WO2018057823A1 - Generation and use in adoptive immunotherapy of stem cell-like memory t cells - Google Patents

Generation and use in adoptive immunotherapy of stem cell-like memory t cells Download PDF

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WO2018057823A1
WO2018057823A1 PCT/US2017/052846 US2017052846W WO2018057823A1 WO 2018057823 A1 WO2018057823 A1 WO 2018057823A1 US 2017052846 W US2017052846 W US 2017052846W WO 2018057823 A1 WO2018057823 A1 WO 2018057823A1
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cells
population
antigen
specific
human
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French (fr)
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Richard John O'REILLY
Aisha Nasreen HASAN
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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Priority to EP17784722.5A priority Critical patent/EP3515461A1/en
Priority to CA3037002A priority patent/CA3037002A1/en
Priority to US16/333,651 priority patent/US10857182B2/en
Priority to JP2019515991A priority patent/JP2019530449A/ja
Priority to AU2017330379A priority patent/AU2017330379B2/en
Publication of WO2018057823A1 publication Critical patent/WO2018057823A1/en
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    • 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
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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Definitions

  • TSC M cells stem cell-like memory T cells
  • T cells that are selected for expansion and adoptive transfer have been identified as a critical factor that determines the persistence of transferred cells.
  • Antigen-specific T cells in the presence of infections or cancer can expand and differentiate into effector T cells devoted to rapidly clearing of the pathogens as well as memory T cells that can persist long-term and defend against recurrence of disease.
  • the memory T cell compartment is heterogeneous and encompasses multiple subsets with distinctive properties.
  • Current evidence indicates that central memory T cells (T CM cells) that express high levels of CD62L and CCR7 are less differentiated, while CD62L ⁇ CCR7 ⁇ effector memory T cells (T EM cells) represent committed progenitor cells that undergo terminal differentiation (Berger et al, 2008, J Clin Invest 118:294-305).
  • TSC M cells stem cell-like memory T cells that express CD45RA, CCR7 and CD62L, like naive T cells, but also express CD95.
  • Human TSC M cells have been expanded in vitro (Gattinoni et al., 2011, Nat Med 17: 1290-1297; Cieri et al., 2013, Blood 121 :573-584). When compared with other memory T cell populations, human T S C M cells have exhibited increased proliferative capacity. TSC M cells transduced to express a mesothelin-specific chimeric antigen receptor have also exhibited greater proliferation and superior antitumor responses following adoptive transfer in a humanized mouse model (Gattinoni et al., 2011, Nat Med 17: 1290-1297).
  • T S C M cells can differentiate into T CM , T em and effector T cells, they also have a marked potential for self-renewal as shown by serial transplantation experiments (Cieri et al., 2013, Blood 121 :573-584). Because of these attributes, TSC M cells have attracted
  • 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, utilizing stem cell-like memory T cells (TSC M cells). Also disclosed are antigen-specific T cells generated by such methods, and methods of treating a human patient using such antigen- specific T cells.
  • TSC M cells stem cell-like memory T cells
  • 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: (a) ex vivo sensitizing a population of human blood cells to one or more antigens of the pathogen or cancer over a period of time in culture, wherein at the initiation of said period of time, the population of human blood cells contains at least 50% stem cell-like memory T cells (TSC M cells); and (b) cryopreserving (i) the ex vivo sensitized population of human blood cells, or (ii) cells derived therefrom that comprise antigen-specific T cells recognizing the one or more antigens of the pathogen or cancer; thereby producing said population of cells comprising antigen-specific T cells.
  • TSC M cells stem cell-like memory T cells
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the ex vivo sensitized population of human blood cells or cells derived therefrom.
  • the aforementioned period of time in culture (termed herein "the Sensitization Culture Time;” i.e., the culture time period over which sensitization occurs) is in the range of 9-21 days.
  • the Sensitization Culture Time is in the range of 9-14 days.
  • the Sensitization Culture Time is 14 days.
  • the ex vivo sensitizing step comprises co-culturing the population of human blood 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 population of human blood 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, such as dendritic cells, cytokine-activated monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus- transformed B-lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells (AAPCs).
  • the antigen presenting cells are AAPCs.
  • 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 recombinantly 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 population of cells comprising antigen-specific T cells comprises antigen-specific T cells that endogenously express a public T cell receptor (TCR) recognizing the one or more antigens.
  • TCR public T cell receptor
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that recombinantly express a public TCR recognizing the one or more antigens.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises transducing the population of human blood cells with a nucleic acid encoding a public TCR (e.g., at a time when the population of human blood cells has been cultured for 3-5 days).
  • a public TCR e.g., at a time when the population of human blood cells has been cultured for 3-5 days.
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a complementarity determining region (CDR)3 of
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of CASSPKTGAVYGYTF (SEQ ID NO:4).
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that recombinantly express a chimeric antigen receptor (CAR) recognizing the one or more antigens.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises transducing the population of human blood cells with a nucleic acid encoding a CAR (e.g., at a time when the population of human blood cells has been cultured for 3-5 days).
  • 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 transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing one or more antigens of the pathogen or cancer at a time when the population of human blood cells has been cultured for 3-5 days, wherein the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of CAS SPQTGAS YGYTF (SEQ ID NO:3).
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of CAS SPQTGAS YGYTF (SEQ ID NO:3).
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom. In a specific embodiment, the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • 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 CMV infection comprising transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing CMV pp65 at a time when the population of human blood cells has been cultured for 3-5 days, wherein the public TCR comprises a ⁇ - chain comprising a variable domain, which comprises a CDR3 of CAS SPQTGAS YGYTF (SEQ ID NO:3), and wherein the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • 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 CMV infection comprising transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing CMV pp65 at a time when the population of human blood cells has been cultured for 3-5 days, wherein the public TCR comprises a ⁇ - chain comprising a variable domain, which comprises a CDR3 of CASSPKTGAVYGYTF (SEQ ID NO:4), and wherein the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • 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 transducing a population of human blood cells with a nucleic acid encoding a CAR recognizing one or more antigens of the pathogen or cancer at a time when the population of human blood cells has been cultured for 3-5 days, wherein the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • the population of human blood cells used in accordance with the methods of generating a population of cells comprising antigen-specific T cells described herein contains at least 50% TSC M cells. In a specific embodiment, the population of human blood cells contains at least 90% TSC M cells. In another specific embodiment, the population of human blood cells contains at least 95% TSC M cells. In another specific embodiment, the population of human blood cells contains at least 99% TSC M cells. In another specific embodiment, the population of human blood cells contains 100% TSC M cells.
  • the population of human blood cells contains less than 10% T N cells. In a specific embodiment, the population of human blood cells contains less than 5% T N cells. In another specific embodiment, the population of human blood cells contains less than 1%) T N cells. In another specific embodiment, the population of human blood cells contains no T N cells.
  • the method of generating a population of cells comprising antigen-specific T cells described herein further comprises a step of deriving the population of human blood cells from a human cell sample.
  • the deriving step comprises enriching for T S C M cells from the human cell sample.
  • the enriching step comprises selecting for cells that are CD3 + CD62I/CD45RO " CD95 .
  • the step of deriving the population of human blood cells from a human cell sample comprises sorting TSC M cells from the human cell sample by fluorescence-activated cell sorting (FACS).
  • the population of human blood cells is derived from a human donor that is seropositive for the one or more antigens.
  • 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 one or more peptides or proteins derived from the one or more antigens.
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of the pathogen.
  • the pathogen can be a virus, bacterium, fungus, helminth or protist.
  • the pathogen is a virus.
  • the virus is cytomegalovirus (CMV).
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.
  • BKV BK virus
  • JCV John Cunningham virus
  • ADV herpesvirus
  • HAV human immunodeficiency virus
  • influenza virus ebola virus
  • poxvirus poxvirus
  • rhabdovirus or paramyxovirus
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of the cancer.
  • the cancer can be a blood cancer.
  • the cancer is multiple myeloma or plasma cell leukemia.
  • the one or more antigens of the cancer is Wilms tumor 1 (WTl).
  • the cancer can also be a solid tumor cancer, such as, 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.
  • a solid tumor cancer such as, 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.
  • kits for treating a human patient having a pathogen or cancer comprising: (i) generating a population of cells comprising antigen-specific T cells according to a method described herein; and (ii) administering the population of cells comprising antigen-specific T cells to the human patient.
  • kits for treating a human patient having a pathogen or cancer comprising administering a population of cells comprising antigen-specific T cells to the human patient, wherein the population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen- specific T cells according to a method described herein.
  • the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted by an HLA allele shared with the diseased cells in the human patient to be treated.
  • the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells share 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 the diseased cells in the human patient to be treated.
  • the population of human blood cells is derived from a human donor that is allogeneic to the human patient.
  • the human patient has been the recipient of a transplant from a transplant donor, and the human donor is a third-party donor that is different from the transplant donor.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In a specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 5 x 10 4 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 4 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 5 x 10 3 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 3 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the step of administering comprises administering the population of cells comprising antigen-specific T cells to the human patient at the dose described above weekly.
  • the step of administering is by bolus intravenous infusion.
  • the step of administering comprises administering at least 2 doses of the population of cells comprising antigen-specific T cells to the human patient. In specific embodiments, the step of administering comprises administering 2, 3, 4, 5, or 6 doses of the population of cells comprising antigen-specific T cells to the human patient.
  • the step of administering comprises administering a first cycle of one dose per week of the population of cells comprising antigen-specific T cells for 3 consecutive weeks followed by a washout period during which no dose of the population of cells comprising antigen-specific T cells is administered, followed by a second cycle of said one dose per week of the population of cells comprising antigen-specific T cells for 3 consecutive weeks.
  • the step of administering comprises administering two, three, four, five, or six cycles of one dose per week of the population of cells comprising antigen- specific T cells for three consecutive weeks, each cycle separated by a washout period during which no dose of the population of cells comprising antigen-specific T cells is administered.
  • the washout period is about 1, 2, 3, or 4 weeks. In a preferred embodiment, the washout period is about 3 weeks.
  • the step of administering of the population of cells comprising antigen-specific T cells does not result in any graft-versus-host disease (GvHD) in the human patient.
  • GvHD graft-versus-host disease
  • populations of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, wherein the population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen-specific T cells according to a method described herein.
  • 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, wherein the population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen-specific T cells according to a method described herein, and wherein the population of cells comprising antigen-specific T cells is cryopreserved.
  • a cell bank comprising a plurality of populations of cells comprising antigen-specific T cells described herein.
  • FIG. 1 Isolation and characterization of T N , TSC M , TC M and T EM populations from human peripheral blood. Flow cytometry was used to isolate the TN, TSC M , C M and T EM subsets from peripheral blood mononuclear cell (PBMCs). Lymphocytes were first gated within PBMCs by forward scatter (FSC) and side scatter (SCC) and analyzed for CD45RO, CD95, CD62L and CD3 expression. CD3 lymphocytes were then gated for CD45RO CD62I7 T CM and CD45RO + CD62L " T EM , CD45RO " CD62L + CD95 + T SCM cells and CD45RO " CD62L + CD95 " T N cells.
  • FSC forward scatter
  • SCC side scatter
  • FIG. 1 Phenotypic characterization of naive and memory (T N , T S CM ? T C M and TEM)-derived CMV-specific CD8 + T cells upon antigen stimulation.
  • A), (B) and (C) Isolated T cells from T N , TSC M , T CM and T EM cell populations were sensitized with artificial antigen presenting cells (AAPCs) expressing CMVpp65 peptide and HLA-A:0201.
  • AAPCs artificial antigen presenting cells
  • CD3+ T lymphocytes derived from each cell population were evaluated at 7 (A), 14 (B) and 30 days (C) post- stimulation.
  • CD8 + single, live T cells were gated from CD3+ T lymphocytes.
  • HLA-A:0201-NLV Tetramer (Tet) + and LV-Tet " T cells were then gated within CD8 + T cells (left panel).
  • D Representative FACS plots of the CD57 expression in CD8 + T cells.
  • E and
  • FIG. 4 Enrichment of CMV-specific CD8 + T cells results from rapid expansion of early memory T cells.
  • A In-vitro proliferation of A2- LV-Tet + T cells was evaluated after in-vitro sensitization with CMVpp65 antigen by EdU as shown in a
  • CD3 + T cells were sensitized with artificial antigen presenting cells (AAPCs) expressing CMVpp65 peptide and HLA-A*0201. The enrichment of LV-Tet + T cells after stimulation was evaluated as shown on the left panel. Percentage of EdU incorporation gated on single, live CD8 + T lymphocytes is shown in the middle panel. Percentage of EdU incorporation gated on LV-Tet + CD8 + T cells is shown on the right panel. (B) Phenotypic analysis of LV- Tet + T cells (right panel) in comparison to NLV- Tet + T cells with EdU incorporation (left panel) is shown for a representative donor. (White: CD45RO + CD62L " T EM ; Light Grey: CD45RO + CD62L + T CM ; Dark Grey: CD45RO "
  • CD45RO " CD62L + CD95 " T n cells were not detected post antigen stimulation.
  • C Fold expansion of A2- LV-Tet + T cells after 14 days of antigen-specific T cell stimulation was evaluated within LV-Tet + CD8 + T cells within the T N -, TSC M -, TC M - and T EM - derived cells.
  • FIG. 5 Functional cytokine profile and cytotoxic activity of in vitro expanded CMV-specific T cells derived from naive (T N ) and memory T cells (TSCM, CM and T EM ).
  • T N naive
  • TSCM, CM and T EM memory T cells
  • the percentage of CD8 + T cells expressing CD137 are shown on the first 2 panels (on Figure 5A-1) with or without peptide stimulation. The proportion of CD8+ T cells expressing CD137 and secreting IFN- ⁇ or TNF-a cytokine is shown in the next 2 panels (on Figure 5A-2). CD8 + T cells secreting both IFN- ⁇ and TNF-a cytokine are shown in the last panel (on Figure 5 A-2).
  • B Cytotoxic activity was evaluated by CD 107a degranulation assay. The percentage of CD8 + T cells expressing CD 107a with (middle panel) or without (left panel) peptide stimulation is shown. The percentage of CD8 + T cells expressing both CD137 and CD107a is shown in the right panel.
  • TCR sequencing wa s p er formed 0 n sorted NLV-Tet + T cells derived from Naive (T N ), and memory (TSC M , T CM and T EM ) subsets 1 from a separate donor (D) before, (E) after 15 days and, (F) after 30 days of in vitro stimulation, and compared for their similarities. Values shown are proportions of overlap.
  • TCR sequencing analysis for this donor at the same time points (pre-, day 15 and day 30 post-stimulation) is summarized in (G).
  • T cells from HLA-A:0201 + and A:2402 + CMV seropositive donors were sensitized with artificial antigen presenting cells (AAPCs) expressing HLA-A:0201 or HLA-A:2402 and CMVpp65 protein.
  • AAPCs artificial antigen presenting cells
  • NLV-Tet + or QYD-Tet + T cells Phenotypic analysis of NLV-Tet + or QYD-Tet + T cells was performed to evaluate the proportion of TSC M , TC M and T EM -derived cells after antigen specific stimulation (CD45RO + CD62L + T CM : Light Gray; CD45RO + CD62L " T EM : White;
  • CD45RO CD62L + CD95 + T SCM : Dark Gray). There were no Tet + T cells with T N phenotype CD45RO " CD62L + CD95 " post-sensitization.
  • T cells labeled with EdU were used to evaluate and compare the proportion of proliferating T cells within NLV-Tet + and the proportion of proliferating T cells within QYD-Tet + T cells, within T S C M -, T CM - and T EM -derived cells 4,5,7 and 8 days post stimulation ( Figure 7B-1).
  • 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, antigen-specific T cells generated by such methods, and methods of treating a human patient using such antigen-specific T cells.
  • stem cell-like memory T cells are a more suitable source of T cells for the generation of antigen- specific T cells for adoptive immunotherapy, relative to naive T cells (T N cells), central memory T cells (TC M cells) and effector memory T cells (T EM cells), because they allow rapid, persistent and selective in vitro expansion of antigen-specific T cells that recognize dominant epitopes of antigens of pathogens found in human blood and are the principal and persistent reservoir for rapid repopulation of immunodominant T cells in vivo.
  • T N cells naive T cells
  • TC M cells central memory T cells
  • T EM cells effector memory T cells
  • 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: (a) ex vivo sensitizing a population of human blood cells to one or more antigens of the pathogen or cancer over a period of time in culture, wherein at the initiation of said period of time, the population of human blood cells contains at least 50% stem cell-like memory T cells (T S C M cells); and (b) cryopreserving (i) the ex vivo sensitized population of human blood cells, or (ii) cells derived therefrom that comprise antigen-specific T cells recognizing the one or more antigens of the pathogen or cancer; thereby producing said population of cells comprising antigen-specific T cells.
  • T S C M cells stem cell-like memory T cells
  • the aforementioned period of time in culture (termed herein "the Sensitization Culture Time;” i.e., the culture time period over which sensitization occurs) is in the range of 9-21 days.
  • the Sensitization Culture Time is in the range of 9-14 days.
  • the Sensitization Culture Time is 9 days.
  • the Sensitization Culture Time is 10 days.
  • the Sensitization Culture Time is 11 days. In another specific embodiment, the Sensitization Culture Time is 12 days. In another specific embodiment, the Sensitization Culture Time is 13 days. In another specific embodiment, the Sensitization Culture Time is 14 days. In another specific embodiment, the Sensitization Culture Time is 15 days. In another specific embodiment, the Sensitization Culture Time is 16 days. In another specific embodiment, the Sensitization Culture Time is 17 days. In another specific embodiment, the Sensitization Culture Time is 18 days. In another specific embodiment, the Sensitization Culture Time is 19 days. In another specific embodiment, the Sensitization Culture Time is 20 days. In another specific embodiment, the Sensitization Culture Time is 21 days.
  • 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 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; Koehne et al., 2015, Biol Blood
  • the ex vivo sensitizing step comprises co-culturing the population of human blood cells with one or more immunogenic peptides or proteins derived from the one or more antigens (preferably also in the presence of antigen presenting cells).
  • the ex vivo sensitizing step comprises co-culturing the population of human blood cells with antigen presenting cells that present the one or more antigens.
  • the ex vivo sensitizing step preferably comprises first supplementing the culture with IL-15 and IL-7 (e.g., starting from day 4 after the initiation of the Sensitization Culture Time, or earlier), and then supplementing the culture with IL-2 (e.g., at least 7 days after the initiation of the
  • Sensitization Culture Time optionally together with IL-15 and IL-7.
  • IL-15 and IL-7 help to maintain stem cell-like phenotype of the TSC M cells, and are preferably added to the culture within the first seven days of culturing in the ex vivo sensitizing step and then again for multiple times.
  • IL-2 helps to boost expansion of the antigen-specific T cells, and is preferably added to the cell culture on a culture date that is later than the initial addition of IL-15 and IL-7 to the cell culture in the ex vivo sensitizing step (e.g., IL-2 is added at least 7 days after the initiation of the Sensitization Culture Time).
  • the ex vivo sensitizing step comprises supplementing the culture with IL-15 and IL-7 on day 4 and day 7 after the initiation of the Sensitization Culture Time, and then supplementing the culture with IL-15, IL-7 and IL-2 every other day starting after day 12 after the initiation of the Sensitization Culture Time.
  • 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, such as dendritic cells, cytokine-activated monocytes, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus- transformed B-lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells (AAPCs).
  • the antigen presenting cells are dendritic cells.
  • the antigen presenting cells are PBMCs.
  • the antigen presenting cells are EBV-BLCL cells.
  • the antigen presenting cells are AAPCs.
  • the antigen presenting cells are derived from the donor of the population of human blood cells.
  • 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; Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678, or International Patent Application Publication No.
  • 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; Hasan et al., 2009, J Immunol 183 : 2837-2850; and International Patent Application Publication No. WO 2016/073550.
  • the antigen presenting cells are genetically engineered to recombinantly 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 recombinantly 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 step of cryopreserving comprises steps of: (c) recovering from culture the ex vivo sensitized population of human blood cells or cells derived therefrom; (d) combining with a cryopreservative the ex vivo sensitized population of human blood cells or cells derived therefrom; and (e) freezing the ex vivo sensitized population of human blood cells, or cells derived therefrom, combined with the cryopreservative.
  • Cells derived from the ex vivo sensitized population of human blood cells and comprising antigen- specific T cells which recognize the one or more antigens of the pathogen or cancer can be a fraction of the ex vivo sensitized population of human blood cells (e.g., a CD3 + T cell population enriched from the ex vivo sensitized population of human blood cells, or a CD8 + cytotoxic T cell population enriched from the ex vivo sensitized population of human blood cells) or an expanded population of the ex vivo sensitized population of human blood cells.
  • Freezing of cells is ordinarily destructive. On cooling, water within the cell freezes. Injury then occurs by osmotic effects on the cell membrane, cell dehydration, solute
  • Cryopreservative which can be used in accordance with the present invention can be, but is not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, or inorganic salts.
  • the cryopreservative which is used in accordance with the present invention is DMSO.
  • DMSO freely permeates the cell and protects intracellular organelles by combining with water to modify its freezability and prevent damage from ice formation. Addition of plasma, fetal calf serum, or human albumin can augment the protective effect of DMSO. After addition of DMSO, cells should be kept at 0 °C until freezing, since DMSO concentrations of about 1% are toxic at temperatures above 4 °C.
  • a controlled slow cooling rate is also critical.
  • Different cryopreservatives (Rapatz, G., et al., 1968, Cryobiology 5(1): 18-25) and different cell types have different optimal cooling rates ⁇ see, e.g. , Rowe and Rinfret, 1962, Blood 20:636; Rowe,, 1966, Cryobiology 3 : 12-18; Lewis et al, 1967, Transfusion 7: 17-32; and Mazur, 1970, Science 168:939-949) .
  • the heat of fusion phase where water turns to ice should be minimal.
  • the cooling procedure can be carried out by use of, e.g., a programmable freezing device or a methanol bath procedure.
  • a controlled rate freezer is used in the cryopreserving step to bring the temperature of the vial of cells to ⁇ -90 °C or less, at a rate ranging from -0.3 to -2 °C per minute.
  • the following program can be used: 1) wait for chamber is 4 °C and sample is 6.0 °C; 2) ramp at 1.0 °C/min. until sample is -6.0 °C; 3) ramp at 25 °C/min. until chamber is - 45 °C; 4) ramp at 10 °C/min. until chamber is -14 °C; 5) ramp at 1.0 °C/min. until chamber is - 40 °C; 6) ramp at 10 °C/min. until chamber is -90 °C; and 7) transfer to liquid nitrogen.
  • the cells can be placed in a Mr. FrostyTM or other alcohol/polystyrene insulated freezing chamber pre-conditioned at -20 °C and frozen overnight by transferring the chamber to a -80 °C freezer, prior to transfer to liquid nitrogen storage.
  • the ex vivo sensitized population of human blood cells or cells derived therefrom can be rapidly transferred to a long-term cryogenic storage vessel.
  • the ex vivo sensitized population of human blood cells or cells derived therefrom can be cryogenically stored in liquid nitrogen (-196 °C) or its vapor (-165 °C).
  • the cells are stored at -80 °C for 2 days and then transferred to liquid nitrogen.
  • a plurality of the populations of cells comprising antigen- specific T cells are generated and stored as described herein, thereby producing a cell bank.
  • the cryopreserving step is performed as follows: First, a freeze mix is prepared containing 90% fetal calf serum and 10% DMSO (in sterile, 15 ml tubes, 9 ml of heat inactivated and filtered fetal calf serum is mixed with sterile, filtered 1 ml of DMSO). Aliquots of this freeze mix are prepared under sterile conditions and then stored at -20 °C for use as a suspension medium for freezing cells. T cells suspended at 1 x 10 6 /ml in Tcell medium are centrifuged in 15 or 50 ml tubes at 1500 rpm for 5 mins. The supernatant is gently discarded, cells are washed a second time with phosphate buffered saline (PBS), and cell pellet is then suspended in the thawed freeze mix at 10 x 10 6 cells/ ml.
  • PBS phosphate buffered saline
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the ex vivo sensitized population of human blood cells or cells derived therefrom.
  • Frozen cells are preferably thawed quickly ⁇ e.g., in a water bath maintained at 37-41 °C) and chilled immediately upon thawing.
  • the vial containing the frozen cells can be immersed up to its neck in a warm water bath; gentle rotation will ensure mixing of the cell suspension as it thaws and increase heat transfer from the warm water to the internal ice mass. As soon as the ice has completely melted, the vial can be immediately placed in ice.
  • cryopreservative if toxic in humans, can be removed prior to therapeutic administration, and the removal is preferably accomplished upon thawing.
  • the cryopreservative is DMSO
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that recombinantly express a protein of interest, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • This can be achieved by transducing the population of human blood cells during their time in culture with a nucleic acid encoding the protein of interest.
  • the nucleic acid preferably is a vector in which a nucleic acid sequence encoding the protein of interest is operably linked to a promoter.
  • the transducing preferably occurs during days 3-5 in culture, since, as shown by the example section herein (i.e., Section 6), this time is when the TSC M cells exhibit the highest proliferative capacity.
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that endogenously express a public T cell receptor (TCR) recognizing the one or more antigens.
  • TCR public T cell receptor
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that recombinantly express a public TCR recognizing the one or more antigens.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises transducing the population of human blood cells with a nucleic acid encoding a public TCR (e.g., at a time when the population of human blood cells has been cultured for 3-5 days), for example, using a transducing method as described in Section 5.1.2, infra.
  • the transducing step can be performed before, during, or after the ex vivo sensitizing step, which reduces alloreactivity of the population of cells comprising antigen- specific T cells.
  • Public TCRs are peptide-specific TCRs with highly homologous sequences detected in multiple individuals (Li et al, 2012, Cell Res 22:33-42). Public TCRs for a variety of human viruses have been described (Argaet et al, 1994, J Exp Med 180:2335-2340; Wang et al, 2012, Sci Transl Med 4: 128ral42; Nguyen et al, 2014, J Immunol 192:5039-5049; Trautmann et al, 2005, J Immunol 175:6123-6132).
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a complementarity determining region (CDR)3 of CASSPQTGASYGYTF (SEQ ID NO:3).
  • CDR complementarity determining region
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of CAS SPKTGAVYGYTF (SEQ ID NO:4).
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of S* n TG* n GY (SEQ ID NO: 16; *n indicates any amino acid sequence of any length and any amino acid combination).
  • the population of cells comprising antigen-specific T cells comprises antigen-specific T cells that recombinantly express a chimeric antigen receptor (CAR) recognizing the one or more antigens ⁇ see Section 5.1.3, infra, for more details regarding CAR).
  • the method of generating a population of cells comprising antigen-specific T cells further comprises transducing the population of human blood cells with a nucleic acid encoding a CAR ⁇ e.g., at a time when the population of human blood cells has been cultured for 3-5 days), for example, using a transducing method as described in Section 5.1.3, infra.
  • the transducing step can be performed before, during, or after the ex vivo sensitizing step, which reduces alloreactivity of the population of cells comprising antigen-specific T cells.
  • 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 transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing one or more antigens of the pathogen or cancer (preferably at a time when the population of human blood cells has been cultured for 3-5 days), wherein the population of human blood cells contains at least 50% TSCM cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the transducing preferably occurs during days 3-5 in culture, since, as shown by the example section herein ⁇ i.e., Section 6), this time is when the TSCM cells exhibit the highest proliferative capacity.
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of
  • the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of CASSPKTGAVYGYTF (SEQ ID NO:4).
  • the public TCR comprises a ⁇ - chain comprising a variable domain, which comprises a CDR3 of S* n TG* n GY (SEQ ID NO: 16; *n indicates any amino acid sequence of any length and any amino acid combination).
  • 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 CMV infection comprising transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing CMV pp65 (preferably at a time when the population of human blood cells has been cultured for 3-5 days), wherein the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of
  • 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 CMV infection comprising transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing CMV pp65 (preferably at a time when the population of human blood cells has been cultured for 3-5 days), wherein the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of
  • CAS SPKTGAVYGYTF SEQ ID NO:4
  • the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen- specific T cells.
  • 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 CMV infection comprising transducing a population of human blood cells with a nucleic acid encoding a public TCR recognizing CMV pp65 (preferably at a time when the population of human blood cells has been cultured for 3-5 days), wherein the public TCR comprises a ⁇ -chain comprising a variable domain, which comprises a CDR3 of S* n TG* n GY (SEQ ID NO: 16; *n indicates any amino acid sequence of any length and any amino acid combination), and wherein the population of human blood cells contains at least 50% TSC M cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • the cryopreserving and thawing steps can be performed as described in Section 5.1.1, supra.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of expanding in culture the transduced population of human blood cells or cells derived therefrom, wherein the transduced population of human blood cells or cells derived therefrom has not been
  • TCR is a cell surface molecule on T cells that is responsible for recognizing antigen peptide-bound major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the population of human blood cells transduced with a nucleic acid encoding a TCR can be generated by any method known in the art, for example, as described in Stauss et al., 2015, Curr Opin Pharmacol 24: 113-118; Sharpe and Mount, 2015, Dis Model Mech 8:337-350; Kunert et al., 2013, Front Immunol 4: 363; Stone et al., 2012, Methods Enzymol 503 : 189-222; or Park et al., 2011, Trends Biotechnol 29:550-557.
  • the nucleic acid encoding a TCR can be DNA, RNA, or a nucleic acid analog. In specific embodiments, such a nucleic acid may be part of a vector.
  • the vector is an expression vector that is capable of directing the expression of a nucleic acid encoding a polypeptide of the TCR described herein in T cells.
  • expression vectors suitable for directing the expression of a nucleic acid encoding a polypeptide of the TCR described herein include, but are not limited to, plasmids and viral vectors, such as synthetic vectors, lentiviral vectors, replication-defective retroviral vectors, autonomously replicating plasmids.
  • an expression vector used for directing the expression of a nucleic acid encoding a polypeptide of the TCR described herein includes one or more regulatory sequences operably linked to the nucleic acid to be expressed.
  • "Operably linked" is intended to mean that a nucleic acid of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid in T cells.
  • Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • a nucleic acid encoding a polypeptide of the TCR described herein can be transduced into host cells via conventional transformation or transfection (such as, transfection by a virus, e.g., a retrovirus or lentivirus) techniques.
  • transformation or transfection such as, transfection by a virus, e.g., a retrovirus or lentivirus
  • Such techniques include, but are not limited to, calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation.
  • Cells containing a nucleic acid encoding a polynucleotide of the TCR described herein may be selected using one or more selectable markers known in the art.
  • 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 transducing a population of human blood cells with a nucleic acid encoding a CAR recognizing one or more antigens of the pathogen or cancer (preferably at a time when the population of human blood cells has been cultured for 3-5 days), wherein the population of human blood cells contains at least 50% TSCM cells; thereby producing said population of cells comprising antigen-specific T cells.
  • the transducing preferably occurs during days 3-5 in culture, since, as shown by the example section herein (i.e., Section 6), this time is when the TSCM cells exhibit the highest proliferative capacity.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of cryopreserving the transduced population of human blood cells or cells derived therefrom.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of cryopreserving, steps of thawing and optionally expanding in culture the transduced population of human blood cells or cells derived therefrom.
  • the cryopreserving and thawing steps can be performed as described in Section 5.1.1, supra.
  • the method of generating a population of cells comprising antigen-specific T cells further comprises, after the step of transducing, a step of expanding in culture the transduced population of human blood cells or cells derived therefrom, wherein the transduced population of human blood cells or cells derived therefrom has not been
  • CARs are engineered receptors that provide both antigen binding and immune cell activation functions (Sadelain et al., 2013, Cancer Discovery 3 :388-398). They usually comprise an antigen-binding domain (e.g., derived from a monoclonal antibody or the extracellular domain of a receptor), a transmembrane domain, an intracellular domain, and optionally a co-stimulatory domain. CARs can be used to graft the specificity of an antigen-binding domain onto an immune cell such as a T cell.
  • the population of human blood cells transduced with a nucleic acid encoding a CAR can be generated by any method known in the art, for example, as described in Stauss et al., 2015, Curr Opin Pharmacol 24: 1 13-1 18; Sharpe and Mount, 2015, Dis Model Mech 8:337-350; or Park et al., 201 1, Trends Biotechnol 29:550-557.
  • the nucleic acid encoding a CAR can be DNA, RNA, or a nucleic acid analog. In specific embodiments, such a nucleic acid may be part of a vector.
  • the vector is an expression vector that is capable of directing the expression of a nucleic acid encoding a polypeptide of the CAR described herein in T cells.
  • expression vectors suitable for directing the expression of a nucleic acid encoding a polypeptide of the CAR described herein include, but are not limited to, plasmids and viral vectors, such as synthetic vectors, lentiviral vectors, replication-defective retroviral vectors, autonomously replicating plasmids.
  • an expression vector used for directing the expression of a nucleic acid encoding a polypeptide of the CAR described herein includes one or more regulatory sequences operably linked to the nucleic acid to be expressed.
  • "Operably linked” is intended to mean that a nucleic acid of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid in T cells.
  • Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • a nucleic acid encoding a polypeptide of the CAR described herein can be transduced into host cells via conventional transformation or transfection (such as, transfection by a virus, e.g., a retrovirus or lentivirus) techniques.
  • transformation or transfection such as, transfection by a virus, e.g., a retrovirus or lentivirus
  • Such techniques include, but are not limited to, calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation.
  • Cells containing a nucleic acid encoding a polynucleotide of the CAR described herein may be selected using one or more selectable markers known in the art.
  • the population of human blood cells contains at least 50% TSC M cells. In a specific embodiment, the population of human blood cells contains at least 60% TSC M cells. In another specific embodiment, the population of human blood cells contains at least 70% TSC M cells. In another specific embodiment, the population of human blood cells contains at least 80% T S C M cells. In another specific embodiment, the population of human blood cells contains at least 90% TSC M cells. In another specific embodiment, the population of human blood cells contains at least 95% TSC M cells. In another specific specific
  • the population of human blood cells contains at least 99% TSC M cells. In another specific embodiment, the population of human blood cells contains 100% T S C M cells. In another specific embodiment, the population of human blood cells contains about 50- 75% TSC M cells. In another specific embodiment, the population of human blood cells contains about 75- 90% TSC M cells. In another specific embodiment, the population of human blood cells contains about 90- 100%) TSC M cells. In another specific embodiment, the population of human blood cells contains about 95- 100% T SCM cells.
  • T SCM cells are CD3 + CD62L + CD45RO " CD95 + .
  • TSC M cells are CD3 + CD62L + CD45RA + CD95 + .
  • TSC M cells are CD3 + CCR7 + CD45RA + CD95 + .
  • T SCM cells are CD3 + CD62L + CD45RO " CD95 + .
  • T SCM cells are CD3 + CCR7 + CD28 + CD45RO " CD95 + .
  • T SCM cells are CD3 + CCR7 + CD28 + CD45RO " CD95 + .
  • T SCM cells are CD3 + CD62L + CCR7 + CD45RA + CD95 + .
  • T SCM cells are CD3 + CD62L + CCR7 + CD45RA + CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD28 + CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD28 + CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD45RO " CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD45RO " CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD127 + CD95 + .
  • T SCM cells are CD3 + CCR7 + CD45RA + CD127 + CD95 + .
  • T SCM cells are CD3 + CD62L + CCR7 + CD45RA + CD27 + CD127 + CD45RO " CD95 + .
  • T SCM cells are CD3 + CD62L + CCR7 + CD28 + CD45RA + CD27 + CD127 + CD103 " CD45RO " CD95 + .
  • the cell surface marker expression of TSC M cells satisfies the following: (i) CD3 + CD95 + ; (ii) CD45RO " or CD45RA + , or a combination thereof; and (iii) CD62L + , or CCR7 + , or CD127 + , or a combination thereof; and optionally (iv) CD28 + , or CD27 + , or CD 103 " , or a combination thereof.
  • the population of human blood cells contains less than 50% T N cells. In a specific embodiment, the population of human blood cells contains less than 40% TN cells. In another specific embodiment, the population of human blood cells contains less than 30% T N cells. In another specific embodiment, the population of human blood cells contains less than 20%) T N cells. In another specific embodiment, the population of human blood cells contains less than 10%> T N cells. In another specific embodiment, the population of human blood cells contains less than 5% T N cells. In another specific embodiment, the population of human blood cells contains less than 2% T N cells. In another specific embodiment, the population of human blood cells contains less than 1%> T N cells. In another specific embodiment, the population of human blood cells contains no T N cells. In another specific embodiment, the population of human blood cells contains about 50-30%> T N cells. In another specific specific
  • the population of human blood cells contains about 30-20%> T N cells. In another specific embodiment, the population of human blood cells contains about 20-10%> T N cells. In another specific embodiment, the population of human blood cells contains about 10-0%> T N cells. In another specific embodiment, the population of human blood cells contains about 10- 5% T N cells. In another specific embodiment, the population of human blood cells contains about 5-0% T N cells.
  • T N cells are CD3 + CD62L + CD45RO " CD95 " .
  • TN cells are CD3 + CD62L + CD45RA + CD95 " .
  • TN cells are CD3 + CCR7 + CD45RA + CD95 " .
  • T N cells are CD3 + CCR7 + CD28 + CD45RO " CD95 " .
  • T N cells are CD3 + CD62L + CCR7 + CD45RA + CD95 " .
  • T N cells are CD3 + CCR7 + CD45RA + CD28 + CD95 " .
  • T N cells are CD3 + CCR7 + CD45RA + CD45RO " CD95 " .
  • T N cells are CD3 + CCR7 + CD45RA + CD45RO " CD95 " .
  • T N cells are CD3 + CCR7 + CD45RA + CD45RO " CD95 " .
  • T N cells are
  • T N cells are CD3 + CCR7 + CD45RA + CD127 + CD95 " .
  • T N cells are CD3 + CCR7 + CD45RA + CD127 + CD95 " .
  • T N cells are CD3 + CD62L + CCR7 + CD45RA + CD27 + CD127 + CD45RO " CD95 " .
  • T N cells are CD3 + CD62L + CCR7 + CD28 + CD45RA + CD27 + CD127 + CD103 " CD45RO " CD95 " .
  • the cell surface marker expression of T N cells satisfies the following: (i)
  • the method of generating a population of cells comprising antigen-specific T cells described herein further comprises a step of deriving the population of human blood cells from a human cell sample.
  • the human cell sample can be any cell sample that contains TSC M cells or cells that can be induced in culture to become TSC M cells, such as, but is not limited to, a hematopoietic cell sample, a blood cell sample, a fractionated or unfractionated whole blood sample, a fractionated or unfractionated apheresis collection (e.g., a leukapheresis collection, such as leukopak), PBMCs, or a T cell population (e.g., T cells enriched for from PBMCs).
  • a hematopoietic cell sample e.g., a blood cell sample, a fractionated or unfractionated whole blood sample, a fractionated or unfractionated apheresis collection (e.g., a leukapheresis collection, such
  • the human cell sample is PBMCs.
  • PBMCs can be isolated from the blood sample 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; or as described in Section 6.2, infra.
  • the human cell sample is a population enriched in T cells from PBMCs. T cells can be enriched for from the PBMCs by any method known in the art to enrich for T cells from a blood sample or PBMCs.
  • T cells can be enriched for from PBMCs.
  • T cells can be enriched for from
  • PBMCs by sorting the PBMCs using an anti-CD3 antibody and/or depleting from the PBMCs adherent monocytes and natural killer cells.
  • the step of deriving the population of human blood cells from a human cell sample can employ any known method in the art to produce a population of human blood cells that contains at least 50% T S C M cells from the human cell sample, such as, but is not limited to, sorting the human cell sample to select for TSC M cells or in vitro reprogramming cells in the human cell sample to turn them into TSC M cells.
  • the step of deriving the population of human blood cells from a human cell sample comprises affinity selection for cells that express cell surface markers of T S C M cells (e.g., using antibodies to the cell surface markers).
  • the step of deriving the population of human blood cells from a human cell sample comprises sorting TSC M cells from the human cell sample by fluorescence-activated cell sorting (FACS). In other embodiments, the step of deriving the population of human blood cells from a human cell sample comprises sorting T S C M cells from the human cell sample by magnetic separation.
  • FACS fluorescence-activated cell sorting
  • the deriving step comprises enriching for TSC M cells from the human cell sample.
  • TSC M cells exhibit a set of cell surface markers that can be used to distinguish them from other T cell subsets, thus the enriching step can comprise selecting for TSC M cells based on their markers.
  • the enriching step comprises selecting for TSC M cells that are CD3 + CD62L + CD45RO " CD95 + .
  • the enriching step comprises selecting for TSC M cells that are CD3 D62I/CD45RA D95 .
  • the enriching step comprises selecting for TSC M cells that are CD3 + CCR7 + CD45RA + CD95 + .
  • the enriching step comprises selecting for T SCM cells that are CD3 + CCR7 + CD28 + CD45RO " CD95 + .
  • the enriching step comprises selecting for TSC M cells that are
  • the enriching step comprises selecting for T SCM cells that are CD3 + CCR7 + CD45RA + CD28 + CD95 + . In another specific embodiment, the enriching step comprises selecting for T S C M cells that are
  • the enriching step comprises selecting for T SCM cells that are CD3 + CCR7 + CD45RA + CD127 + CD95 + . In another specific embodiment, the enriching step comprises selecting for TSC M cells that are
  • the enriching step comprises selecting for TSC M cells that are
  • the enriching step comprises selecting for cells whose cell surface marker expression satisfies the following: (i) CD3 + CD95 + ; (ii) CD45RO " or CD45RA + , or a
  • the deriving step comprises depleting T N cells from the human cell sample.
  • Naive T cells T N cells are distinguished from TSC M cells by the expression of cell surface marker CD95.
  • the depleting step comprises selecting against ⁇ i.e., depleting, or excluding) cells that are CD3 + CD62L + CD45RO " CD95 " .
  • the depleting step comprises selecting against ⁇ i.e., depleting, or excluding) cells that are CD3 + CD62L + CD45RA + CD95 " .
  • the depleting step comprises selecting against ⁇ i.e., depleting, or excluding) cells that are
  • the depleting step comprises selecting against ⁇ i.e., depleting, or excluding) cells that are CD3 + CCR7 + CD28 + CD45RO " CD95 " .
  • the depleting step comprises selecting against ⁇ i.e., depleting, or excluding) cells that are CD3 + CD62L + CCR7 + CD45RA + CD95 " .
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are CD3 + CCR7 + CD45RA + CD28 + CD95 " .
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are CD3 + CCR7 + CD45RA + CD28 + CD95 " .
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells that are CD3 + CD62L + CCR7 + CD28 + CD45RA + CD27 + CD127 + CD103 " CD45RO " CD95 " .
  • the depleting step comprises selecting against (i.e., depleting, or excluding) cells whose cell surface marker expression level satisfies the following: (i)
  • the population of human blood cells is derived from a human donor that is seropositive for the one or more antigens. In certain embodiments, the population of human blood cells is derived from a human donor that is seronegative for the one or more antigens.
  • the human donor can be an adult (at least age 16), an adolescent (age 12-15), a child (under age 12), or a fetus. In a specific embodiment, the human donor is an adult.
  • kits for treating a human patient having a pathogen or cancer comprising: (i) generating a population of cells comprising antigen-specific T cells according to a method described in Section 5.1, supra; and (ii) administering the population of cells comprising antigen-specific T cells to the human patient.
  • kits for treating a human patient having a pathogen or cancer comprising administering a population of cells comprising antigen-specific T cells to the human patient, wherein the population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen- specific T cells according to a method described in Section 5.1, supra.
  • the population of human blood cells is derived from a human donor that is allogeneic to the human patient.
  • the human patient has been the recipient of a transplant from a transplant donor, and the human donor is a third-party donor that is different from the transplant donor.
  • the human patient has been the recipient of a transplant from a transplant donor, and the human donor is the transplant donor.
  • the transplant is a hematopoietic stem cell transplantation (HSCT), such as a peripheral blood stem cell transplantation, a bone marrow transplantation, or a cord blood transplantation.
  • the transplant is a solid organ transplant, such as a kidney transplant, a liver transplant, a heart transplant, an intestinal transplant, a pancreas transplant, a lung transplant, or a small bowel transplant.
  • the step of administering of the population of cells comprising antigen-specific T cells does not result in any graft-versus-host disease (GvHD) in the human patient.
  • GvHD graft-versus-host disease
  • the route of administration of the population of cells comprising antigen-specific T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the route of administration of the population of cells comprising antigen-specific T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the route of administration of the population of cells comprising antigen-specific T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the route of administration of the population of cells comprising antigen-specific T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the route of administration of the population of cells comprising antigen-specific T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the route of administration of the population of cells
  • administration is intravenous.
  • the administering step is by infusion of the population of cells comprising antigen-specific T cells.
  • the infusion is bolus intravenous infusion.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In a specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 5 x 10 4 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 4 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 5 x 10 3 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is less than or equal to about 1 x 10 3 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose of about 1 x 10 3 to 5 x 10 3 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose of about 5 x 10 3 to 1 x 10 4 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose of about 1 x 10 4 to 5 x 10 4 cells of the population of cells comprising antigen- specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose of about 5 x 10 4 to 1 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is at least 1 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 5 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 1 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 2 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 3 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 4 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 5 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 6 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 1 x 10 7 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 1 x 10 5 to 5 x 10 5 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 5 x 10 5 to 1 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 1 x 10 6 to 2 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient. In another specific embodiment, the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 2 x 10 6 to 5 x 10 6 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the administering step comprises administering the population of cells comprising antigen-specific T cells to the human patient, at a dose that is about 5 x 10 6 to 1 x 10 7 cells of the population of cells comprising antigen-specific T cells per kg of the human patient.
  • the step of administering comprises administering the population of cells comprising antigen-specific T cells to the human patient at the dose described above weekly. In certain embodiments, the step of administering comprises administering the population of cells comprising antigen-specific T cells to the human patient at the dose described above twice weekly. In certain embodiments, the step of administering comprises administering the population of cells comprising antigen-specific T cells to the human patient at the dose described above biweekly. In certain embodiments, the step of administering comprises administering the population of cells comprising antigen-specific T cells to the human patient at the dose described above every three weeks.
  • the step of administering comprises administering at least 2 doses of the population of cells comprising antigen-specific T cells to the human patient.
  • the step of administering comprises administering 2, 3, 4, 5, or 6 doses of the population of cells comprising antigen-specific T cells to the human patient.
  • the step of administering comprises administering 2 doses of the population of cells comprising antigen-specific T cells to the human patient.
  • the step of administering comprises administering 3 doses of the population of cells comprising antigen-specific T cells to the human patient.
  • the step of administering comprises administering 4 doses of the population of cells comprising antigen- specific T cells to the human patient.
  • the step of administering 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 comprising antigen-specific T 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 comprising antigen-specific T cells is administered.
  • at least two cycles e.g., 2, 3, 4, 5, or 6 cycles
  • the at least two consecutive weeks are 2 consecutive weeks. In a preferred 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. In another specific embodiment, the step of administering comprises administering two, three, four, five, or six cycles of one dose per week of the population of cells comprising antigen-specific T cells for three consecutive weeks, each cycle separated by a washout period during which no dose of the population of cells comprising antigen-specific T cells is administered.
  • the step of administering comprises administering a first cycle of one dose per week of the population of cells comprising antigen-specific T cells for 3 consecutive weeks followed by a washout period during which no dose of the population of cells comprising antigen-specific T cells is administered, followed by a second cycle of said one dose per week of the population of cells comprising antigen-specific T cells for 3 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 a preferred 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).
  • the step of administering of the population of cells comprising antigen-specific T cells comprises continuously administering the population of cells comprising antigen-specific T cells at a dose described herein weekly (i.e., there is no week during which the population of cells comprising antigen-specific T cells is not administered, and thus there is no washout period).
  • 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 a preferred 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 method of treating a human patient having a pathogen or cancer as described above further comprises, after administering to the human patient a first population of cells comprising antigen-specific T cells generated according to a method described in Section 5.1, supra, administering to the human patient a second population of cells comprising antigen-specific T cells generated according to a method described in Section 5.1, supra, wherein the antigen-specific T cells in the second population of cells comprising antigen- specific T cells are restricted by a different HLA allele (different from the HLA allele by which antigen-specific cells contained in the first population of cells comprising antigen-specific T cells are restricted) shared with the diseased cells in the human patient.
  • a different HLA allele different from the HLA allele by which antigen-specific cells contained in the first population of cells comprising antigen-specific T cells are restricted
  • the method of treating a human patient having a pathogen or cancer comprises administering a first cycle of one dose per week of the first population of cells comprising antigen-specific T cells, for at least two consecutive weeks ⁇ e.g., 2, 3, 4, 5, or 6 consecutive weeks), optionally followed by a washout period during which no dose of any population of cells comprising antigen-specific T cells is administered, and 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). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks.
  • the washout period is about 2 weeks. In a preferred 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 and prior to administering the second population of cells comprising antigen-specific T cells.
  • the first and second populations of cells comprising antigen-specific T cells can each be administered by any route and any dosage regimen as described in Section 5.2.1, supra.
  • two populations of cells comprising antigen-specific T cells that are each restricted ⁇ i.e., antigen-specific T cells in the two populations of cells are each restricted) by a different HLA allele shared with the diseased cells in the human patient are administered serially.
  • three populations of cells comprising antigen- specific T cells that are each restricted ⁇ i.e., antigen-specific T cells in the three populations of cells are each restricted) by a different HLA allele shared with the diseased cells in the human patient are administered serially.
  • four populations of cells comprising antigen-specific T cells that are each restricted i.e., antigen-specific T cells in the four populations of cells are each restricted
  • a different HLA allele shared with the diseased cells in the human patient are administered serially.
  • more than four populations of cells comprising antigen-specific T cells that are each restricted i.e., antigen- specific T cells in the more than four populations of cells are each restricted
  • a different HLA allele shared with the diseased cells in the human patient are administered serially.
  • the method of treating a human patient having a pathogen or cancer further comprises concurrently treating the human patient with a second therapy for the pathogen or cancer, which second therapy is not treatment with a population of cells comprising antigen-specific T cells according to the invention, for example, at about the same time, the same day, or same week, or same treatment period (treatment cycle) during which the population of cells comprising antigen-specific T cells is administered, or on similar dosing schedules, or on different but overlapping dosing schedules.
  • no second therapy for the pathogen or cancer is concurrently administered to the human patient over a period of time over which the population of cells is repeatedly administered to the human patient.
  • the method of treating a human patient having a pathogen or cancer further comprises, before the administering step, a step of treating the human patient with a second therapy for the pathogen or cancer, which is not treatment with a population of cells comprising antigen-specific T cells according to the invention.
  • isolated populations of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, wherein the isolated population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen-specific T cells according to a method described in Section 5.1, supra.
  • an isolated population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, wherein the isolated population of cells comprising antigen-specific T cells is the product of a method comprising generating the population of cells comprising antigen-specific T cells according to a method described in Section 5.1, supra, and wherein the population of cells comprising antigen-specific T cells is cryopreserved.
  • the isolated population of cells comprising antigen-specific T cells comprises CD8+ T cells. In specific embodiments, the isolated population of cells comprising antigen-specific T cells comprises CD4+ T cells. In specific embodiments, the isolated population of cells comprising antigen-specific T cells comprises both CD8+ and CD4+ T cells.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra preferably (1) exhibits substantial cytotoxicity toward fully or partially HLA-matched (relative to the human donor of the population of human blood cells) antigen presenting cells that are loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer; (2) lacks substantial alloreactivity; and/or (3) is restricted ⁇ i.e., the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted) by an HLA allele shared with the diseased cells in the human patient, and/or shares ⁇ i.e., the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells share) at least 2 HLA alleles ⁇ e.g., at least 2 out of 8 HLA alleles) with the diseased cells in the human patient.
  • cytotoxicity, alloreactivity, information as to which HLA allele(s) the population of cells comprising antigen-specific T cells is restricted ⁇ i.e., to which HLA allele(s) the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted
  • the HLA assignment of the population of cells comprising antigen- specific T cells i.e., the HLA assignment of the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells
  • a method known in the art before administration to a human patient for example, such 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
  • a cell bank comprising a plurality of isolated populations of cells comprising antigen-specific T cells described herein.
  • information as to cytotoxicity, alloreactivity, and/or HLA restriction and and/or assignment, as described herein, is ascertained for each of the plurality of isolated populations of cells comprising antigen-specific T cells contained in the cell bank, and linked to the identifier of the corresponding population of cells comprising antigen-specific T cells, so as to facilitate the selection of a suitable population of cells comprising antigen-specific T cells from the plurality for therapeutic administration to a human patient.
  • the cytotoxicity of a population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra, toward fully or partially HLA-matched (relative to the human donor of the population of human blood cells) antigen presenting cells can be determined by any assay known in the art to measure T cell mediated cytotoxicity.
  • the assay can be performed using the population of cells comprising antigen-specific T cells directly, an aliquot thereof, or a precursor cell population that indicates the cytotoxicity of the population of cells comprising antigen-specific T cells.
  • the cytotoxicity is determined by a standard 51 Cr release assay 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 generated by a method described in Section 5.1, supra 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 one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • 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 comprising antigen-specific T cells generated by a method described in Section 5.1, supra 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 one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • the population of cells comprising antigen- specific T cells generated by a method described in Section 5.1, supra 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 one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • 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).
  • PHA phytohemagglutinin
  • AAPCs artificial antigen presenting 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.
  • 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.
  • Alloreactivity of a population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra 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 5.3.1, supra, but with antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer, and/or HLA-mismatched (relative to the human donor of the population of human cells) antigen presenting cells.
  • 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 5.3.1, supra, but with antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer, and/or HLA-mismatched (relative to
  • the assay can be performed using the population of cells comprising antigen-specific T cells directly, an aliquot thereof, or a precursor cell population that indicates the alloreactivity of the population of cells comprising antigen- specific T 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.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • antigen-presenting cells are fully or partially HLA-matched antigen presenting cells (relative to the human donor of the population of human blood cells) ⁇ e.g., antigen presenting cells derived from the human donor of the population of human blood cells).
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra 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 one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lyses less than or equal to 10% of antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra, lyses less than or equal to 5% of antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lacks substantial cytotoxicity in vitro toward HLA-mismatched (relative to the human donor of the population of human blood cells) antigen presenting cells.
  • such antigen-presenting cells are loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • such antigen-presenting cells are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra, lyses less than or equal to 15%, 10%, 5%, 2%, or 1% of HLA-mismatched (relative to the human donor of the population of human blood cells) antigen presenting cells.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lyses less than or equal to 10% of HLA-mismatched (relative to the human donor of the population of human blood cells) antigen presenting cells.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lyses less than or equal to 5% of HLA-mismatched (relative to the human donor of the population of human blood cells) antigen presenting cells.
  • the population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express one or more peptides or proteins derived from the one or more antigens of the pathogen or cancer, 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).
  • PHA phytohemagglutinin
  • AAPCs artificial antigen presenting cells
  • the HLA assignment ⁇ i.e., the HLA loci type) of a population of cells comprising antigen-specific T cells generated by a method described in Section 5.1, supra, ⁇ i.e., the HLA assignment of the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells) and/or the HLA assignment of the diseased cells in the human patient to be treated can be ascertained ⁇ i.e., typed) by any method known in the art for typing HLA alleles.
  • the assignment can be performed using the population of cells comprising antigen-specific T cells directly, an aliquot thereof, or a precursor cell population that indicates the HLA
  • 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 transplantation.” in Leffell et al., eds., 1997, Handbook of Human Immunology, Boca Raton: CRC Press; Dunn, 2011, Int J Immunogenet 38:463-473; Erlich, 2012, Tissue Antigens, 80: 1-11; Bontadini, 2012, Methods, 56:471-476; and Lange et al., 2014, BMC Genomics 15: 63.
  • At least 4 HLA loci are typed.
  • 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 diseased cells in the human patient to be treated is ascertained by typing the origin of the diseased cells (e.g., the human patient or a transplant donor for the human patient, as the case may be).
  • the origin of the diseased cells 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 generated by a method described in Section 5.1, supra, 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 116:5045- 5049; Hasan et al., 2009, J Immunol, 183 :2837-2850; Doubrovina et al., 2012, Blood 120: 1633- 1646; or International Patent Application Publication No.
  • the determination can be performed using the population of cells comprising antigen-specific T cells directly, an aliquot thereof, or a precursor cell population that indicates the HLA allele by which the population of cells comprising antigen-specific T cells is restricted (i.e., the HLA allele by which the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted).
  • the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted by an HLA allele shared with the diseased cells in the human patient to be treated.
  • the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells share 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 the diseased cells in the human patient to be treated.
  • the antigen-specific T cells contained in the population of cells comprising antigen-specific T cells are restricted by an HLA allele shared with diseased cells in the human patient to be treated, and share 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 the diseased cells in the human patient to be treated.
  • compositions comprising a therapeutically effective amount of an isolated population of cells comprising antigen-specific T cells described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is in a cryopreserved form.
  • the pharmaceutical acceptable carrier can be any physiologically-acceptable solution suitable for the storage and/or therapeutic administration of T cells, for example, a saline solution, a buffered saline solution, or a bio-compatible solution comprising one or more cryopreservatives (e.g., phosphate-buffered saline containing 7% DMSO, 5 % dextrose and 1% dextran; hypothermosol containing 5% DMSO and 5% human serum albumin; normal saline containing 10% DMSO and 16% human serum albumin; or normal saline containing 10% DMSO and 15% human serum albumin).
  • cryopreservatives e.g., phosphate-buffered saline containing 7% DMSO, 5 % dextrose and 1% dextran
  • hypothermosol containing 5% DMSO and 5% human serum albumin e.g., phosphate-buffered saline containing 7% DMSO, 5
  • the population of cells comprising antigen-specific T cells can be stored in the pharmaceutical composition at any concentration desirable for its long-term storage and convenience of storage and handling.
  • the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a
  • the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a
  • the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 20 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 50 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 100 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 200 x 10 6 cells/mL.
  • the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 500 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 1 to 10 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 10 to 100 x 10 6 cells/mL. In another specific embodiment, the population of cells comprising antigen-specific T cells is stored in the pharmaceutical composition at a concentration of about 100 to 1000 x 10 6 cells/mL.
  • kits comprising in one or more containers the
  • kits further comprise a second pharmaceutical composition comprising a second compound or biological product for treating the pathogen infection or cancer.
  • Optionally associated with such one or more containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • compositions and kits encompassed herein can be used in accordance with the methods of treating a human patient as provided in this disclosure.
  • the one or more antigens of a pathogen can be one or more peptides or proteins whose expressions are unique to the pathogen.
  • the pathogen can be a virus, bacterium, fungus, helminth or protist.
  • the pathogen is a virus (such as a virus that has a latency).
  • the virus is cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the one or more antigens of CMV is CMV pp65, CMV IE1, or a combination thereof.
  • the one or more antigens of CMV is CMV pp65.
  • the virus is Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the one or more antigens of EBV is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMPl, LMP2, or a combination thereof.
  • the one or more antigens of EBV is EBNA1, LMPl, LMP2, or a combination thereof.
  • the virus is BK virus (BKV), John Cunningham virus (JCV), herpesvirus (such as human herpesvirus-6 or human herpesvirus-8), adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.
  • BKV BK virus
  • JCV John Cunningham virus
  • herpesvirus such as human herpesvirus-6 or human herpesvirus-8
  • HAV hepatitis B virus
  • HCV hepatitis C virus
  • HSV herpes simplex virus
  • VZV varicella zoster virus
  • HPV human papillomavirus
  • MMV Merkel cell polyomavirus
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of the pathogen described herein.
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of CMV.
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of EBV.
  • the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of BKV, JCV, herpesvirus (such as human herpesvirus-6 or human herpesvirus-8), adenovirus, human immunodeficiency virus, influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.
  • herpesvirus such as human herpesvirus-6 or human herpesvirus-8
  • adenovirus human immunodeficiency virus
  • influenza virus ebola virus
  • poxvirus poxvirus
  • rhabdovirus or paramyxovirus
  • the human patient has an infection of the pathogen.
  • the human patient has a CMV infection ⁇ e.g., CMV viremia, CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV-positive meningoma, or CMV-positive glioblastoma multiforme).
  • CMV infection e.g., CMV viremia, CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV-positive meningoma, or CMV-positive glioblastoma multiforme.
  • the human patient has an EBV-positive lymphoproliferative disorder (EBV-LPD) (for example, an EBV-positive post-transplant lymphoproliferative disorder) resulting from EBV infection, such as B-cell hyperplasia, lymphoma (such as, B-cell lymphoma , non-Hodgkin lymphoma ⁇ e.g., diffuse large B-cell lymphoma, for example in the elderly), T-cell lymphoma, EBV- positive Hodgkin's lymphoma, Burkitt lymphoma), polymorphic or monomorphic EBV-LPD, autoimmune lymphoproliferative syndrome, or mixed PTLD (post-transplant
  • EBV-LPD EBV-positive lymphoproliferative disorder
  • the human patient has an EBV-positive nasopharyngeal carcinoma. In other embodiments of the methods of treating a human patient described herein, the human patient has an EBV-positive gastric cancer. In other embodiments of the methods of treating a human patient described herein, the human patient has an EBV+ leiomyosarcoma. In other
  • the human patient has an EBV-positive NK/T lymphoma.
  • the pathogen is a bacterium, such as mycobacterium tuberculosis.
  • the one or more antigens of a cancer can be one or more peptides or proteins whose expressions are higher in cancerous cells (of the corresponding type of cancer) relative to non-cancerous cells, or one or more peptides or proteins which are uniquely expressed in cancerous cells (of the corresponding type of cancer) relative to non-cancerous cells.
  • the cancer can be a blood cancer, such as, 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 multiple myeloma or plasma cell leukemia.
  • the one or more antigens of the cancer is Wilms tumor 1 (WT1).
  • the cancer can also be a solid tumor cancer, including, but is not limited to, a sarcoma, a carcinoma, a lymphoma, a germ cell tumor, or a blastoma.
  • the solid tumor cancer that can be, such as, 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 ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to one or more antigens of the cancer described herein. In a specific embodiment of the methods of generating a population of cells comprising antigen-specific cells described herein, the ex vivo sensitizing step comprises ex vivo sensitizing the population of human blood cells to WT1.
  • the human patient has a cancer described herein.
  • the human patient has multiple myeloma or plasma cell leukemia (e.g., a WT1 -positive multiple myeloma or plasma cell leukemia).
  • 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).
  • the human patient treated with a method described herein has failed a previous therapy for the pathogen or cancer, which previous therapy is not treatment with a population of cells comprising antigen-specific T cells according to the invention, due to resistance to or intolerance of the previous therapy.
  • a disease is considered resistant to a therapy, if it has no response, or has an incomplete response (a response that is less than a complete remission), or progresses, or relapses after the therapy.
  • the previous therapy could be an antiviral agent known in the art (e.g., an antiviral drug or antibody), or an anti-cancer therapy known in the art (e.g., a chemotherapy or a radiotherapy), as the case may be. 6.
  • TSC M cells stem cell-like memory T cells
  • Tet + TSC M cells rather than Tet + T N cells are the principal reservoir for rapid repopulation of immunodominant T cells in the circulation.
  • T N cell naive T cell
  • TSC M cell stem cell-like memory T cell
  • TC M cell central memory T cell
  • T EM cell effector memory T cell
  • Tet + T cells were regularly generated from CD62L + CD45RO " CD95 " T N cells and from CD62L + CD45RO " CD95 + T SCM cells, as well as TC M cells and T EM cells.
  • Tet + T cells derived from each of the T N , TSC M , TC M and T EM subsets generated IFN- ⁇ , T F- ⁇ and granzyme B.
  • Tet + T cells derived from each subset also expressed similar levels of PD-1 and KLRG-1. However, Tet + T cells derived from the T N subset and those derived from the TSC M subset expressed higher levels of CD27 and lower levels of CD57 than those derived from the T CM or T EM subset.
  • Tet + T cells derived from the T S C M subset were distinguished from those derived from the T N , TC M and T EM subsets by a significantly greater level of proliferation and by their rapid and selective expansion of LV-specific T cells bearing TCRs identical in sequence to those expressed by T EM and TC M in the blood.
  • the example described herein suggests that Tet + T S C M cells rather than Tet + T N cells are the principal reservoir for rapid repopulation of immunodominant T cells in the circulation. 6.2. Materials and Methods
  • PBMCs Peripheral blood mononuclear cells
  • T cell populations representing T N , TSC M , C M and T EM cells were gated and sorted on a BD FACS Aria-II SORT (BD Biosciences) based on the following markers: TSC M population as CD3 + CD45RO ⁇ CD62L + CD95 + , T N population as CD3 + CD45RO ⁇ CD62L + CD95 ⁇ , and the TC M and T EM populations as
  • CD3 + CD45RO + CD62L + and CD3 + CD45RO + CD62L were then sensitized with irradiated HLA-A0201 + and CMVpp65 + artificial antigen presenting cells (AAPCs) (O.
  • T cells were supplemented with IL-7 (5 ng/ml), and IL-15 (5 ng/ml) on day 4 and day 7 after culture initiation and were re-stimulated every 10 days with AAPCs and autologous PBMC feeders. After day 12, IL-7 (5 ng/ml), IL-15 (5 ng/ml), and IL-2 (20U/ml) were supplemented to T cell cultures every other day. 6.2.3. Quantitation of antigen-specific CD8+ T cells by tetramer analysis
  • CMVpp65 specific T cells responsive to the LV peptide within the cultured T cells were enumerated by Tetramer analysis as previously described (Hasan et al., 2009, J Immunol 183 :2837-2850).
  • Commercially available CMVpp65 MHC -peptide tetramers for HLA-A*0201 and A*2402-bearing peptide sequences LVPMVATV (SEQ ID NO: l) and QYDPVAALF (SEQ ID NO:2) (Beckman Coulter) were used.
  • T cells were incubated with CD3 FITC, CD8 PE, CD4 PerCP (BD Biosciences) and an APC-conjugated tetrameric complex for 20 mins on ice, washed and analyzed by FACS using a FACSCalibur flow cytometer with dual laser for four-color capability. Data were analyzed using FlowJo software (Tree Star). T cells were gated on CD3- and CD8-positive cells to determine the percentage of tetramer-positive CD8 + T lymphocytes.
  • T cell subsets were further characterized by flow cytometry using specific T cell memory and co-stimulatory markers.
  • T cells were labeled with fluorescent antibodies against CCR7 PE (BD Biosciences), CD27 FITC (Miltenyi Biotec), CD57 FITC (Miltenyi Biotec), CD 127 PE (Miltenyi Biotec), CD28 PECy7 (BD Biosciences), KLRGl PE (Miltenyi Biotec) and PD1 PECy7 (eBioscience), and analyzed by FACS. Doublet exclusion for lymphocytes was achieved by gating on forward scatter (FSC) vs side scatter (SSC) followed by FSC (height) versus FSC (area).
  • FSC forward scatter
  • SSC side scatter
  • T cells Functional activity of T cells was evaluated after short secondary stimulation using several parameters including secretion of intracellular cytokines (IFN- ⁇ and TNF-a), activation marker expression (CD 137), and cytotoxicity (CD 107a) by intracellular fluorescence staining. All antibodies were purchased from BD bioscience. Irradiated autologous B-lymphoblastoid cell line cells (BLCL cells) loaded with NLVPMVATV (SEQ ID NO: 1) peptide were co-incubated with T cells in an effector to target ratio of 1 :5 for 16 hours in the presence of 1 ⁇ g/ml brefeldin A (Sigma-Aldrich).
  • Co-cultured T cells were then labeled with anti-CD3 APC and anti CD8 PE for 15 mins at room temp, washed and then permeabilized with Perm solution (BD Biosciences) and then co-incubated with anti-IFN- ⁇ PECy7 (BD Biosciences) and anti-TNF-a APC (Miltenyi Biotec), or anti-CD 137 PE (BD Biosciences), or anti-CD 107a FITC (BD Biosciences).
  • EdU labeling (Therm oFisher) was used to evaluate T cell proliferation. 10 ⁇ EdU was added to the culture media for lh at 37 °C. Labeled cells were washed with PBS and resuspended in T cell culture media. T cells were then analyzed by flow cytometry, and the proportion of proliferating T cells was determined by the percentage of EdU + gated T cells using the FlowJo software (Treestar). Apoptotic T cells were defined by Annexin V staining (BD Biosciences).
  • T cell receptor ⁇ (TCRVP) chain hypervariable complementarity-determining region 3 was amplified and sequenced from DNA extracted from NLV-Tet + T cell subsets (T N , TSC M , T CM , and T EM ) isolated by fluorescence-activated cell sorting (FACS) (purity > 95%) or LV-Tet + T cells derived from these subsets, from days 0, 15 or 30 post-stimulation using the immunoSEQ platform at Adaptive Biotechnologies. Rearranged CDR3 sequences were classified as nonproductive if they included insertions or deletions resulting in frameshift or premature stop codons, and were excluded from subsequent analyses, according to the immunoSEQ validated algorithm.
  • FACS fluorescence-activated cell sorting
  • TCR clonality and sample overlap were determined using the immunoSEQ Analyzer 2.0. within a range of 0 to 1, where a low number indicates higher diversity, while a high number indicates higher clonality within the sample.
  • Sample overlap indicates the percent of similar clones within a pair of sample types.
  • T SCM cells within human PBMCs as a specific subset of memory T cells with high proliferative potential that when modified to express a tumor-specific chimeric antigen receptor, also exhibits superior functional activity.
  • This example described herein was designed to test whether in vivo primed virus- specific human TSC M cells also exhibit these attributes. T cells were isolated from PBMCs of 12 healthy HLA A0201 + CMV seropositive donors.
  • TSC M population was identified as CD3 + CD45RO ⁇ CD62L + CD95 + cells, T N population as CD3 + CD45RO " CD62L + CD95 " cells, and T CM and T EM populations as CD3 + CD45RO + CD62L + and CD3 + CD45RO + CD62L ⁇ cells respectively ( Figure 1).
  • CMVpp65 As a model antigen, the memory phenotype of the antigen-specific T cells was then examined. Accordingly, the CMVpp65-specific T cell populations in the blood of 11 of 12 donors were evaluated. In each of the donors tested, a discrete population of antigen- specific T cells could be identified using HLA peptide tetramers that were responsive to the well known CMVpp65 epitope LVPMVATV (SEQ ID NO: l) presented by HLA-A0201. Further analysis of these tetramer positive T cells demonstrated that the majority of memory T cells in peripheral blood that recognize LV epitope bear either a TC M or T EM phenotype (0.9%-31% and 15.
  • the sorted T cell subsets were sensitized with artificial antigen presenting cells (AAPCs) exclusively expressing HLA-A*0201, CMVpp65 and T cell costimulatory molecules as previously described (Hasan et al., 2009, J Immunol 183 :2837-2850). Then this approach was modified to include supplementation of the cultures with IL-7 and IL-15 every 2 days beginning at day 4 as previously described (Cieri et al., 2013, Blood 121 :573-584).
  • AAPCs artificial antigen presenting cells
  • HLA-A*0201 NLV- tetramer + CD8 + T cells could be enriched up to 5.5 % by 7 days from 0.6 % at day 0 as shown for one representative donor ( Figure 2A).
  • Figure 2A A similar enrichment of HLA-A*0201 LV-tetramer + T cells was also observed within the TC M - and T EM -derived CD8 + T cells (6.1% and 4.1% respectively) (Figure 2A).
  • Tsc M -derived cells Over a period of 4 weeks after antigen-specific in vitro sensitization, Tsc M -derived cells gradually acquired T CM and T EM phenotype within both the LV-Tet + as well as LV-Tet " T cell populations ( Figures 2B and 2C). Strikingly, within the NLV-Tet + T cells derived from the TSC M subset, a proportion of cells with a CD45RA + and CD62L + TSC M phenotype could be detected for up to 14 days in culture. By this time, both TcM-and TEM- derived cells had converted to T EM phenotype ( Figure 2B).
  • T N cells CMV-specific T cells derived from CD95 " T N cells also were generated. Regardless of the detection of NLV-Tet + T cells in the sorted T N cells within the peripheral blood before antigen stimulation, NLV-Tet + T cells could be generated from 6 of 6 different donors by this method of sensitization from sorted T N cells.
  • T N cells upregulated the CD95 expression within 2 days and converted to a TSC M phenotype.
  • T N cells also maintained a less differentiated memory phenotype, with expression of CD62L and CCR7 within a proportion of NLV-Tet + T cells for a longer duration during antigenic stimulation than the T CM - and T EM -derived cells.
  • T N -derived cells demonstrated CCR7 expression in 12% of NLV-Tet + T cells after 14 days of continuous antigenic stimulation, in comparison to 7.5%, 0.8% and 0.5% CCR7 expressing NLV-Tet + T cells derived from TSC M , TC M and T EM subsets respectively.
  • the differential expression of CCR7 in T N -derived LV-Tet + T cells was significantly higher than that observed in TC M - and T EM -derived LV-Tet + T cells (p ⁇ 0.01) (Figure 2D).
  • the enrichment of LV-Tet + populations was less pronounced within the T N -derived cells (0.4%, 0.4% and 3.6% at Day 7, 14 and 30 as shown in Figures 2A, 2B and 2C) than within T cells derived from the memory T cell populations (T S C M , T CM and T EM cells).
  • the slow enrichment of CMV-specific cells might result from the extremely low precursor frequencies in the T N subset in the peripheral blood.
  • CD27 is constitutively expressed as a co-stimulatory molecule on naive and memory T cells. Its expression increases upon T cell activation, and is lost at the fully differentiated effector phase (Hintzen et al., 1993, J Immunol 151 :2426-2435). Consistent with its role in T cell activation and proliferation, a recent study also demonstrated that CD27 co-stimulation improves the function of chimeric antigen receptor (CAR) modified T cells (Song et al., 2012, Blood 119:696-706).
  • CAR chimeric antigen receptor
  • CD57 which has been described to be indicative of replicative senescence and antigen-induced apoptosis in HIV-specific CD8 + T cells (Brenchley et al., 2003, Blood 101 :2711-2720), was evaluated. After antigen-specific stimulation, a significantly lower proportion of CD57 expressing cells was found within T N -derived NLV-Tet + T cells, compared to TC M - and T EM -derived NLV-Tet + T cells as shown for 6 donors tested (**p ⁇ 0.005 and *p ⁇ 0.05) ( Figures 3C and 3D).
  • Tsc M -derived cells expressed lower levels of CD57 than TC M - and T EM -derived cells, though this difference was not statistically significant for the donors tested. Nonetheless, this data demonstrate a trend towards increasing levels of CD57 expression with increasing T cell differentiation (T N ⁇ T S C M ⁇ T CM ⁇ T EM )-
  • Tsc M -derived T cells demonstrate higher proliferative capacity leading to superior expansion of antigen-specific T cells
  • TSC M cells maintain a less differentiated memory phenotype during antigen-specific activation, and a higher level of CD27 expression which sustains a greater proliferative potential.
  • TSC M cells might serve as a durable reservoir of antigen-experienced T cells for memory immune response upon antigen re- challenge.
  • the in vitro proliferative capacity of different memory T cell subsets upon antigen-specific stimulation with AAPCs expressing CMVpp65 was evaluated, and the phenotype of proliferating T cells was examined using EdU labeling ⁇ see Section 6.2, supra). Within total T cell populations stimulated, vigorous T cell proliferation was observed starting at 3 days after stimulation.
  • TSC M cells appear to reach their maximum proliferative capacity on days 3-5 in culture, suggesting that this is the optimal time to carry out transduction of the TSC M cells, if desired, particularly transduction with a vector ⁇ e.g., a retroviral vector) that requires cell proliferation for integration.
  • a vector ⁇ e.g., a retroviral vector
  • CD137 has been described as a marker of antigen-specific CD8 + T cells that correlates with functional activity including production of cytokines TNF-a and IFN- ⁇ , as well as cytotoxicity activity (evaluated by CD 107a degranulation assay) (Wolfl et al., 2007, Blood 1 10:201-210).
  • T cells capable of secreting both TNF-a and IFN- ⁇ were observed in T cells derived from all subsets including T N and T S CM- TO demonstrate cytotoxicity activity of T cells derived from different subsets, the CD 107a degranulation assay was performed. Complementing their capacity to secrete cytokines, T N - and TscM-derived cells also demonstrated functional cytotoxic activity against peptide loaded autologous targets. The percentage of CD107a-expressing cells within the T N - and TscM-derived CD8 + T cell population was 20% and 22% respectively (Figure 5B).
  • epitope specific T cells can be expanded in vitro from T N and T S CM cells by stimulation with antigen-expressing AAPCs in the presence of IL-7 and IL-15. These epitope specific T N - and TSCM- derived cells, after expansion in vitro, demonstrate a less differentiated memory phenotype than TCM- or T E M-derived epitope specific T cells expanded in vitro.
  • T N - and T S cM-derived cells are functional as evidenced by their ability to release cytokines and degranulate in response to antigen stimulation.
  • T S cM-derived CMV-specific T cells exhibit an oligoclonal repertoire of public TCRs that is similar to that of TC M - and T EM -derived populations
  • NLV-Tet + T cells derived from naive as well as memory T cell subsets were examined.
  • TCR sequences within NLV-Tet T cells TN-, TSC M -, TC M - and TEM-derived NLV-specific T cells from two CMV seropositive donors were evaluated. It was first evaluated if there were common TCR sequences within CMV-specific T cells derived from T N , TSC M , TC M and T EM cells.
  • T N -, TSC M - , TC M - and T EM - derived T cells were expanded in vitro using artificial antigen presenting cells for 30 days, and the NLV-Tet + T cells and NLV-Tet " T cells were sorted. Then TCR repertoire analysis was performed by next-generation sequencing on the sorted Tet + T cells in comparison to the Tet " T cells. TCR sequencing data were analyzed for similarities in nucleotide sequences by sample overlap. This analysis demonstrates a high overlap between TCR sequences for TC M - and T EM -derived NLV-Tet + T cells post-stimulation (96% overlap in Figure 6C).
  • T S c M -derived cells demonstrated high similarities with T N - and T CM -derived cells, and less similarities with T EM -derived cells (T N : 84%; TC M : 91%; and T EM: 57% in Figure 6C).
  • T N -derived Tet + cells also had a high degree of TCR similarities/overlap with Tet + Tc M -derived cells (87%)) but very little overlap with T EM -derived cells (14%). No TCR overlap was detected within NLV-Tet " T cells derived from either naive or memory T cells (data not shown).
  • Tet + T cells recognizing the NLV epitope that are derived from the naive like T N subset demonstrate a high degree of overlap between TCR sequences expressed by TSC M - and Tc M -derived cells. While the TCR sequence detected in TSC M and TC M derived T cells were less frequently detected in the T EM -derived cells, nevertheless, there was a significant number of specific public TCRs common to T cells derived from all memory T cell compartments.
  • T N -, TSC M -, TC M - and T EM -derived NLV-Tet + T cells were sorted from another donor prior to stimulation and the TCR sequence was evaluated for clonality and overlap.
  • TCR clonality describes the degree to which one or a few clones dominate the repertoire, with 0 being a flat distribution and 1 being an entirely oligoclonal sample.
  • T EM NLV-Tet + T cells demonstrated a high degree of clonality, with a clonality index of 0.66 ( Figure 6A).
  • Tsc M -derived Tet + T cells developed a restricted TCR repertoire within 15 days post-stimulation, which is highly similar to Tc M -and T EM -derived Tet + T cells (T S C M TC M : 86% and T S C M T EM : 95% in Figure 6E).
  • T S C M TC M 86%
  • T S C M T EM 95% in Figure 6E.
  • T N -derived Tet + T cells demonstrated very different TCR sequences compared to TC M - and T EM -derived Tet + T cells with an overlap of 38% and 3.8%) respectively (Figure 6E).
  • T SC M constitute the principal and immediate source for replenishing immunodominant LV-specific TC M and T EM populations in the circulation.
  • the T N -derived Tet + T cells also contained unique sequences that that were not shared with Tet + T cells derived from other subsets.
  • the sequence CAS S YVTGTGNYGYTF (SEQ ID NO: 5) was only detected in T N -derived Tet + T cells post-stimulation in high frequencies (89% and 87 % at day 15 and 30 respectively, Table 2). This suggests that T N -derived Tet + T cells can ultimately potentially expand upon antigen exposure to populate the immunodominant memory T cell pool, and also expand unique clones recognizing the antigen.
  • Table 2 Predominant clonotypes represented within naive and memory A2- LV-specific CD8 + T cells in CMV seropositive donors
  • Tsc M -derived T cells demonstrate higher proliferative capacity generating superior expansion of antigen-specific T cells.
  • the phenomenon of epitope-specific T cell immunodominance could be a consequence of either over-representation of or preferential proliferation of TSC M cells within T cells responding to immunodominant compared to subdominant epitopes.
  • the in vitro T cell proliferative capacity of T S C M , T CM , and T EM subsets, respectively, from a CMV seropositive donor co-inheriting HLA-A*0201 and A*2402, upon antigen specific stimulation was measured.
  • the A24-QYD-Tet + T cells contained a higher percentage of Annexin V + cells compared to A2-NLV-Tet + T cells ( Figure 7B). These data suggest that a higher level of T cell apoptosis within subdominant epitope- specific T cells might promote the preferential enrichment of immunodominant T cells.
  • This example described herein characterized HLA-A0201 -restricted T N , TSC M , TC M and T EM cells specific for the immunodominant NLV peptide of CMVpp65 that were isolated by immunoadsorption to tetramers from the blood of healthy seropositive donors, and T cells derived from these subsets after sequential intervals of in vitro sensitization.
  • the Tet + and Tet " T cells from the same T cell subset population were also compared. The results suggest a major role for TSC M cells as a durable reservoir of T cell memory in the recall immune response.
  • Tet + TSC M in the blood exhibit a CD45RA + CCR7 + and CD62L + phenotype similar to T N cells, but also express CD95.
  • Tet + CD95 Tet + CD95
  • Tet + T S C M cells expressed significantly higher levels of the co-stimulatory marker CD27 than detected on Tet + TC M and Tet + T EM cells; Tet + TSC M cells demonstrated somewhat higher expression of the activation marker CD127.
  • Tet + T N and Tet + TSC M cells expressed lower levels of the senescence marker CD57; Tet + T N cells expressed a significantly lower level of CD28.
  • Tet + T cells derived from all subsets generated a similarly high proportion of cells expressing PD-1, which differed significantly from the low proportion of PD-1 + cells in the Tet " T cells derived from these subsets.
  • the killer-cell lectin like receptor Gl (KLRG1) which is usually considered to be a marker of late differentiation, was expressed to a similar degree in the NLV-peptide responding T N -, TSC M -, TC M - and T EM -derived T cells. In contrast, this marker was expressed at a minimal level in the Tet " T N -derived T cells.
  • Tet + T N - and T S cM-derived T cells still differentially express the CD45RA, CCR7 and CD62L markers associated with earlier stages of maturation and the capacity for targeted migration and longer survival.
  • Tet + T N -, TSC M -, TC M - and T EM -derived T cells all contained cells capable of secreting TNF-a and IFN- ⁇ , and degranulating in response to antigen
  • Tet + CD95 T N cells on the basis of their proliferative response to antigen stimulation. Indeed, their proliferative response also significantly exceeded that of Tet + TC M and Tet + T E M cells.
  • TCR sequencing analysis in the example described herein has shown that in the peripheral circulation, the T N and TSC M cells recognizing the same epitope LV are more diverse than TC M and T EM cells. This broad TCR repertoire of T N and T S C M cells recognizing the same pathogen could be hypothesized to provide better control of virus infection.
  • TSC M cells generate T cells that are oligoclonal within 15 days after antigen encounter, and further that these T cells express a repertoire closely related to that of Tet + T EM and Tet + TC M cells that are dominant in the circulation.
  • T S c M -derived CMV-specific T cells share high overlap of TCR usage with TC M and T EM -derived CMV-specific T cells early post- stimulation, provides evidence that Tsc M -derived T cells are particularly effective in
  • T N cells serve as a pool of precursor cells capable of selecting peptide-specific T cells with a broader spectrum of binding characteristics, from which memory T cells of optimal avidity can be selected for expansion if required to control viral variants.
  • T cell responses to latent viruses often focus on a small number of the available antigenic epitopes and use a narrow TCR repertoire, a phenomenon termed
  • immunodominance A number of factors have been reported to influence immunodominance, including antigen presentation, peptide-MHC binding affinity and stability or TCR avidity and the frequency of precursors in the naive T cell pool (Khan et al., 2007, J Immunol 178:4455- 4465). In the two donors tested in the example herein, an overrepresentation or a preferential proliferation of Tsc M -derived T cells responding to immunodominant epitopes was not found, compared to those responding to subdominant epitopes. Instead, a higher level of T cell apoptosis was observed within subdominant epitope-specific T cells, which might promote the preferential enrichment of immunodominant T cells.
  • Tet + T cells specific for the NLV HLA-A0201 epitope generated from TSC M cells was their expression of T cell receptor with amino acid sequences identical to sequences previously reported for public T cell receptors specific for the NLV epitope (Yang et a/., 2015, J Biol Chem 290:29106-29119).
  • Public T cell receptors are peptide- specific TCRs with highly homologous sequences detected in multiple individuals (Li et a/., 2012, Cell Res 22:33-42).
  • Public TCRs have been described in T cells responding to a variety of human viruses (Argaet et a/., 1994, J Exp Med 180:2335-2340).
  • NLV pepude-specific T cell repertoires have been shown to exhibit a high prevalence of public TCRs (Wang et a/., 2012, Sci Transl Med 4: 128ral42; Nguyen et a/., 2014, J Immunol 192:5039-5049; Trautmann et a/., 2005, J Immunol 175:6123-6132). Indeed, seven public CDR3a and six CDR3 motifs account for -70% of the total NLV-specific TCR response (Wang et a/., 2012, Sci Transl Med 4: 128ral42).
  • the example herein also identified a common TCR CDR3 represented within T N , TSC M , T M and T EM -derived Tet + T cells post-stimulation that bears the S* n TG* n GY (SEQ ID NO: 16; *n indicates any amino acid sequence of any length and any amino acid combination) motif which has the highest reported frequency among the other published motifs.
  • antigen-specific T S C M - derived T cells exhibit and maintain a less differentiated phenotype than those derived from other memory T cell populations responding to a single epitope, displaying an expansion profile of tissue homing, co-stimulatory and senescence markers that is intermediate between T N and TC M - derived cells.
  • Tet + CMVpp65 LV-specific T cells were regularly detected cells derived from the CD95 " T N cell compartment.
  • antigen-specific TSC M cells differ from Tet + T N cells as well as Tet + TC M and Tet + T EM cells in that they proliferate to a much higher degree in response to antigen stimulation.
  • Tet + T N and Tet + TSC M - derived cells unlike Tet " T N and Tet " T S cM-derived cells, were found to express PD-1 and KLRG- 1 at levels similar to those expressed by Tet + TC M - and Tet + T EM -derived cells. They also exhibit effector function, indicating the capacity to generate TNF-a, IFN- ⁇ and granzyme B.
  • TSC M cells in response to antigen stimulation, TSC M cells not only undergo marked proliferation but also rapidly select T cell clones specific for the CMVpp65 NLV epitope presented by HLA-A*0201 that, by TCR sequencing, are public TCRs identical to those expressed by NLV/HLA-A*0201 specific T EM and T CM cells that are immunodominant in the blood of the HLA-A*0201 seropositive donors.
  • Tet + TSC M cells Tet + T N cells do not rapidly select clones expressing the immunodominant TCRs detected in T EM and TC M population in the blood.
  • Tet + T N cells maintain a distinct repertoire that is markedly more varied than that detected in NLV-specific T CM and T EM cells in the blood, as that detected in T S C M - derived cells upon 15 days of in vitro stimulation.
  • the example herein suggests that while the expansion of TSC M , TC M and T EM cells proliferating in response to the immunodominant epitope NLV presented by HLA-A*0201 is similar to those proliferating in response to the subdominant QYD epitope presented by HLA-A*2402 expressed by the same donor, the proportions of immunodominant LV-specific TSC M , C M , and T EM -derived cells that undergo apoptosis are markedly smaller than that of the subdominant QYD-specific counterparts.
  • TSC M subset serves as the major durable reservoir for repopulating immunodominant T EM and TC M cells in the circulation upon secondary antigenic challenge.
  • Adoptive transfer of virus-specific T cell lines enriched for T S C M could potentially provide better disease control by providing immunodominant virus-specific T cells capable of rapid and extensive proliferation and enhanced persistence.

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