is tolerated by the individual (i.e., the conditional status is tolerance of the donor transplant in said individual).
  • the reference biological sample is obtained from the donor transplant, either before or after the subject has received the donor transplant.
  • the baseline value of the microvesicles and/or the at least one biomarker is determined by quantifying the amount and/or relative level of the microvesicles and/or at least one microvesicle biomarker from a reference biological sample obtained from the subject or obtained from a reference individual or obtained from the donor transplant, either before or after the subject has received the donor transplant.
  • the baseline value of the microvesicles and/or the at least one biomarker is determined by quantifying the amount and/or relative level of the microvesicles and/or at least one microvesicle biomarker from a reference biological sample obtained from the subject, e.g., before rejection of the donor transplant occurs.
  • the reference biological sample is obtained from the subject pre-transplant.
  • pre-transplant includes any of the days before the subject receives the donor transplant.
  • the reference biological sample can be obtained on day 30 or fewer pre-transplant, on day 25 or fewer pre-transplant, on day 20 or fewer pre-transplant, on day 15 or fewer pre- transplant, on day 10 pretransplant, on day 9 pre-transplant, on day 8 pre-transplant, on day 7 pre-transplant, on day 6 pre-transplant, on day 5 pre-transplant, on day 4 pre-transplant, on day 3 pre-transplant, on day 2 pre-transplant, or on day 1 pre-transplant.
  • pre-transplant also includes the day of transplant but prior to the subject receiving the donor transplant.
  • the reference biological sample is obtained from the subject post-transplant, such as on the day of transplant after the subject has received the donor transplant, or within about 1 43 53258724.1 Attorney Docket No.047162-7412WO1(02347) day to within about 5 days post-transplant.
  • multiple reference biological samples are obtained and analyzed and the results therefrom are averaged to obtain the baseline value of the amount and/or relative level of the immune cell and/or donor cell microvesicles and/or at least one immune cell and/or donor cell microvesicle biomarker.
  • the at least one reference biological sample is the same type of biological sample as the at least one biological sample used for assessing the conditional status.
  • Non-limiting examples of biological samples include biological material obtained from the subject, including a biological fluid, e.g., blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, biliary fluid and combinations thereof.
  • the at least one biological sample comprises plasma.
  • the at least one reference biological sample comprises plasma.
  • the at least one biological sample comprises plasma and the at least one reference biological sample comprises plasma.
  • the at least one immune cell microvesicle biomarker and/or the at least one donor cell microvesicle biomarker is a protein and/or a nucleic acid or portion thereof.
  • nucleic acids include DNA, cDNA, mRNA, tRNA, miRNA, snoRNA, scaRNA, lncRNA, and piRNA.
  • the biomarkers of the present invention reside on the surface of or within the immune cell microvesicles and/or the donor cell microvesicles.
  • the at least one immune cell microvesicle biomarker and/or the at least one donor cell microvesicle biomarker is a protein, a messenger RNA (mRNA), and/or a micro RNA (miRNA).
  • the immune cell microvesicle biomarker is a protein.
  • the immune cell microvesicle biomarker is an mRNA.
  • the immune cell microvesicle biomarker is a micro RNA.
  • the donor cell microvesicle biomarker is a protein.
  • the donor cell microvesicle biomarker is an mRNA. In some embodiments, the donor cell microvesicle biomarker is a micro RNA. In certain embodiments of the methods disclosed herein, the quantified amount of the 44 53258724.1 Attorney Docket No.047162-7412WO1(02347) immune cell microvesicle biomarker and/or the donor cell microvesicle biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the immune cell microvesicle biomarker and/or the donor cell microvesicle biomarker.
  • the control biomarker is generally selected based on the same type of molecule as the immune cell microvesicle biomarker or donor cell microvesicle biomarker that is being analyzed (i.e., a protein, an mRNA, or a micro RNA).
  • the control biomarker has a relatively constant expression level that is not affected by transplant rejection events.
  • Those of skill in the art will be able to select the appropriate control biomarker for use in the methods disclosed herein.
  • Non-limiting examples of an immune cell microvesicle control biomarker include canonical exosome biomarkers such as CD63, flotillin-1, and TSG101, or, e.g., micro RNA hsa miR 26.
  • the immune cell and/or donor cell microvesicles are characterized by a diameter of about 30 nm to about 400 nm. In some embodiments, the immune cell microvesicles and/or the donor cell microvesicles are characterized by a diameter of 100 nm or less.
  • the donor transplant comprises one or more organ(s), tissue(s), and/or cells.
  • the donor transplant is selected from the group consisting of heart, lung(s), kidney(s), liver, pancreas, pancreatic islet tissue(s), and pancreatic beta cells.
  • the donor transplant is heart.
  • the donor transplant is lung.
  • the donor transplant comprises pancreatic islet tissue(s).
  • the donor transplant comprises pancreatic beta cells.
  • the pancreatic beta cells are cultured pancreatic beta cells.
  • the method comprises quantifying the amount and/or relative level of the immune cell microvesicles and comparing the amount and/or relative level of the immune cell microvesicles to the baseline value, wherein an increase of at least about 40% in the amount and/or relative level of the immune cell microvesicles compared to the baseline value indicates rejection of the donor transplant in the 45 53258724.1 Attorney Docket No.047162-7412WO1(02347) subject.
  • the donor transplant is heart and the method comprises quantifying the amount and/or relative level of the immune cell microvesicles and comparing the amount and/or relative level of the immune cell microvesicles to the baseline value, wherein an increase of at least about 40% in the amount and/or relative level of the immune cell microvesicles compared to the baseline value indicates rejection of the donor transplant in the subject.
  • the at least one immune cell microvesicle biomarker is selected from the group consisting of miR let7i, miR 21a, miR 101b, CD4 protein, CD4 mRNA, CD8 protein, CD8 mRNA, donor-specific T cell receptor (TCR) protein, interferon gamma (IFN ⁇ ) mRNA, CD19 mRNA, CD38 mRNA, CD38 protein, HLA-DR mRNA, HLA-DR protein, ICOSL mRNA, CD20 mRNA, IL6-R mRNA, and BAFF-R mRNA, or any combination thereof.
  • the at least one immune cell microvesicle biomarker is miR let7i.
  • the at least one immune cell microvesicle biomarker is IFN ⁇ mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is CD19 mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is CD38 mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is CD38 protein. In some embodiments, the at least one immune cell microvesicle biomarker is HLA-DR mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is HLA-DR protein. In some embodiments, the at least one immune cell microvesicle biomarker is ICOSL mRNA.
  • the at least one immune cell microvesicle biomarker is CD20 mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is IL6-R mRNA. In some embodiments, the at least one immune cell microvesicle biomarker is BAFF-R mRNA. In some embodiments of the methods disclosed herein, the at least one immune cell 46 53258724.1 Attorney Docket No.047162-7412WO1(02347) microvesicle biomarker is miR let7i, miR 21a, and/or miR 101b, and the control biomarker is hsa miR 26.
  • the at least one immune cell microvesicle biomarker is donor-specific TCR protein, and the conditional status of the donor transplant is rejection when the relative level of the donor-specific TCR protein is at least about 2.2-fold greater than the baseline value.
  • the donor transplant is heart, and the at least one donor cell microvesicle biomarker is selected from the group consisting of Troponin T protein, Troponin T mRNA, Troponin I protein, and Troponin I mRNA, or any combination thereof.
  • the donor transplant is heart, and the at least one donor cell microvesicle biomarker is Troponin T protein.
  • the donor transplant is heart, and the at least one donor cell microvesicle biomarker is Troponin T mRNA. 47 53258724.1 Attorney Docket No.047162-7412WO1(02347) In some embodiments, the donor transplant is heart, and the at least one donor cell microvesicle biomarker is Troponin I protein. In some embodiments, the donor transplant is heart, and the at least one donor cell microvesicle biomarker is Troponin I mRNA. In some embodiments of the methods disclosed herein, the donor transplant is heart, the at least one donor cell microvesicle biomarker is Troponin T protein, and the control biomarker is TSG101 protein.
  • the donor transplant is heart
  • the at least one donor cell microvesicle biomarker is Troponin T mRNA
  • the control biomarker is GAPDH mRNA.
  • the at least one donor cell microvesicle biomarker is Troponin T protein
  • the conditional status of the donor transplant is rejection when the relative level of the Troponin T protein is at least about 40% less than the baseline value.
  • the at least one donor cell microvesicle biomarker is Troponin T mRNA
  • the conditional status of the donor transplant is rejection when the relative level of the Troponin T mRNA is at least about 60% less than the baseline value.
  • the donor transplant is lung(s), the immune cell is a T cell, and the at least one immune cell microvesicle biomarker is CD8 mRNA, IFN ⁇ mRNA, HLA-DR mRNA, or any combination thereof.
  • the donor transplant is lung(s), the immune cell is a B cell and the at least one immune cell microvesicle biomarker is CD19 mRNA, CD20 mRNA, CD38 mRNA, IL6-R mRNA, BAFF-R mRNA, IFN ⁇ mRNA, HLA-DR mRNA, ICOSL mRNA, or any combination thereof.
  • kits for assessing the conditional status of a donor transplant in a subject, the kit comprising a means (e.g., capturing agent, reagent, technological platform, or combinations thereof) for detecting and/or isolating immune cell microvesicles and/or at least one immune cell microvesicle biomarker from at least one biological sample obtained from the subject.
  • the conditional status indicates rejection of the donor transplant, tolerance of the 48 53258724.1 Attorney Docket No.047162-7412WO1(02347) donor transplant, or recovery from rejection of the donor transplant (e.g., after treatment of a rejection event).
  • the donor transplant is selected from the group consisting of heart, lung(s), kidney(s), liver, pancreas, pancreatic islet tissue(s), and pancreatic beta cells.
  • the donor transplant is heart.
  • the donor transplant is lung.
  • the donor transplant comprises pancreatic islet tissue(s).
  • the donor transplant comprises pancreatic beta cells.
  • the pancreatic beta cells are cultured pancreatic beta cells.
  • Types of kits include, but are not limited to, packaged probe and primer sets (e.g.
  • kits can comprise a pair of oligonucleotide primers suitable for polymerase chain reaction (PCR) or nucleic acid sequencing, for detecting one or more biomarker(s) to be identified.
  • a pair of primers can comprise nucleotide sequences complementary to a biomarker, and be of sufficient length to selectively hybridize with said biomarker.
  • the complementary nucleotides can selectively hybridize to a specific region in close enough proximity 5' and/or 3' to the biomarker position to perform PCR and/or sequencing.
  • Multiple biomarker-specific primers can be included in the kit to simultaneously assay a large number of biomarkers.
  • the kit can also comprise one or more polymerases, reverse transcriptase and nucleotide triphosphates (e.g., dNTPs), wherein the nucleotide triphosphates can be further detectably labeled.
  • a primer can be at least about 10 nucleotides or at least about 15 nucleotides or at least about 20 nucleotides in length and/or up to about 200 nucleotides or up to about 150 nucleotides or up to about 100 nucleotides or up to about 75 nucleotides or up to about 50 nucleotides in length.
  • the oligonucleotide primers can be immobilized on a solid surface or support, for example, on a nucleic acid microarray, wherein the position of each oligonucleotide primer bound to the solid surface or support is known and identifiable.
  • kits can comprise at least one nucleic acid probe, suitable for in situ hybridization or fluorescent in situ hybridization, for detecting the biomarker(s) to be identified.
  • kits will generally comprise one or more oligonucleotide probes that have specificity for various biomarkers. 49 53258724.1 Attorney Docket No.047162-7412WO1(02347)
  • a kit can comprise at least one antibody for immunodetection of the biomarker(s) to be identified.
  • Antibodies, both polyclonal and monoclonal, specific for a biomarker can be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • the immunodetection reagents of the kit can include detectable labels that are associated with, or linked to, the given antibody or antigen itself
  • detectable labels include, for example, chemiluminescent or fluorescent molecules (rhodamine, fluorescein, green fluorescent protein, luciferase, Cy3, Cy5, or ROX), radiolabels (3H, 35S, 32P, 14C, 131I) or enzymes (alkaline phosphatase, horseradish peroxidase).
  • the biomarker-specific antibody can be provided bound to a solid support, such as a column matrix, an array, or well of a microtiter plate. Alternatively, the support can be provided as a separate element of the kit.
  • a kit can comprise one or more primers, probes, microarrays, or antibodies suitable for detecting one or more biomarkers.
  • the set of biomarkers set forth above can constitute at least 10 percent or at least 20 percent or at least 30 percent or at least 40 percent or at least 50 percent or at least 60 percent or at least 70 percent or at least 80 percent of the species of markers represented on the microarray.
  • a biomarker detection kit can comprise one or more detection reagents and other components (e.g., a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide triphosphates, and in the case of Sanger type DNA sequencing reactions, chain terminating nucleotides, positive control sequences, negative control sequences, and the like) necessary to carry out an assay or reaction to detect a biomarker.
  • a kit can also include additional components or reagents necessary for the detection of a biomarker, such as secondary antibodies for use in immunohistochemistry.
  • a kit can further include one or more other biomarkers or reagents for evaluating other prognostic factors, e.g., stage of rejection.
  • a biomarker detection kit can comprise one or more reagents and/or tools for isolating donor organ- or tissue-specific extracellular microvesicles from a biological sample.
  • a biomarker detection kit can comprise one or more reagents and/or tools for isolating subject T cell-specific, subject B cell- 50 53258724.1 Attorney Docket No.047162-7412WO1(02347) specific, donor organ-specific, or donor tissue-specific extracellular microvesicles from a biological sample.
  • a kit can also include reagents necessary for isolating the protein and/or nucleic acids from the isolated extracellular microvesicles.
  • a kit can further contain means for comparing the biomarker with a reference standard, and can include instructions for using the kit to detect the biomarker of interest.
  • the instructions describes that the change in the level and/or presence of a biomarker, set forth herein, is indicative that the donor transplant in a subject is being rejected and/or is injured.
  • the instructions describe that the change in the level and/or presence of a biomarker, set forth herein, is indicative that the subject is rejecting the donor transplant.
  • the instructions describe that the change in the level and/or presence of a biomarker, set forth herein, is indicative of the level of effectiveness of a therapy, e.g., a therapy for treating rejection of a donor transplant, such as immunosuppressive therapy.
  • a therapy e.g., a therapy for treating rejection of a donor transplant, such as immunosuppressive therapy.
  • Reports, Programmed Computers, and Systems The results of a test (e.g., the conditional state of a transplanted organ, tissue, or cells in a subject), or an individual’s predicted drug responsiveness (e.g., response to immunosuppressive therapy), based on assaying one or more biomarkers, and/or any other information pertaining to a test, can be referred to herein as a "report".
  • a tangible report can optionally be generated as part of a testing process (which can be interchangeably referred to herein as “reporting,” or as “providing” a report, “producing” a report, or “generating” a report).
  • Examples of tangible reports can include, but are not limited to, reports in paper (such as computer-generated printouts of test results) or equivalent formats and reports stored on computer readable medium (such as a CD, USB flash drive or other removable storage device, computer hard drive, or computer network server, etc.).
  • Reports can be part of a database, which can optionally be accessible via the internet (such as a database of patient records or genetic information stored on a computer network server, which can be a "secure database” that has security features that limit access to the report, such as to allow only the patient and the patient's medical practitioners to view the report while preventing other unauthorized individuals from viewing the report, for example).
  • reports can also be displayed on a 51 53258724.1 Attorney Docket No.047162-7412WO1(02347) computer screen (or the display of another electronic device or instrument).
  • a report can include, for example, an individual's medical history, or can just include size, presence, absence or levels of one or more biomarkers (for example, a report on computer readable medium such as a network server can include hyperlink(s) to one or more journal publications or websites that describe the medical/biological implications, such as increased or decreased disease risk, for individuals having certain biomarkers or levels of certain biomarkers).
  • the report can include risk or other medical/biological significance (e.g., drug responsiveness, suggested prophylactic treatment, etc.) as well as optionally also including the biomarker information, or the report can just include biomarker information without including disease risk or other medical/biological significance (such that an individual viewing the report can use the biomarker information to determine the associated disease risk or other medical/biological significance from a source outside of the report itself, such as from a medical practitioner, publication, website, etc., which can optionally be linked to the report such as by a hyperlink).
  • risk or other medical/biological significance e.g., drug responsiveness, suggested prophylactic treatment, etc.
  • the report can just include biomarker information without including disease risk or other medical/biological significance (such that an individual viewing the report can use the biomarker information to determine the associated disease risk or other medical/biological significance from a source outside of the report itself, such as from a medical practitioner, publication, website, etc., which can optionally be linked to the report such as by a hyperlink).
  • a report can further be "transmitted” or "communicated” (these terms can be used herein interchangeably), such as to the individual who was tested, a medical practitioner (e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.), a healthcare organization, a clinical laboratory, and/or any other party or requester intended to view or possess the report.
  • the act of "transmitting” or “communicating” a report can be by any means known in the art, based on the format of the report.
  • "transmitting" or “communicating” a report can include delivering a report ("pushing") and/or retrieving ("pulling”) a report.
  • reports can be transmitted/communicated by various means, including being physically transferred between parties (such as for reports in paper format) such as by being physically delivered from one party to another, or by being transmitted electronically or in signal form (e.g., via e-mail or over the internet, by facsimile, and/or by any wired or wireless communication methods known in the art) such as by being retrieved from a database stored on a computer network server, etc.
  • parties such as for reports in paper format
  • signals form e.g., via e-mail or over the internet, by facsimile, and/or by any wired or wireless communication methods known in the art
  • the disclosed subject matter provides computers (or other apparatus/devices such as biomedical devices or laboratory instrumentation) programmed to carry out the methods described herein.
  • the disclosed subject matter provides a computer programmed to receive (i.e., as input) the identity of the one 52 53258724.1 Attorney Docket No.047162-7412WO1(02347) or more biomarkers disclosed herein, alone or in combination with other biomarkers, and provide (i.e., as output) the risk (e.g., risk of organ or tissue rejection) or other result (e.g., organ or tissue rejection diagnosis or prognosis, drug responsiveness, etc.) based on the level or identity of the biomarker(s).
  • the risk e.g., risk of organ or tissue rejection
  • other result e.g., organ or tissue rejection diagnosis or prognosis, drug responsiveness, etc.
  • Such output can be, for example, in the form of a report on computer readable medium, printed in paper form, and/or displayed on a computer screen or other display.
  • the system is controlled by the individual and/or their medical practitioner in that the individual and/or their medical practitioner requests the test, receives the test results back, and (optionally) acts on the test results to reduce the individual's disease risk, such as by implementing a disease management system.
  • Example 1 Circulating T cell specific extracellular microvesicle profiles in cardiac allograft acute cellular rejection Materials and Methods Study Design: Schematic of the study design is shown in FIG.1.
  • mice All animals were purchased from Jackson Laboratory (Bar Harbor, ME). C57BL/6 (MHC H2-K b ) immunocompetent wild type and C57BL/6 immunodeficient (MHC H2-Kb Prkdc SCID) mice served as recipients. BALB/c (MHC H2-K d ) and C57BL/6 (for syngeneic control) mice served as heart donors. The study included Rejection, Maintenance, and Control arms. In the Rejection arm, full MHC mismatch (BALB/c into C57BL/6) heart transplants were performed, resulting in ACR with allograft asystole by day 12 to 14.
  • Recipient abdominal aorta and inferior vena cava was exposed, and the donor heart pulmonary artery to vena cava and donor aorta to recipient aorta anastomoses were performed to complete the allograft/ isograft implantation. Allograft was assessed daily via percutaneous palpation and heart rate monitoring until the animal was sacrificed or until asystole.
  • Mouse plasma sEV isolation sEVs were isolated from 250 ul to 300 ul of C57BL/6 mouse and BALB/c mouse plasma samples using size exclusion chromatography along with ultracentrifugation (Vallabhajosyula, et al., J Clin Invest.2017;127(4): 1375-1391; Habertheuer, et al., J Thorac Cardiovasc Surg.2018;155(6); Habertheuer, et al., Transplantation. 2022;106(4)). Based on recommendations per MISEV 2018 position statement, 17 EVs investigated in this study are categorized as small EVs ⁇ 200 nm (sEVs).
  • T cell sEVs the distinct subpopulation of CD3-bearing small EVs enriched based on anti-CD3 antibody bead incubation with whole plasma sEVs.
  • Plasma was centrifuged at 500g for 10 minutes to eliminate cell debris and the clear supernatant passed through a Sepharose 2B column (Sigma-Aldrich, St. Louis, MO) and the eluent was collected in 100 ⁇ l fractions.
  • sEV fractions were pooled after monitoring absorbance at 280 nm. The pooled fractions were filtered through a 100 kilodalton cut-off membrane and then ultracentrifuged at 120,000 g for 2 hours at 4 °C.
  • the pelleted sEV fraction was resuspended in 1X PBS and after quality assessment by nanoparticle detector analysis and Western blot for exosome marker expression, it was utilized for downstream applications.
  • sEVs were characterized by protein content (transmembrane proteins CD63, CD81), cytosolic proteins recovered in sEVs (TSG101, flotillin- 1, alix), and sEV purity control assessing for absence of apolipoprotein and cytochrome c 55 53258724.1 Attorney Docket No.047162-7412WO1(02347) (Thery, et al., J Extracell Vesicles.2018;7(1)).
  • sEVs were quantified by total protein amount and by total particle number on the nanoparticle detector.
  • Electron microscopy Transmission electron microscopy analysis was performed to identify sEVs isolated from mouse plasma, Jurkat cell culture supernatant, and human plasma sEVs containing 250 ⁇ g/ ⁇ l of protein equivalent in 10 ⁇ l volume was applied onto 200-mesh copper grids (Quantifoil R1.2/1.3). Images were recorded on Falcon III direct electron detector at a magnification of 25,000x.
  • Antibodies Anti-mouse CD3 antibody (Santa Cruz Biotechnologies Inc., Santa Cruz, CA) was utilized for nanoparticle detector (Nanosight NS300) fluorescence staining.
  • Anti-rabbit, anti-mouse, rabbit IgG, and mouse IgG were also purchased from Santa Cruz Biotechnologies.
  • Anti-rabbit, and anti-mouse conjugated quantum dot (605 nm) were purchased from Life Technologies (Grand Island, NY) and utilized per manufacturer’s protocol for nanoparticle detector fluorescence analysis.
  • Antibodies used in this study for sEV protein analysis were purchased from various manufacturers and used according to the manufacturer suggested dilutions and conditions for Western blot analysis (FIG.11).
  • Enrichment of CD3-expressing small sEVs from mouse and human plasma Anti-CD3 monoclonal antibody was conjugated to N-hydroxysuccinamide (NHS) magnetic beads (ThermoFisher, MA).
  • NHS N-hydroxysuccinamide
  • Anti-CD3 ⁇ antibody (50 ⁇ g for 1x10 10 sEVs) was added to the prewashed beads in 1 X TBS working volume of 500 microliters. Beads were rotated for 2-3 hours at room temperature, and the unbound antibody was separated using magnetic stand. Antibody bound beads were washed one time with 1 X TBS to remove the unbound antibody completely and the bound beads were re-diluted in 1XTBS. Antibody conjugated NHS-beads were added to total sEVs in 1.5 ml Eppendorf tube and rotated for 3 56 53258724.1 Attorney Docket No.047162-7412WO1(02347) hours to overnight at 4 o C.
  • Eppendorf tubes were placed on magnetic stand to remove unbound sEVs and saved as unbound sEVs fraction. Specific sEVs bound to beads were washed one more time with 1XTBS and used for downstream protein and RNA cargo analysis. Bead bound EVs elution. sEVs bound to the beads were incubated with elution buffer (2M Glycine-pH 2.5) in 1.5 ml Eppendorf tube for 5 minutes at room temperature and neutralized with 1M Tris (pH 8.0) in a ratio of 1:10. Eppendorf tube placed on the magnetic stand to collect the released sEVs from beads and applied for downstream applications.
  • elution buffer 2M Glycine-pH 2.5
  • 1M Tris pH 8.0
  • T cell sEVs Nanoparticle detector tracking analysis
  • sEVs were analyzed on the NanoSight NS300 nanoparticle detector on the light scatter mode for quantification and size distribution (Malvern Instruments Inc., Westborough, MA). Experimental sample analysis was performed using NTA3.4 software. T cell specific surface marker detection on EVs was performed using the fluorescence mode.
  • sEVs were lysed in 1X RIPA buffer with 1X protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO). Protein concentration was measured using bicinchoninic acid assay. For each sample 500 ⁇ l of Trizol and 100 ⁇ l chloroform was added and centrifuged at 12,800g for 10 minutes. After centrifugation, the top clear solution was mixed with 2.5 volumes of isopropanol and stored at -80 °C for 1 hour to overnight. Next, sample was centrifuged at 12,800g for 10 minutes and the RNA pellet was washed with 75% ethanol and dissolved in RNAse free water.
  • RNA concentration was estimated 57 53258724.1 Attorney Docket No.047162-7412WO1(02347) on NanoDrop One (ThermoScientific, Waltham, MA).
  • Western blot analysis sEVs were lysed and separated on polyacrylamide gel, transferred onto nitrocellulose membrane (Life Technologies). The blot was blocked, incubated with desired antibody as per manufacturer protocol. Horseradish peroxidase coupled secondary antibody (Santa Cruz Biotechnologies) was added for 1 hour and target protein was detected by chemiluminescence using Image quant LAS 400 Phospho-Imager (GE). Quantification of the bands was performed using Image Quant software.
  • RT-PCR Reverse transcription PCR
  • Total RNA 25 ng to 50 ng was reverse transcribed with the SuperScript III one-step RT-PCR system (Life Technologies) for gene expression validation per manufacturer protocol using the following primers shown in Table 1: Table 1: Primer sequences Gene Forward Reverse C D3 ⁇ AAGTCGAGGACAGTGGCTACTAC CATCAGCAAGCCCAGAGTGATACA (SEQ ID NO: 1) (SEQ ID NO: 2) I FN- ⁇ TCAAGTGGCATAGATGTGGAAGAA TGGCTCTGCAGGATTTTCATG (SEQ ID NO: 3) SEQ ID NO: 4) C XCL10 GGATGGCTGTCCTAGCTCTG ATAACCCCTTGGGAAGATGG (SEQ ID NO: 5) (SEQ ID NO: 6) F oxp3 CAGCTGCCTACAGTGCCCCTAG CATTTGCCAGCAGTGGGTAG (SEQ ID NO: 7) (SEQ ID NO: 8) I L2 CCTGAGCAGGATGGA
  • the miR-Amp cDNA 58 53258724.1 Attorney Docket No.047162-7412WO1(02347) template product was diluted 10-fold and 5 ul were used for PCR assay using Taqman Fast Advanced Master mix (2X) and Taqman Advanced specific miRNA assay (20X) in 15 ul volume of reaction per well. Reactions were performed in Quant Studio 3 qPCR system (Applied biosystems), using 40 cycles, at the following conditions: 95°C for 1 second and anneal/extend at 60°C for 20 seconds. The Ct values of individual miRNAs in the TaqMan Advanced miRNA were derived using the TaqMan system software. All the RT-qPCR reactions were performed in duplicate.
  • Ct values were analyzed by the 2-(DDCt) method
  • the levels of each miRNA were normalized by the level of has-miR-26b as internal control. Normalized expression in the Rejection arm samples were expressed as fold difference compared to normalized expression in time matched syngeneic controls set at a value of 1.
  • Next generation sequencing (NGS) of T cell sEV miRNA cargoes RNA extracted from specific sEVs bound to NHS-CD3 was used for NGS at University of Pennsylvania RNA Core Facility. Quality control tests of the total RNA samples were done by using the Agilent Bioanalyzer and Nanodrop spectrophotometry at the RNA Core Facility. Library preparation and sequencing.
  • sEV RNA samples were assayed for quantity and quality with Agilent 2100 Bioanalyzer and the Agilent RNA 6000 Pico Kit (Agilent Technologies, no.5067-1513). Libraries were prepared using the QIAseq miRNA Library Kit (Qiagen, catalogue no.331502) per protocol in the sample preparation guide. Libraries were assayed for overall quality and quantified using the High Sensitivity DNA Kit for the Agilent 2100 Bioanalyzer (Agilent Technologies, no.5067-4626). Samples were multiplexed for sequencing, then 100-base-pair (bp) single-read sequencing of a multiplexed pool of samples was conducted on an Illumina HiSeq 4000 sequencer.
  • Illumina bcl2fastq v.2.20.0.422 software was used to convert bcl to demultiplexed fastq files. Trimming adaptor sequence and unique molecular identifier extraction.
  • the library preparation kit when sequenced to 100 bp, produces reads with a unique molecular identifier, as well as fixed or nearly fixed sequences.
  • the program ‘cutadapt’ was used to remove the trailing adaptor AGATCGGAAGAGCACACGTCT (SEQ ID NO: 15), with settings ‘-m 36 –max-n 1’.
  • the putative small RNA sequence was extracted using a custom R script.
  • Hierarchical clustering using Euclidean distance and complete-linkage clustering was performed for the 31 up and downregulated microRNAs (
  • Gene targets of the differentially expressed miRNAs were determined by at least 3 validated experiments using miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/). miRNA gene targets were evaluated for fit to Gene Ontology Biological Process (GO: BP) pathways using STRING (https://string-db.org/). Significantly enriched apoptotic or immune-related pathways were then selected by false discovery rates (FDR) ⁇ 0.05 using REVIGO [Supek, et al., PLoS One, 2011. 6(7): p. e21800].
  • Fluorescence activated cell sorting Blood samples were collected using EDTA tubes and subsequently stained with the following anti-mouse cell-surface markers: CD3 (BioLegend, CA), CD4 (BioLegend), CD8 (BioLegend), CD19 (BD Pharmingen), and B220 (BioLegend). For each sample, 10 ⁇ L of whole blood was incubated with a mastermix containing the antibodies for 20 minutes at room temperature in the dark. Samples were analyzed on BD LSR Fortessa flow cytometer (BD Biosciences, NJ), first gated using FSC-A by SSC-A, then single cells using FSC-A by FSC-H.
  • BD LSR Fortessa flow cytometer BD Biosciences, NJ
  • Cardiomyocyte isolation and culture Primary cardiomyocytes were isolated from whole heart preparations according to previously described protocols [Bustamante, et al., Can Med Assoc J, 1982.126(7): p.791-3; Tian, et al., STAR Protoc, 2020.1(2): p.100045].
  • Apoptosis assay was performed using Click-iTTM Plus TUNEL assay for in situ apoptosis detection, using Alexa FluorTM 647 dye kit (C10619-ThermoFisher, MA) following the manufacturer suggested 60 53258724.1 Attorney Docket No.047162-7412WO1(02347) protocol.
  • Primary cardiomyocytes were isolated from whole heart preparations. Briefly, specimen was mechanically dissociated using sharp dissection at room temperature into 5 mm 2 myocardial pieces and further digested using collagenase type II (275 u/mL), protease XXIV (1.2 u/mL) in an oxygenated reaction chamber maintained at 37°C with continuous agitation.
  • cardiomyocyte containing supernatant was strained through a 250 ⁇ M nylon cell strainer and further purified with centrifugation at 30g for 5 minutes at room temperature. Cardiomyocytes were then resuspended in myocyte growth media and plated on a laminin coated culture vessel at a density of 2-6 x 10 4 cells/cm 2 and incubated at 37 ⁇ C, 5% CO2. Non-cardiomyocyte cell types were selected against by addition of 10 ⁇ M cytarabine to culture medium.
  • Apoptosis assay was performed by using Click-iTTM Plus TUNEL assay for in situ apoptosis detection, using Alexa FluorTM 647 dye kit (C10619-ThermoFisher Scientific) following the manufacturer suggested protocol. Briefly, cardiomyocytes from C57BL/6 (syngeneic) mouse and BALB/c (allogeneic) mouse were maintained on 20 mm coverslips coated with laminin and incubated with Control (syngeneic) arm T cell specific EVs or Rejection arm T cell specific EVs (250 ng protein equivalent EVs per well) for 24 hours.
  • TUNEL assay Plasma from 7 rejection arm animals and 4 control arm animals was used for TUNEL assay experiments.1X PBS was used as background control. Cells were washed and fixed with 3.7% formaldehyde. TUNEL assay was conducted with the in-situ Cell Death Detection Kit, to assess for in-situ DNA fragmentation and quantified by fluorescence microscopy. The cells with TUNEL-labelled nuclei (red color) represented as TUNEL-positive cells, and blue stain DAPI labelled all cell nuclei. The fraction of apoptotic nuclei was then measured for each experimental condition and expressed as the frequency of TUNEL+ nuclei in three, randomly selected and non-overlapping microscope fields.
  • EV characterization was 61 53258724.1 Attorney Docket No.047162-7412WO1(02347) performed as previously described [Ratajczak and Ratajczak, Leukemia, 2020, 34: 3126-3135; Sedgwick and D’Souza-Schorey, Traffic, 2018.19(5): 319–327].
  • Isolation and Western blot analysis of T cell sEVs from human plasma Affinity antibody coupled bead purification method was adopted for T cell specific sEV isolation.
  • RNA cargo from circulating T cell EVs was enriched using anti-CD3 antibody conjugated bead methodology, and cDNA synthesis was performed using TaqMan miRNA reverse transcription kit (4366596, Applied Biosystem) for (RT specific) hsa-let-7i (ID: 002221), hsa-miR-21a (ID:000397), and hsa-miR-101b (ID:002253) according to the manufacturer suggested conditions. Then, the reverse transcription product was mixed with specific TaqMan miRNA assay TM primers in TaqMan universal PCR master mix, no AmpErase-UNG (4324018) for amplification.
  • TaqMan miRNA reverse transcription kit 4366596, Applied Biosystem
  • the PCR reaction was conducted at 95°C for 5 min, 50 repeated cycles of 95°C for 5 seconds, and then 60°C for 1 minute. All RT- qPCR Ct values were analyzed by the 2-(DDCt) method. The levels of each miRNA were normalized by the expression level of hsa-miR-26b as internal control provided in the TaqMan miRNA assay.
  • Human T cell receptor sequencing from T cell specific sEVs Circulating T cell EV RNA was extracted using Trizol reagent (Life technologies) followed by phenol-chloroform extraction.
  • RNA was converted to complementary DNA (cDNA) using the protocol described in high-capacity cDNA reverse transcription kit (Applied Biosystems, ref 4368814), and the synthesized cDNA was submitted to Adaptive Biotechnologies for TCR sequencing (immunoSEQ hsTCRB-v4b).
  • cDNA complementary DNA
  • Applied Biosystems, ref 4368814 high-capacity cDNA reverse transcription kit
  • the synthesized cDNA was submitted to Adaptive Biotechnologies for TCR sequencing (immunoSEQ hsTCRB-v4b).
  • Adaptive Biotechnologies for TCR sequencing immunoSEQ hsTCRB-v4b.
  • ACR were chosen for TCR sequencing to assess whether TCR mRNA inside T cell EVs can represent functional clones with intact complementarity determining region 3.
  • Statistical Analysis First, data were checked for distribution.
  • Jurkat T cells release sEVs carrying T cell specific surface markers in vitro Jurkat T cells were cultured in EV-free media, and supernatant was collected for sEV isolation. sEV phenotype was characterized by NTA (FIG.2A), electron microscopy (FIG.2B), and protein marker expression (FIG.2C) per MISEV guidelines. On Western blot, in addition to exosome marker expression (CD63, flotillin-1, and TSG101), T cell markers CD4, CD8, and T cell receptor (TCR) were also detected (FIG.2C).
  • T cell sEVs were isolated at several time points in the three study arms and quality checked for size distribution on NTA (FIG.3A), electron microscopy (FIG.3B), and protein expression (FIG.3C) per MISEV guidelines. That whole plasma EV profiles are similar in Rejection versus Maintenance arms was previously demonstrated [Habertheuer, A., et al., J Thorac Cardiovasc Surg, 2018.155(6): p. 2479-2489].
  • T cell sEV subpopulation was quantified within the total plasma sEV pool using anti-CD3 antibody conjugated quantum dots on NTA fluorescence mode.
  • FIGs. 3D, 3E, 3F Representative signals for T cell EV subpopulation for the Control, Maintenance, and Rejection arms are shown (FIGs. 3D, 3E, 3F).
  • FIGs. 3D, 3E, 3F Representative signals for T cell EV subpopulation for the Control, Maintenance, and Rejection arms are shown (FIGs. 3D, 3E, 3F).
  • serial blood samples from single animal were analyzed over several time points to assess if CD3 sEV signal varied with ACR.
  • CD3 sEV signal remained near baseline levels over follow-up (FIGs.3D, 3E).
  • T cell sEV 63 53258724.1 Attorney Docket No.047162-7412WO1(02347) signal increased by day 4, peaked around day 7-8, and decreased to baseline levels by day 15 post-transplant (FIG.3F) when allograft rejection was complete.
  • FOG.3F day 15 post-transplant
  • T cell sEV levels peaked from baseline values between days 4 to 7, cargo analysis was performed at these 2 time points.
  • T cell sEVs were purified using antibody conjugated bead technology (FIG.5A).
  • the unbound sEV fraction was analyzed for absence of T cell markers, CD3, CD4, and CD8, by Western blot (FIG.5B).
  • bead-bound sEVs were eluted and checked on NTA for detection of nanoparticles in the sEV range (FIG.5C).
  • Anti- CD3 antibody bead-bound sEVs showed expression of CD4, TCR, and immunoregulatory markers, including IFN ⁇ , FoxP3, and Serca-2 (FIG.5D). These markers were upregulated in day 7 compared to day 4 samples in Rejection arm but remained unchanged at low levels in Control arm.
  • T cell sEV mRNA cargoes also showed increased expression of CD3 ⁇ , TCR, IFN ⁇ , and IL2 by RT-PCR on day 7 time point in Rejection arm but not Control arm (FIGs.5E – 5F).
  • T cell sEV miRNA cargo profiles NGS of miRNA cargoes of circulating T cell sEVs was performed to: (i) assess for candidate biomarkers of ACR, and (ii) provide mechanistic insights into potential functional roles of T cell sEVs.
  • FIG.6A Differential expression analysis of T cell sEV cargo was performed (FIG.6B) and identified top 31 differentially expressed miRNAs in days 4 versus 7 (FIG.6C). To determine their biological significance, experimentally validated miRNA gene targets were identified with miRTarBase and pathway analysis was performed which showed changes in immune regulatory processes in accordance with the temporal flow of the ACR process (FIG.6D). Day 4 miRNA cargoes, representing early stages of ACR, showed selective targeting of immune pathways involved in regulation of B cell activation, T cell differentiation, adaptive immune response, lymphoid organ development, and lymphocyte activation (FIGs.6D, 6E).
  • T cell sEV miRNAs serve as candidate biomarkers of ACR.
  • BALB/c (donor) or C57BL/6 (recipient) cardiomyocytes were incubated with either total plasma sEVs, T cell sEVs, CD3 antibody unbound sEVs, or plasma secretome in sEV preparations of day 7 samples from Rejection or Control arms (FIG. 8A). Cytotoxicity was assessed by TUNEL assay. Control arm sEV samples and secretome did not mediate apoptosis of either BALB/c (allogeneic) or C57BL/6 (syngeneic) cells (FIGs.8B, 8E) compared to DNase I treated positive controls (FIGs.8D, 8G).
  • T cell sEVs express these markers.
  • Fs.8I, 8J Western blot
  • CD38 and MHC II markers of T cell activation status, were also upregulated in Rejection arm T cell sEV subset.
  • Circulating T cell sEVs can be detected in clinical heart transplantation.
  • this platform was investigated in heart transplant patients.
  • 3 patients with ACR episodes as diagnosed by endomyocardial biopsy time-matched blood samples were obtained for T cell sEV profiling (FIG.12).
  • Grade >2 ACR is clinically significant, mandating treatment with additional immunosuppression and repeating endomyocardial biopsy to confirm reversal of grade 66 53258724.1
  • Plasma sEVs were characterized by electron microscopy (FIG.
  • FIG.9A 9A
  • NTA FIG.9B
  • Western blot for EV markers FIG.9C
  • Methodology for enrichment of CD3-expressing sEVs in mouse model was translated to clinical setting.
  • Anti-CD3 antibody bead-bound sEVs were eluted and analyzed by NTA to confirm enrichment of intact sEVs (FIG.9D).
  • This fraction was studied by Western blot for confirmation of expression of T cell markers, with their absence in the unbound fraction (FIGs.9E, 9F).
  • T cell intra-sEV cargoes showed upregulation of IFN ⁇ , granzyme B, perforin, Fas ligand, human leukocyte antigen class II, and CD38 by Western blot at time points of grade 2 ACR (FIGs.9G-9I). Resolution of grade 2 ACR led to corresponding decrease in expression of these activation markers in the T cell sEV subset.
  • grade 2 ACR grade 2 ACR
  • T cell clones with intact complementarity determining region 3 sequences can be sequenced from the circulating T cell EV subset in the clinical setting.
  • productive TCR sequences were successfully identified, with 38 unique functional clones (Table 2).
  • Clustering analysis of these 38 sequences against a large reference dataset containing 20 million TCRs demonstrated that only one TCR sequence clustered, indicating that the other 37 TCRs were unique to this cohort (Table 3). This suggested that T cell EVs may carry clone-specific TCRs, potentially facilitating targeted injury to the donor cardiomyocytes via functional TCR expressed as part of their cargo.
  • Table 2 Past-filter TCR sequences with intact complementarity determining region 3 in 4 heart transplant patients with moderate grade 2 ACR events for 11 studied time points is shown.
  • TCR 67 53258724.1 Attorney Docket No.047162-7412WO1(02347) sequences with non-productive CDR3, failed TRBV calls or missing conserved motifs were removed.
  • TRB Clonal CDR3 Sequence Variable- F requen Rank Sample Name gene cy CASSQSRPIIHHNSPLHF TRBV3- 0.2222222222 1 Patient 2_POD- (SEQ ID NO: 17) 1*01 22222 0_NO_REJ_TCRB.tsv CASRSILQHARNTIYF TRBV21- 0.0769230769 1 Patient (SEQ ID NO: 18) 1*01 230769 2_POD13_MOD_REJ_TCRB.
  • T cell EV platform will also enable noninvasive monitoring of efficacy of treatment of clinically significant grade >2 ACR episodes that require additional immunosuppressive therapy, thereby replacing repeat EMB as the standard of care method to confirm successful treatment of ACR.
  • T cell EVs play a role in the pathophysiology associated with ACR: (a) time specific changes in circulating T cell EV profiles are seen in Rejection arm only; (b) increased expression of proinflammatory / allo-stimulatory markers inside T cell EVs (IL2, CXCL10, IFN ⁇ , CD38, MHC II, TCR); (c) pathway analysis of T cell EV miRNA cargoes showed sequential upregulation of immune regulatory pathways and apoptotic pathways on days 4 and 7 post-transplant; (d) miR target analysis showed regulation of genes involved in apoptosis pathways, including HDAC4 [Paroni, et al., Mol Biol Cell, 2004.
  • T cell EVs express proteins important in mediating targeted apoptosis by cytotoxic T cells, including TCR, Fas L, granzyme B, and perforin;
  • TCR cytotoxic T cells
  • Fas L cytotoxic T cells
  • granzyme B granzyme B
  • alloreactive T cell clones may potentiate donor tissue inhibition/ injury and cell death even before they migrate to the allograft via their EVs carrying donor alloreactive clone specific TCRs.
  • the concept of targeted cytotoxicity mediated via T cell EVs contemplated herein is consistent with an interesting observation noted in previous studies of donor tissue EV profiles during ACR.
  • donor tissue specific EVs collect in secondary lymphoid organs such as regional lymph node and promote alloreactive T cell activation via semi-direct, indirect, and direct pathways [DeWolf and Sykes, J Clin Invest, 2017.127(7): p.2473-2481; Barry and Bleackley, Nat Rev Immunol, 2002.2(6): p.401-9; Chavez-Galan, et al., Cell Mol Immunol, 2009.6(1): p.15-25]. While specific alloreactive T cell clones proliferate before migration to the allograft, their activation leads to release of T cell EVs expressing TCR specific to donor tissue into peripheral circulation.
  • Donor specific T cell EVs migrate to allograft and initiate targeted injury/ apoptosis of donor cells, which leads to inhibition of donor EV release. They also directly bind donor EVs in circulation leading to a decrease in the donor EV signal. This is reflected as a decrease in circulating donor EVs early during ACR, before graft infiltration by T cells. This enables the adaptive immune arm to mediate injury/ inhibition of donor tissue even before alloreactive T cells can infiltrate into the allograft. T cell EVs also carry cytokines such as IFN ⁇ , and CXCL10 which facilitate creation of a proinflammatory microenvironment in the donor tissue and may promote donor specific T cell migration to the allograft and potentiate the alloreactive response.
  • cytokines such as IFN ⁇ , and CXCL10 which facilitate creation of a proinflammatory microenvironment in the donor tissue and may promote donor specific T cell migration to the allograft and potentiate the alloreactive response.
  • T cell EVs are mediated via the expressed TCR, like their cellular counterparts. Accordingly, specific T cell EV TCR clonal subpopulations activated during ACR are studied and characterized.
  • Example 2 Circulating Tissue Specific Extracellular Microvesicles for Noninvasive Diagnosis of Grade 2 Acute Cellular Rejection in Clinical Heart Transplantation Materials and Methods
  • EMB endomyocardial biopsy
  • Circulating donor heart EVs and recipient T cell EVs were profiled and compared to EMB.
  • Donor heart EVs were enriched using anti-donor HLA I specific antibody and its cargo was analyzed for cardiac specific troponin T protein and mRNA expression by Western blot and quantitative reverse transcriptase PCR (RT-qPCR).
  • Recipient T cell EVs were purified using anti-CD3 antibody, and its cargo for CD4, CD8, and T cell receptor (TCR) proteins were quantified by Western blot.
  • T cell EV specific miRNA (miR) cargoes let7i, miR 101b, and miR 21a were quantified by stem loop RT-qPCR.
  • samples were collected at pretransplant, post operative days (POD) 1, 2, or 3, and weekly thereafter until time of discharge.
  • Sample collection was coordinated with times of routine blood draw for clinical monitoring to minimize patient discomfort, with single EDTA tube containing 5 ml of patient blood drawn for plasma isolation.
  • single EDTA tube was drawn from the venous access site (internal jugular vein or common femoral vein) placed to obtain endovascular access to perform allograft biopsy. In all cases, EDTA tube was transported at room temperature to the investigator’s laboratory for plasma isolation and downstream EV analysis.
  • Sample collection was performed in 14 consenting consecutive heart transplant patients without any exclusion criteria; of these 5 patients (15.7%) had 10 grade 2 ACR episodes.
  • the 5 patients with grade 2 ACR episodes were matched with 5 patients with similar EMB time points without grade 2 ACR to serve as comparative controls (Patients 1-10).
  • EV analysis was completed in these 10 patients.
  • 61 EMBs were performed over the follow-up period in the 10 patients, with 10 grade 2 ACR episodes, 14 grade 1 ACR episodes, and 37 grade 0 ACR events. All grade 2 ACR episodes occurred within the first 51 days post-transplant, and 8 out of 10 events occurred in the first 30 days post-transplant. There were no exclusion criteria for this study and all the performed EMB time points were included.
  • circulating donor heart EVs and T cell EVs were purified to validate whether other tissue specific intra-EV cargoes may be altered during grade 2 ACR episodes.
  • Candidate biomarkers tested in EV cargoes Eight potential candidate biomarkers from cargoes of circulating donor heart EVs and recipient T cell EVs were selected and tested.
  • cardiac troponin T is a known bona fide cardiomyocyte specific marker, its specific protein and mRNA expression was assessed in donor heart EVs.
  • EV methodologies established in the mouse model were modified and validated for translation to the clinical setting, as described in Example 1. Sensitivity, specificity, and diagnostic accuracy for each candidate biomarker to distinguish > grade 2 ACR was calculated, setting EMB result as the gold standard. In Patient 11, other potential tissue specific markers were tested to assess whether the EV platform may be investigated for other candidate biomarkers to further improve diagnostic accuracy – cardiac specific troponin I protein and mRNA in donor heart EVs, and CD4, CD8, and IFN ⁇ mRNA in T cell EVs. EV cargo signal quantitation and established threshold cut-off for distinguishing > grade 2 ACR. Because the clinical consequence of failing to diagnose grade > 2 ACR can impart marked morbidity to patient, threshold cut-off was optimized to maximize sensitivity for all candidate biomarkers.
  • candidate biomarker signal expression was normalized to internal control marker expression.
  • cardiac troponin T expression was normalized to expression of canonical exosome marker TSG101 by Western blot.
  • the signal for POD 1 time point (POD 1 troponin T/ TSG101 ratio) was set to a value of 1, and relative expression of troponin T signal for other POD time points were calculated to assess for differences in expression.
  • Reduction in relative expression of troponin T protein by > 40% from POD 1 baseline was set as threshold cut-off for > grade 2 ACR.
  • Troponin T mRNA expression in donor heart EVs was analyzed along with expression of GAPDH mRNA as internal control by RT-qPCR.
  • the normalized expression of troponin T (troponin T/ GAPHDH mRNA expression) on POD 1 was set at value of 1, and relative expression compared to POD 1 value was quantified, with > 60% reduction in signal as cut-off for distinguishing > grade 2 ACR.
  • CD4, CD8, and TCR marker expression was normalized internally to expression of canonical exosome marker TSG101. Relative expression for the POD time points was quantified compared to the normalized pretransplant expression of the specific marker set at value of 1.
  • Plasma was processed from peripheral blood sample as previously described. Sample was spun at 2000 RPM x 20 minutes at 4°C and the supernatant representing plasma was collected and stored at -80°C for EV isolation. EV isolation was performed as previously reported. Tissue specific EV purification. Donor heart EVs were enriched from whole plasma EV pool using magnetic beads coupled to anti-donor human leukocyte antigen (HLA) I specific antibodies, with the bead bound EVs representing allograft EV subset.
  • HLA human leukocyte antigen
  • T cell EVs were purified using anti-CD3 antibody conjugated beads.
  • RT-qPCR of EV mRNA cargoes Biomarkers tested cardiac troponin T, CD4, CD8, and IFN ⁇ .
  • Stem loop RT-qPCR of EV miRNA cargoes The expression pattern of these three T cell EV miRNAs (let 7i, miR 101b, and miR 21a) was analyzed by stem loop quantitative RT-PCR assay, with normalization to internal miRNA control, hsa miR 26.
  • Statistical Analysis Statistical analysis was performed to assess whether biomarker levels can differentiate between Acute Cellular Rejection (ACR) and no ACR.
  • biomarkers were analyzed: CD4, CD8, TCR, Troponin T protein, Troponin T mRNA, miRNA Let 7i, miRNA 101b, and miRNA 21a.
  • the level of each biomarker on a specific day was considered as the ratio of the measurement on that day to the pre-transplant measurement or the first post-transplant measurement when the pre-transplant measurement was absent.
  • Wilcoxon rank sum tests were conducted to compare biomarker levels at time points with ACR and without ACR.
  • Generalized estimating equations were used to assess the biomarker effects on the ACR event, accounting for the correlations among biomarker levels on different days within the same patient.
  • ROC curves were constructed for each biomarker using both logistic regression and generalized estimating equations.
  • Optimal cutoff points for each biomarker were determined using the Youden method. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were calculated at the optimal cutoff points. To evaluate whether biomarker levels can detect the 76 53258724.1 Attorney Docket No.047162-7412WO1(02347) relief of rejection, the Spearman correlations between biomarker levels and grades of ACR were calculated for measures after the first observation of an ACR event for each patient.
  • grade > 2 ACR leads to dynamic changes in cargoes of circulating donor heart EVs and T cell EVs - marked suppression of donor heart EV signal secondary to ACR mediated allograft injury and marked increase in T cell EV signal due to activation of alloreactivity during ACR.
  • Clinical Data Overall, none of the patients developed decrease in heart function before or after grade 2 ACR episodes, as confirmed by echocardiography. Maintenance immunosuppression comprised of tacrolimus therapy in all patients. Grade 2 ACR episodes were primarily treated with bolus intravenous steroids, along with selective addition of mycophenolate mofetil and thymoglobulin therapy. There were zero antibody mediated rejection (AMR) episodes or grade > 2 ACR episodes.
  • AMR antibody mediated rejection
  • Plasma EV sample concentration and size distribution was assessed by nanoparticle tracking analysis. Size distribution of EVs was consistent with nanoparticles in the exosome size range (median particle size 100 nm). Plasma EV size distribution and concentration were similar at time points of grade 2 ACR versus grade 0 ACR. In all samples, plasma EVs protein fraction was analyzed for expression of canonical exosome markers CD63 and TSG101, and for absence of cytochrome c (mitochondrial and apoptotic body marker) as part of quality control. Electron microscopy of plasma EVs also showed nanoparticles in the exosome size range. Quality control of tissue specific EV purification was also performed to validate optimal capture.
  • anti-donor HLA I specific antibody conjugated beads were prepared, quality tested for specificity and utilized for purification of donor heart EVs.
  • the unbound fraction representing recipient specific EVs was assessed for absence of expression of donor specific HLA I and cardiac troponin T.
  • the unbound EV fraction was tested for absence of CD3 by Western blot. Tissue specific EV cargo analysis.
  • grade 2 ACR resulted in >40% reduction in relative expression of protein in 8 out of 10 episodes.
  • Grade 1 ACR events showed variable reduction in cardiac troponin T protein expression compared to POD 1 baseline, suggesting the dynamic nature of circulating tissue specific EVs.
  • CD4, CD8, and TCR signals were detected in all patients suggesting that a constitutive circulating T cell EV pool exists in peripheral blood, and upon initiation of immunosuppression at time of heart transplantation, the signal does not change in post-transplant period if there is no > moderate acute cellular rejection episode.
  • grade 2 ACR episodes persistent elevation above threshold was observed for CD4, CD8, and TCR protein signals.
  • 2 grade 0 ACR events were falsely positive, both occurring at time points of repeat EMB after treatment of grade 2 ACR episodes.50% of mild ACR episodes (7 out of 14) also showed CD4 expression levels higher than threshold cut-off.
  • CD8 protein expression was also markedly elevated at all time points of moderate acute rejection (10 out of 10).
  • T cell EV miRNA cargo analysis In the 5 patients without any grade 2 ACR episodes, 79 53258724.1 Attorney Docket No.047162-7412WO1(02347) miR let 7i, miR 101b, and miR 21a expression remained stable at all time points, including grade 1 ACR events by EMB. In the 5 subjects with moderate rejection, T cell EV miRNA cargoes were analyzed for 8 EMB-matched time points with grade 2 ACR – all 3 miRNAs showed time specific elevation in signal at the 8 time points.
  • miRNAs Compared to T cell protein marker analysis, miRNAs showed much higher fold increase in signal with grade 2 ACR, along with more rapid decrease back to baseline upon resolution of grade 2 ACR by EMB. There was high level of consistency in signal changes for each candidate miRNA biomarker in correlation with grade 2 ACR episodes. For miR let 7i, there were no false negative results, and a single false positive result – in Patient 3, repeat EMB on POD 44 (grade 0 ACR) after treatment of grade 2 ACR episode on POD 30 showed signal higher than threshold, although the signal had decreased over 5-fold at this time point compared to POD 30.
  • T cell EV miRNA markers were highly sensitive for grade 2 ACR.
  • Grade 2 ACR may lead to alterations in other tissue specific markers.
  • cardiac troponin T and I proteins are measured in peripheral blood as highly sensitive and specific markers of myocardial infarction.
  • CD4, CD8, and IFN ⁇ mRNAs were upregulated in T cell EVs at the time point of grade 2 ACR specifically, correlating in expression pattern to the other T cell EV markers detailed above. Diagnostic accuracy of the candidate biomarkers. Sensitivity, specificity, and diagnostic accuracy for candidate biomarkers from donor heart EVs and T cell EVs were assessed. For cardiac troponin T protein in donor heart EVs, sensitivity and specificity were 80% and 95.1% respectively, with diagnostic accuracy of 92.2%. There were 2 false negative results, both in Patient 1, and 2 false positive results (Patients 1 POD 7 and Patient 7 POD 7) occurring at times points of grade 1 ACR, where repeat EMB 7-8 days later showed grade 2 ACR.
  • Cardiac troponin T mRNA RT-qPCR assay showed 100% sensitivity, 97.2% specificity, and accuracy of 97.8%. There was one false positive result in Patient 7 at POD 7 time point, where EMB showed grade 1 ACR; troponin T protein and miR 21a markers also met threshold cut-offs for grade 2 ACR at this time point (FIGs.46B, 46F). Surveillance EMB on POD 15 was positive for grade 2 ACR in this patient. CD4, CD8, and TCR protein expression in T cell EVs showed 100% sensitivity for grade 2 ACR detection, with specificity ranging from 82.4% to 88.2% (FIGs.37C – 37E).
  • miR 101b was >36-fold higher than baseline on POD 16 time point, which showed grade 2 ACR.
  • EMB on POD 25 showed grade 1 ACR, but miR 101b levels, though trending on, were 7.4-fold higher than baseline. This was the single false positive result with miR 101b cargo analysis.
  • Sensitivity and specificity for miR 21a expression was 100% and 90.9%.
  • T cell EV miRNA cargo profiles showed improved diagnostic accuracy compared to T cell protein markers, and much higher fold changes in expression at grade 2 ACR time points were observed with miRNAs.
  • Statistical Analysis was performed to assess whether biomarker levels can differentiate between Acute Cellular Rejection (ACR) and no ACR.
  • the eight biomarkers (CD4, CD8, TCR, Troponin T protein, Troponin T mRNA, miRNA Let 7i, miRNA 101b, and miRNA 21a) were analyzed as described in the methods. Scatter plots of the biomarker levels are shown in FIGs.24A – 24H. Box plots of the biomarker levels across ACR grades are shown in FIGs.25A – 25H.
  • the level of each biomarker on a specific day was considered as the ratio of the measurement on that day to the pre-transplant measurement or the first post-transplant measurement when the pre-transplant measurement was absent.
  • Wilcoxon rank sum tests were conducted to compare biomarker levels at time points with ACR and without ACR.
  • Generalized estimating equations were used to assess the biomarker effects on the ACR event, accounting for the correlations among biomarker levels on different days within the same patient.
  • ROC curves were constructed for each biomarker using both logistic regression (FIGs.26A – 26H) and generalized estimating equations (FIGs.27A – 27E).
  • EVs were purified from the patient’s plasma as described in Examples 1 and 2, and B-cell specific EVs were isolated using anti-CD19 and anti- CD38 antibody coated beads. Results B cell EV biomarkers predict antibody-mediated transplant rejection. B cell EVs were analyzed by qPCR for mRNA levels of biomarkers CD19 and CD38 to assess for their ability to detect and/or predict antibody-mediated rejection events. Increases in CD19 and CD38 mRNA levels above baseline were observed on POD 11 and POD 57, prior to AMR detection by biopsy on POD 71, and returned to baseline levels after the patient was treated with rituximab.
  • B cell EV biomarkers are able to predict AMR events, allowing for treatment before the donor heart is damaged.
  • 83 53258724.1 Attorney Docket No.047162-7412WO1(02347)
  • Example 4 Circulating Tissue Specific Extracellular Vesicles as Biomarkers of Acute Cellular Rejection in Clinical Heart Transplantation Materials and Methods Abbreviations.
  • ACR acute cellular rejection
  • AMR antibody mediated rejection
  • AR acute rejection
  • cTnI cardiac isoform Troponin I
  • cTnT cardiac isoform Troponin T
  • EMB endomyocardial biopsy
  • EVs extracellular vesicles
  • HLA Human leukocyte antigen
  • miRNA/ miR microRNA
  • NPV negative predictive value
  • NTA nanoparticle tracking analysis
  • PPV positive predictive value
  • POD postoperative day
  • RT-PCR reverse transcription polymerase chain reaction
  • RT-qPCR reverse transcription- quantitative polymerase chain reaction
  • TCR T cell receptor. Study design and sample collection.
  • Venous blood (5-6 ml EDTA tube) was obtained from 12 patients to post-operative day (POD) 112 at timepoints - pretransplant, PODs 0, 1, 2, or 3, and time points of EMBs. In total, 70 EMB-matched samples were analyzed (average 5.83 biopsies per patient). Characterization from plasma sEVs. Institutional Review Board (IRB) approvals from University of Pennsylvania, Yale University school of Medicine, were obtained along with the consents from patients undergoing heart transplantation.
  • IRB Institutional Review Board
  • Endomyocardial biopsy tissue sections were processed in the histopathology laboratory at Yale University for POD 57 and POD 85 timepoints. EMB tissue was fixed in 10% neutral buffered formalin. Paraffin-embedded sections (5 ⁇ m) were stained for the presence of C4d protein by immunohistochemistry, along with hematoxylin and eosin staining using standard 84 53258724.1 Attorney Docket No.047162-7412WO1(02347) protocols. Images were captured using Leica microscope. Horse radish peroxidase conjugated anti-rabbit secondary antibody was used, and immunoreactivity was observed with the diaminobenzidine chromogen reaction. Antibodies.
  • Antibodies were purchased from different vendors and followed their data sheet suggested dilutions and conditions for Western blot analysis. Antibodies utilized for Western blot and sEV analysis are shown in Table 6. In this study, human specific anti-CD3 (Santa Cruz Biotechnologies Inc., Santa Cruz, CA) and donor HLA I specific antibodies (One Lambda Inc., Canoga Park, CA) were used for NHS-magnetic bead conjugation. Secondary antibodies conjugated to HRP (anti-rabbit, anti-mouse, anti-goat) were purchased from Santa Cruz Biotechnologies Inc, and used for Western blot analysis.
  • HRP anti-rabbit, anti-mouse, anti-goat
  • Donor HLA–specific antibodies, T cell specific (anti-CD3) antibody, and B cell specific (anti-CD19) antibody was covalently conjugated to N-hydroxysuccinamide (NHS) magnetic beads (ThermoFisher, MA) using manufacturer’s protocol, and detailed previously (Example 1 herein and Korutla, et al., Am J Transplant.2024 Mar;24(3):419-435).1-2x10 10 nanoparticles of sEVs were incubated with 86 53258724.1 Attorney Docket No.047162-7412WO1(02347) antibody–magnetic beads complex overnight at 4°C. The bead bound and unbound sEV fractions were separated on magnetic stand.
  • NHS N-hydroxysuccinamide
  • MISEV Extracellular Vesicles
  • sEV subpopulations were purified using beads conjugated to the following 87 53258724.1 Attorney Docket No.047162-7412WO1(02347) antibodies: anti-donor human leukocyte antigen (HLA) I specific antibody (donor heart sEVs), anti-CD3 antibody (T cell sEVs), and anti-CD19 antibody (B cell sEVs).
  • HLA human leukocyte antigen
  • T cell sEVs anti-CD3 antibody
  • B cell sEVs anti-CD19 antibody
  • RNA and protein cargo analysis Total RNA was extracted from sEVs by using Trizol reagent. sEVs were lysed with 500 ul of Trizol, extracted with 100 ul chloroform and centrifuged at 12,800g for 15 minutes.
  • RNA quality and concentration was measured on NanoDrop One (ThermoScientific, MA).
  • bound sEVs were lysed in 1X RIPA buffer containing 1X protease inhibitor cocktail (Sigma-Aldrich Co., MO), followed by the calorimetric assay (BCA) to estimate the protein concentration.
  • RNA was separated on NuPAGE 4-12% Bis-Tris gel and transferred onto iBlot 2 nitrocellulose membrane. The blot was blocked with 5% milk, incubated with desired antibody as per the vendor suggested protocol. HRP conjugated secondary antibody (Santa Cruz Biotechnologies) in 1:5000 dilution was added for 1 hour and target protein was detected by chemiluminescence using Image quant LAS 400 Phospho-Imager (GE). Image Quant software was used to quantify the protein signals. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). sEV RNA (25-50 ng) was reverse transcribed using SuperScript III system (Life Technologies, CA) per manufacturer protocol.
  • RT-PCR Reverse transcription PCR
  • RT-qPCR quantitative RT-PCR
  • cDNA amplification was performed using TaqMan Advanced cDNA synthesis kit (Applied Biosystems, MA). Ct values were analyzed by delta-delta Ct method (Livak and Schmittgen, Methods, 2001.25(4): p.402-8). Primers used are shown in Table 7.
  • RNA 50-100 ng was utilized for cDNA synthesis and amplification using a High-capacity cDNA reverse transcription kit (4368814) according to the supplier instructions (Applied Biosystems, Foster, CA).
  • a High-capacity cDNA reverse transcription kit (4368814) according to the supplier instructions (Applied Biosystems, Foster, CA).
  • 2 ul of synthesized cDNA along with forward and reverse primers was mixed with PowerUp SYBR Green master mix (Applied Biosystems) in each reaction.
  • the RT-qPCR was performed on the QuantStudio 3 real time PCR system (Applied Biosystems) using reaction conditions; 95°C for 2 min, 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds.
  • cDNA was synthesized from sEV RNA 89 53258724.1 Attorney Docket No.047162-7412WO1(02347) using TaqMan miRNA reverse transcription kit (4366596, Applied Biosystem) for (RT specific) hsa-let-7i (ID: 002221), hsa-miR-21a (ID:000397), and hsa-miR-101b (ID:002253). Then, the reverse transcription product was mixed with specific TaqMan miRNA assay TM primers in TaqMan universal PCR master mix, no AmpErase-UNG (4324018) for amplification.
  • TaqMan miRNA reverse transcription kit 4366596, Applied Biosystem
  • the PCR reaction was carried out at 95°C for 5 min, 50 repeated cycles of 95°C for 5 seconds, and then 60°C for 1 minute. All RT-qPCR Ct values were analyzed by the 2 -ddCt method (Livak and Schmittgen, Methods, 2001.25(4): p.402-8). The levels of each miRNA were normalized by the expression level of hsa-miR-26b as internal control provided in the TaqMan miRNA assay. sEV cargo signal quantitation. In all cases, candidate biomarker signal was normalized to internal control. For proteins, quantitation was performed by densitometry on Western blot, and biomarker expression was normalized to TSG101 (canonical exosome marker) expression.
  • AR events in the 12 patients are shown (FIG.31).
  • Receiver operating characteristic (ROC) curves were constructed using both logistic regression and generalized estimating equations. Optimal cutoff points for each biomarker were determined using Youden method. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were calculated at the optimal cutoff points. To evaluate whether biomarker levels could detect effective treatment of moderate ACR, Spearman correlations between biomarker levels and grades of ACR were calculated for measures after the first observation of moderate ACR event for each patient. Results Clinical Data. Donor-recipient demographics, immunosuppressive regimen, and pertinent echocardiographic data are shown in Tables 8-10. Donor recipient HLA profiles are shown in Table 11.
  • ACR Acute cellular Patient rejection
  • AMR Treatment echo- echoc- Demographics
  • AMR treatment rejection suppression treatment
  • pAMR1 pAMR2 timepoints #1
  • Postoperative POD11 LVEF Donor: 35-year-old
  • ACR2 POD8 day (POD) 8 70%, RV normal male, Caucasian, no Solumedrol POD3: Left size & function smoking, no chest 500mg ventricle (LV) trauma, head intravenous (IV) ejection fraction trauma, BMI 39.16 daily x 3 (EF) 60%
  • right Recipient 37-year- POD23: ventricle (RV) & POD30: LVEF old male, Caucasian
  • ACR2 POD22 Prednisone LV normal 70%, mild LV history of smoking, 100mg daily x 5 function hypertrophy, RV body mass index normal size & (
  • Escalation immunosuppression treatment for moderate ACR episodes along with pertinent echocardiographic findings at time points of moderate ACR is shown.
  • Escalation immunosuppression in Patient 7 at time points of pAMR2 is also shown, along with echocardiographic data.
  • Table 11 Recipient and respective donor HLA profiles
  • Recipient-Donor Human Leukocyte Antigen (HLA) profiles are shown.
  • Anti-donor HLA I specific antibody utilized for enrichment of donor heart sEVs is shown in bold and underlined.
  • Plasma sEVs were characterized per MISEV guidelines (Thery, et al., J Extracell Vesicles.2018;7(1)). By electron microscopy, small EVs were detected (FIG.32A), confirmed by nanoparticle tracking analysis (NTA) (FIG.32B). Overall, sEV size distribution and concentration were similar at different time points (FIG.32B). sEV size distribution or concentration when comparing by ACR grade were similar (FIG.32C).
  • CD3 antibody bead- bound fraction was eluted and analyzed by NTA to validate enrichment of intact sEVs (FIG. 33A), and the unbound sEVs were studied for CD3 absence by Western blot (FIG.33B).
  • CD19 antibody bead-bound fraction was eluted for sEV characterization by NTA (FIG.33C) and the unbound sEVs were assessed for CD19 absence by Western blot (FIG.33D)
  • donor heart sEVs were eluted and confirmed by NTA for enrichment of intact sEVs (FIG.33E) and the unbound sEVs were analyzed for absence of troponins by Western blot (FIG.33F).
  • Donor heart and T cell sEV cargo analysis Expression of the 8 candidate biomarkers for EMB-matched time points is shown.
  • FIGs.34A-34D Representative data for Patients 1-6 in whom there were moderate ACR episodes are shown in FIGs.34A-34D, FIGs.35A-35D, FIGs.36A-36D, FIGs. 37A-37D, FIGs.38A-38D, and FIGs.39A-39F, respectively.
  • sEV profiles in Patient 7 with AMR are shown in FIGs.45A-45G.
  • sEV cargo profiles for Patients 8-12 in whom there were no moderate ACR or AMR episodes are shown in FIGs.40A-40D, FIGs.41A-41D, FIGs.42A- 42D, FIGs.43A-43D, and FIGs.44A-44D, respectively.
  • donor heart sEV cTnT protein expression by Western blot is shown, along with cTnT mRNA expression by RT- qPCR.
  • T cell sEVs CD4, CD8, and TCR protein expression by Western blot is shown, along with quantitation of miRs let 7i, 101b, and 21a by stem loop RT-qPCR.
  • Donor heart sEV analysis Overall, in 5 patients without moderate ACR episodes, there were 3 mild ACR episodes and other EMBs showed zero ACR (FIGs.40A-40D, FIGs.41A- 41D, FIGs.42A-42D, FIGs.43A-43D, and FIGs.44A-44D, respectively).
  • cTnT protein signal remained steady or above POD 1 baseline at all time points (FIG.40A, FIG.41A, FIG.42A, FIG.43A, and FIG.44A, respectively).
  • In the 6 subjects with 11 moderate ACR episodes (Patients 1-6), there were also 11 mild ACR events (FIG.34A, FIG.35A, FIG.36A, FIG.37A, FIG.38A, and FIG.39A, respectively).
  • Patient 1 had 4 moderate ACR and 2 mild ACR episodes, requiring escalation of immunosuppression 4 times (FIGs.34A-34D).
  • Patient 3 had 4 99 53258724.1 Attorney Docket No.047162-7412WO1(02347) mild and 2 moderate ACR events (FIGs.36A-36D).
  • moderate ACR resulted in reduction in cTnT expression in 8 out of 11 episodes.
  • There were 2 false positive events that were mild ACR by EMB one in Patient 1 (FIG.34A) and the other in Patient 4 (FIG.37A); repeat EMB 7-8 days later in both patients showed moderate ACR.
  • cTnT mRNA cargo analysis by RT-qPCR showed stable signal in the 5 patients without moderate ACR events (FIG.40B, FIG.41B, FIG.42B, FIG.43B, and FIG.44B, respectively).
  • FIG.34C Western blots for CD4, CD8, TCR, and TSG101 proteins and their relative expression in T cell sEVs are shown (FIG.34C, FIG.35C, FIG.36C, FIG.37C, FIG.38C, FIG.39D, FIG.45E, FIG.40C, FIG.41C, FIG.42C, FIG.43C, and FIG.44C).
  • T cell protein markers remained stable (FIG. 40C, FIG.41C, FIG.42C, FIG.43C, and FIG.44C, respectively).
  • CD4, CD8, and TCR protein signals increased with moderate ACR episodes, and reversal of ACR led to decrease in the signals back towards baseline (FIG.34C, FIG.35C, FIG.36C, FIG.37C, FIG.38C, and FIG.39D, respectively).
  • CD8 expression was elevated at all time points of moderate ACR (11 out of 11).
  • T cell sEV miRNA cargo analysis In 5 patients without moderate ACR episodes, let 7i, miR 101b, and miR 21a expression remained stable (FIG.40D, FIG.41D, FIG.42D, FIG.43D, and FIG.44D, respectively).
  • T cell EV miRs showed strong correlation with increase in expression levels with moderate ACR episodes. Moderate ACR may lead to changes in other cell specific markers. Whether other cell- specific markers may also be altered secondary to ACR was analyzed. First, cardiac troponin I (cTnI) protein and mRNA expression were assessed in donor heart sEVs.
  • cTnI cardiac troponin I
  • AMR would lead to dynamic changes in circulating B cell sEV cargoes (FIG.28).
  • surveillance EMB on POD 57 demonstrated pAMR1, with repeat biopsy on POD 71 showing pAMR2 (FIG.45A), treated with escalation in tacrolimus + azathioprine, and intravenous methylprednisolone.
  • EMB on POD 85 showed persistent pAMR2 requiring further escalation to rituximab and plasmapheresis therapy started on POD 90.
  • cTnT and cTnI protein and mRNA cargoes were downregulated in donor sEVs at time points of pAMR2 (FIGs.45B-45D).
  • T cell sEV markers CD8 and TCR were unchanged, but CD4 was upregulated (FIG.45E).
  • HLA-DR major histocompatibility complex II
  • CD38 proteins were noted at time points of pAMR (FIG.45F).
  • CD38 mRNA and CD19 mRNA expression was also upregulated (FIG.45G).
  • ROC curves were constructed for each biomarker using logistic regression (FIG.46B) and generalized estimating equations (FIGs.47A-47E). Area under curve values were highest for cTnT mRNA (0.99) and for T cell sEV miRNAs (> 0.97) (FIG.46B). Optimal threshold cut-offs for each biomarker along with sensitivity, specificity, PPV, and NPV are shown (FIG.46C). cTnT mRNA demonstrated highest diagnostic accuracy, with sensitivity of 100%, specificity of 97.4%, NPV of 100%, and PPV of 91.7%.
  • sEV RNA signatures showed higher diagnostic accuracy compared to 101 53258724.1 Attorney Docket No.047162-7412WO1(02347) protein markers. Spearman correlations between biomarker signals and ACR grades were calculated after observation of moderate ACR event to assess if sEV biomarkers may enable noninvasive monitoring of ACR treatment efficacy. This demonstrated good to strong correlation of changes in RNA signatures in donor heart sEVs and T cell sEVs with reversal of moderate ACR, especially with cTnT mRNA (FIG.46D).
  • Enrichment of sEV subpopulations improves the signal to noise ratio of biomarker signature compared to peripheral blood sample that represents sEV contribution from all cell types in the body (FIGs.33A-33F).
  • the results of the present study support this hypothesis.
  • Another important aspect of sEV platform may be its utility for ACR diagnosis in the peri-transplant period (first 30 to 45 days).
  • the present study had no exclusion criteria other than patient age. Ten out of 11 moderate ACR episodes in the present study occurred during the first 38 days post-transplant.
  • donor sEVs are constitutively released into peripheral blood immediately after transplantation, reaching steady state under conditions of allograft quiescence. Similar trend was noted with circulating T cell sEVs (Example 1 herein and Korutla, et al., Am J Transplant.2024 Mar;24(3):419-435). At present, it is unclear whether sEV profiles change over long-term follow-up.
  • Example 5 B cell extracellular vesicle biomarkers for detection of antibody mediated rejection in lung transplantation Materials and Methods Sex as a biological variable: Sex was not considered as a biological variable for this study. Antibodies: Antibodies were purchased and recommended dilutions in the manufacturer’s data sheets for Western blot analysis (ST1) were followed.
  • Monoclonal anti-CD3 (SC-1179), and anti-CD19 (SC-18896) antibodies were used to conjugate with the N-hydroxysuccinamide (NHS) magnetic beads (88827) from Pierce Inc (Waltham, MA).
  • NHS N-hydroxysuccinamide
  • Horse radish peroxidase-conjugated secondary anti-rabbit and anti-mouse antibodies for western blot analysis were purchased from Santa Cruz Biotechnologies Inc.
  • Isolation and 104 53258724.1 Attorney Docket No.047162-7412WO1(02347) characterization of small EVs was performed with modification of previously published methodologies (Habertheuer, et al., Transplantation.2022;106(4):754-766; Habertheuer, et al., Am J Transplant.2022;22(7):1909-1918; Example 1 herein and Korutla, et al., Am J Transplant. 2024;24(3):419-435). With the aim of translating the sEV methodologies to the clinical setting, the isolation protocol was modified to performing ultracentrifugation as the primary modality, excluding size exclusion chromatography techniques which are very time consuming and resource intensive, and more difficult to achieve wide application.
  • extensive quality testing was performed to ensure that the sEV cargo analysis experiments were not compromised.
  • Plasma sample ultracentrifugation methodologies were adopted to isolate small EVs by spinning the plasma at 130,000 x g twice for 2 hours and the sEV pellet was resuspended in phosphate buffered saline. Protein concentration was obtained using absorbance spectrometry and 5 ug of sEV protein equivalent was run by nanoparticle tracking analysis (NS300, Malvern Instruments, Westborough, MA) to obtain nanoparticle size distribution and concentration.
  • the mean particle size distribution was ⁇ 100 nm, and in any preparations that the mean size was over 100 nm, the sample was not utilized for further downstream sEV applications.
  • Western blot was performed to assess for expression of exosome markers, CD63, CD81, TSG101, flotillin-1, and alix-1.
  • Western blot was performed to check for absence of calnexin (endoplasmic reticulum marker) and cytochrome c (mitochondria marker), along with appropriate positive control.
  • apolipoproteins in plasma can be in the same size range as sEVs
  • Western blot for absence of apolipoprotein in the sEV preparation was also performed along with total human plasma protein as positive control.
  • sEVs (1.5 to 2.0 x 10 10 particles based on sample availability) were incubated with antibody–magnetic beads complex overnight at 4°C. Using the magnetic stand, bead bound sEVs and unbound EVs were separated.
  • the bound fraction representing the T cell EV subsets, or B cell subsets were washed with phosphate buffered saline and used for the downstream applications (RNA and protein 105 53258724.1 Attorney Docket No.047162-7412WO1(02347) cargo).
  • the bound sEV fraction was tested by Western blot for expression of cell-specific markers (T cell or B cell based on the antibody used) and the unbound sEV fraction was assessed for absence of cell-specific markers and presence of exosome marker, flotillin-1; along with appropriate positive control.
  • RNA and protein cargo analysis Total RNA was extracted from T cell specific and B cell specific small EVs by using Trizol reagent (Ambien, life technologies) followed by phenol chloroform extraction methodology (Example 1 herein and Korutla, et al., Am J Transplant.2024;24(3):419-435). RNA quality and concentration (ng/ ⁇ l) was measured on NanoDrop One (ThermoScientific, Waltham, MA). Bound sEVs were lysed using radioimmunoprecipitation assay buffer and separated on NuPAGE 4-12% Bis-Tris gel and transferred onto iBlot 2 nitrocellulose membrane (Invitrogen).
  • RNAs expression analysis using RT-qPCR RNA enriched from anti-CD3 bound sEVs or from anti-CD19 bound sEVs was used for amplification of cDNA using Vilo cDNA reverse transcription kit (11754050) (Thermo Fisher Scientific).
  • RT-qPCR reactions were performed using synthesized cDNA along with forward and reverse primers (Table 13) mixed with PowerUp SYBR Green master mix (Thermo Fisher Scientific) in each reaction.
  • the RT-qPCR reactions were carried out on the QuantStudio 3 real time qPCR system (Thermo Fisher Scientific).
  • expression levels were normalized to internal control b-actin mRNA expression. Obtained relative quantitative values from cycle threshold (Ct) were normalized to ⁇ -actin expression for that sample and quantified using the 2 ⁇ C T method (Livak, et al., Methods.2001; 25(4):402-408).
  • B cell sEV mRNA cargoes via RT-qPCR quantification of B cell sEV mRNA cargoes via RT-qPCR was performed for 8 markers of B cell activation (i.e., CD19, CD20, IL6-R, BAFF-R, IFN ⁇ , HLA-DR, CD38, ICOS ligand (ICOSL) (Heeger, et al., Nature Rev Nephrology.2024;20:218-232).
  • AMR Antibody-mediated rejection
  • ACR acute cellular rejection
  • Patient-1 developed mixed AMR and ACR; Patients-2 and -3 did not have ACR or AMR.
  • Peripheral blood was collected on postoperative days (PODs) 1, 3, and 30, with surveillance transbronchial lung biopsies performed on POD-30.
  • PODs postoperative days
  • DSAs de novo donor-specific antibodies
  • FVC functional vital capacity
  • FEV1 forced expiratory volume in one second
  • Plasma sEVs enriched with exosomes were isolated using serial ultracentrifugation, and sEVs were characterized per the Society of Extracellular Vesicles guidelines (Welsh, et al., J Extracell Vesicles.2024;13(5):e12451), including expression of exosome markers CD63, flotillin-1, TSG101, alix-1, CD81 on Western blot, along with absence of calnexin (endoplasmic reticulum marker), cytochrome c (mitochondria marker), and apolipoprotein E (plasma lipoprotein marker) (FIG.49A, Table 12).
  • NTA nanoparticle tracking analysis
  • B cell activation protein markers including CD20, class II human leukocyte antigen (HLA)-DR, CD38, IFN ⁇ , IL6 receptor (IL6-R), BAFF receptor (BAFF-R)
  • HLA human leukocyte antigen
  • IL6-R IL6 receptor
  • BAFF-R BAFF receptor
  • T cell sEV mRNA cargoes were quantified for markers of T cell alloreactivity (CD8, IFN ⁇ , HLA-DR, CD4) (Table 13). Relative expression at POD-3 and POD- 30 was compared to POD-1 baseline.
  • Patient-1 with mixed ACR/AMR showed upregulation of T cell markers CD8, IFN ⁇ , and HLA-DR on POD-30 (FIG.50H), while Patients-2 and -3 exhibited marker expression levels similar to baseline across all timepoints (FIG.50H).
  • B cell sEV mRNA cargoes were quantified for 8 markers of B cell activation: CD19, CD20, IL6-R, BAFF-R, IFN ⁇ , HLA-DR, CD38, ICOS ligand (ICOSL).
  • RT-qPCR assays were established for these sEV RNA markers.
  • these markers of B cell activation were upregulated at POD-30 timepoint compared to POD-1 and POD-3 timepoints, coinciding with AMR/ACR diagnosis and treatment (FIG.50I).
  • Patients-2 and -3 showed unchanged B cell sEV marker expression across all timepoints (FIG.50I).
  • Embodiment 11 The method of any one of embodiments 1-10, wherein the method further comprises: (i) detecting and/or isolating donor cell microvesicles and/or at least one donor cell microvesicle biomarker from the at least one biological sample; (ii) quantifying the amount and/or relative level of the donor cell microvesicles and/or 111 53258724.1 Attorney Docket No.047162-7412WO1(02347) at least one donor cell microvesicle biomarker; and (iii) comparing the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample to a baseline value of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker; wherein the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker in the at least one biological sample compared to the baseline
  • Embodiment 26 The method of embodiment 25, wherein the immune cell microvesicles are T cell microvesicles and/or B cell microvesicles.
  • Embodiment 27 The method of embodiment 25 or embodiment 26, wherein obtaining and/or having obtained at least one biological sample from the subject comprises obtaining and/or having obtained biological samples from the subject at multiple timepoints post-transplant.
  • Embodiment 28 The method of any one of embodiments 25-27, wherein the baseline value is determined by quantifying the amount and/or relative level of the immune cell microvesicles and/or at least one immune cell microvesicle biomarker from a reference biological sample 115 53258724.1 Attorney Docket No.047162-7412WO1(02347) obtained from the subject pre-transplant, on transplant day, or within about 1 day to within about 5 days post-transplant.
  • Embodiment 29 The method of any one of embodiments 25-28, wherein the quantified amount of the immune cell microvesicle biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the immune cell microvesicle biomarker.
  • Embodiment 30 The method of any one of embodiments 25-29, wherein the at least one biological sample comprises plasma.
  • Embodiment 31 The method of any one of embodiments 25-30, wherein: (a) the immune cell is a T cell and the rejection is acute cellular rejection (ACR); or (b) the immune cell is a B cell and the rejection is antibody-mediated rejection (AMR).
  • Embodiment 32 The method of embodiment 31, wherein the ACR is at least grade 2 ACR.
  • Embodiment 33 The method of embodiment 31 or embodiment 32, wherein: (a) the rejection is ACR and the rejection therapy comprises any one or more of bolus intravenous steroids, mycophenolate mofetil, and thymoglobulin therapy; or (b) the rejection is AMR and the rejection therapy comprises an anti-CD20 antibody (e.g., rituximab or ofatumumab).
  • the rejection is ACR and the rejection therapy comprises any one or more of bolus intravenous steroids, mycophenolate mofetil, and thymoglobulin therapy
  • the rejection is AMR and the rejection therapy comprises an anti-CD20 antibody (e.g., rituximab or ofatumumab).
  • Embodiment 34 The method of any one of embodiments 25-33, wherein: (a) the immune cell is a T cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD3 antibody; and/or (b) the immune cell is a B cell and the detecting and/or isolating immune cell microvesicles comprises use of an anti-CD19 antibody and/or an anti-CD38 antibody.
  • Embodiment 36 The method of embodiment 35, wherein the baseline value is determined by quantifying the amount and/or relative level of the donor cell microvesicles and/or at least one donor cell microvesicle biomarker from a reference biological sample obtained from the subject pre-transplant, on transplant day, or within about 1 day to within about 5 days post-transplant.
  • Embodiment 37 The method of embodiment 35 or embodiment 36, wherein the quantified amount of the donor cell biomarker is normalized to a quantified amount of a control biomarker to obtain the relative level of the donor cell biomarker.
  • Embodiment 38 The method of any one of embodiments 25-37, wherein the at least one immune cell microvesicle biomarker and/or the at least one donor cell microvesicle biomarker is a protein, a messenger RNA (mRNA), and/or a micro RNA.
  • Embodiment 39 The method of any one of embodiments 25-38, wherein the immune cell microvesicles and/or the donor cell microvesicles are characterized by a diameter of about 30 nm to about 400 nm, preferably wherein the diameter is 100 nm or less.
  • Embodiment 40 The method of any one of embodiments 25-39, wherein the donor transplant is 117 53258724.1 Attorney Docket No.047162-7412WO1(02347) selected from the group consisting of heart, lung(s), kidney(s), liver, pancreas, pancreatic islet tissue(s), and pancreatic beta cells.
  • Embodiment 41 The method of embodiment 40, wherein the donor transplant is heart or lung(s).
  • Embodiment 42 The method of any one of embodiments 25-41, wherein the method comprises quantifying the amount and/or relative level of the immune cell microvesicles and comparing the amount and/or relative level of the immune cell microvesicles to the baseline value, wherein an increase of at least about 40% in the amount and/or relative level of the immune cell microvesicles compared to the baseline value indicates rejection of the donor transplant in the subject.
  • Embodiment 43 The method of any one of embodiments 25-42, wherein the at least one immune cell microvesicle biomarker is selected from the group consisting of miR let7i, miR 21a, miR 101b, CD4 protein, CD4 mRNA, CD8 protein, CD8 mRNA, donor-specific T cell receptor (TCR) protein, interferon gamma (IFN ⁇ ) mRNA, CD19 mRNA, CD38 mRNA, CD38 protein, HLA-DR mRNA, HLA-DR protein, ICOSL mRNA, CD20 mRNA, IL6-R mRNA, and BAFF-R mRNA, or any combination thereof.
  • TCR T cell receptor
  • IFN ⁇ interferon gamma
  • Embodiment 44 The method of embodiment 43, wherein: (a) the at least one immune cell microvesicle biomarker is miR let7i, miR 21a, and/or miR 101b, and the control biomarker is hsa miR 26; and/or (b) the at least one immune cell microvesicle biomarker is CD4 protein, CD8 protein, and/or donor-specific TCR protein, and the control biomarker is TSG101 protein.
  • Embodiment 45 The method of embodiment 43 or embodiment 44, wherein: (a) the at least one immune cell microvesicle biomarker is miR let7i and/or miR 21a, and wherein the conditional status of the donor transplant is rejection when the relative level of the miR let7i and/or the miR 21a is at least about 4-fold greater than the baseline value; (b) the at least one immune cell microvesicle biomarker is miR 101b, and wherein the 118 53258724.1 Attorney Docket No.047162-7412WO1(02347) conditional status of the donor transplant is rejection when the relative level of the miR 101b is at least about 3-fold greater than the baseline value; (c) the at least one immune cell microvesicle biomarker is CD4 protein, and wherein the conditional status of the donor transplant is rejection when the relative level of the CD4 protein is at least about 2.2-fold greater than the baseline value; (d) the at least one immune cell microvesicle biomarker is CD8 protein
  • Embodiment 46 The method of any one of embodiments 35-45, wherein the donor transplant is heart, and wherein the at least one donor cell microvesicle biomarker is selected from the group consisting of Troponin T protein, Troponin T mRNA, Troponin I protein, and Troponin I mRNA, or any combination thereof.
  • Embodiment 47 The method of embodiment 46, wherein: (a) the at least one donor cell microvesicle biomarker is Troponin T protein and the control biomarker is TSG101 protein; (b) the at least one donor cell microvesicle biomarker is Troponin T mRNA and the control biomarker is GAPDH mRNA.
  • Embodiment 48 The method of embodiment 46 or embodiment 47, wherein: (a) the at least one donor cell microvesicle biomarker is Troponin T protein, and wherein the conditional status of the donor transplant is rejection when the relative level of the Troponin T protein is at least about 40% less than the baseline value; and/or (b) the at least one donor cell microvesicle biomarker is Troponin T mRNA, and wherein the conditional status of the donor transplant is rejection when the 119 53258724.1 Attorney Docket No.047162-7412WO1(02347) relative level of the Troponin T mRNA is at least about 60% less than the baseline value.
  • Embodiment 49 The method of any one of embodiments 1-48, wherein the donor transplant is lung(s), further wherein: (a) the immune cell is a T cell and the at least one immune cell microvesicle biomarker is CD8 mRNA, IFN ⁇ mRNA, HLA-DR mRNA, or any combination thereof; and/or (b) the immune cell is a B cell and the at least one immune cell microvesicle biomarker is CD19 mRNA, CD20 mRNA, CD38 mRNA, IL6-R mRNA, BAFF-R mRNA, IFN ⁇ mRNA, HLA-DR mRNA, ICOSL mRNA, or any combination thereof.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Physiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des méthodes d'évaluation de l'état conditionnel, et de traitement du rejet, d'une greffe de donneur chez un sujet. Les méthodes comprennent la détection et/ou l'isolement et la quantification de microvésicules de cellules immunitaires et/ou d'au moins un biomarqueur de microvésicule de cellules immunitaires à partir d'un ou de plusieurs échantillons biologiques obtenus du sujet.
PCT/US2024/052821 2023-10-27 2024-10-24 Profilage de microvésicules de cellules immunitaires pour la surveillance et le traitement d'un rejet de greffe de donneur Pending WO2025090771A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363593761P 2023-10-27 2023-10-27
US63/593,761 2023-10-27
US202463643572P 2024-05-07 2024-05-07
US63/643,572 2024-05-07

Publications (1)

Publication Number Publication Date
WO2025090771A1 true WO2025090771A1 (fr) 2025-05-01

Family

ID=95516421

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/052821 Pending WO2025090771A1 (fr) 2023-10-27 2024-10-24 Profilage de microvésicules de cellules immunitaires pour la surveillance et le traitement d'un rejet de greffe de donneur

Country Status (1)

Country Link
WO (1) WO2025090771A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes
US20160237496A1 (en) * 2013-10-02 2016-08-18 Hitachichemical Company Ltd. Methods for assessing status of post-transplant liver and determining and administering specific treatment regimens
US20190250147A1 (en) * 2015-10-13 2019-08-15 The Trustees Of The University Of Pennsylvania Methods for using enriched exosomes as a platform for monitoring organ status
WO2020097440A1 (fr) * 2018-11-09 2020-05-14 University Of Maryland, Baltimore Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes
US20160237496A1 (en) * 2013-10-02 2016-08-18 Hitachichemical Company Ltd. Methods for assessing status of post-transplant liver and determining and administering specific treatment regimens
US20190250147A1 (en) * 2015-10-13 2019-08-15 The Trustees Of The University Of Pennsylvania Methods for using enriched exosomes as a platform for monitoring organ status
WO2020097440A1 (fr) * 2018-11-09 2020-05-14 University Of Maryland, Baltimore Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées

Similar Documents

Publication Publication Date Title
Feng et al. Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants
de Leur et al. Characterization of donor and recipient CD8+ tissue-resident memory T cells in transplant nephrectomies
Xu et al. Role of osteopontin in amplification and perpetuation of rheumatoid synovitis
Samson et al. Involvement and prognosis value of CD8+ T cells in giant cell arteritis
Spivey et al. Gene expression profiling in acute allograft rejection: challenging the immunologic constant of rejection hypothesis
JP2020126067A5 (fr)
US10385397B2 (en) Biomarkers for determining an allograft tolerant phenotype
Dugast et al. Biomarkers and possible mechanisms of operational tolerance in kidney transplant patients
Chancharoenthana et al. Molecular immune monitoring in kidney transplant rejection: a state-of-the-art review
US20120142550A1 (en) Vanin 1 as a Peripheral Blood Oxidative Stress Sensor
KR20200103747A (ko) 메토트렉세이트 유도성 면역 관용의 바이오마커
Kennel et al. Longitudinal profiling of circulating miRNA during cardiac allograft rejection: a proof-of-concept study
Chen et al. Single-Cell RNA sequencing reveals immune cell dynamics and local intercellular communication in acute murine cardiac allograft rejection
Gambella et al. Caveolin-1 in kidney chronic antibody-mediated rejection: an integrated immunohistochemical and transcriptomic analysis based on the Banff Human Organ Transplant (B-HOT) gene panel
Korutla et al. Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection
JP2013526862A (ja) 腎移植寛容を決定する方法
Zhao et al. Donor-recipient mismatch at the SIRPA locus adversely affects kidney allograft outcomes
Zarkhin et al. Microarrays: monitoring for transplant tolerance and mechanistic insights
JP2010004750A (ja) 自己免疫疾患による組織傷害の発症または発症可能性の診断方法、およびその利用
WO2025090771A1 (fr) Profilage de microvésicules de cellules immunitaires pour la surveillance et le traitement d'un rejet de greffe de donneur
Subramanian et al. HERV1-env induces unfolded protein response activation in autoimmune liver disease: a potential mechanism for regulatory T cell dysfunction
US20240255520A1 (en) Hla-deleted glomerular endothelial cells and diagnostic method using thereof
Liu et al. Cis-interaction between CD52 and T cell receptor complex interferes with CD4+ T cell activation in acute decompensation of cirrhosis
US20130183686A1 (en) Method of evaluating immunosuppression
Srivastava et al. Association studies of Toll‐like receptor gene polymorphisms with allograft survival in renal transplant recipients of North India

Legal Events

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

Ref document number: 24883332

Country of ref document: EP

Kind code of ref document: A1