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WO2017156365A1 - Procédés de génération de lymphocytes t spécifiques à un antigène pour une immunothérapie adoptive - Google Patents

Procédés de génération de lymphocytes t spécifiques à un antigène pour une immunothérapie adoptive Download PDF

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Publication number
WO2017156365A1
WO2017156365A1 PCT/US2017/021730 US2017021730W WO2017156365A1 WO 2017156365 A1 WO2017156365 A1 WO 2017156365A1 US 2017021730 W US2017021730 W US 2017021730W WO 2017156365 A1 WO2017156365 A1 WO 2017156365A1
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Prior art keywords
cells
human patient
population
administering
cancer
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Quenther KOEHNE
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4242—Transcription factors, e.g. SOX or c-MYC
    • A61K40/4243—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

  • Antigen-specific T cells can be used in adoptive immunotherapy to treat infections and cancer, such as cytomegalovirus (CMV) infections, EBV-associated lymphoproliferative disorder (EBV-LPD), and WT1 (Wilms tumor l)-positive leukemia (see, e.g., Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678; O'Reilly et al., 2012, Seminars in
  • Antigen-specific T cells are usually generated from peripheral blood mononuclear cells (PBMCs) collected before mobilization with granulocyte colony-stimulating factor (G-CSF) from peripheral blood stem cell transplant (PBSCT) donors (Clancy et al., 2013, Biol Blood Marrow Transplant 19:725- 734).
  • PBMCs peripheral blood mononuclear cells
  • G-CSF granulocyte colony-stimulating factor
  • PBSCT peripheral blood stem cell transplant
  • PBSCT donors are administered G-CSF so as to increase the number of circulating hematopoietic stem cells (i.e., the donors are G-CSF mobilized) so that peripheral blood of the donors can be used for hematopoietic reconstitution (via PBSCT).
  • G-CSF mobilization has been shown to impair T cell function (Bunse et al, 2013, PloS One 8:e77925).
  • PBSCT donors need to donate blood for a second time, to generate antigen-specific T cells, either before the G-CSF mobilization or after the G-CSF mobilization at a time (e.g., 6 weeks) when they are no longer G-CSF mobilized, thereby increasing the costs, and increasing the medical risk and pain to the donors associated with blood donation.
  • PBMCs exposed to G-CSF can be used to generate CMV- specific T cells that retain functionality, but the method takes an unfractionated portion of the apheresis collection to generate CMV-specific T cells, thereby reducing the number of CD34 + cells available for hematopoietic reconstitution of the transplant recipient (Clancy et al., 2013, Biol Blood Marrow Transplant 19:725-734).
  • the method thereby also has the disadvantage that it uses only apheresis collections that have a threshold number of CD34+ cells. Id.
  • the present invention provides methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer using a CD34 " fraction of an apheresis collection from G-CSF mobilized donors. Also disclosed are methods of treating a human patient using antigen-specific T cells generated by such methods, and methods of assessing antigen-specific T cells for suitability for therapeutic administration to a human patient.
  • kits for generating a population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer comprising ex vivo sensitizing T cells derived from a CD34 " cell population to one or more antigens of the pathogen or cancer, wherein the CD34 " cell population is the product of a method comprising separating CD34 + cells from CD34 " cells in an apheresis collection that comprises T cells from a human donor who is G-CSF mobilized, thereby producing the CD34 " cell population.
  • the methods further comprise, prior to the ex vivo sensitizing step, a step of separating CD34 + cells from CD34 " cells in the apheresis collection, to produce the CD34 " cell population.
  • the apheresis collection is a leukapheresis collection.
  • the separating step comprises sorting the apheresis collection using an anti-CD34 antibody.
  • the anti-CD34 antibody is coupled to magnetic beads, and the sorting of the apheresis collection using the anti-CD34 antibody is performed by magnetic separation.
  • the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized.
  • the step of administering G-CSF comprises administering G-CSF to the human donor for 5 to 6 consecutive days.
  • the step of administering G- CSF comprises administering G-CSF to the human donor once daily at 10 mcg/kg per dose for multiple consecutive days.
  • the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection.
  • the subjecting step comprises subjecting blood from the human donor to apheresis daily on the last two days of G-CSF administration.
  • the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized
  • the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection.
  • the methods further comprise, between the separating step and the ex vivo sensitizing step, a step of isolating peripheral blood mononuclear cells (PBMCs) from the CD34 " cell population.
  • PBMCs peripheral blood mononuclear cells
  • the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with one or more immunogenic peptides or proteins derived from the one or more antigens.
  • the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with antigen presenting cells that present the one or more antigens.
  • the methods further comprise, after the step of isolating PBMCs, a step of enriching T cells from the PBMCs.
  • the step of enriching T cells from the PBMCs comprises sorting the PBMCs using an anti-CD3 antibody.
  • the ex vivo sensitizing step comprises co-culturing the enriched T cells with one or more immunogenic peptides or proteins derived from the one or more antigens.
  • the ex vivo sensitizing step comprises co-culturing the enriched T cells with antigen presenting cells that present the one or more antigens.
  • the antigen presenting cells used in the ex vivo sensitizing step can be any antigen presenting cells suitable for presenting the one or more antigens of the pathogen or cancer, such as dendritic cells, cytokine-activated monocytes, PBMCs, Epstein-Barr virus-transformed B- lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells.
  • the antigen presenting cells used in the ex vivo sensitizing step are dendritic cells.
  • the antigen presenting cells used in the ex vivo sensitizing step are EBV-BLCL cells.
  • the antigen presenting cells are loaded with one or more immunogenic peptides or proteins derived from the one or more antigens.
  • the antigen presenting cells are genetically engineered to express one or more immunogenic peptides or proteins derived from the one or more antigens.
  • the one or more immunogenic peptides or proteins are a pool of overlapping peptides derived from the one or more antigens.
  • the pool of overlapping peptides is a pool of overlapping pentadecapeptides.
  • the one or more immunogenic peptides or proteins are one or more proteins derived from the one or more antigens.
  • the human donor is allogeneic to the human patient.
  • the methods further comprise, after the separating step, recovering the separated CD34 + cells and using the separated CD34 + cells in a peripheral blood stem cell transplantation (PBSCT).
  • PBSCT peripheral blood stem cell transplantation
  • the human patient is the recipient of the separated CD34 + cells in the PBSCT, and the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone the PBSCT. In other embodiments, the human patient is not the recipient of the separated CD34 + cells in the PBSCT.
  • methods of treating a human patient having or suspected of having a pathogen or cancer comprising: (i) generating a population of cells comprising antigen-specific T cells for therapeutic administration to the human patient according to a method described above; and (ii) administering the population of cells comprising antigen- specific T cells to the human patient.
  • the administering step is by bolus intravenous infusion.
  • the administering step comprises administering at least about 1 x 10 5 cells of the population of cells per kg per dose per week to the human patient.
  • the administering step comprises administering about 1 x 10 6 to about 5 x 10 6 cells of the population of cells per kg per dose per week to the human patient.
  • the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient, and a washout period of at least one week between two consecutive doses, wherein no dose of the population of cells is administered during the washout period.
  • the washout period is about 1, 2, 3, or 4 weeks.
  • kits for assessing a population of cells comprising antigen-specific T cells ex vivo sensitized to one or more antigens of a pathogen or cancer, for suitability for therapeutic administration to a human patient having the pathogen or cancer comprising determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype, wherein the predominant effector memory phenotype is CD62L " CCR7 " CD45RA " CD28 + CD27 +/” CD57 +/” , and wherein determining that the antigen-specific T cells do not exhibit a predominant effector memory phenotype indicates that the population of cells is not suitable for therapeutic administration to the human patients.
  • the methods of assessing a population of cells comprising antigen-specific T cells further comprise determining whether CD137 is upregulated in the antigen-specific T cells, wherein determining that CD137 is not upregulated indicates that the population of cells is not suitable for therapeutic administration to the human patient.
  • the human patient has or is suspected of having a pathogen.
  • the pathogen can be a virus, bacterium, fungus, helminth, or protist. In certain embodiments, the pathogen is a virus.
  • the virus is cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the human patient has or is suspected of having a CMV infection.
  • the human patient has a CMV infection.
  • the one or more antigens of CMV in the methods described in this disclosure is CMV pp65, CMV IE1, or a combination thereof.
  • the virus is Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the one or more antigens of EBV in the methods described in this disclosure is EBNAl, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMPl, LMP2, or a combination thereof.
  • the human patient has or is suspected of having an EB V-positive lymphoproliferative disorder (EBV-LPD).
  • the human patient has an EBV-LPD.
  • the EBV-LPD is an EB V-positive lymphoma.
  • the one or more antigens of EBV is EBNAl, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMPl, LMP2, or a combination thereof.
  • the human patient has or is suspected of having an EB V-positive nasopharyngeal carcinoma.
  • the human patient has an EB V-positive nasopharyngeal carcinoma.
  • the one or more antigens of EBV is EBNAl, LMPl, LMP2, or a combination thereof.
  • the virus is polyoma BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.
  • BKV polyoma BK virus
  • JCV John Cunningham virus
  • ADV herpesvirus
  • HAV human immunodeficiency virus
  • influenza virus ebola virus
  • poxvirus poxvirus
  • rhabdovirus or paramyxovirus.
  • the human patient has or is suspected of having a cancer. In some embodiments, the human patient has a cancer.
  • the cancer is a blood cancer.
  • the cancer is a cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin.
  • the one or more antigens of the cancer is WT1 (Wilms tumor 1).
  • the cancer is multiple myeloma or plasma cell leukemia.
  • the present invention provides methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer.
  • T-cell depleted peripheral blood stem cell transplantation TCD-PBSCT
  • T-cell depletion is usually carried out by selecting the CD34 + fraction of the apheresis collection from a G-CSF mobilized transplant donor for use in TCD-PBSCT (O'Reilly et al., 2010, Seminars in Immunology 22: 162-172). While the CD34 + cells are used for transplantation, the CD34 " cells are normally discarded.
  • the normally discarded CD34 " fraction of the apheresis collection from a G-CSF mobilized donor is used to generate functional antigen-specific T cells suitable for therapeutic administration to a human patient to provide an immune response against an undesirable pathogen or a cancer in the patient or suspected of being in the patient.
  • CD34 " fraction of the apheresis collection donors do not need to undergo additional blood collection prior to the G-CSF mobilization or when they are no longer G-CSF mobilized, thereby avoiding the costs, medical risk, and pain associated with additional blood collections.
  • the methods according to the invention do not reduce the number of CD34 + cells, they also avoid the risk associated with possibly insufficient CD34 + cell content to provide hematopoietic reconstitution of transplant recipients, and can be performed independent of any threshold CD34 + cell count in the apheresis collection used to generate the T cells for therapeutic use.
  • kits for generating a population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer comprising ex vivo sensitizing T cells derived from a CD34 " cell population to one or more antigens of the pathogen or cancer, wherein the CD34 " cell population is the product of a method comprising separating CD34 + cells from CD34 " cells in an apheresis collection that comprises T cells from a human donor who is G-CSF mobilized, thereby producing the CD34 " cell population.
  • the methods further comprise, prior to the ex vivo sensitizing step, a step of separating CD34 + cells from CD34 " cells in the apheresis collection, to produce the CD34 " cell population.
  • the separating step also comprises collecting the CD34 " cell population;
  • the methods further comprise, after the separating step, a step of collecting the CD34 " cell population.
  • the apheresis collection is a leukapheresis collection.
  • the separating step comprises sorting the apheresis collection using an anti-CD34 antibody.
  • the sorting of the apheresis collection using an anti-CD34 antibody is performed by Fluorescence Activated Cell Sorting (FACS).
  • the anti-CD34 antibody is coupled to magnetic beads, and the sorting of the apheresis collection using the anti-CD34 antibody is performed by magnetic separation.
  • the sorting of the apheresis collection using the anti-CD34 antibody is performed by a CliniMACS® system, e.g., the CliniMACS® Cell Selection System.
  • the sorting of the apheresis collection using the anti-CD34 antibody is performed using the CliniMACS® CD34 Reagent System.
  • the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized.
  • the step of administering G-CSF is by subcutaneous administration.
  • the step of administering G-CSF comprises administering G-CSF to the human donor for 3 to 8 consecutive days.
  • the step of administering G- CSF comprises administering G-CSF to the human donor for 4 to 6 consecutive days.
  • the step of administering G-CSF comprises administering G-CSF to the human donor for 5 to 6 consecutive days.
  • the step of administering G-CSF comprises administering G-CSF to the human donor for 4 consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor for 5 consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor for 6 consecutive days.
  • the step of administering G-CSF comprises administering G-CSF to the human donor at 5-20 mcg/kg per day. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor at 10 mcg/kg per day. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor daily. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor twice daily. In a specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor once daily at 5-20 mcg/kg per dose for multiple consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor once daily at 10 mcg/kg per dose for multiple consecutive days.
  • the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis (e.g., leukapheresis) to produce the apheresis collection.
  • apheresis e.g., leukapheresis
  • the subjecting step is within 6 weeks of the step of administering G-CSF.
  • the subjecting step is within 1 month of the step of administering G-CSF.
  • the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is within three weeks of the step of administering G- CSF.
  • the subjecting step is after the step of administering G-CSF, in specific
  • the subjecting step is within two weeks of the step of administering G-CSF.
  • the subjecting step is within one week of the step of administering G-CSF.
  • the subjecting step is multiple days (e.g., 3-7 days) after the step of administering G-CSF.
  • the subjecting step is two days after the step of administering G-CSF.
  • the subjecting step is one day after the step of administering G-CSF.
  • the subjecting step is on the same day as the step of administering G-CSF.
  • the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration.
  • apheresis e.g., leukapheresis
  • the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration.
  • the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration.
  • the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration.
  • apheresis e.g., leukapheresis
  • the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration and on the day after.
  • apheresis e.g., leukapheresis
  • the methods comprise administering G-CSF to the human door (e.g., once daily at 10 mcg/kg per dose) for 4 to 6 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) on the last day, or the last two days, or the next to last day, of G-CSF administration to produce the apheresis collection.
  • apheresis e.g., leukapheresis
  • the methods comprise administering G-CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 4 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection.
  • apheresis e.g., leukapheresis
  • the methods comprise administering G- CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 5 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection.
  • the methods comprise administering G-CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 6 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection.
  • Apheresis can be performed by any method known in the art.
  • apheresis is performed by centrifugation, for example, by continuous flow centrifugation or intermittent flow centrifugation.
  • apheresis is performed by filtration.
  • apheresis is performed by using an automated apheresis machine.
  • the ex vivo sensitizing step can be performed by any method known in the art to stimulate T cells to be antigen-specific ex vivo, such as the method described in the example in Section 5 herein, or a method as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123-1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360-4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 1480- 1492;
  • the methods further comprise, between the separating step and the ex vivo sensitizing step, a step of isolating peripheral blood mononuclear cells (PBMCs) from the CD34 " cell population.
  • PBMCs can be isolated from the CD34 " cell population by any method known in the art to isolated PBMCs from a blood sample, such as by Ficoll-Hypaque centrifugation as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; and the example in Section 5 herein.
  • the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with antigen presenting cells that present the one or more antigens.
  • the methods further comprise, after the step of isolating PBMCs, a step of enriching T cells from the PBMCs.
  • T cells can be enriched from the PBMCs by any method known in the art to enrich T cells from a blood sample or PBMCs.
  • Non-limiting exemplary methods for enriching T cells can be found in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Hasan et al., 2009, J Immunol 183 : 2837-2850; and Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678.
  • the step of enriching T cells from the PBMCs comprises sorting the PBMCs using an anti-CD3 antibody. In specific embodiments, the step of enriching T cells from the PBMCs comprises depleting of adherent monocytes and natural killer cells from the PBMCs. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with antigen presenting cells that present the one or more antigens.
  • the antigen presenting cells used in the ex vivo sensitizing step can be any antigen presenting cells suitable for presenting the one or more antigens of the pathogen or cancer, such as dendritic cells, cytokine-activated monocytes, PBMCs, Epstein-Barr virus-transformed B- lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells.
  • the antigen presenting cells used in the ex vivo sensitizing step are dendritic cells.
  • the antigen presenting cells used in the ex vivo sensitizing step are EBV-BLCL cells.
  • the antigen presenting cells are derived from the human donor.
  • the antigen presenting cells can be obtained by any method known in the art, such as the method(s) described in Koehne et al., 2000, Blood 96: 109-117; Koehne et al., 2002, Blood 99: 1730-1740; Trivedi et al., 2005, Blood 105:2793-2801; O'Reilly et al., 2007, Immunol Res 38:237-250; Hasan et al., 2009, J Immunol 183 : 2837-2850; Barker et al., 2010, Blood 116:5045-5049; O' Reilly et al., 2011, Best Practice & Research Clinical Haematology 24:381-391; Doubrovina et al., 2012, Blood 120: 1633-1646; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678.
  • the antigen presenting cells are loaded with one or more immunogenic peptides or proteins derived from the one or more antigens.
  • Non-limiting exemplary methods for loading antigen presenting cells with peptide(s) derived from antigen(s) can be found in Trivedi et al., 2005, Blood 105:2793-2801; and Hasan et al., 2009, J Immunol 183 : 2837-2850.
  • the antigen presenting cells are genetically engineered to express one or more immunogenic peptides or proteins derived from the one or more antigens.
  • Any appropriate method known in the art for introducing nucleic acid vehicles into cells to express proteins can be used to genetically engineer the antigen presenting calls to express the one or more immunogenic peptides or proteins derived from the one or more antigens.
  • the one or more immunogenic peptides or proteins are a pool of overlapping peptides derived from the one or more antigens.
  • the pool of overlapping peptides is a pool of overlapping pentadecapeptides.
  • the one or more immunogenic peptides or proteins are one or more proteins derived from the one or more antigens.
  • the human donor is seropositive for the one or more antigens of the pathogen or cancer.
  • the human donor is an adult.
  • the human donor is allogeneic to the human patient; thus the population of cells is allogeneic to the human patient.
  • the human donor is a donor for a peripheral blood stem cell transplantation (PBSCT).
  • the methods further comprise, after the separating step, recovering the separated CD34 + cells and using the separated CD34 + cells in the PBSCT.
  • the human patient is the recipient of the separated CD34 + cells in the PBSCT, and the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone the PBSCT. In other embodiments, the human patient is not the recipient of the separated CD34 + cells in the PBSCT.
  • the apheresis collection that is employed in a method of the invention, to generate antigen-specific T cells is selected for use independent of CD34 + cell count in the apheresis collection.
  • the apheresis collection has equal to or less than 2.5 x 10 6 CD34 + cells per kg of the PBSCT recipient's weight.
  • the apheresis collection has equal to or less than 2 x 10 6 CD34 + cells per kg of the PBSCT recipient's weight.
  • the apheresis collection has equal to or less than 1.5 x 10 6 CD34 + cells per kg of the PBSCT recipient's weight.
  • the apheresis collection has equal to or less than 1 x 10 6 CD34 + cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 5 x 10 5 CD34 + cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10 5 CD34 + cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 5 x 10 4 CD34 + cells per kg of the PBSCT recipient's weight.
  • the apheresis collection has equal to or less than 1 x 10 4 CD34 + cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 2.5 x 10 4 CD34 + cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10 4 CD34 + cells per kg of the PBSCT recipient's weight.
  • the apheresis collection has less than 10 9
  • the apheresis collection has less than 5 x 10 8 mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 1 x 10 8 mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 5 x 10 7 mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 1 x 10 7 mononuclear cells per kg of the PBSCT recipient's weight.
  • the methods further comprise, after the ex vivo sensitizing step, cryopreserving a cell population comprising the ex vivo sensitized T cells for storage.
  • the methods further comprise, after the ex vivo sensitizing step, cryopreserving a cell population comprising the ex vivo sensitized T cells for storage, storing the cell population, thawing the cell population, and optionally expanding the ex vivo sensitized T cells of the cell population in vitro to generate the population of cells for therapeutic
  • the methods can further comprise a step of administering the thawed cell population, or thawed and expanded cell population, to the human patient.
  • the methods further comprise, after the ex vivo sensitizing step, expanding the sensitized T cells in vitro to generate the population of cells for therapeutic administration, wherein the sensitized T cells have not been cryopreserved for storage.
  • kits for treating a human patient having or suspected of having a pathogen or cancer comprising: (i) generating a population of cells comprising antigen-specific T cells for therapeutic administration to the human patient according to a method described in Section 4.1; and (ii) administering the population of cells comprising antigen-specific T cells to the human patient.
  • the administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the administration is intravenous. In certain embodiments, the administering step is by infusion of the population of cells. In specific embodiments, the infusion is bolus intravenous infusion.
  • the administering step comprises administering at least about 1 x 10 5 cells of the population of cells per kg per dose per week to the human patient. In specific embodiments, the administering step comprises administering about 1 x 10 6 to about 1 x 10 7 cells of the population of cells per kg per dose per week to the human patient. In specific
  • the administering step comprises administering about 1 x 10 6 to about 5 x 10 6 cells of the population of cells per kg per dose per week to the human patient.
  • the administering step comprises administering about 1 x 10 6 to about 2 x 10 6 cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 1 x 10 6 cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 2 x 10 6 cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 3 x 10 6 cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 5 x 10 6 cells of the population of cells per kg per dose per week to the human patient.
  • the administering step comprises administering at least 2 doses of the population of cells to the human patient. In specific embodiments, the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient. In a specific embodiment, the administering step comprises administering 2 doses of the population of cells to the human patient. In another specific embodiment, the administering step comprises administering 3 doses of the population of cells to the human patient. In another specific embodiment, the administering step comprises administering 4 doses of the population of cells to the human patient [0058] In certain embodiments, the administering step comprises a washout period between two consecutive doses, wherein no dose of the population of cells is administered during the washout period.
  • the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks.
  • the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient, and a washout period of at least one week between two consecutive doses, wherein no dose of the population of cells is administered during the washout period.
  • the washout period is about 1, 2, 3, or 4 weeks.
  • the washout period is about 2 weeks.
  • the washout period is about 3 weeks.
  • the washout period is about 4 weeks.
  • the administering step comprises administering at least two cycles (e.g., 2, 3, 4, 5, or 6 cycles) of one dose per week of the population of cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks), each cycle separated by a washout period during which no dose of the population of cells is administered.
  • at least two cycles e.g., 2, 3, 4, 5, or 6 cycles
  • each cycle separated by a washout period during which no dose of the population of cells is administered.
  • the at least two consecutive weeks are 2 consecutive weeks. In another specific embodiment, the at least two consecutive weeks are 3 consecutive weeks. In another specific embodiment, the at least two consecutive weeks are 4 consecutive weeks.
  • the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks.
  • an additional cycle is administered only when the previous cycle has not exhibited toxicity (for example, no grade 3-5 serious adverse events, graded according to NCI CTCAE 4.0).
  • a first dosage regimen described herein is carried out for a first period of time, followed by a second and different dosage regimen described herein that is carried out for a second period of time, wherein the first period of time and the second period of time are optionally separated by a washout period.
  • the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks.
  • the second dosage regimen is carried out only when the first dosage regimen has not exhibited toxicity (for example, no grade 3-5 serious adverse events, graded according to NCI CTCAE 4.0).
  • the methods of treating a human patient having or suspected of having a pathogen or cancer as described above further comprise, after administering to the human patient a first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1, administering to the human patient a second population of cells comprising antigen-specific T cells, wherein the second population of cells is restricted by a different HLA allele (different from that of the first population of cells) shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, and the antigen-specific T cells in the second population of cells are sensitized to an antigen of the pathogen or cancer.
  • HLA allele different from that of the first population of cells
  • the methods of treating a human patient having or suspected of having a pathogen or cancer comprise administering a first cycle of one dose per week of the first population of cells comprising antigen-specific T cells, generated according to a method described in Section 4.1, for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks) followed by a washout period during which no dose of the first or second population of cells is administered, followed by a second cycle of one dose per week of the second population of cells comprising antigen-specific T cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks).
  • the washout period is at least about 1 week (e.g., about 1-6 weeks).
  • the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks.
  • the human patient has no response, an incomplete response, or a suboptimal response (i.e., the human patient may still have a substantial benefit from continuing treatment, but has reduced chances of optimal long-term outcomes) after administering the first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1 and prior to administering the second population of cells comprising antigen-specific T cells.
  • the methods of treating a human patient having or suspected of having a pathogen or cancer as described above further comprise, before administering to the human patient a first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1, administering to the human patient a second population of cells comprising antigen-specific T cells, wherein the second population of cells is restricted by a different HLA allele (different from that of the first population of cells) shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, and the antigen-specific T cells in the second population of cells are sensitized to an antigen of the pathogen or cancer.
  • HLA allele different from that of the first population of cells
  • the methods of treating a human patient having or suspected of having a pathogen or cancer comprise administering a first cycle of one dose per week of the second population of cells comprising antigen-specific T cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks) followed by a washout period during which no dose of the first or second population of cells is administered, followed by a second cycle of one dose per week of the first population of cells comprising antigen-specific T cells, generated according to a method described in Section 4.1, for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks).
  • the washout period is at least about 1 week (e.g., about 1-6 weeks).
  • the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks.
  • the human patient has no response, an incomplete response, or a suboptimal response (i.e., the human patient may still have a substantial benefit from continuing treatment, but has reduced chances of optimal long-term outcomes) after administering the second population of cells comprising antigen-specific T cells and prior to administering the first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1.
  • the second population of cells comprising antigen-specific T cells can be generated by a method as described in Section 4.1 or any method as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123-1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360-4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 1480-1492; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663
  • the second population of cells comprising antigen-specific T cells can be any suitable cell
  • two populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the two populations of cells is generated by a method as described in Section 4.1.
  • three populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous are administered serially, wherein at least one of the three populations of cells is generated by a method as described in Section 4.1.
  • four populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous are administered serially, wherein at least one of the four populations of cells is generated by a method as described in Section 4.1.
  • more than four populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the more than four populations of cells is generated by a method as described in Section 4.1.
  • kits for assessing a population of cells comprising antigen-specific T cells ex vivo sensitized to one or more antigens of a pathogen or cancer, for suitability for therapeutic administration to a human patient having the pathogen or cancer comprising determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype, wherein the predominant effector memory phenotype is CD62L " CCR7 " CD45RA " CD28 + CD27 +/” CD57 +/” , and wherein determining that the antigen-specific T cells do not exhibit a predominant effector memory phenotype indicates that the population of cells is not suitable for therapeutic administration to the human patients.
  • the step of determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype upon the ex vivo sensitizing is performed by FACS.
  • the methods of assessing a population of cells comprising antigen-specific T cells further comprise determining whether CD137 is upregulated in the antigen-specific T cells, wherein determining that CD137 is not upregulated indicates that the population of cells is not suitable for therapeutic administration to the human patient.
  • the step of determining whether CD137 is upregulated in the antigen- specific T cells upon the ex vivo sensitizing is performed by FACS.
  • ex vivo sensitizing can be performed as described in Section 4.1.
  • the population of cells comprising antigen-specific T cells as described in this disclosure preferably (1) exhibits substantial cytotoxicity toward fully or partially ULA-matched (relative to the human donor) antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient; (2) lacks substantial alloreactivity; and/or (3) is restricted by an ULA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient, or shares at least 2 ULA alleles with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous.
  • cytotoxicity, alloreactivity, information as to which ULA allele(s) the population of cells is restricted, and/or the ULA assignment of the population of cells are measured by a method known in the art before administration (for example, as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123- 1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360- 4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 14
  • the cytotoxicity of a population of cells toward fully or partially HLA-matched (relative to the human donor) antigen presenting cells can be determined by any assay known in the art to measure T cell mediated cytotoxicity.
  • the cytotoxicity is determined by a standard 51 Cr release assay as described in the example in Section 5 herein or as described in Trivedi et al., 2005, Blood 105:2793-2801 or Hasan et al., 2009, J Immunol 183 : 2837-2850.
  • the population of cells comprising antigen-specific T cells exhibits substantial cytotoxicity in vitro toward ⁇ e.g., exhibits substantial lysis of) fully or partially HLA matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • the fully or partially HLA-matched antigen presenting cells are fully HLA- matched antigen presenting cells ⁇ e.g., antigen presenting cells derived from the human donor).
  • the population of cells exhibits lysis of greater than or equal to 20%, 25%, 30%), 35%), or 40% of the fully or partially HLA-matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • the population of cells exhibits lysis of greater than or equal to 20%) of the fully or partially HLA-matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • Antigen presenting cells that can be used in the cytotoxicity assay include, but are not limited to, dendritic cells, phytohemagglutinin (PHA)-lymphoblasts, macrophages, B-cells that generate antibodies, EBV-BLCL cells, and artificial antigen presenting cells (AAPCs).
  • the antigen presenting cells used in the cytotoxicity assay are dendritic cells.
  • the fully or partially HLA-matched antigen presenting cells used in the cytotoxicity assay are loaded with a pool of peptides derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • the pool of peptides can be, for example, a pool of overlapping peptides (e.g., pentadecapeptides) spanning the sequence of the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • Alloreactivity can be measured using a cytotoxicity assay known in the art to to measure T cell mediated cytotoxicity, such as a standard 51 Cr release assay, as described in Section 4.4.1, but with antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient, and/or HLA-mismatched (relative to the human donor) antigen presenting cells.
  • a population of cells comprising antigen- specific T cells that lacks substantial alloreactivity results generally in the absence of graft- versus-host disease (GvHD) when administered to a human patient.
  • GvHD graft- versus-host disease
  • the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • antigen-presenting cells are fully or partially HLA-matched antigen presenting cells (relative to the human donor) ⁇ e.g., antigen presenting cells derived from the human donor).
  • the population of cells lyses less than or equal to 15%, 10%, 5%, 2%, or 1% of antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In a specific embodiment, the population of cells lyses less than or equal to 15% of antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward HLA- mismatched (relative to the human donor) antigen presenting cells.
  • such antigen-presenting cells are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • such antigen-presenting cells are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.
  • the population of cells lyses less than or equal to 15%, 10%, 5%), 2%), or 1%) of HLA-mismatched (relative to the human donor) antigen presenting cells. In a specific embodiment, the population of cells lyses less than or equal to 15% of HLA-mismatched (relative to the human donor) antigen presenting cells.
  • the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient, as described above, and lacks substantial cytotoxicity in vitro toward HLA-mismatched antigen presenting cells as described above.
  • Antigen presenting cells that can be used in the alloreactivity assay include, but are not limited to, dendritic cells, phytohemagglutinin (PHA)-lymphoblasts, macrophages, B-cells that generate antibodies, EBV-BLCL cells, and artificial antigen presenting cells (AAPCs).
  • the antigen presenting cells used in the alloreactivity assay are dendritic cells.
  • the HLA assignment ⁇ i.e., the HLA loci type) of the population of cells and/or the human donor can be ascertained ⁇ i.e., typed) by any method known in the art.
  • Non-limiting exemplary methods for ascertaining the HLA assignment can be found in ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and Immunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2 (2007), American Society for Histocompatibility and Immunogenetics; Hurley, "DNA-based typing of HLA for
  • HLA loci preferably HLA-A, HLA-B, HLA-C, and HLA-DR
  • 4 HLA loci preferably HLA-A, HLA-B, HLA-C, and HLA-DR
  • 6 HLA loci are typed.
  • 8 HLA loci are typed.
  • high-resolution typing is preferable for HLA typing.
  • the high-resolution typing can be performed by any method known in the art, for example, as described in ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and
  • the HLA assignment of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient is ascertained by typing the origin of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous (e.g., the human patient or a transplant donor for the human patient, as the case may be).
  • the origin of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous can be determined by any method known in the art, for example, by analyzing variable tandem repeats (VTRs) (which is a method that uses unique DNA signature of small DNA sequences of different people to distinguish between the recipient and the donor of a transplant), or by looking for the presence or absence of chromosome Y if the donor and the recipient of a transplant are of different sexes (which is done by cytogenetics or by FISH
  • the HLA allele by which the population of cells comprising antigen-specific T cells is restricted can be determined by any method known in the art, for example, as described in Trivedi et al., 2005, Blood 105 :2793-2801 ; Barker et al., 2010, Blood 1 16:5045-5049; Hasan et al., 2009, J Immunol, 183 :2837-2850; or Doubrovina et al., 2012, Blood 120: 1633-1646.
  • the population of cells comprising antigen-specific T cells is restricted by an HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient.
  • the population of cells comprising antigen-specific T cells shares at least 2 HLA alleles (for example, at least 2 out of 8 HLA alleles, such as two HLA-A alleles, two HLA-B alleles, two HLA-C alleles, and two HLA-DR alleles) with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient.
  • HLA alleles for example, at least 2 out of 8 HLA alleles, such as two HLA-A alleles, two HLA-B alleles, two HLA-C alleles, and two HLA-DR alleles
  • the population of cells comprising antigen-specific T cells is restricted by an HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient, and shares at least 2 HLA alleles (for example, at least 2 out of 8 HLA alleles, such as two HLA-A alleles, two HLA-B alleles, two HLA-C alleles, and two HLA- DR alleles) with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient.
  • HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient.
  • the human patient has or is suspected of having a pathogen.
  • the human patient has the pathogen.
  • the human patient has a disorder (e.g., cancer) associated with the pathogen (e.g., resulting from infection with the pathogen).
  • the human patient is suspected of having the pathogen.
  • the human patient is seropositive for the pathogen, and has symptoms of an infection by the pathogen.
  • the human patient is seropositive for the pathogen, and has symptoms of a disorder (e.g., cancer) associated with the pathogen (e.g., resulting from infection with the pathogen).
  • the pathogen can be a virus, bacterium, fungus, helminth, or protist.
  • the pathogen is a virus.
  • the virus is cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the human patient has or is suspected of having a CMV infection. In specific embodiments, the human patient has or is suspected of having a CMV infection subsequent to the human patient having undergone an HSCT. In specific embodiments, the human patient has a CMV infection. In a specific embodiment, the human patient has or is suspected of having CMV viremia. In another specific embodiment, the human patient has CMV viremia. In another specific embodiment, the human patient has or is suspected of having CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV- positive meningoma, or CMV-positive glioblastoma multiforme. In another specific specific
  • the human patient has CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV-positive meningoma, or CMV- positive glioblastoma multiforme.
  • the one or more antigens of CMV in the methods described in this disclosure is CMV pp65, CMV IE1, or a combination thereof.
  • the one or more antigens of CMV in the methods described in this disclosure is CMV pp65.
  • the virus is Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the one or more antigens of EBV in the methods described in this disclosure is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, or a combination thereof.
  • the human patient has or is suspected of having an EBV-positive lymphoproliferative disorder (EBV-LPD) (for example, an EBV-positive post-transplant lymphoproliferative disorder).
  • EBV-LPD EBV-positive lymphoproliferative disorder
  • the human patient has an EBV-LPD (for example, an EBV-positive post-transplant lymphoproliferative disorder).
  • the EBV-LPD can be, but is not limited to, B-cell hyperplasia, B-cell lymphoma (for example, diffuse large B- cell lymphoma), T-cell lymphoma, polymorphic or monomorphic EBV-LPD, EBV-positive Hodgkin's lymphoma, Burkitt lymphoma, autoimmune lymphoproliferative syndrome, or mixed PTLD (post-transplant lymphoproliferative disorder).
  • the EBV-LPD is an EBV-positive lymphoma (for example, and EBV-positive B-cell lymphoma).
  • the EBV-LPD is present in the central nervous system of the human patient.
  • the EBV-LPD is present in the brain of the human patient.
  • the one or more antigens of EBV is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, or a combination thereof.
  • the human patient has or is suspected of having an EBV-positive nasopharyngeal carcinoma.
  • the human patient has an EBV-positive nasopharyngeal carcinoma.
  • the one or more antigens of EBV is EBNA1, LMP1, LMP2, or a combination thereof.
  • the virus is polyoma BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.
  • BKV polyoma BK virus
  • JCV John Cunningham virus
  • ADV herpesvirus
  • HAV human immunodeficiency virus
  • influenza virus ebola virus
  • poxvirus poxvirus
  • rhabdovirus or paramyxovirus
  • the virus is BKV.
  • the virus is JCV.
  • the virus is ADV.
  • the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).
  • the disorder is not responsive to a therapy for the disorder previously administered to the human patient, such as chemotherapy (e.g., combination chemotherapy), radiation therapy, or a combination thereof).
  • chemotherapy e.g., combination chemotherapy
  • radiation therapy e.g., radiation therapy, or a combination thereof
  • the pathogen is a virus and the human patient has an infection associated with the virus
  • the infection is not responsive to a previous antiviral (small molecule) drug therapy.
  • the human patient has or is suspected of having a cancer.
  • the human patient has a cancer.
  • the human patient is suspected of having a cancer.
  • the human patient who is suspected of having the cancer is seropositive for one or more antigens of the cancer, and has symptoms normally associated with the cancer.
  • the cancer is not associated with a pathogen.
  • the cancer is associated with a pathogen.
  • An antigen of a cancer can be a cancer-specific or cancer- associated antigen, and thus can be a peptide or protein whose expression is higher in the cancer tissue or cancer cells than in non-cancerous tissues or non-cancerous cells, or a peptide or protein which is uniquely expressed in the cancer tissue or cancer cells relative to non-cancerous tissues or non-cancerous cells.
  • the cancer is a blood cancer.
  • a blood cancer that can be treated using a population of cells comprising antigen-specific T cells described in this disclosure can be, but is not limited to: acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, Large granular lymphocytic leukemia, adult T-cell leukemia, plasma cell leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, or multiple myeloma.
  • the cancer is a solid tumor cancer.
  • the solid tumor cancer can be a sarcoma, a carcinoma, a lymphoma, a germ cell tumor, a blastoma, or a combination thereof.
  • a solid tumor cancer that can be treated using a population of cells comprising antigen- specific T cells described in this disclosure can be, but is not limited to: a cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin.
  • the one or more antigens of the cancer is WT1 (Wilms tumor 1).
  • the cancer is multiple myeloma or plasma cell leukemia.
  • the cancer is relapsed/refractory multiple myeloma (RRMM), which can be, for example, primary refractory multiple myeloma, relapsed multiple myeloma, or relapsed and refractory multiple myeloma.
  • RRMM relapsed/refractory multiple myeloma
  • the cancer is primary plasma cell leukemia.
  • the cancer is secondary plasma cell leukemia.
  • the cancer is not responsive to an anti-cancer therapy previously administered to the human patient, such as chemotherapy (e.g., combination chemotherapy), radiation therapy, or a combination thereof.
  • chemotherapy e.g., combination chemotherapy
  • radiation therapy e.g., radiation therapy, or a combination thereof.
  • the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone a hematopoietic stem cell transplantation (HSCT), such as a peripheral blood stem cell transplantation, a bone marrow transplantation, or a cord blood transplantation.
  • HSCT hematopoietic stem cell transplantation
  • the human donor is the donor of the HSCT.
  • the human donor can be a related donor or unrelated donor of the HSCT.
  • the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone a PBSCT, and the human donor is the donor of the PBSCT.
  • the human donor is a third-party donor that is different from the donor of the HSCT.
  • the human patient has not been the recipient of an HSCT.
  • the human patient is an adult (at least age 16). In another specific embodiment, the human patient is an adolescent (age 12-15). In another specific embodiment, the patient is a child (under age 12). 5. EXAMPLE
  • CD34 fraction of an apheresis collection from a G-CSF mobilized donor, normally discarded, is a suitable source to generate antigen-specific T cells for adoptive immunotherapy.
  • each normal human donor received 10 mcg/kg of G-CSF, administered subcutaneously daily for 6 days.
  • the donor underwent daily leukapheresis designed to provide a minimum of 10 9 mononuclear cells/kg of the transplant recipient's weight.
  • apheresis product Aliquots of the apheresis product were collected. The apheresis product was prepped for the CliniMACS® Cell Selection System. The mechanism of action of the CliniMACS Cell Selection System is based on magnetic-activated cell sorting, which can select or remove specific cell types depending on the cell-specific immunomagnetic label used.
  • the apheresis product was first co-incubated with the CliniMACS® CD34 reagent (antibody-coated paramagnetic particles). Prior to and during incubation of the anti-CD34 beads with the G-CSF mobilized apheresis collection, intravenous gammaglobulin was added to the incubation fluid at a concentration of 1.5 mg/ml.
  • the cells were passed through a high-gradient magnetic separation column in the CliniMACS® clinical cell selection device. Magnetically labeled CD34 + cells were retained in the magnetized column, and CD34 " cells flowed through as the effluent fraction. The CD34 + cells retained in the column were eluted by removing the magnetic field from the column, then washing the cells through the column and collecting them. The final CD34 + cell enriched product was concentrated by centrifugation and tested before final release for administration for PBSCT as per SOPs (Standard Operating Procedures) from the MSKCC Cytotherapy Lab Manual.
  • SOPs Standard Operating Procedures
  • the CD34 + cells were washed in normal saline for intravenous infusion containing 1% human serum albumin, and suspended in a volume of 25-50 ml for intravenous administration. Aliquots of the product were taken for in-process and final product testing were performed as per SOPs from the Cytotherapy Lab Manual.
  • CMV CTLs cytotoxic T lymphocytes
  • PBMCs were isolated after Ficoll-Hypaque centrifugation with 10 ml taken from 8 separate unrelated donor CD34 " apheresis collections.
  • lxlO 6 cells/mL of unmodified and cryopreserved PBMCs were stimulated with 0.5xl0 5 /mL 6000 CGy irradiated donor-derived dendritic cells (DCs) or 0.5xl0 5 /mL irradiated donor-derived Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs), both pulsed with the pool of overlapping pentadecapeptides of CMVpp65.
  • DCs CGy irradiated donor-derived dendritic cells
  • BLCLs Epstein-Barr virus-transformed B lymphocyte cell lines
  • CMV CTLs were able to be generated and expanded from all 8 donor-derived CD34 " specimens.
  • CMV-specific T cell were expanded from 8/8 donors for the BLCL group, but only 5/8 in the DC group, which may reflect the previously described impairment of DC function after G-CSF mobilization.
  • cultures from the BLCL sensitized T cells were predominantly CD8 + (95%) and were specific for CMV (64%) as well as EBV (24%) antigens
  • the DC sensitized cultures consisted of CD8 + (68%) and CD4 + (32%) T cells with CMV specificity of 42% and 18%, respectively.
  • CMV and EBV specific CD8 + T cells were multifunctional expressing high levels of CD 107a, T Fa and IFN- ⁇ .
  • CMV-specific CD4+ T cells also produced IL-2 and up-regulated CD 154, suggesting their potential to sustain T and B cell expansion.
  • Degranulation observed by flow cytometry correlated with high levels of cytotoxicity, assessed by the standard 4h 5 Chromium assay, against antigen loaded DCs. No alloreactivity, NK cell expansion or
  • CD4 + CD25 + CD127 low FOXP3 + Tregs were present at the end of any cultures.
  • HLA-A*0201 and B*0702 for which tetramers to NLV and TPR sequences of pp65- protein were available, high proportions of responses were confirmed to be directed to these epitopes.
  • Further characterization of the differentiation status of CMV CTLs identified in cultures by up-regulation of CD 137 upon peptide stimulation, revealed a predominant effector memory phenotype (CD62L " CCR7 " CD45RA " CD28 + CD27 +/” CD57 +/” ).
  • CD62L CCR7
  • CD45RA CD28 + CD27 +/
  • CD57 +/ a predominant effector memory phenotype
  • expanded cells expressed high levels of PD-1 but no relevant expression of the other markers (LAG-3, TIM-3 or CTLA-4).

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Abstract

La présente invention concerne des procédés de génération de lymphocytes T spécifiques à un antigène pour une administration thérapeutique à un patient humain atteint ou soupçonné d'être atteint d'un pathogène ou d'un cancer à l'aide de la fraction CD34 d'une collection d'apharèse de donneurs de G-CSF mobilisés. L'invention concerne également des procédés de traitement d'un patient humain à l'aide de lymphocytes T spécifiques à un antigène générés par ces procédés et des procédés d'évaluation des lymphocytes T spécifiques à un antigène pour leur adéquation pour une administration thérapeutique à un patient humain.
PCT/US2017/021730 2016-03-11 2017-03-10 Procédés de génération de lymphocytes t spécifiques à un antigène pour une immunothérapie adoptive Ceased WO2017156365A1 (fr)

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* Cited by examiner, † Cited by third party
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CN108251368A (zh) * 2017-12-20 2018-07-06 中国科学院遗传与发育生物学研究所 一种建立nk和/或t细胞系的方法
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Cited By (4)

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US10300090B2 (en) 2017-03-15 2019-05-28 Orca Biosystems, Inc. Compositions of hematopoietic stem cell transplants
US10857183B2 (en) 2017-03-15 2020-12-08 Orca Biosystems, Inc. Method of hematopoietic stem cell transplants
US12011461B2 (en) 2017-03-15 2024-06-18 Orca Biosystems, Inc. Compositions and methods of hematopoietic stem cell transplants
CN108251368A (zh) * 2017-12-20 2018-07-06 中国科学院遗传与发育生物学研究所 一种建立nk和/或t细胞系的方法

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