WO2022056285A1 - Cocktails de cytokines pour expansion sélective de sous-ensembles de lymphocytes t - Google Patents

Cocktails de cytokines pour expansion sélective de sous-ensembles de lymphocytes t Download PDF

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WO2022056285A1
WO2022056285A1 PCT/US2021/049902 US2021049902W WO2022056285A1 WO 2022056285 A1 WO2022056285 A1 WO 2022056285A1 US 2021049902 W US2021049902 W US 2021049902W WO 2022056285 A1 WO2022056285 A1 WO 2022056285A1
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cells
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cell
cov
sars
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Patrick Hanley
Christopher LAZARSKI
Michael Keller
Catherine BOLLARD
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Children's National Medical Center
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Priority to US17/537,816 priority Critical patent/US20220160767A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2318—Interleukin-18 (IL-18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)

Definitions

  • the disclosure relates to methods of culturing and expanding CD4+ and/or CD8+ T cells in culture.
  • the methods include expanding, proliferating and storing lymphocytes in tissue culture by exposing the lymphocytes to a combination of cytokines and/or nucleic acids expressing cytokines (or functional fragments or variants thereof).
  • the disclosure further relates to methods of generating and manufacturing CD4+ and/or CD8+ T cells that are specific to one or a plurality of viral antigens.
  • VSTs virusspecific T cells
  • Adoptive immunotherapy with virus-specific T cells therefore can be an alternative and effective way to treat viral infections that lack any effective treatment options.
  • T cells specific to coronavirus antigens, specifically antigens from SARS-CoV-2 can be an alternative and effective way to treat COVID-19.
  • IL-6 which has been proposed to enhance Thl7 development [9]
  • IL-7 which promotes T cell homeostatic survival [10-12]
  • IL-21 which also promotes the activity of CD8+ T cells and formation or maintenance of central memory [13-15]
  • IL- 2 is a canonical T cell growth cytokine, which continues to be used in clinical trials due to its demonstrated effectiveness in expanding T cells derived from tumor infiltrating lymphocytes [16]
  • engraftment and survival of sufficient numbers of highly differentiated memory T cell subsets upon adoptive transfer is difficult to achieve; Bush, D. H., et al., , Role of memory' T cell subsets for adoptive immunotherapy.
  • T cell based therapies are limited by rapid acquisition of tolerant phenotypes of T cells or limited by low percentages of memory T cells produced in vitro.
  • T cell culture systems have been shown to expand T cells specific for a virus using specific cytokines, there is a need for a safer, more reliable, simpler, clinically scalable method for inducing, expanding, or recovering clinically relevant numbers of virus-specific or tumor-specific T cells, especially for T cells having phenotypes useful for sustained prevention or treatment of immunosuppressed patients at risk of opportunistic viral infections or for cancer relapse.
  • the disclosure relates to certain cytokine compositions as well as the same compositions that induce or stimulate growth and proliferation of CD4+ and CD8+ T cell subpopulations after exposure for a time period sufficient to induce the growth or proliferation.
  • Tire method allows for a scientist to choose how and when to grow more cytotoxic T cells instead of helper ( 1)4 T cells. Essentially, this allows one of ordinary skill to toggle between CD4 and CD8 dominance by choosing a different cytokine mixture, which ultimately creates T' cell products of different compositions. Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed methods and compositions.
  • the disclosure also relates to a tissue culture system comprising a plurality of lymphocytes positioned within at least one vessel, cell culture media and a composition comprising at least two cytokines from Table 1 or functional fragments or variants thereof, wherein the functional fragments thereof or the variants thereof comprises at least about 75% sequence identity to the sequences identified in Table I,
  • the disclosure relates to a method of selectively growing memory effector T cells from a cell composition comprising naive T cells comprising: contacting one or plurality of lymphocyt.es comprising the naive T cells with at least two peptides or nucleic acids encoding peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100% or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4
  • the disclosure relates to a method of selectively growing memory' effector T cells from a cell composition comprising naive T cells comprising: contacting one or plurality of lymphocytes comprising the naive T cells with at least two peptides or nucleic acids encoding peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4, functional fragment
  • the disclosure also relates to a method of inducing an antigen-specific immune response against a viral antigen, the method comprising: (a) contacting one or plurality of lymphocytes with at least two peptides or nucleic acids encoding peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period
  • the viral antigen is from a virus of the family Coronaviridae. In some embodiments, the viral antigen is from a coronavirus. In some embodiments, the viral antigen comprises at least a portion of a coronavirus membrane protein. In some embodiments, the viral antigen comprises at least a portion of a coronavirus envelope protein. In some embodiments, the viral antigen comprises at least a portion of a coronavirus spike protein. In some embodiments, the viral antigen comprises at least a portion of a coronavirus nucleocapsid protein. In some embodiments, the viral antigen is from SARS-CoV-2, In some embodiments, the viral antigen comprises at least a portion of SARS-CoV-2 membrane protein.
  • the viral antigen comprises at least a portion of SARS-CoV-2 envelope protein. In some embodiments, the viral antigen comprises at least a portion of SARS-CoV-2 spike protein. In some embodiments, the viral antigen comprises at least a portion of SARS-CoV-2 nucleocapsid protein.
  • the methods disclosed herein may be used to produce T cells which recognize other opportunistic pathogens such as bacterial (mycobacterium including Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellular e, or Mycobacterium kansasii; Salmonella), fungal (Candida, Cocci diodomy cosis, Ciyptococcus, Histoplasmosis, Pneumocystis pneumonia), tumor (invasive cervical cancer), or parasite (Ciyptosporidiosis, Toxoplasmosis) pathogens.
  • T cell immunity is often directed against intracellular pathogens which may be targeted by T cells produced as disclosed herein.
  • Opportunistic infections which are infections that are generally of lower virulence within a healthy host but cause more severe and frequent disease in immunosuppressed individuals, typically occur in the period 1 month to 1 year after transplantation.
  • Some nonlimited examples of opportunistic viral infections include cytomegalovirus (CMV), herpes simplex virus (HSV, type 1 or type 2, HHV-8, or HIV.
  • the antigen-specific T cells are used to treat infections by neonatal, congenital, and/or intrauterine pathogens including rubella, cytomegalovirus (CMV), parvovirus Bl 9, varicella-zoster (VZV), enteroviruses, HIV, HTLV-1, hepatitis A, hepatitis B, hepatitis C, Lassa Fever, and Japanese Encephalitis.
  • Perinatal and neonatal infections agents include Herpes Simplex Virus (including Human Herpes Simplex types 1 and 2), VZV, Enteroviruses, HIV, Hepatitis B, Hepatitis C, and HTLV-1.
  • pathogens include respiratory syncytial virus (RSV), metapneumovirus (hMPV), rhinovirus, parainfluenza (PIV), and human coronavirus, norovirus, Herpes simplex virus (HSV), SARS-1, SARS2, Zika virus, and encephalitis viruses.
  • RSV respiratory syncytial virus
  • hMPV metapneumovirus
  • PIV parainfluenza
  • HSV Herpes simplex virus
  • SARS-1 SARS2
  • Zika virus Zika virus
  • encephalitis viruses encephalitis viruses.
  • the disclosure further relates to a method of generating, culturing and/or manufacturing CD4+ and/or CD8+ effector memory cells comprising: contacting one or plurality of lymphocytes with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation of the one or
  • the disclosure further relates to a method of generating, culturing and/or manufacturing CD4+ and/or CD8+ effector memory cells comprising: contacting one or plurality' of lymphocytes with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality' of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation
  • the disclosure relates to a method of expanding a population of CD4+ and/or CD8+ memory effector T cells in a composition of cultured cells comprising contacting one or plurality of lymphocytes with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality' of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation of the
  • the disclosure relates to a method of expanding a population of CD4+ and/or CD8+ memory effector T cells in a composition of cultured cells comprising contacting one or plurality' of lymphocytes with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation
  • the disclosure relates to a method of culturing a composition comprising a population of one or a plurality of T cells comprising contacting the one or plurality of T cells with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation of the one or plurality
  • the disclosure relates to a method of culturing a composition comprising a population of one or a plurality'' of T cells comprising contacting the one or plurality of T cells with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4, functional fragments or variants thereof for a time period sufficient to stimulate growth and proliferation of the one
  • the disclosure also relates to a T cell composition comprising from about 2% to about 23% CD4+ and/or CD8+ memory effector cells manufactured or grown from a population of naive T cells or lymphocytes by any of the disclosed methods provided herein.
  • the disclosure further relates to a tissue culture system comprising: lymphocytes from a subject, cell culture media, and a composition comprising at least two polypeptide or nucleic acid encoding peptides chosen from a polypeptide that comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two polypeptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
  • the disclosure also relates to a T cell composition comprising from about 2% to about 23% CD4+ and/or CD8+ memory' effector cells manufactured or grown from a population of naive T cells or lymphocytes by any of the disclosed methods provided herein.
  • the disclosure further relates to a tissue culture system comprising: lymphocytes from a subject, cell culture media, and a composition comprising at least two polypeptide or nucleic acid encoding peptides chosen from a polypeptide that comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to human IL-18, IL-15, IL-6, IL-7, IL-4 or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two polypeptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%
  • any of the disclosed methods further comprise wherein the one or plurality of T cells are naive prior to step of contacting and the one or plurality of T cells are CCR7+ CD45RO+ after performing the step of contacting.
  • the disclosure relates to methods of treating a Coronaviridae infection comprising administering one or a plurality of compositions comprising a therapeutically effective amount of T cells of the disclosure stimulated with: one or a plurality of cytokine compositions disclosed herein; and/or one or a plurality of viral antigens that comprise from about 70% to about 100% sequence identity to SEQ ID: 11, 12, 13, and/or 14, or antigen fragments thereof.
  • the Coronaviridae infection is a COVID-19 infection.
  • the disclosure also relates to a method of treatment, wherein the step of administering comprises intravenous or parenteral administration injection of cells.
  • FIG. 1 depicts the experimental design described in Example 1.
  • Peripheral blood was collected from consenting donors and cells were purified by Ficoll gradient isolation. 1 x 10 5 cells were plated in each well on Day 0 with CMV peptide pools (IE1 and pp65) and the indicated cytokine concentration alone or in combination. On Day 7, plates were split equally into fresh media and with fresh cytokines. On Day 10, plates were re-stimulated with or without additional peptides for 6 hours, fixed, and stained for phenotype (CD3, CD56, CD4, CD8), viability, and effector cytokine secretion (IFNy, TNFa).
  • phenotype CD3, CD56, CD4, CD8
  • IFNy effector cytokine secretion
  • FIG. 2A, 2B, 2C and 2D depict the plate layouts described in Example I. 1 x 10 5 cells were plated in each well on Day 0 with CMV peptide pools encompassing IE1 and pp65 and the indicated cytokine concentration alone or in combination for plate layout 3 (FIG. 2A). On Day 7, plates were split equally into fresh media and with fresh cytokines. On Day 10, plates were restimulated with or without peptides for 6 hours, fixed, and stained for phenotype (CD3, CD56, CD4, CD8), viability, and effector cytokine secretion (IFNy, TNFa).
  • phenotype CD3, CD56, CD4, CD8
  • IFNy effector cytokine secretion
  • FIG. 3 depicts the gating strategy described in Example 1.
  • a representative sample was analyzed in Flowjo for staining and gating of effector lymphocyte populations.
  • Cells were separated from debris by gating forward scatter vs. side scatter.
  • Viable cells were identified by low' staining of Live Dead viability dye.
  • T cells were identified by CD3+ staining, and effector subsets were subsequently characterized by CD4+ vs CD8+ staining.
  • Cells were then evaluated for cytokine production after culture with peptide pools by comparing staining of IFNy and TNFa with wells cultured in media alone.
  • FIG. 4 depicts a summary-- of representative heat map data obtained from the screening of additional cytokine combinations described in Example 1.
  • 1 x 10 5 cells from Sample 3 and Sample 4 w'ere plated in each well on Day- 0 with CMV peptide pools encompassing IE1 and pp65 and the indicated cytokine alone or in combination for plate layout 3.
  • Cells w'ere analyzed for the total count of viable CD3+ T cells and the frequency of effector cytokine secretion over background (IFNy, TNFa),
  • FIG. 5A and 5B depict identification of superior cy tokine combinations via high throughput flow cytometry analysis.
  • FIG. 5A Quantifications of phenotype and function of two plates from Sample 4 were analyzed by flow-- cytometry on Day 10, with entire contents of wells collected and visualized by heat map. The total count of viable CD3+ T cells were quantified from w-ells restimulated with media alone. The frequency of IFNy+ CD3+ cells was derived from the frequency of IFNy+ CD3+ cells after re-stimulation with IE1 and pp65 peptide pools and subtracted from media alone control wells.
  • FIG. 5A Quantifications of phenotype and function of two plates from Sample 4 were analyzed by flow-- cytometry on Day 10, with entire contents of wells collected and visualized by heat map. The total count of viable CD3+ T cells were quantified from w-ells restimulated with media alone. The frequency of IFNy+ CD3+ cells was derived from the frequency of I
  • FIG. 5B Wells containing the highest concentration of cytokines were compared between Samples 1-4 when cultured using plate layouts 1 and 2. The total recovered viable CD3 + count and the frequency of CMV specific CD3+ IFNy+ cells (n > 8) was compared across each sample.
  • FIG. 6 A, 6B and 6C show that culture in IL- 15 and IL-6 stimulates expansion of CMV specific CD3+ T cells equal or better than IL-4 and IL-7.
  • the median total count of viable CD3+ CD4+ cells, CD3+ CD8+ cells, and the ratio of CD4+/CD8+ cells were calculated from wells and shown in FIG. 6B. From wells re-stimulated with IE1 and pp65 peptide pools, the median frequency of CD3+ TFNy+ cells and CD3+ IFNy+ TNFa+ cells were calculated from wells and shown in FIG. 6C, and the frequency of IFNy+ cells within CD4+ CD3+ and CD8+ CD3+ subtypes were analyzed.
  • FIG. 7A, 7B, 7C and 7D shown that T cell therapy products are effector memory in phenotype (CCR7- CD45RO+).
  • Cells were analyzed for the expression of memory markers CCR7 and CD45RO and divided into four populations both pre and post-culture with cytokines.
  • the preculture memory phenotype of viable cells was quantified for all samples (FIG. 7 A) according to the layout presented for representative Sample 1 (FIG. 7B).
  • samples were analyzed again for memory markers CCR7 and CD45RO and averaged samples cultured in IL- 15/IL-6 and IL-4/IL-7 growth conditions (FIG. 7C) and analyzed using 2-way ANOVA with Tukey’s correction (* corresponds to p ⁇ 0.05).
  • One representative sample was examined for the memory' phenotype of CD3+ cells which were positive or negative for IFNy after re-stimulation with CMV specific peptides (FIG. 7D).
  • FIG. 8A and 8B shown that cells cultured in Grex-10 vessels with IL-15/IL-6 produce equivalent levels of IFNy compared with culture in IL-4/IL-7.
  • Cells were grown in Grex-10 culture vessels with IL-15 and IL-6 or IL-4 and IL-7 for 10 days and tested in ELISPOT assays for IFNy production when re-stimulated with media alone, actin (I pg/mL), IEI and pp65 peptide pools (1 pg/mL), or SEB (0.5 pg/mL).
  • a representative sample is given in FIG. 8A and the mean of four different samples was compared using 2-way ANOVA with Tukey’s correction (FIG. 8B).
  • FIG. 9 depicts short amino acid sequences from viruses used in the production of amino acid mixes exposed to the cells of the disclosure.
  • FIG. 10 A, 10B, IOC, 10D and 10E show T-cell recognition of SARS-CoV-2 viral antigens.
  • SFC Spot forming units
  • FIG. 10E Phenotype of the expanded cells was accessed by flow cytometry with markers for T-cells (CD3, CD4, CD8, TCRap, TCRyS), NK cells (CD16/CD56), and B-cells (CD 19).
  • FIG. 11 A, 11B, 11C and 11D show T-cell recognition of epitopes within membrane protein.
  • FIG. HA T-cell epitope mapping of membrane protein was performed using 17 mini-pools containing 5-12 peptides each, with responses measured via IFN-y ELISpot (SFC: spot forming units).
  • FIG. 11 AB Epitope mapping identified responses to four peptides within AA 145-173 and 192-222 of the C -terminal intravirion domain. Intracellular cytokine staining demonstrated a predominant CD4-mediated response to membrane peptides 37-38 (FIG. 11C) as well as peptides 44-45 (FIG. HD).
  • SEB :: staphylococcal enterotoxin beta.
  • FIG. 12A and 12.B show clinical characteristics of convalescent COVID-19 patients.
  • FIG. 12A Flow diagram of illness seventy (based on WHO classifications), T-cell and antibody immune response to SARS-CoV-2, and basis of COVID-19 diagnosis.
  • FIG. 12B Ribbon diagram of primary clinical symptoms of the 23 convalescent patients, as well as timing of PCR testing and research evaluation.
  • FIG. 13A and 13B show SARS-CoV-2 antibody testing of normal controls and convalescent patients. Testing for antibodies to nucleocapsid (FIG. 13A) and spike proteins (FIG. 13B) was performed via luciferase immunoprecipitation assay. Positivity thresholds (dotted lines) were set based on previous data using unexposed normal control samples.
  • FIG. 14 A and 14B show T-cell extended phenotyping of coronavirus-specific T-cells.
  • FIG. 14A T-cell populations following expansion were determined via flow cytometry. T-cells were classified as naive (CD45RO-/CCR7+/CD95-), central memory (CD45RO+CCR7+/CD95+), effector memory (CD45RO+CCR7-), and stem cell memory' (CD45RO-/CCR7+/CD95+), and terminal effector (CD45RO-/CCR7-).
  • FIG, 14B Gating strategy for T-cell memory/naive subsets.
  • FIG. 15 shows detection of T-cell responses to SARS CoV-2 proteins from peripheral blood.
  • Peripheral blood mononuclear cells PBMC
  • convalescent patients triangles
  • unexposed controls circles
  • results are reported as spot forming colonies (SFC) per 1x10 5 cells per well.
  • PBMC alone and actin stimulation were utilized as negative controls.
  • Peptide libraries from cytomegalovirus pp65 and IE1 as well as adenovirus hexon and penton were utilized as additional viral controls.
  • FIG. 16 shows T-cell responses to SARS-CoV-2 versus illness severity in convalescent patients.
  • Expanded coronavirus-specific T-cells were tested for specificity to SARS-CoV-2 structural protein libraries on day 10 of culture via IFN-y ELISpot. Control unexposed donors (circles) and convalescent patients with mild disease (upward triangles) or moderate to severe disease (downward triangles) by WHO criteria were tested. Expanded cells alone (CTL alone) and actin stimulated cells were used as negative controls. Results are reported as spot forming colonies (SFC) per 1x10 5 cells/well.
  • SFC spot forming colonies
  • FIG. 17 shows that wells containing the highest concentration of cytokines were compared between Samples 1-4 when cultured using plate layouts 1 and 2.
  • FIG. 18A, I8B and 18C show that cells were re-stimulated with CMV peptide for 6 hour and evaluated for phenotype and function by flow cytometry, with entire well contents analyzed for the top dilutions of IL-15 and IL-6, IL-4 and IL-7, IL- 15 alone, and no cytokine controls. Individual replicates (n>8) were averaged across experiments and individual patient samples (n-6), and statistics were analyzed by 2-way ANOVA with Tukey’s correction with **** p ⁇ 0.0001.
  • FIG. 19 show's 1 x 10 5 cells from Sample 3 and Sample 4 were plated in each well on Day 0 with CMV peptides IEI and pp65 and the indicated cytokine concentration alone or in combination for plate layout 3. Cells were analyzed for the total count of viable CD3+ T cells and the frequency of effector cytokine secretion over background (IFNy, TNFa).
  • FIG, 21 A-21D show' the specificity of ex vivo expanded CST. Following 10-12 days of culture, specificity' of CD4 and CD8 T-cell populations for membrane, spike, and nucleocapsid proteins was assessed by intracellular cytokine staining for IFN-y and TNFa (FIG. 21 A). Subject 2 demonstrates a CD4-predominant response targeting structural proteins. Summary' data of the response of expanded CD4 + T-cells (FIG. 21 B), CD8 + T-cells (FIG. 21C) and ⁇ T cells (FIG.
  • FIG. 23A-23C show SARS-CoV-2 epitope mapping of CSTs.
  • T-cell epitope mapping of structural proteins was performed using mini-pools containing 5-12 peptides each, with responses measured via IFN-y ELISpot (SFC: spot forming units per 1x10 5 cells (FIG. 23A).
  • Epitopes within membrane protein were identified within the C-terminus at AA 144-163 and 173-192, which were recognized by 8 and 6 donors respectively (FIG. 23B).
  • Mapping of spike epitopes demonstrated three regions at AA 57-75, 205-224, and 449-463, which were recognized by 3 donors (FIG. 23B).
  • Mapping of nucleocapsid epitopes showed two regions at AA 257-271 and 313-335 were recognized by 3 donors (Fig. 23C).
  • FIG. 25A-25C show the epitope locations within SARS-CoV-2 structural proteins.
  • FIG. 25B Within spike proteins, epitopes were found within the SI region, including one epitope within the receptor binding domain (RED).
  • FIG. 25C In nucleocapsid protein, epitopes were identified in the region of the dimerization domain (DD).
  • FIG. 26A-26C show ICS (CD4+ and CD8+) and ELISpot specificities.
  • FIG. 26A shows significantly increased CD4+ specificity using IL-15/7 compared to IL-4/7 in Grex validation.
  • FIG. 26B shows increased CD8+ specificity using IL-15/7 compared to IL-4/7 in Grex validation.
  • FIG. 26C shows increased specificity using IL- 15/7 compared to IL-4/7 in Grex -ELISpot.
  • IL-15/7 showed increased CD4+ and CD8+ specificity compared to IL-4/7.
  • cytokine For example, if a cytokine is disclosed and discussed and a number of modifications that can be made to a number of molecules including the polypeptide or nucleic acid expressing the polypeptides are discussed, each and every combination and permutation of cytokines and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary'.
  • A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combmation A-D.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are if there are a variety' of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint unless the context specifically indicates otherwise.
  • a reference to “A and/or B,” when used in conjunction with open- ended language such as “comprising” can refer, in some embodiments, to A without B (optionally including elements other than B); in another embodiments, to B without A (optionally including elements other than A); in yet another embodiments, to both A and B (optionally including other elements); etc.
  • At least prior to a number or series of numbers (e.g. “at least two”) is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
  • at least is present before a senes of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
  • antigen refers to any substances or molecules, such as polypeptides, peptides, or glyco- or lipo-peptides, that are recognized by the immune system, such as by the cellular or humoral arms of the human immune system, to elicit an immune response.
  • antigenic determinants such as peptides with lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acid residues that bind to major histocompatibility' complex (MHC) molecules, form parts of MHC Class I or II complexes, or that are recognized when complexed with such molecules.
  • MHC major histocompatibility' complex
  • the terms “’activate,” “stimulate,” “enhance” “increase” and/or “induce” are used interchangeably to generally refer to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.
  • “Activate” in context of an immunotherapy refers to a primary' response induced by ligation of a cell surface moiety.
  • such stimulation entails the ligation of a receptor and a subsequent signal transduction event. Further, the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule.
  • activating CD8+ T cells or “CD8+ T ceil activation” refer to a process (e.g., a signaling event) causing or resulting in one or more cellular responses of a CD8+ T cell (CTL), selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • CTL CD8+ T cell
  • an “activated CD8+ T cell” refers to a CD8+ T cell that has received an activating signal, and thus demonstrates one or more cellular responses, selected from proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure CD8+ T cell activation are known in the art and are described herein.
  • allogeneic refers to medical therapy in which the donor and recipient are different individuals of the same species.
  • autologous refers to medical therapy in which the donor and recipient are the same person.
  • Coding sequence or “encoding nucleic acid” as used herein refers to a nucleic acid (RNA, DNA, or RNA/DNA hybrid molecule) that comprises a nucleotide sequence which encodes a protein.
  • the coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to whom the nucleic acid is administered.
  • “Complement” or “complementary” as used herein may mean a nucleic acid may mean Watson-Crick (e.g, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • cytokine as used herein has its normal meaning in the art.
  • Non-limiting examples of cytokines used in the disclosure include interleukin-2 (IL-2), IL-4, IL, -6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27.
  • cytotoxic T cell or “cytotoxic T lymphocyte” as used herein is a type of immune cell that bears a CD8+ antigen and that can kill certain cells, including foreign cells, tumor cells, and cells infected with a virus. Cytotoxic T cells can be separated from other blood cells, grown ex vivo, and then given to a patient to kill tumor or viral cells.
  • a cytotoxic T cell is a type of white blood cell and a type of lymphocyte.
  • DC dendritic cell
  • effector cell describes a cell that can bind to or otherwise recognize an antigen and mediate an immune response.
  • Tumor-, virus-, or other antigen-specific T cells and NKT cells are examples of effector cells.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • epitopope or “antigenic determinant” as used herein refers to the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • a functional fragment means any portion of a polypeptide that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the wild-type polypeptide upon which the fragment is based.
  • a functional fragment of a polypeptide is a polypeptide that comprises or possesses at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any polypeptides disclosed in Table 1 and has sufficient length to retain at least partial binding affinity to one or a plurality of ligands that bind to the polypeptides in Table 1.
  • a functional fragment of a nucleic acid is a nucleic acid that comprises or possesses at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any nucleic acid to which it is being compared and has sufficient length to retain at least partial function related to the nucleic acid to which it is being compared.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 10, about 20, about 30, about 40, about 50 , about 60, about 70, about 80, about 90, or about 100 contiguous amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 50 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 100 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 150 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 200 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 300 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 350 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 400 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 450 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 500 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 550 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 600 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 650 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 700 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 750 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 800 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 850 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 900 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of at least about 950 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1050 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1750 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2250 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2500 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 10, about 20, about 30, about 40, about 50 , about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 50 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 100 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of no more than about 150 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 200 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 300 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 350 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 400 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 450 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 550 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 600 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of no more than about 650 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 700 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 800 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 850 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 900 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 950 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 1000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 1050 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 1250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of no more than about 1500 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 1750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 2000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of no more than about 2250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 2500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 2750 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of no more than about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 2750 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 2500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 2250 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 2000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 1750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 1500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 1250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 1000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 950 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length from about 10 to about 850 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 800 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 700 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 650 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 600 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 550 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 450 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 400 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 350 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 300 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 200 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 150 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 100 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 90 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 80 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 70 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 60 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 50 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 40 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 30 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 20 ammo acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 10 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 20 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 30 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 40 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 50 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 60 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 70 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 80 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 90 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 100 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 150 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 200 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 250 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 300 to about 3000 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 350 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 400 to about 3000 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 450 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 500 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 550 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 600 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 650 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 700 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 750 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 800 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 850 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 900 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed m Table 1 and has a length from about 950 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 1000 to about 3000 ammo acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 1050 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 1250 to about 3000 amino acids.
  • the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 1500 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 1750 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 2000 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 2250 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length from about 2500 to about 3000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a. length from about 2750 to about 3000 amino acids.
  • heterologous refers to a nucleic acid sequence that is operably linked to another nucleic acid sequence to which it is not operably linked in nature, or to which it is operably linked at a different location in nature.
  • a protein-coding nucleic acid sequence operably linked to a promoter which is not the native promoter of this protein-coding sequence is considered to be heterologous to the promoter.
  • the heterologous sequence comprises a plasmid or episome.
  • a “naive” T cell or other immune effector cell as used herein is one that has not been exposed to or primed by an antigen or to an antigen-presenting cell presenting a peptide antigen capable of activating that cell.
  • a “naive” donor is an individual whose immune system has not been exposed to, immunized with, or challenged with a particular epitope, antigen, or microorganism and thus substantially lacks immunological memory to an epitope, antigen or microorganism.
  • non-engineered when referring to the cells of the compositions means a cell that does not contain or express an exogenous nucleic acid or ammo acid sequence.
  • the cells of the compositions do not express, for example, a chimeric antigen receptor.
  • a “peptide library” or “overlapping peptide library” as used herein within the meaning of the application is a complex mixture of peptides which in the aggregate covers the partial or complete sequence of a protein antigen. Successive peptides within the mixture overlap each other, for example, a peptide library may be constituted of peptides 15 amino acids in length which overlapping adjacent peptides in the library by 11 amino acid residues and which span the entire length of a protein antigen.
  • Peptide libraries may be commercially available or may be custom- made for particular antigens,
  • a “peripheral blood mononuclear- cell” or “PBMC” as used herein is any peripheral blood cell having a round nucleus. These cells consist of lymphocytes (T cells, B-cells, NK cells) and monocytes. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, and a small percentage of dendritic cells.
  • precursor ceil refers to a ceil which can differentiate or otherwise be transformed into a particular- kind of cell.
  • a “T cell precursor cell” or “pre-T cell” can differentiate into a T cell and a “dendritic precursor cell” can differentiate into a dendritic cell.
  • a “T cell population” or “T cell subpopulation” can include thymocytes, immature T- lymphocytes, mature T-lymphocytes, resting T-lymphocytes and activated T-lymphocytes.
  • the T cell population or subpopulation can include aP T cells, including CI)4+ T cells, CD8+ T cells, yb T cells, natural killer T cells, or any other subset of T cells.
  • Types of T cells include helper T cells (CD4), CD4 TREGS cells (e.g. CTLA-A GITR + PD-1 + CCR + CCR4+ CXCR4+ GITR + LAG3+ OX40+ ICOS), cytotoxic T cells (CD8), CD8 mucosal associated invariant T cells (Rearranged TCRp chains with Vp gene segments), CD8 memory T cells (e.g, CD45RO hl and CD95 + CD45RO lOW subsets), B cells, or gamma/delta T cells markers.
  • helper T cells CD4
  • CD4 TREGS cells e.g. CTLA-A GITR + PD-1 + CCR + CCR4+ CXCR4+ GITR + LAG3+ OX40+ ICOS
  • CD8 mucosal associated invariant T cells Rearranged TCRp chains with Vp gene segments
  • CD8 memory T cells e.g, CD45RO hl and CD95 + CD45RO
  • T cell phenotypes include cells with one or more of the following markers: CD4+, CD8+, CD4+/CD25+, CD45RO+, CD27+, CD28+, and/or PD1.
  • T cell phenotypes include CD4+CD8+; CD27+CD28+ and CD4+, CD45RO+ and CD27+.
  • T cell phenotypes, transcription factors, functions, and other features comprise those described by, and incorporated by reference to, Dong, G. and Martinez, G. J., T cells: the usual subsets, NATURE REVIEWS, 2010, hypertext transfer protocol ://www.nature.com/reviews/posters/Tcellsubsets.
  • cytotoxic T cells surface phenotype: aP TCR, CD3, CD8; effector molecules secreted: perform, granzynie, IFN ⁇ ), exhausted T cells (surface phenotype: CD3, CD8, PD1, TIM3, IB11, LAG3), anergic T cells (surface phenotype: aP TCR, CD3, BTLA; effector factors: GRAIL, CBL-B, ITCH, NEDD4), Tr 1 cells (surface phenotype: ub TCR, CD3, CD4; effector molecules secreted: IL-10), Natural TR eg T cells (surface phenotype: ⁇ TCR, CD3, CD4, CD25, CTLAA4, GITR; effector molecules secreted: IL- 10, TGF ⁇ , IL-35), inducible T Reg cells (surface phenotype: ⁇ TCR, CD3, CD4, CD25, CTLAA4, GITR; effector molecules secreted: IL- 10, TGFP), NKT cells (surface
  • T cells or T cell populations as disclosed above such as those produced by the disclosed methods may be performed using cell sorting or by antibody -based removal or ablation of cells bearing particular markers.
  • Naive human CD4 T cells express CD45RA, CCR7, CD62L and CD27 and may be enriched or recovered using these markers
  • Antibodies recognizing T and B cell markers are commercially available and incorporated by reference to MATER METHODS 2016:6: 1502. Kits and materials for isolation of T ceils are commercially available, for example, from STEMCELL Technologies, hypertext transfer protocol secure://www. stemcell. com/cell-and- tissue-ty'pes/popular-cell-and-tissue-types/t-cells/cell-isolation.html (incorporated by reference).
  • Cell sorting methods including single-cell sorting, fluorescent-activated cell sorting, magnetic- activated cell sorting, and cell sorting by microfluidic devices, axe known and may be used to isolated T cells by phenotype: hypertext transfer protocol secure://en.wikipedia.org/wiki/Cell__sorting (incorporated by reference, last accessed August 20, 2021).
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the disclosed compositions are administered with at least one pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compositions of the present disclosure to subjects.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in cany mg or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include, but not limited to, sugars, such as lactose, glucose and sucrose: starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository' waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic acid,
  • Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin, which is incorporated herein by reference in its entirety’.
  • the pharmaceutically acceptable carrier is sterile and pyrogen-free water.
  • the pharmaceutically acceptable carrier is Ringer’s Lactate, sometimes known as lactated Ringer’s solution.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • sequence identity As used herein, “sequence identity,” “percent identity” or “percent homology'” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accehys, San Diego, Calif.)) using its default parameters. “Identical” or “identity” as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same o ver a specified region.
  • Tire percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity’.
  • the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • thymine (T) and uracil (U) may be considered equivalent.
  • BLAST high scoring sequence pair
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.
  • the Blast algorithm parameters W, T and X determine the sensitivity' and speed of the alignment.
  • the Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci.
  • a nucleic acid is considered similar to another if the smallest sum probability’ in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001.
  • Two single-stranded polynucleotides are “the complement” of each other if their sequences can be aligned in an anti-parallel orientation such that every' nucleotide in one polynucleotide is opposite its complementary' nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5’ or the 3’ end of either sequence.
  • a polynucleotide is “complementary” to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions.
  • a polynucleotide can be complementary' to another polynucleotide without being its complement.
  • the term “subject” is used throughout the specification to describe an animal from which a cell sample is taken or an animal to which a disclosed cell or nucleic acid sequences have been administered.
  • the animal is a human.
  • the term “patient” may be interchangeably' used.
  • the term “patient” will refer to human patients suffering from a particular disease or disorder.
  • the subject may be a human suspected of having or being identified as at risk to develop cancer of the blood.
  • the subject may' be diagnosed as having cancer of the blood or being identified as at risk to develop cancer of the blood.
  • the subject is suspected of having or has been diagnosed with requiring a bone marrow transplant.
  • the subject may be a human suspected of having or being identified as at risk to develop bone marrow transplants.
  • the subject may be a mammal which functions as a source of the endothelial cell sample.
  • the subject may be a non-human animal from which an endothelial cell sample is isolated or provided.
  • the term “mammal” encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, caprmes, and porcines.
  • Nucleic acid or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together. 'The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may' hybridize to a target sequence under stringent hybridization conditions.
  • nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. In some embodiments, the nucleic acid is isolated from an organism.
  • “Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control.
  • the distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • Promoter may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-natural amino acids or chemical groups that are not amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a natural source e.g., cells
  • contaminating materials from the natural source e.g., soil particles, minerals, chemicals from the environment, and/or cellular materials from the natural source, such as but not limited to cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and/or lipids present in cells.
  • substantially free of natural source materials refers to preparations of a compound or agent that has been separated from the material (e.g., cellular components of the cells) from which it is isolated.
  • a compound or agent that is isolated includes preparations of a compound or agent having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materials and/or contaminating materials.
  • an “isolated” nucleic acid sequence or nucleotide sequence is one which is separated from other nucleic acid molecules which are present in a natural source of the nucleic acid sequence or nucleotide sequence.
  • an “isolated”, nucleic acid sequence or nucleotide sequence, such as a cDNA molecule can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors when chemically synthesized.
  • an “’isolated” nucleic acid sequence or nucleotide sequence is a nucleic acid sequence or nucleotide sequence that is recombinantly expressed in a heterologous cell.
  • Stringent hybridization conditions may mean conditions under which a first nucleic acid sequence (e.g, probe) will hybridize to a second nucleic acid sequence (e.g, target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5- 10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50%> of the probes are occupied at equilibrium).
  • Tm thermal melting point
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g, about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50%> formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0,1 % SDS at 65°C,
  • hybridization or “hybridizes” as used herein refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form duplexes via Watson- Crick base pairing. Two nucleotide sequences are “complementary” to one another when those molecules share base pair organization homology. “Complementary” nucleotide sequences will combine with specificity to form a stable duplex under appropriate hybridization conditions.
  • two sequences need not have perfect homolog ⁇ 7 to be “complementary.”
  • two sequences are sufficiently complementary when at least about 90% (preferably at least about 95%) of the nucleotides share base pair organization over a defined length of the molecule.
  • “Substantially complementary” as used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or ammo acids, or that the two sequences hybridize under stringent hybridization conditions.
  • “Substantially identical” as used herein may mean that, in respect to a first and a second sequence, a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical over a region of 8, 9, 10, I I , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
  • therapeutic effect as used herein is meant to refer to some extent of relief of one or more of the symptoms of a disorder (e.g, S ARS-CoV-2 infection) or its associated pathology
  • a “therapeutically effective amount” as used herein is meant to refer to an amount of an agent which is effective, upon single or multiple dose administration (such as a first, second and/or third booster) to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment.
  • a “therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the present disclosure employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
  • a therapeutically effective dose may also be determined from human data.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered agent. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
  • General principles for determining therapeutic effectiveness which may be found in Chapter 1 of Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.
  • Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability' of the active ingredient in the two products are not significantly different under statable test conditions.
  • treat refers to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., a viral infection).
  • the terms “treat,” “treated,” “treating,” “treatment,” and the like refer to the beneficial effects that a subject derives from a therapy, such as, but not limited to, the reduction or inhibition of the progression, spread and/or duration of a disease or disorder, the reduction or amelioration of the severity’ of a disease or disorder, amelioration of one or more symptoms of a disease or disorder, and/or the reduction in the duration of one or more symptom of a disease or disorder resulting from the administration of one or more therapies.
  • such terms in the context of viral infection include, but are not limited to, one, two, or three or more results following the administration of a therapy to a subject: (1) a reduction in the growth of virus in the body by measuring serum levels of vims; (2) a reduction in the number of virus-bearing cells; (3) an eradication, removal, or control of cell number expressing virus; (4) a reduction in mortality; (5) an increase in survival rate; (6) an increase in length of survival; (7) an increase in the number of patients with latent viral infection; (8) a decrease in hospitalization rate; (9) a decrease in hospitalization lengths; and (10) the maintenance in the numbers of virus in serum so that it does not increase by more than 10%, or by more than 8%, or by more than 6%, or by more than 4%; preferably the size of the tumor does not increase by more than 2% from a sample of a subject.
  • the terms “prevent,” “preventing” and “prevention” in the context of the adminis tration of a therapy to a subject refer to the inhibition of the onset or recurrence of a disease or disorder in a subject.
  • a subject is administered one or more therapies to “manage” a disease or disorder so as to prevent the progression or worsening of symptoms associated with a disease or disorder.
  • any of the nucleic acids disclosed herein can encode variants of any of the polypeptides disclosed herein.
  • “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
  • Variant with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity, such as the biological activity of the one or combination of cytokines presented herein.
  • Variant may also mean a protein with an ammo acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at. least one biological activity.
  • a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, m part, by considering the hydropathic index of amino acids, as understood m the art.
  • the hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that ammo acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ⁇ 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity' of ammo acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S.
  • Nucleic acid molecules or nucleic acid sequences of the disclosure include those coding sequences comprising one or more of nucleic acid sequences encoding any of the amino acid sequences disclosed herein, such as those identified in Table 1, and functional fragments thereof that possess no less than about 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the coding sequences of the amino acid sequences disclosed herein.
  • Vector used herein means, in respect to a nucleic acid sequence, a nucleic acid sequence comprising a regulatory' nucleic acid sequence that controls the replication or expression of an expressible gene.
  • a vector may' be either a self-replicating, extrachromosomal vector or a vector which integrates into a host genome. Alternatively, a vector may also be a vehicle comprising the aforementioned nucleic acid sequence.
  • a vector may be a plasmid, bacteriophage, viral particle (isolated, atenuated, recombinant, ere.).
  • a vector may comprise a double-stranded or singlestranded DNA, RNA, or hybrid DNA/RNA sequence comprising double-stranded and/or singlestranded nucleotides.
  • the vector is a viral vector that comprises a nucleic acid sequence that is a viral packaging sequence responsible for packaging one or plurality of nucleic acid sequence that encode one or a plurality of polypeptides.
  • the vector comprises a viral particle comprising a nucleic acid sequence operably linked to a regulatory' sequence, wherein the nucleic acid sequence encodes a fusion protein comprising one or a plurality of structural viral polypeptides or fragments thereof.
  • the disclosure relates to any vector comprising one or a plurality' of nucleic acid sequences encoding any two of the disclosed cytokines of Table 1, and/or any functional fragment or variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the disclosure relates to the vectors comprising, consisting of, or consisting essentially of SEQ ID NO:
  • the disclosure relates to the vectors comprising a nucleic acid that encodes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or a functional fragment thereof. In some embodiments, the disclosure relates to the vectors comprising variants or functional fragments of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.
  • the disclosure relates to the vectors comprising variants or functional fragments of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 that comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.
  • the functional fragment is a subunit of the cytokine known to have biological effect without association with another subunit.
  • “Viral vector” as disclosed herein means, in respect to a vehicle, any virus, virus-like particle, virion, viral particle, or pseudotyped virus that comprises a nucleic acid sequence that directs packaging of a nucleic acid sequence in the virus, virus-like particle, virion, viral particle, or pseudotyped virus.
  • the virus, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and/or between host cells.
  • the virus, virus-like particle, virion, viral particle, or pseudotyped virus is capable of transferring a vector (such as a nucleic acid vector) into and/or between target cells, such as a endothelial cell or hematopoietic cell in culture.
  • a vector such as a nucleic acid vector
  • T cell compositions for the treatment of disorders can be administered as a single composition comprising T cell subpopulations stimulated by the methods disclosed herein.
  • the T cell compositions are stimulated with a combination of cytokines disclosed herein for a time period sufficient to stimulate growth and proliferation of the one or plurality of ⁇ 1)4 and/or CD8+ T cells.
  • the T cell compositions are stimulated with a combination of cytokines disclosed herein and are exposed to one or more tumor-associated antigens or viral antigens for a time period sufficient to stimulate growth and proliferation of the one or plurality of CD4+ and/or CD8+ T cells that are specific to the tumor-associated antigens or viral antigens used for stimulation.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is from about 1 day to about 12 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8 + T cells is from about 2 to about 10 days.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is from about 3 to about 7 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is from about 4 to about 8 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is from about 5 to about 7 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8v T cells is about 1 day.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 2 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+- T cells is about 3 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 4 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 5 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 6 days.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 7 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 8 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 9 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 10 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 11 days. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is about 12 days.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 5% of CD8+ or CD4+ T cells produce either IFNy or TNFa. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 6% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 7% of CD8+- or CD4+ T cells produce either IFNy or TNFa. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 8% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 9% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 10% of CD8+ or CD4- ⁇ - T cells produce either IFNy or TNFa, In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 11% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 12% of CD8+ or CD4+ T ceils produce either IFNy or TNFa. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 13% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 14% of CD8+ or CD4+ T cells produce either IFNy or TNFa. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 15% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 16% of CD8+ or CD4+ T cells produce either IFNy or TNFa, In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 17% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4- ⁇ - and/or CD8+ T cells is a time period in which no less than about 18% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 19% of CD8+ or CD4+ T cells produce either IFNy or TNFa, In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 20% of CD8+ or CD4+ T cells produce either IFNy or TNFa.
  • the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 21% of CD8+ or CD4+ T cells produce either IFNy or TNFa. In some embodiments, the time period sufficient to stimulate growth or proliferation of the one or plurality of ( i)4 and/or CD8+ T cells is a time period in which no less than about 22% of CD8+ or CD4+ T cells produce either IFNy or TNFa,
  • the subpopulations of T cells are derived through the ex vivo expansion of a single population of T cells, wherein the single population of T cells are exposed to a pool of one or more antigenic peptides (epitopes) of each of the viral antigens in the presence of a combination of two or more cytokines chosen from: IL-18, IL-15, IL-6, IL-7 and IL-4.
  • the combination of cytokines is IL-15 and IL-6.
  • the combination of cytokines is IL-15 and IL-7
  • the combination of cytokines is IL-7 and IL-4.
  • the disclosure relates to T cell compositions comprising memory effector T cells that are antigen-specific for one or more viral antigens.
  • the T cell compositions are activated to recognize one or a plurality of viral antigen peptides provided in Figures 8 or peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequences depicted in Figures 8.
  • the T cell compositions are activated to recognize one or more viral antigens of a virus of the family Coronaviridae.
  • the T cell compositions are activated to recognize one or more viral antigens of a coronavirus.
  • the T cell compositions are activated to recognize one or more viral antigens of SARS-CoV-2.
  • Coronaviruses are a group of viruses that cause diseases in mammals and birds. Coronaviruses were first discovered in the 1960s. The earliest virus from the family of Coronaviridae discovered were infectious bronchitis virus in chickens and two viruses from the nasal cavities of human patients with the common cold that were subsequently named human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43). Other members of this family have since been identified, including SARS-CoV in 2003, HCoV NL63 in 2004, HKU1 in 2005, MERS-CoV in 2012, and SARS-CoV-2 (formerly known as 2019-nCoV or novel coronavirus 2019, which caused the global COVID- 19 pandemic). Most of these have involved serious respiratory' tract infections.
  • SARS-CoV-2 is the virus strain that causes the coronavirus disease 2019 (COVID-19) pandemic, which infected millions of people and caused hundrens of thousand of death worldwide.
  • SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucieocapsid) proteins.
  • the N protein holds the viral RNA genome, and the S, E, and M proteins together create the viral envelope.
  • the complete genome of SARS-CoV-2 has been sequenced and the sequence is publically available in the GenBank database under the accession No. NC_045512.
  • the spike (S) protein is the protein responsible for allowing the vims to attach to and fuse with the membrane of a host cell and has the ammo acid sequence of SEQ ID NO: 11 (GenBank Accession No. QHD43416).
  • the envelope (E) protein of SARS-CoV-2 has the amino acid sequence of SEQ ID NO: 12 (GenBank Accession No. QHD43418).
  • the membrane (M) protein of SARS-CoV-2 has the amino acid sequence of SEQ ID NO:
  • the nucleocapsid (N) protein of SARS-CoV-2 has the amino acid sequence of SEQ ID NO:
  • T cell compositions activated to recognize one or more viral antigens of SARS-CoV-2 can be prepared by exposing or pulsing antigen presenting cells or artificial antigen presenting cells with one or more peptides or epitopes from SARS-CoV-2.
  • the peptide segments can be generated by making overlapping peptide fragments of the SARS-CoV-2 antigen, as provided for example in commercially available overlapping peptide libraries or “PepMixTM.”
  • the overlapping peptide libraries include peptides that are about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more amino acids long and overlapping one another by about 5, 6, 7, 8, 9, 10 , 11 or more amino acids.
  • the peptides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or more amino acids in length, for example, and there is overlap of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids in length.
  • the SARS-CoV-2 specific T cell compositions are generated using one or more antigenic peptides to SARS-CoV-2, or a modified or heteroclitic peptide derived from a SARS-CoV-2 antigenic peptide.
  • SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides (for example, peptides that are from about 10 to about 20 ammo acids in length) from one or a combination of the structural proteins of SARS-CoV-2 disclosed herein, such as 15-mers peptides containing amino acids overlap (for example 11 amino acids of overlap) between each peptide formed by scanning the proteins having the ammo acid sequences of SEQ ID NO: 11, SEQ ID NO:
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: 11, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: 12, or functional fragments or variants thereof.
  • the SARS- CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO:
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen lbrary comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 12, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 13, or functional fragments or variants thereof.
  • the SARS- CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the ammo acid sequences of SEQ ID NO: 11 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS- CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the ammo acid sequences of SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS- CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in the protein of SEQ ID NO: 11. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 12 to about 524 of the protein of SEQ ID NO: 11. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 12 to about 331 of the protein of SEQ ID NO: 1 I.
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in ammo acid residue from about 57 to about 75 of the protein of SEQ ID NO: 11. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in ammo acid residue from about 205 to about 224 of the protein of SEQ ID NO: I I. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in ammo acid residue from about 331 to about 524 of the protein of SEQ ID NO: 11 .
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 449 to about 463 of the protein of SEQ ID NO: 11. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes selected from Table A.
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in the protein of SEQ ID NO: 12.
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in the protein of SEQ ID NO: 13. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 100 to about 222 of the protein of SEQ ID NO: 13. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 144 to about 192 of the protein of SEQ ID NO: 13.
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 144 to about 163 of the protein of SEQ ID NO: 13. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 163 to about 192 of the protein of SEQ ID NO: 13. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality' of epitopes located in amino acid residue from about 173 to about 192 of the protein of SEQ ID NO: 13. In some embodiments, the SARS- CoV-2 specific T cell compositions are generated using one or a plurality of epitopes selected from Table B.
  • the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in the protein of SEQ ID NO: 14. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 257 to about 361 of the protein of SEQ ID NO: 14. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes located in amino acid residue from about 257 to about 271 of the protein of SEQ ID NO: 14. In some embodiments, the SARS-CoV-2 specific T cell compositions are generated using one or a plurality of epitopes selected from Table C.
  • the T cell compositions can be generated through the ex vivo expansion of a first T cell population exposed to one or more antigenic peptides of each of the selected viral antigens separately, wherein following activation and expansion of lymphocytes, the first T cell population is combined with a second T cell population stimulated by a cytokine composition disclosed herein into a single composition for administration.
  • the T cell compositions can be derived through the ex vivo expansion of a first and second T cell populations exposed to one or more antigenic peptides of each of the selected viral antigens separately, wherein following activation and expansion, the separate T cell populations are each individually administered simultaneously or sequentially to the subject.
  • the first and second T cell populations are derived from the same donor source.
  • the first or second T cell populations are derived from one or more different donor sources.
  • the cells are contacted sequentially or simultaneously with any of the cytokine combinations disclosed herein and one or more polypeptides or nucleic acid sequences encoding polypeptides chosen from one or a combination of peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to the ammo acid sequences depicted in Figures 8 or variants or functional fragments thereof.
  • the cells are contacted sequentially or simultaneously with any of the cytokine combinations disclosed herein and one or more peptides (for example, peptides that are from about 10 to about 20 ammo acids in length) from one or a combination of SARS-CoV-2 antigens having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • peptides for example, peptides that are from about 10 to about 20 ammo acids in length
  • SARS-CoV-2 antigens having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • the T cell compositions described herein may be derived from a population of cells from an autologous source, an allogeneic source, for example a healthy donor not suffering from a disorder, or cord blood.
  • the methods disclosed herein use PBMCs, stem cells, pre-T cells, or cord blood, from a partially histocompatible sibling, parent, son or daughter, grandparent, grandson or grand daughter, first or second cousin, or other blood relative.
  • T cells may be obtained from autologous cells.
  • HLA type-I genes e.g, HLA-A, HLA-B, or HLA-C
  • HLA type II gene e.g. HLA-DR or HLA-DQB1
  • antigen-specific T cells are produced from non-naive or naive cells that share at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 HLA alleles (e.g, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA- DQB1, HLA-DRA, and HLA-DRB1) with a prospective donor.
  • HLA alleles e.g, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA- DQB1, HLA-DRA, and HLA-DRB1
  • the T cells compositions described herein may be derived from a population of cells from a subject diagnosed with or suspected of having a viral infection. In some embodiments, the T cells compositions described herein may be derived from a population of cells from a subject diagnosed with or suspected of having coronavirai infection. In some embodiments, the T cells compositions described herein may be derived from a population of cells from a subject diagnosed with or suspected of having infection of SARS-CoV-2. In some embodiments, the T cells compositions described herein may be derived from a population of cells from a subject diagnosed with or suspected of having COVID-19.
  • Non-limiting exemplary methods of generating ex vivo primed and expanded T cells capable of recognizing at least one antigenic peptide of a tumor antigen can be found in Shafer et al,, Leuk Lymphoma (2010) 51(5):870-880; Cruz et al., Clin Cancer Res., (2011) 17(22): 7058-7066; Quintarelli et al., Blood (2011) 117(12): 3353-3362; Chapuis et al., Sci TransI Med (2013) 5(174): 174ra27; and US 2017/0037369, all incorporated herein by reference in their entireties.
  • one or more antigenic peptides (epitopes) from the target viral antigen is used in addition to the cytokine compositions disclosed herein.
  • a single antigenic peptide, multiple antigenic peptides, or a library of antigenic peptides can be used to prime and activate a T cell subpopulation targeting each of the specific viral antigens.
  • the peptide segments can be generated by making overlapping peptide fragments of viral antigen that are from about 5 to about 15 ammo acids in length as discussed above.
  • generation of the T cell composition can be accomplished through the ex vivo priming and activation of the T cell subpopulations with selected antigenic epitopes of the targeted viral antigen, for example, a single epitope or multiple specific epitopes of the viral antigen.
  • the T cell subpopulation is activated and primed with pooled peptides to a viral antigen, wherein the pooled peptides include a library' of overlapping peptides from the viral antigen (peptide mix) which has been enriched by additionally including one or more specific known, identified, or heteroclitic epitopes of the viral antigen.
  • the peptides used to prime the T cells are the same length.
  • the peptides are of vary ing lengths. In other embodiments, the peptides included in the pool for priming the T cells substantially only include known viral antigenic epitopes. In some embodiments, the T cell subpopulation is primed with one or more antigenic peptides expressed by a patient’s tumor. In some embodiments, the T cell subpopulation is primed with one or more neoantigens. In some embodiments, the neoantigen is a mutated form of an endogenous protein derived through a single point mutation, a deletion, an insertion, a frameshift mutation, a fusion, misspliced peptide, or intron translation of the targeted tumor cells comprising the viral antigens.
  • the T cell composition is derived through the ex vivo expansion of separate T cell populations (a first T cell population and a second T cell population), wherein the T cell composition includes T cell subpopulations primed separately to a pool of viral peptides, wherein each T cell subpopulation is specific for a single viral antigenic peptide.
  • the pooled viral peptides are comprised of overlapping peptides derived from viral peptides selected from those provided in FIG. 9.
  • individual or mixed peptides may be obtained commercially, for example, from JPT (hypertext transfer protocol secure//shop.jpt.com).
  • PepMixTM SARS-CoV-2 (Spike B.1,1.7), PepMixTM SAR.S- CoV (Spike Glycoprotein): PepMixTM SARS-CoV-2 (AP3A); PepMix'TM SARS-CoV-2 (C+NSP11); PepMixTM SARS-CoV-2 (NCAP); PepMixTM SARS-CoV-2 (NCPMUT); PepMixTM SARS-CoV-2 (NS6); PepMixTM SARS-CoV-2 (NS7a); PepMixTM SARS-CoV-2 (NS7B); PepMixTM SARS-CoV-2 (NS8); PepMixTM SARS-CoV-2 (Nspl); PepMixTM SARS-CoV-2 (Nsp2- Nspl6) as well as the other SARS-CoV-2, coronavirus, and viral peptides and pepmixes described in the JPT catalog (last accessed September 8, 2021 ,
  • the pooled viral peptides are comprised of overlapping peptides derived from viral peptides chosen from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • the pooled viral peptides are comprised of overlapping peptides derived from the viral antigen of SEQ ID NO: 11, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11.
  • the pooled viral peptides are comprised of overlapping peptides derived from the viral antigen of SEQ ID NO: 12, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12.
  • the pooled viral peptides are comprised of overlapping peptides derived from the viral antigen of SEQ ID NO: 13, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13.
  • the pooled viral peptides are comprised of overlapping peptides derived from the viral antigen of SEQ ID NO: 14, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.
  • the pooled viral peptides derived from the viral antigen of SEQ ID NO: 11, or functional fragments or variants thereof are further enriched with one or more additional peptides derived from SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • the pooled viral peptides derived from the viral antigen of SEQ ID NO: 12, or functional fragments or variants thereof are further enriched with one or more additional peptides derived from SEQ ID NO: 11, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • the pooled viral peptides derived from the viral antigen of SEQ ID NO: 13, or functional fragments or variants thereof are further enriched with one or more additional peptides derived from SEQ ID NO: 11, SEQ ID NO: 12 and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • the pooled viral peptides derived from the viral antigen of SEQ ID NO: 14, or functional fragments or variants thereof are further enriched with one or more additional peptides derived from SEQ ID NO: 11, SEQ ID NO: 12. and/or SEQ ID NO: 13, or functional fragments or variants thereof.
  • the pooled viral peptides are comprised of epitopes derived from viral peptides selected from those provided in Table A, Table B and/or Table C.
  • the T cell composition is derived through the ex vivo expansion of separate T cell populations, wherein the T cell composition includes separate T cell subpopulations primed to a pool of peptides comprising one or more antigenic peptides or epitopes thereof selected from PRAME, Survivin, and WT'l.
  • 1, 2, 3, 4, 5 or more peptides from PRAME, Survivin, and/or WIT may be used to induce or expand T cells (or in some cases excluded).
  • These peptides may comprise, consist essentially of, or consist of the immunologically active peptides used to induce or expand the T cells to specific epitopes.
  • peptides specific to other tumor antigens such as NYESO, MAGE A4, MAGE A3, MAGE Al, neuroelastase, proteinase 3, p53, CEA, claudin6.
  • Histone Hl, Histone H2, Histone H3, Histone H4, MARTI , gplOO, PSA, SOX2, SSX2, Nanog, Oct4, Myc, and Ras may be include or excluded from peptide(s) used to induce or expand T cells.
  • peptides from the following types of classes of tumor antigen may be used (or excluded): Alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), or abnormal products of ras or p53.
  • the pooled peptides are comprised of overlapping peptides derived from antigens selected from PRAME, Survivin, and WT1, or combinations thereof.
  • the pooled viral antigens are further enriched with one or more additional peptides selected from FRAME, Survivin, and WT1 having the following sequences, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity thereto.
  • the epitopes are one or a combination from Table 2:
  • the epitopes are one or a combination from Table 3:
  • the epitopes are one or a combination from Table 4:
  • the pooled peptides include one or more peptides selected from SEQ ID NO: 15 (PRAME), one or more peptides selected from SEQ ID NO: 16 (Survivin), and one or more peptides selected from SEQ ID NO: 17 (WIT), or combinations thereof.
  • the ratio of the T cell subpopulations that comprise the T cell composition is correlated with the tumor expression profile of the subject.
  • compositions of cytokines comprise functional fragments thereof or variants of the disclosed cytokines of Table 1 that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to the amino acids disclosed in Table 1.
  • the amino acid sequence of the immature/precursor form of native human IL-15 which comprises the long signal peptide (residues 1 -2.9. underlined) and the mature human native IL- 15 (residues 30-162, italicized), are provided:
  • nucleotide sequence encoding the immature/precursor form of native human IL- 15, which comprises the nucleotide sequence encoding the long signal peptide (residues 1-29, underlined) and the nucleotide sequence encoding the mature human native IL-15 (residues 30-162, italicized), is provided:
  • IL-7 (NCBI Reference Sequence: NP 000871.1). Signal peptide (underlined) residues 1- 25, mature peptide (italics), residues 26-177.
  • IL-4 (NCBI Reference Sequence: NP_001341919. 1) Signal peptide (underlined) residues 1-24, mature peptide (italics), residues 25-136.
  • IL-18 (NCBI Reference Sequence: NP 001373349.1) IL-18 lacks a signal peptide.
  • the IL- 18 gene encodes a 193 amino acid precursor which accumulates in cell cytoplasm and which is processed intracellularly by caspace 1 or other caspace into its mature 18kDA biologically active form. Region 74-180 shown in italics.
  • the nucleic acid is an isolated or purified nucleic acid.
  • the nucleic acids encode the immature or precursor form of a naturally occurring mammalian IL-15, IL-7, IL-6, IL-4 or IL-18.
  • the nucleic acids encode the mature form of a naturally occurring mammalian IL-15, IL-7, IL-6, IL-4 or IL- 18 free of signal peptide.
  • the nucleic acids encoding native IL-15, IL-7, IL-6, IL-4 or IL-18 encode the precursor form of naturally occurring human IL- 15, IL-7, IL-6, IL-4 or IL-18.
  • the nucleic acids encoding native IL-15, IL-7, IL-6, IL-4 or IL-18 encode the mature form of naturally occurring human IL-15, IL-7, IL-6, IL-4 or IL-18.
  • the nucleic acids encoding IL-15, IL-7, IL-6, IL-4 or IL-18 comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or functional fragments or variants thereof.
  • the nucleic acids encoding IL-15, IL-7, IL-6, IL-4 or IL- 18 comprise the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the immature or precursor form of a naturally occurring mammalian IL- 15, or functional fragments or variants thereof. In some embodiments, the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the mature form of a naturally occurring mammalian IL-15, or functional fragments or variants thereof. In some embodiments, the naturally occurring mammalian IL-15 is a human IL- 15.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL-15 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: I, or functional fragments or variants thereof.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL- 15 comprising the amino acid sequence of SEQ ID NO: 1, or functional fragments or variants thereof
  • the terms ’‘IL-15 variant,’' “interleukin- 15 variant,” or “functional fragment” or “variant” of IL-15 in the context of proteins or polypeptides, refer to: (a) a polypeptide that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a native mammalian IL-15 polypeptide, such as the IL-15 polypeptide of SEQ ID NO: 1; (b) a polypeptide encoded by a nucleic acid sequence that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleic acid sequence encoding a native mammalian IL-15 polypeptide, such as the IL-15 coding nucleic acid of SEQ ID NO: 2; (c) a polypeptide that contains 1, 2,
  • IL-15 variants also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL- 15 polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL- 15 variant is a derivative of a native human IL-15 polypeptide, such as the IL-15 polypeptide of SEQ ID NO: 1.
  • an IL-15 variant is a derivative of an immature or precursor form of naturally occurring human IL- 15 polypeptide, such as the IL- 15 polypeptide of SEQ ID NO: 1.
  • an IL-15 variant is a derivative of a mature form of naturally occurring human IL-15 polypeptide, such as the IL- 15 polypeptide of SEQ ID NO: 1 without the signal peptide.
  • an IL-15 variant is isolated or purified.
  • IL- 15 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-15 polypeptide to bind IL-15Ra polypeptide, as measured by assays well known in the art, e.g., ELISA, Biacore, co-immunoprecipitation.
  • IL-15 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-15 polypeptide to induce IL-15-mediated signal transduction, as measured by assays well- known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the immature or precursor form of a naturally occurring mammalian IL-7, or functional fragments or variants thereof. In some embodiments, the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the mature form of a naturally occurring mammalian IL-7, or functional fragments or variants thereof. In some embodiments, the naturally occurring mammalian IL-7 is a human IL-7.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL- 7 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the ammo acid sequence of SEQ ID NO: 3, or functional fragments or variants thereof.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL-7 comprising the amino acid sequence of SEQ ID NO: 3, or functional fragments or variants thereof.
  • IL-7 variant refers to: (a) a polypeptide that has at least about 40%, 45%, 50%, 5.5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9.5%, 98% or 99% sequence identity to a native mammalian IL-7 polypeptide, such as the IL-7 polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a nucleic acid sequence that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleic acid sequence encoding a native mammalian IL-7 polypeptide, such as the IL-7 coding nucleic acid of SEQ ID NO: 4; (c) a polypeptide that contains 1, 2, 3, 4, 5,
  • IL-7 variants also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL-7 polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL-7 vanant is a derivative of a native human IL-7 polypeptide, such as the IL- 7 polypeptide of SEQ ID NO: 3.
  • an IL-7 variant is a derivative of an immature or precursor form of naturally occurring human IL-7 polypeptide, such as the IL-7 polypeptide of SEQ ID NO: 3.
  • an IL-7 variant is a derivative of a mature form of naturally occurring human IL-7 polypeptide, such as the IL-7 polypeptide of SEQ ID NO: 3 without the signal peptide.
  • an IL-7 variant is isolated or purified.
  • IL-7 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-7 polypeptide to bind IL-7R polypeptide, as measured by assays well known in the art, e.g., ELISA, Biacore, coimmunoprecipitation.
  • IL-7 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-7 polypeptide to induce IL-7 -mediated signal transduction, as measured by assays well-known in the art, e.g, electromobility shift assays, ELISAs and other immunoassays
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the immature or precursor form of a naturally occurring mammalian IL-6, or functional fragments or variants thereof. In some embodiments, the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the mature form of a naturally occurring mammalian IL-6, or functional fragments or variants thereof. In some embodiments, the naturally occurring mammalian IL-6 is a human IL-6.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL- 6 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity' to the amino acid sequence of SEQ ID NO: 5, or functional fragments or variants thereof.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL-6 comprising the ammo acid sequence of SEQ ID NO: 5, or functional fragments or variants thereof.
  • IL-6 variant refers to: (a) a polypeptide that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a native mammalian IL-6 polypeptide, such as the IL-6 polypeptide of SEQ ID NO: 5; (b) a polypeptide encoded by a nucleic acid sequence that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleic acid sequence encoding a native mammalian IL-6 polypeptide, such as the IL-6 coding nucleic acid of SEQ ID NO: 6; (c) a polypeptide that contains 1, 2, 3, 4,
  • IL-6 variants also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL-6 polypeptide and a heterologous signal peptide ammo acid sequence.
  • an IL-6 variant is a derivative of a native human IL-6 polypeptide, such as the IL-6 polypeptide of SEQ ID NO: 5.
  • an IL-6 variant is a derivative of an immature or precursor form of naturally occurring human IL-6 polypeptide, such as the IL-6 polypeptide of SEQ ID NO: 5.
  • an IL-6 variant is a derivative of a mature form of naturally occurring human IL-6 polypeptide, such as the IL-6 polypeptide of SEQ ID NO: 5 without the signal peptide.
  • an IL-6 variant is isolated or purified.
  • IL-6 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-6 polypeptide to bind IL-6R polypeptide, as measured by assays well known in the art, e.g, ELISA, Biacore, coimmunoprecipitation.
  • IL-15 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-6 polypeptide to induce IL-6-mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELIS As and other immunoassays.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the immature or precursor form of a naturally occurring mammalian IL-4, or functional fragments or variants thereof. In some embodiments, the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the mature form of a naturally occurring mammalian IL-4, or functional fragments or variants thereof. In some embodiments, the naturally occurring mammalian IL-4 is a human IL-4.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL- 4 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the ammo acid sequence of SEQ ID NO: 7, or functional fragments or variants thereof.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL-4 comprising the amino acid sequence of SEQ ID NO: 7, or functional fragments or variants thereof
  • IL-4 variant,’' “interleukin-4 variant,'’ or “functional fragment” or “variant” of IL-4 in the context of proteins or polypeptides, refer to: (a) a polypeptide that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a native mammalian IL-4 polypeptide, such as the IL-4 polypeptide of SEQ ID NO: 7; (b) a polypeptide encoded by a nucleic acid sequence that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleic acid sequence encoding a native mammalian IL-4 polypeptide, such as the IL-4 coding nucleic acid of SEQ ID NO: 8; (c) a polypeptide that contains 1, 2, 3,
  • IL-4 variants also include a polypeptide that comprises the ammo acid sequence of a naturally occurring mature form of a mammalian IL-4 polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL-4 variant is a derivative of a native human IL-4 polypeptide, such as the IL-4 polypeptide of SEQ ID NO: 7.
  • an IL-4 variant is a derivative of an immature or precursor form of naturally occurring human IL-4 polypeptide, such as the IL-4 polypeptide of SEQ ID NO: 7.
  • an IL-4 variant is a derivative of a mature form of naturally occurring human IL-4 polypeptide, such as the IL-4 polypeptide of SEQ ID NO: 7 without the signal peptide.
  • an IL-4 variant is isolated or purified.
  • IL-4 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-4 polypeptide to bind IL-4R polypeptide, as measured by assays well known in the art, e.g., ELISA, Biacore, co- immunoprecipitation.
  • IL-4 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammahan IL-4 polypeptide to induce IL-4-mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the immature or precursor form of a naturally occurring mammalian IL- 18, or functional fragments or variants thereof. In some embodiments, the methods of the disclosure comprise exposing cells to a composition of cytokines comprising the mature form of a naturally occurring mammalian IL- 18, or functional fragments or variants thereof. In some embodiments, the naturally occurring mammalian IL-18 is a human IL-18.
  • the methods of the disclosure comprise exposing cells to a composition of cytokines comprising an IL-18 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, or functional fragments or variants thereof.
  • the methods of the disclosure comprise exposing ceils to a composition of cytokines comprising an IL- 18 comprising the amino acid sequence of SEQ ID NO: 9, or functional fragments or variants thereof.
  • the terms “lL-18 variant,” “in terleukin- 18 variant,” or “functional fragment” or “variant” of IL- 18, in the context of proteins or polypeptides refer to: (a) a polypeptide that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a native mammalian IL-18 polypeptide, such as the IL-18 polypeptide of SEQ ID NO: 9; (b) a polypeptide encoded by a nucleic acid sequence that has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleic acid sequence encoding a native mammalian IL-18 polypeptide, such as the IL- 18 coding nucleic acid of SEQ ID NO: 10; (c) a polypeptide that contains I,
  • IL-18 variants also include a polypeptide that comprises the amino acid sequence of a naturally occurring mature form of a mammalian IL- 18 polypeptide and a heterologous signal peptide amino acid sequence.
  • an IL-18 variant is a derivative of a native human IL-18 polypeptide, such as the IL-18 polypeptide of SEQ ID NO: 9.
  • an IL- 18 variant is a derivative of an immature or precursor form of naturally occurring human IL-18 polypeptide, such as the IL-18 polypeptide of SEQ ID NO: 9.
  • an IL-18 variant is a derivative of a mature form of naturally occurring human IL-18 polypeptide, such as the IL- 18 polypeptide of SEQ ID NO: 9 without the signal peptide.
  • an IL-18 variant is isolated or purified.
  • IL-18 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL-18 polypeptide to bind IL-18R polypeptide, as measured by assays well known in the art, e.g, ELISA, Biacore, coimmunoprecipitation.
  • IL-18 variants retain at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a native mammalian IL- 18 polypeptide to induce IL-18-mediated signal transduction, as measured by assays well-known in the art, e.g, electromobility shift assays, ELISAs and other immunoassays.
  • the disclosure relates to a method of expanding CD8+ and/or CD4+ lymphocytes in an vitro culture comprising contacting the lymphocytes with at least two polypeptides, one or a plurality of nucleic acids encoding at least two polypeptides; or a combination of a nucleic acid and a polypeptide; wherein the at least two polypeptides are cytokines chosen from: IL- 15, IL-6, IL-7, IL-4, IL-18 and/or functional fragments or variants thereof.
  • the method relates to contacting one or a plurality' of lymphocytes in an in vitro culture with at least one combination of cytokines chosen from a combination of: (i) IL-15 and IL-6, or functional fragments or variants thereof; (ii) IL-15 and IL-7, or functional fragments or variants thereof; (iii) IL-7 and IL-4, or functional fragments or variants thereof; and/or (iv) IL- 15 and IL-18, or functional fragments or variants thereof.
  • cytokines chosen from a combination of: (i) IL-15 and IL-6, or functional fragments or variants thereof; (ii) IL-15 and IL-7, or functional fragments or variants thereof; (iii) IL-7 and IL-4, or functional fragments or variants thereof; and/or (iv) IL- 15 and IL-18, or functional fragments or variants thereof.
  • Any combination of cytokines in nucleic acid form or protein form may be exposed to one or
  • the cell is a hematopoietic stem cell or a hematopoietic progenitor cell.
  • the methods disclosed herein comprise a multistep process of differentiating a naive T cell into a memory' effector T cell that is either CD4+ or CD8+ and then, subsequently or simultaneously or prior to differentiating the naive T cells, stimulating the naive T cells with one or a plurality of antigens, such as viral antigens.
  • the method of differentiating the endothelial cells disclosed herein comprises exposing the naive cells to at least one cytokine composition, such as but not limited to one comprising IL-15, at a concentration and for a time period sufficient to cause growth and proliferation of CD8 and a change in character from a naive cell to a memory effector cell.
  • the method of differentiating the endothelial cells disclosed herein comprises exposing the naive cells to at least one cytokine composition, such as but not limited to one comprising IL-6, at a concentration and for a time period sufficient to cause growth and proliferation of CD8 and a change in character from a naive cell to a memory effector cell.
  • the method of differentiating the endothelial cells disclosed herein comprises exposing the naive cells to at least one cytokine composition, such as but not limited to one comprising IL-18, at a concentration and for a time period sufficient to cause growth and proliferation of CD8 and a change in character from a naive cell to a memory effector cell.
  • the method of differentiating the endothelial cells disclosed herein comprises exposing the naive cells to at least one cytokine composition, such as but not limited to one comprising IL-4, at a concentration and for a time period sufficient to cause growth and proliferation of CD4 and a change in character from a naive cell to a memory effector cell.
  • the method of differentiating the endothelial cells disclosed herein comprises exposing the naive cells to at least one cytokine composition, such as but not limited to one comprising IL-7, at a concentration and for a time period sufficient to cause growth and proliferation of CD4 and a change in character from a naive cell to a memory effector cell.
  • at least one cytokine composition such as but not limited to one comprising IL-7
  • the naive T cell is exposed to one or a combination of any of the cytokines listed in Table I, at a concentration and for a time period sufficient to induce expression of CCR7 and CD45RO.
  • the composition comprising lymphocytes is exposed to one or a combination of any of the cytokines listed in Table 1 , optionally after exposure to the one or combination of tumor or viral antigens, at a concentration and for a time period sufficient to alter the change a population of lymphocytes cell to T cell effector memory cells.
  • the disclosure relates to methods by which T cells can be differentiated into memory T cells.
  • Reprogramming of the T ceils may be accomplished by exposing the isolated naive T ceils to a composition comprising one or a plurality' of cytokines disclosed herein for a time sufficient to sequentially activate or induce expression of CCR7 and CD45RO.
  • these cells can also be exposed to tumor antigens for priming and eventual introduction or administration into mammals having cancer comprising the tumor antigens.
  • These cells can also be exposed to viral antigens for priming and eventually introduction or administration into mammals having viral infection by the virus from which the viral antigens are used for priming.
  • CCR7 C-C motif chemokine receptor 7
  • This receptor was identified as a gene induced by the Epstein-Barr vims (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. This receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. It has been shown to control the migration of memory' T cells to inflamed tissues, as well as stimulate dendritic cell maturation.
  • the chemokine (C-C motif) ligand 19 (CCL19/ECL) has been reported to be a specific ligand of this receptor.
  • CCR7 from Homo sapiens has the following sequence (GenBank Accession No.
  • PTPRC Protein tyrosine phosphatase, receptor type, C also known as PTPRC is an enzyme that, in humans, is encoded by the PTPRC gene.
  • PTPRC is also known as CD45 antigen (CD stands for cluster of differentiation), which was originally called leukocyte common antigen (LCA).
  • CD45 protein family consists of multiple members that are all products of a single complex gene. CD45RO, the shortest CD45 isoform, which lacks all three of the A, B, and C regions. This shortest isoform facilitates T cell activation.
  • Receptor-type tyrosine-protein phosphatase C isoform 5 precursor from Homo sapiens has the following sequence (GenBank Accession No. NP__001254727); MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGFILQAEEQGSQSKSPNLKSREAD
  • reprogramming of the T cells according to the present disclosure may be accomplished by exposing the isolated naive T cells to a composition comprising one or a plurality of cytokines disclosed herein for a time sufficient to sequentially activate or induce expression of CCR7 comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 48 and CD45RO comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 49.
  • the disclosure relates to a method of expanding viral antigen specific CD8+ and/or CD4+ T cells in an in vitro culture comprising contacting the antigen presenting cells, such as lymphocytes, with one or a plurality of viral antigens in the presence of one or a plurality of cytokines chosen from: IL-15, IL-6, IL-7, IL-4, IL-18 and/or functional fragments or variants thereof.
  • the antigen presenting cells such as lymphocytes
  • cytokines chosen from: IL-15, IL-6, IL-7, IL-4, IL-18 and/or functional fragments or variants thereof.
  • At least two cytokines are present in the in vitro culture chosen from a combination of: (i) IL- 15 and IL-6, or functional fragments or variants thereof; (ii) IL- 15 and IL-7, or functional fragments or variants thereof; (iii) IL-7 and IL-4, or functional fragments or variants thereof; and/or (iv) IL-15 and IL-18, or functional fragments or variants thereof.
  • Any combination of cytokines in nucleic acid form or protein form may be exposed to one or more cells.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells comprise at least about 70%, 75%, 86%.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells are chosen from the viral antigen peptides provided in Figures 8.
  • the disclosure relates to a method of expanding viral antigen specific CD8+ and/or CD4+ T cells in an in vitro culture comprising contacting the antigen presenting cells, such as lymphocytes, with one or a plurality of viral antigens of a virus of the family Coronaviridae in the presence of one or a plurality of cytokines chosen from: IL-15, IL-6, IL-7, IL-4, IL-18 and/or functional fragments or variants thereof.
  • At least two cytokines are present in the in vitro culture chosen from a combination of: (i) IL-15 and IL-6, or functional fragments or variants thereof; (ii) IL- 15 and IL-7, or functional fragments or variants thereof; (iii) IL-7 and IL-4, or functional fragments or variants thereof; and/or (iv) IL- 15 and IL- 18, or functional fragments or variants thereof.
  • Any combination of cytokines in nucleic acid form or protein form may be exposed to one or more cells.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells are from a coronavirus.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells are from SARS-CoV-2 including SARS-CoV-2 delta variant”.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells are peptide fragments of the SARS-CoV-2 antigens comprising the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • the one or plurality of viral antigens used in the methods of the disclosure for priming the cells are in form of a SARS-CoV-2 antigen library comprising a pool of peptides (for example, peptides that are from about 10 to about 20 ammo acids in length) from one or a combination of the structural proteins of SARS-CoV-2 disclosed herein, such as 15-mers peptides containing amino acids overlap (for example 11 amino acids of overlap) between each peptide formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • a SARS-CoV-2 antigen library comprising
  • the SARS-CoV -2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: I I, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ) ID NO: 11.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: 12, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ) ID NO: 12.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the protein having the amino acid sequence of SEQ ID NO: 13, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 13.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV -2 antigen library comprising a pool of peptides formed by scanning the protein having the ammo acid sequence of SEQ ID NO: 14, or functional fragments or variants thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 14.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 12, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SAR.S-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 13, or functional fragments or variants thereof
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13, or functional fragments or variants thereof.
  • the SARS- CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the ammo acid sequences of SEQ ID NO: 12 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific I' cell compositions are generated using a SARS-CoV -2 antigen library comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 1 1, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS-CoV-2 antigen library comprising a pool of peptides formed by scanning the proteins having the ammo acid sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using a SARS- CoV-2 antigen library' comprising a pool of peptides formed by scanning the proteins having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or functional fragments or variants thereof.
  • the SARS-CoV-2 specific T cell compositions are generated using one or plurality' of epitopes chosen from those disclosed in Table A, Table B, and/or Table C or from identical or corresponding epitopes of SARS-COV-2.
  • a corresponding epitope may contain one or more substitutions or deletions to the amino acid sequence of SEQ ID NO: 11 or to a peptide epitope thereof: D614G, T478K, L452R, P681R, T19R, A157-158, D950N and K417N; N439K, Y453F, A69-70; E484K, E484Q, E484P, E484A, E484D, E484G or E484K; K444E, G446V, L452R or F490S; S477G, S477N or S477R; N148S, K150R, K150E, K150T, K150Q or S151P; A69-70 (RDR1), Al 41- - -
  • the disclosure relates to a T cell composition
  • a T cell composition comprising T cells that specifically bind to a Coronaviridae peptide having at least about a 80%, 85%, 90%, 95% sequence identity to SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • the T cells of the disclosed composition specifically bind to one or a plurality' of epitopes located in the protein of SEQ) ID NO: 11, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 12 to about 524 of the protein of SEQ ID NO: 11, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 12 to about 331 of the protein of SEQ ID NO: 11, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality' of epitopes located at amino acid residue from about 57 to about 75 of the protein of SEQ ID NO: 11, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 205 to about 224 of the protein of SEQ ID NO: I I, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 331 to about 524 or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 449 to about 463 of the protein of SEQ ID NO: 11. or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a. plurality of epitopes selected from Table A. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located in the protein of SEQ ID NO: 12, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located in the protein of SEQ ID NO: 13, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 100 to about 2.2.2 of the protein of SEQ ID NO: 13, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality' of epitopes located at amino acid residue from about 144 to about 192 of the protein of SEQ ID NO: 13, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 144 to about 163 of the protein of SEQ ID NO: 13, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 163 to about 192 of the protein of SEQ ID NO: 13, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 173 to about 192 of the protein of SEQ ID NO: 13, or functional fragments thereof.
  • the T cells of the disclosed composition specifically bind to one or a plurality of epitopes selected from Table B. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located in the protein of SEQ ID NO: 14, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality of epitopes located at amino acid residue from about 257 to about 361 of the protein of SEQ ID NO: 14, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality' of epitopes located at amino acid residue from about 257 to about 271 of the protein of SEQ ID NO: 14, or functional fragments thereof. In some embodiments, the T cells of the disclosed composition specifically bind to one or a plurality' of epitopes selected from Table C.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 , the concentration required to produce the half-maximal response, in the range of about 0.01 to about 20 ng/mL. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC50 in the range of about 0.05 to about 18 ng/mL.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity' of EC50 in the range of about 0.1 to about 15 ng/mL, In some embodiments, the T cells of the disclosed composition bind to one or plurality' of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 in the range of about 0.2 to about 10 ng/mL. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity’ of EC 5G in the range of about 0.3 to about 8 ng/mL.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 5G in the range of about 0.4 to about 6 ng/mL. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 5G in the range of about 0.5 to about 5 ng/mL. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 in the range of about 0.6 to about 4 ng/mL.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC50 of about or at least 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
  • the above range includes all intermediate subranges and values.
  • the T cells of the disclosed composition bind to one or plurality' of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 from about 0.1 nM to about 100 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality’ of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC50 from about 0.5 nM to about 80 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 from about 0.8 nM to about 60 nM.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 from about 1 nM to about 50 nM. In some embodiments, the T ceils of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC so from about 5 nM to about 40 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality’ of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC50 from about 7.5 nM to about 35 nM.
  • the T cells of the disclosed composition bind to one or plurality' of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity’ of EC50 from about 10 nM to about 30 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 from about 15 nM to about 35 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality’ of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity’ of EC50 from about 17 nM to about 30 nM. In some embodiments, the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity’ of EC 50 from about 15 nM to about 25 nM.
  • the T cells of the disclosed composition bind to one or plurality of the aforementioned proteins, fragments thereof, or epitopes thereof, with an affinity of EC 50 of about or of at least of 0.1 nM, 0.5 nM, I nM, 5 nM, 7.5 nM, 10 nM, 20nM, 30 nM, 40 nM, 50 nM, 60 nM, 70nM, 80 nM, 90 nM, ⁇ 100 nM or 100 nM.
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of T cells that specifically bind to a Coronaviridae peptide having at least about a 80%, 85%, 90%, 95%, 99%, ⁇ 100%, or 100% sequence identity’ to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • the naive T cells in the pharmaceutical compositions may be derived by a biopsy of a donor (optionally frozen after differentiation and harvesting) followed by expansion in culture using the steps disclosed herein.
  • Blood, serum or lymph tissue may be biopsied from a subject.
  • the starting material is composed of three pm punch biopsies collected using standard aseptic practices. Blood may be collected by the treating physician, placed into a vial.
  • the biopsies are shipped at a temperature of about 2, 3, 4, 5, 6, 7 to about 8°C refrigerated shipper back to the manufacturing facility. After arrival at the manufacturing facility, the biopsy is inspected and, upon acceptance, transferred directly to the manufacturing area.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the composition comprising the one or more cytokines may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary' cells, e.g., primaiy human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body’ fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood- derived sample, or is or is derived from an apheresis or leukapheresis product.
  • Exemplary' samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow; thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell hues, e.g, T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, or pig.
  • isolation of the cells includes one or more preparation and/or iion-affmity-based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • cells from the circulating blood of a subject are obtained, e.g, by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions.
  • a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca +4 /Mg 4+ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffmity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result In a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • specific subpopulations of T cells such as cells positive or expressing one or more markers, e.g., CD4+, CD25+, CD2.7+, CD4+, CD8+ or other markers disclosed herein.
  • a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained.
  • the negative fraction then is subjected to negative selection based on expression o, for example, CD 14 and CD45RA, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
  • CD4+ T helper cells are sorted into naive, central memory', and effector cells by identifying cell populations that have cell surface antigens.
  • CD4+ lymphocytes can be obtained by standard methods.
  • naive CD4+ T lymphocytes are CD45RO+, CD45RA+, CD62L+, CD4+ T cells.
  • central memory CD4+ cells are CD62L+ and CD45RO+.
  • a monoclonal antibody cocktail typically includes antibodies to CDI4, CD20, GDI lb, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • a solid support or matrix such as a magnetic bead or paramagnetic bead
  • the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in METHODS IN MOLECULAR MEDICINE, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A, Brooks and U. Schumacher ⁇ Humana Press Inc., Totowa, NJ).
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads).
  • the magnetically responsive material, e.g, particle generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g, that it is desired to negatively or positively select.
  • the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.
  • a specific binding member such as an antibody or other binding partner.
  • Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452.342 B, which are hereby incorporated by reference in their entireties.
  • the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering.
  • the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population.
  • the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • a freezing solution e.g., following a washing step to remove plasma and platelets.
  • Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
  • the cells are then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen
  • the provided methods include cultivation, incubation, culture, and/or genetic engineering steps.
  • the cell populations are incubated in a culture-initiating composition.
  • the incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g, nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • agents e.g, nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • the stimulating conditions or agents include one or more agent, e.g, ligand, which is capable of activating an intracellular signaling domain of a TCR complex.
  • agent e.g, ligand
  • the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell.
  • agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3.
  • the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28,
  • agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines.
  • the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g, at a concentration of at least about 0.5 ng/ml).
  • the isolated cells are part of one or more disclosed compositions of T cells and those T cells are stimulated with a composition of cytokines agents including IL-15 and/or IL-6.
  • the composition of cytokines is free of one or more of: IL-1, IL-12, IL- 4, or IL-7,
  • methods of the disclosure relate to stimulating isolated naive T cell compositions with a composition of at least two cytokines (such as IL-6 and IL- 15) and are free of one or more of: IL-1, IL- 12, IL-4, or IL-7.
  • the methods of expanding, proliferating or stimulating the T cell populations are free of a step of exposing the T cell populations to cytokines one or more of: IL-2, IL-4, IL-4, IL-7, IL-12, IL- 18 and IL-27. In some embodiments, the methods disclosed herein are free of a step of using a composition of feeder cells.
  • T cells to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject.
  • T cell therapies are defined by number of cells per kilogram of body weight. However, because T cells will replicate and expand after transfer, the administered cell dose will not resemble the final steady-state number of cells.
  • a pharmaceutical composition comprising the T cells of the present invention may be administered at. a dosage of I O 4 to 10 9 cells/kg body weight. In another embodiment, a pharmaceutical composition comprising the T cells of the present invention may be administered at a dosage of 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • compositions comprising the T cells of the present invention may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., 1988, New England Journal of Medicine, 319: 1676).
  • the optimal dosage and treatment regimen for a particular subject can be readily determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • compositions embodied herein for the treatment of a viral infection
  • composition of the present invention may be prepared in a manner known in the art and are those suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.
  • pharmaceutically acceptable carrier as used herein means any suitable carriers, diluents or excipients.
  • compositions of the invention may also include other supplementary physiologically active agents.
  • compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intraarticular, intravenous and intradermal administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the compositions comprising a therapeutically effective amount of stimulated T cells or T cells produced by the methods disclosed herein. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.
  • the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration. In another embodiment, the composition is suitable for intraarticular administration.
  • SARS-CoV-2 or other pathogen-specific T cells are administered parenterally, for example, by intravenous infusion, intraperitoneal infusion, or other parenteral mode. T cells may also be infused to a site of SARS- CoV-2 or microbial infection such as into the lungs or upper or lower respiratory system or into or around another infected tissue or organ.
  • compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the invention also contemplates the combination of the T cell composition of the present disclosure with other drugs and/or in addition to other treatment regimens or modalities such as surgery’.
  • the composition of the present invention is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture.
  • the disclosure relates to a system comprising a cell culture unit that is utilized to culture and expand a T cell population described herein.
  • the cell culture unit comprises one or a plurality of cell reactor surfaces housed in at least a first compartment, the one or plurality’ of cell reactor surfaces in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet.
  • the one or plurality of cell reactor surfaces are configured in a cylindrical form with a hollow’ volume fixed within a cylindrical first compartment; wherein the first media line and the second media line are positioned on opposite faces of the cylindrical first compartment.
  • the first media line can be attached to a first sealable aperture configured for sterile attachment of a cell culture media source.
  • the system further comprises a pump and a fluid regulator in operable contact with the first media line, wherein the pump is capable of generating pressure in the first media tine and wherein the fluid regulator is capable of regulating the speed of fluid from the pump through the first compartment and into the second media line.
  • the one or plurality of cell reactor surfaces can have a surface area from about 0.5 m 2 to about 100.0 m 2 , including any value therein, such as about 3 m 2 , about 4 m 2 , about 5 m 2 , about 6 m 2 , about 7 m 2 , about 8 m 2 , about 9 m 2 , about 10 m 2 , about 11 m 2 , about 12 m 2 , about 13 m 2 , about 14 m 2 , about 15 m 2 , about 16 m 2 , about 17 m 2 , about 18 m 2 , about 19 m 2 , about 20 m 2 , about 21 m 2 , about 22 m 2 , about 23 m 2 , about 24 m 2 , about 25 m 2 , about 26 m 2 , about 27 m 2 , about 28 m 2 , about 29 m 2 , about 30 m 2 , about 31 m 2 , about 32 m 2 , about 33 m 2
  • the system further comprises a gas transfer module in operable connection to the one or plurality of cell reactor surfaces.
  • the gas module comprises a gas pump and a gas regulator connected to the first compartment by a first gas line.
  • the first compartment comprises at least one gas outlet.
  • the gas pump is capable of generating air pressure from the pump to the first compartment through the first gas line.
  • the gas outlet can be one or more vents or the gas outlet can be configured for sterile connection to one or more vents.
  • the gas regulator is capable of regulating the speed of gas from the pump through the first compartment.
  • Some embodiments further comprise a first gas inlet in operable connection to the gas transfer module.
  • the first gas inlet is attached to a second sealable aperture configured for sterile attachment of a gas source.
  • the gas source can be any known gas storage and/or delivery 7 system, such as for example a container or a tank.
  • the system can further comprise an apheresis unit in fluid communication with the cell culture unit.
  • Suitable apheresis units include the Spectra Optia Apheresis System (TerumoBCT).
  • the system further comprises a harvesting compartment in fluid communication with the cell culture unit. Suitable harvesting compartments are discussed elsewhere herein.
  • a cell culture system as described herein can be used to expand memory effector T cells from a subject through culturing one or a plurality of T cells in the system and allowing the T cells to grown in the first compartment for a time period sufficient to proliferate.
  • the T cells can be introduced into the system through the system’s first compartment.
  • the T cells are CD4+ T cells.
  • the T cells are CD8+ T cells.
  • the T cells are a mixture of CI)4+ and CD8+ T cells.
  • the T cells are CD45A+ T cells.
  • the disclosure also relates to a system comprising a cell culture unit comprising one or a plurality of cell reactor surfaces housed in a plurality of compartments, each compartment separated by a removable partition first compartment comprising at least one cell reactor surface, at least one cell reactor surface in fluid connection with a first and second media line, the first media line in fluid communication with a first media inlet, the second media line in fluid communication to a first media outlet.
  • the cell culture unit comprises a single cell culture chamber comprising multiple partitions, each partition independently removable and independently in fluid connection with the first and the second media line and each partition or set of partitions defining a distinct compartment.
  • the cell culture unity' comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more compartments, each compartment separated by and/or defined by one or more partitions.
  • the compartments are configured in a grid or linear pattern.
  • each partition separating one compartment from another compartment may be removed such that the cell reactor surface of a first compartment is or becomes contiguous with a cell reactor surface of a second compartment. The removal of one or more partitions allows for an increased surface area onto which cells from one compartment (such as the first compartment) may proliferate and/or grow into another compartment (such as the second compartment) during a method of culturing.
  • the cell culture unit comprises a set of side walls defining a single surface area divided among 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more compartments each compartment with at least one or a plurality of cell reactor surfaces.
  • each compartment has at least a first cell reactor surface.
  • the disclosure relates to a method of growing T cell populations on a tissue culture sy stem disclosed herein, wherein primary sets of lymphocytes are plated at about a concentration of from about 0.001 to about 10 million cells per milliliter into one or more compartments of the cell culture unit and then allowed to grow to a confluent layer on surface area of from about 1 to about 200 squared centimeters.
  • the method further comprises removing one or more partitions to allow the cells to grow' in a second compartment until confluence, when again, optionally, another partition may successively be removed to allow for more surface for expanded culture.
  • the method of culturing further comprises repeating the step of removing a partition for each of the compartments into which cells should grow.
  • the cell culture unit comprises 3, 4, 5, 6, 7, 8, 9, 10, I I, 12 or more partitions each of which corresponding to the physical barrier between a second and third compartment, between a third and fourth compartment, between a fourth and fifth compartment, between a fifth and sixth compartment, between a sixth and seventh compartment, between a seventh and eighth compartment, between an eighth and ninth compartment, between a ninth and tenth compartment, between a tenth and eleventh compartment, and/or between an eleventh and twelfth compartment, respectively.
  • one or more of the partitions comprise an interior portion, a frame portion and an exterior portion.
  • the interior portion of the partition is positioned in the closed portion of the system; the frame portion spans a wall of the culture system separating the interior of the culture system to the exterior of the system; and the exterior portion is positioned outside of the system.
  • a seal operably fits around the frame portion of one or more of the partitions such that removal of the partition does not introduce pathogens to and/or does not expose the environment outside of the tissue culture system to the interior of the tissue culture system.
  • the cell density of each compartment is from about 0.1 to about 10 million cells per ml of cell culture media. In some embodiments, the cell density of each compartment is from about 0.1 to about 10 million cells per mL. of cell culture media. In some embodiments, the cell density of each compartment is from about 0.5 to about 10 million cells per ml., of cell culture media. In some embodiments, the cell density of each compartment is from about 1.0 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 2 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 3 to about 10 million cells per mL of cell culture media.
  • the cell density' of each compartment is from about 4 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 5 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 6 to about 10 million cells per mL. of cell culture media. In some embodiments, the cell density of each compartment is from about 7 to about 10 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about 8 to about 10 million cells per mL. of cell culture media. In some embodiments, the cell density of each compartment is from about 9 to about 10 million cells per mL of cell culture media.
  • the cell density' of each compartment is from about 0.1 to about 20 million cells per mL of cell culture media. In some embodiments, the cell density of each compartment is from about. 0.1 to about 50 million cells per mL of cell culture media. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.01 million to about 10 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density' of from about 0.03 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.07 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.03 million to about 5 million cells per square centimeter.
  • the systems disclosed herein comprise a cell density of from about 0.001 million to about 5 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.002 million to about 4 million cells per square centimeter. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.003 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.004 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.005 million to about 5 million cells per square centimeter of surface area of cell reactor surface.
  • the systems disclosed herein comprise a cell density' of from about 0.006 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.007 million to about 5 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density' of from about 0.001 million to about 4 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.001 million to about 3 million cells per square centimeter of surface area of cell reactor surface. In some embodiments, the systems disclosed herein comprise a cell density of from about 0.003 million to about 3 million cells per square centimeter of surface area of cell reactor surface.
  • VSTs viral-specific T cells
  • Current manufacturing approaches are rapid but growth conditions can still be further improved.
  • a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96 well plate was designed to fully characterize T cell viability, function, growth, and differentiation.
  • Peripheral blood mononuclear cells (PBMC) from six consenting donors were seeded at 100,000 cells/well with pools of CMV peptides from IE-1 and pp65, and combinations of IL-15, IL-6, IL21, IFNa, IL 12, IL 18, IL-4, and IL-7.
  • IL-15/IL-6 and IL-4/IL-7 were optimal for expansion of viral specific CD3-f- T cells, (18-fold and 14-fold respectively compared with unstimulated controls).
  • CD8+ T cells expanded 24-fold in IL-15/IL-6, and 9-fold in IL-4/IL-7 cultures (p ⁇ 0.0001).
  • CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL- 15/IL-6 (p ⁇ 0.0001).
  • CD45RO+ CCR7- effector memory T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/TL-7 cultures respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNy (7.6% producing both TNFa and IFNy), and 18.5% of CD3+ cells grown in IL-4/IL-7 (9% producing both TNFa and IFNy ). This study show's the utility of this single plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10 7 PBMC.
  • Adoptive T cell immunotherapies are increasingly used to treat infection and malignant disease.
  • a common technique involves culturing T cells with antigen presenting cells (APC) exposed to peptide antigens in the presence of a cytokine cocktail.
  • APC antigen presenting cells
  • cytokine cocktail Several immunomodulatory cytokines are currently used to promote T cell division and differentiation, but optimal conditions for the growth and function of peptide stimulated T cell products have yet to be fully defined.
  • Cell products used in clinical trials have typically supplemented T cell cultures with the growlh promoting cytokines IL2, IL-15, IL-4 and IL-7 [1-3], 'The search to optimize culture conditions is, however, limited by the time and labor needed to screen multiple cytokine combinations.
  • a flow' cytometry-based approach was therefore established to rapidly evaluate many cytokine combinations in a single 96 well plate to measure T cell phenotype and potency on a limited numbers of cells.
  • IL-4 enhances survival of resting T cells and induces CD4+ Th2 helper differentiation [5- 7] while IL-15 promotes survival and diversity' of CD8+ memory' T cells [8, 9], IL2 is a canonical T cell growth cytokine which continues to be used in clinical trials due to its effectiveness m expanding T cells derived from tumor infiltrating lymphocytes [10].
  • cytokines also have important functions: IL-6 may enhance Th17 development [11], IL-7 promotes I' cell homeostatic survival [12-14], and IL21 promotes the activity of CD8+ T cells [15-17],
  • the manufacturing methods have transitioned from culturing T cells in 24 well plates with IL2 and APC transduced with viral antigens [18-20] to a simplified culture containing a combination of IL-4 and IL-7 in G-Rex gas permeable devices with soluble mixes of peptides [3, 21, 22].
  • This system rapidly expands functionally competent T cells specific for multiple viruses.
  • Flow cytometric assays minimize culture volume reducing the number of cells needed for validation, while increasing the number of testable conditions that can be applied to donor PBMC.
  • new' cytokine combinations controlling phenotypic diversity, growth and function of viral specific T cells products were identified. It was found that high throughput screening by multicolor flow cytometry is affordable and practical for product development.
  • Peripheral blood was collected from de-identified platelet transfusion filters from donors according to IRB-approved protocol.
  • PBMCs were separated by Ficoll and spun at 800 x g for 25 minutes to purify lymphocytes. Cells were washed twice with complete RPMI, and 2 x 10 7 cells were re-suspended in 5 ml., with 10 ⁇ L of 200 pg/mL of peptide libraries encompassing IE1 and PP65. Cells were incubated at 37°C for 1 hour. Five mL of complete media was added and 100,000 cells/well were plated in 96-well round-bottom plates. Cytokines were added at the indicated concentration in a final volume of 200 pL. Cells were cultured for seven days.
  • Intracellular cytokine staining Plates were spun down at 400 x g for 5 minutes and resuspended in 100 pL of complete media containing the mix of IE1 and PP65 peptide libraries at 1.0 pg/mL final concentration with no peptide controls. Cells were incubated with peptides at 37°C for I hour. Then, 100 uL of complete media containing Brefeldin A or Monensin was added. Cells were cultured for an additional 5 hours, after which cells were stained with antibodies for phenotyping.
  • ELISPOT assay ELISPOT plates were coated with 100 pL of I pg/mL final concentration anti-IFNy mAb (Clone 1-D1K; Mabtech, Cincinnati, OH) in sterile ELISPOT carbonate coating buffer (1.59 g Na 2 CO 3 , 2,93 g NaHCC) 3 per liter of sterile water) overnight at 4°C. Plates were washed twice with 150 pl, of coating buffer, and 100 ⁇ L of compl ete media was added to wells and incubated for 1 hour at 37°C.
  • Cells from G-rex10 culture vessels were plated at the indicated concentrations in 200 pL total volume with actin (1 pg/mL), IE1 and pp65 peptide pools (1 pg/mL), SEB (0,5 pg/mL), or media alone. Plates were incubated for 16 hours at 37°C, after which cells were decanted and plates were washed six times with lx PBS/0.05% Tween 20.
  • biotinylated anti-IFNy mAb at 1 pg/mL 100 ⁇ L of biotinylated anti-IFNy mAb at 1 pg/mL (Clone 7-B6-1; Mabtech) in biotin buffer (2.5 g biotin in 500 ml, lx PBS) was added to each well and incubated at 37°C for 1 hour. Plates were washed six times with lx PBS/0.05% Tween 20. 100 pL of avidin-peroxidase solution in lx PBS/Tween 20 (APC; Millipore Sigma, Darmstadt, Germany) was added per well and incubated for 1 hour at room temperature. Plates were washed three times with lx PBS/0.05% Tween 20 and three times with lx PBS.
  • Markers for staining included CD62L V450 (Biolegend; clone DREG-56), CD4 BV570 (Biolegend; clone RPA-T4), CD45RO BV605 (Biolegend; clone LCHL1), CD8 BV711 (Biolegend; clone SKI), CD56 BV785 (Biolegend; clone 5.1H11), CCR7 FITC (Biolegend; G043H7), CD28 PE (Miltenyi Biotec, San Diego, CA; clone REA612) , CD95 PE Dazzle CF594 (Biolegend; clone DX2), CD3 PerCP Cy5.5 (Biolegend; clone OKT3), TNFa PE Vio770 (Miltenyi Biotec; clone cA2), IFNy APC (Biolegend; clone RS.B3), CD107a APC H7 (Miltenyi Biotec; clo
  • CD3+ CD4+, and CD3+ CD8+ T cells present within the culture were measured, along with CD3- CD56+ NK cells.
  • Viability was measured by vital dye staining using Live Dead Aqua, while cytotoxic function was compared by measuring IFN ⁇ and TNFa intracellular cytokine production in viral wells pulsed with peptide pools over antigen non-specific background wells.
  • surface expression of T cell memory markers were used to judge the differentiation status of cells, including CD45RA, CD45RO, CCR7, CD28, CD95, and CD62L.
  • IL-15 10 ng/mL
  • IL-6 100 ng/mL
  • FIG. 5B It was confirmed that addition of IL-15 or IL-7 was sufficient for expansion of CD3+ T cells, and that the original selection of IL-15/IL-6 was superior to all other combmations.
  • culture in a combination of IL-15/IL-6 consistently promoted CD3+ T cell expansion and IFNy production to levels similar to culture in IL-4/IL-7.
  • cytokine culture imparts bias on the ratio of CD4 vs CD8 cells in viral specific T cell products.
  • Culture in IL-15/IL-6 expanded a median of 17.1 -fold more CD3+ cells compared with no cytokine controls (p ⁇ 0.0001).
  • Culture in IL-4/IL-7 expanded a. median of 13.8-fold more compared with no cytokine control (p ⁇ 0.0001; FIG. 7A).
  • CD3- CD56+ NK cells were present, in cultures expanded with IL-15/IL-6, with a median of 6.6% of total cells recovered (4106 cells: FIG. 6A). Less than 300 NK cells were recovered on average from wells containing IL-4/IL- 7, representing 0.6% of total cells recovered.
  • Both culture conditions expanded CD3+ T cells producing IFNy in response to CMV peptide pool re-stimulation.
  • the proportion of multi-cytokine producing cells was also investigated, as evidence suggests these cells offer superior protection against viral infection when compared with cells producing a single cytokine.
  • VSTs are effector memory in phenotype.
  • the extent of T cell differentiation has been suggested to influence the persistence of adoptively transferred T cells [27, 28].
  • the surface phenotype of VSTs expanded by in vitro culture in IL-I5/IL-6 and IL-4/IL-7 was characterized to identify the proportion of cells expressing different, combinations of T cell memory markers in FIG. 7.
  • Pre-culture CD3+ T cells comprised on average 32% naive/stem cell memory cells, 30.4% effector memory cells, 22% central memory cells and 15.4% terminal effectors.
  • Ten-day VST products had a preponderance of effector-memory CD3+ cells representing 72.3% and 76.9% of cells grown in IL-4/IL-7 and IL-15/IL-6, respectively.
  • Terminal effector ceils lacking both CCR7 and CD45RO represented 1 1.3% in IL-4/IL-7, and 14.3% in IL-15/IL-6.
  • Central memory cells represented a minority of cells after culture, 9,3% and 6.6% in IL-4/IL-7 and 1L-15/TL-6 cultures, respectively.
  • the memory phenotype of antigen specific cells was also compared with antigen non-responsive cells.
  • CD3+ IFNy+ ceils had a predominantly effector memory phenotype, while a small number of naive cells (4.2% in IL-4/IL- 7 and 1.7% in IL-15/IL-6) remained within the IFN-y negative (antigen non-reactive) fraction, suggesting that culture in IL-4/IL-7 was preserving a subset of naive cells within the final product.
  • Process development time substantially reduced by using the IQue Process development of antigen-specific T cells such as VSTs has largely been limited to testing individual conditions in 24-welI plates or Grex-10 devices, which limits the systematic testing of an array of cytokines and growth conditions. Expanding and testing VSTs using such methods requires approximately 20 hours of work and 34 hours of incubation per cytokine condition. In contrast, process development in 96 well plates and a 13 color flow cytometry panel required only 12 hours of work and 8 hours of incubation. To evaluate 40 cytokine conditions would therefore take 2160 hours per sample by existing methods, but only 20 hours using the improved approach disclosed herein (Table 5A).
  • Table 5A Comparison of traditional process development (PD) in culture vessels, traditional PD in plates, and micro assay using flow cytometry'. Optimized cytokine conditions identified in 96-well plate translates to clinical scale manufacturing. Miniaturized cell cultures may not reliably scale-up in a linear fashion. To test whether this system could predict the phenotype and function of clinical size products, whether IL- 15/IL-6 cultures in Grex-10 culture vessels would recapitulate the data from the experiments in 96- well plates was investigated.
  • At least 1 x 10 7 cells were seeded with IE1 and pp65 peptide pools in Grex-10 culture vessels with medium and either IL-4/IL-7 at 400 U/mL IL-4/100 ng/mL IL-7 or 10 ng/mL IL-15/ 100 ng/mL IL-6 (FIG. 8).
  • Cells grown in IL-15/IL-6 produced a mean of 638 ⁇ 297 spots per 100,000 cells in response to CMV peptides, while cells cultured in IL-4 and IL-7 produced a mean of 555 ⁇ 230 spots per 100,000 added cells in response to CMV peptide pool restimulation.
  • the CMV response was antigen specific, as cells produced less than 10 spots on average in response to either actin or no peptide controls per 100,000 added cells. This demonstrated that cells grown in IL-15/IL-6 were functionally equivalent to cells grown in IL-4/IL- 7 when cultured to clinical scale and that the high throughput screening method can reliably optimize product development.
  • IL-6 improved cell expansion in combination with IL-15 without modifying effector function. These results are consistent with knockout mouse experiments showing that IL-6 reduces the threshold for TCR signaling in CD8+ T ceils [30], promoting memory T cell expansion in response to antigen specific peptide re-stimulation.
  • the requirement for including IL-15 or IL-7 for memory T cell expansion is not unexpected as both receptors share homology' with IL2 and use the common y-chain and its associated Jak/STAT signaling proteins [31-33], Recombinant IL-7 has been used clinically to expand T cell subsets in cases of lymphopenia [34, 35], and was included with IL-4 for its pro-survival benefits for T cells [36],
  • the combination of IL-15/IL-6 may provide a more balanced ratio of antigen specific CD4+ to CD8+ T cells during polyclonal expansion of T cell products against not only viral specific antigens, but also other targets, including tumor-associated antigens.
  • this high throughput plate-based flow cytometric assay was shown to be able to effectively and reliably measure T cell growth, function, and phenotype to optimize VST product development.
  • the data here show that IL- 15/IL-6 is equivalent to IL-4/IL-7 in GMP culture conditions.
  • the modular nature of the assay facilitates future investigations to optimize culture conditions with 3 or 4 cytokine combinations using IL-15ZIL-6 and IL-4/IL-7 as a baseline.
  • SARS- CoV-2-specific T cells were expanded from the peripheral blood of 23 convalescent donors by stimulation with peptides spanning the SAR.S-CoV-2 envelope, membrane, nucleocapsid, and spike antigens.
  • SARS-CoV-2 a novel coronavirus first reported in December 2019 from Wuhan, China, is responsible for the ongoing pandemic of coronavirus disease 2019 (COVID- 19)[46],
  • the adaptive immune response to SARS-CoV-2 remains ill-defined, and there is an urgent need to fill this gap in knowledge in order to enable the development of effective vaccines and therapies.
  • antibody responses to the spike and nucleocapsid proteins are well described [45, 47], the characterization of T cell response to SARS-CoV-2 is still limited.
  • T cell epitopes within conserved regions of SARS-CoV-2 structural proteins are presented. These predominately comprise MHC -Il-restricted CD4+ T cell responses, similar to those observed in response to other respiratory viruses.
  • T cell response to SARS-CoV-2 in concert with humoral immunity', this study advances our understanding of the overall adaptive immune response to SARS-CoV-2, facilitating the development of both adoptive T cell therapies and of effective vaccines for the treatment of individuals at risk of infection.
  • PBMCs Peripheral blood mononuclear cells
  • CSTsf Evaluated T cell products included SARS-CoV-2 specific T cells, manufactured from PBMCs of seropositive and seronegative volunteers. VSTs were produced using a rapid expansion protocol previously described. Briefly, PBMCs were pulsed with overlapping peptide pools encompassing viral antigens (1 pg /15 x 10 6 PBMCs) for 30 minutes at 37°C. Peptide libraries of 15-mers with 11 amino acids overlaps encompassing the spike, membrane, nucleocapsid, and envelope proteins were generated (A&A peptide, San Diego, CA, USA) from the SARS-CoV-2 reference sequence (NC 045512.2).
  • IL-4 400 lU/ml; R&D Systems, Minneapolis, MN
  • IL-7 10 ng/mi; R&D Systems
  • CTL media consisting of 45% RPMI (GE Healthcare, Logan, UT), 45% Click’s medium (Irvine Scientific, Santa Ana, CA), 10% fetal bovine serum, and supplemented with 2 mM GlutaMax (Gibco, Grand Island, NY).
  • Cytokines were replenished on day 7. On day 10, cells were harvested and evaluated for antigen specificity and functionality.
  • IFN- ⁇ Enzyme-Linked Immunospot (ELlSpot) Assay Antigen specificity of T cells was measured by IFN- ⁇ ELlSpot (Millipore, Burlington, MA). T cells were plated at 1 x 10 5 /well with no peptide, actin (control), or each of the individual SARS-CoV-2 pepmixes (200 ng/peptide/well). Plates were sent for IFN-y spots forming cells (SFC) counting (Zellnet Consulting, Fort Lee, NJ).
  • VSTs were stained with fluorophore-conjugated antibodies against CD4, CD8, TCRap, TCR ⁇ , and CD56. (Miltenyi Biotec, Bergisch Gladbach, Germany; BioLegend, San Diego, CA).
  • LIPS Luciferase Immunoprecipitation Systems
  • Testing for antibodies to spike and nucleocapsid proteins were performed using a LIPS assay as recently described [45]. Briefly, plasma samples were incubated with spike and nucleocapsid proteins fused to Gaussia and Renilla luciferase, respectively, protein A/G beads were added, the mixture was washed, coelenterazine substrate (Promega) was added, and luciferase activity was measured in light units with a Berthold 165 LB 960 Centro microplate luminometer. Antibody levels were reported as the geometric mean level (GML) with 95% confidence interval (CI).
  • GML geometric mean level
  • CI 95% confidence interval
  • Cut-off limits for determining positive antibodies in the SARS-CoV-2-infected samples were based on the mean plus three standard deviations of the serum values derived from uninfected blood donor controls or by receiver operator characteristics (ROC) analysis. For some of the data percentages for categorical variables, mean and range, geometric mean plus 95% CI were used to describe the data. Wilcoxon signed rank were used for statistical analysis.
  • Sequences of SARS-CoV-2 antigenic peptides are provided in the following Table 5B.
  • the epitopes contained within the peptides described in Table 5B and the other tables herein may be employed in the methods for culturing T cells disclosed herein or for treatment or prevention of disease using T cells recognizing these epitopes.
  • epitopes at AA 173-192 were recognized by 5 patients, and also confirmed to be CD4-restricted (FIG. 11C). These epitopes all lie within the C-terminal domain which is located inside the virion and on intracellular membranes of infected cells that is a conserved region within all known strains of SARS-CoV2 [56],
  • T-cell and humoral responses measured here represent an effective adaptive immune response to SARS-CoV-2.
  • Subjects were not tested longitudinally, and therefore the absence of T-cell responses in 30% of subjects may relate to the timing of T-cell responses following primary infection. Evaluation was limited to structural viral proteins, given their described immunodominance in related coronaviruses, but it is possible that T-cell responses to non-structural proteins may also occur.
  • IL-15/IL-6 Function IL-15/IL-7 Function
  • IL-7 /IL-4 Function IL-15/IL-6 Function
  • CD3+ T cell expansion was also significantly reduced in wells with lower concentrations of IL-15 regardless of the presence of additional cytokines IL-6, IL21, or IFNa. Also, culture with IL-6, IL2I, or IFNa alone was not sufficient to stimulate T cell expansion in the absence of IL-15. CD3+ T cell expansion in the presence of IL-4 and IL-7 (400 U/mL and 10 ng/mL) was less than seen with cells cultured in IL-15 for this sample, with an average of 29,998 CD3+ cells recovered (12.3-fold increase over no cytokine controls).
  • the combination of IL- 15 (10 ng/mL) and IL-6 (100 ng/mL) was selected for further investigation based on the favorable expansion of CD3+ T cells and their cytokine production as seen in four of the samples tested compared with other cytokine combinations (FIG. 17).
  • the plate layout was further modified to investigate additional replicates of IL-15 and IL-6, new cytokine combinations with IL12 and 1L18 while removing IL21 and IFNa cytokines, and new combinations of IL-4, IL-15, IL-6, and IL-7 with each other (FIG. 19).
  • cytokine culture imparts bias on the ratio of CD4 vs CDS cells in viral specific T cell products.
  • IL- 15 and IL-6 expanded VST cells was comparable to that seen when cells were cultured in IL-4 and IL-7.
  • IL-15/IL-6 expanded 18.8-fold more CD3+ cells on average as compared with no cytokine controls (FIG. 18A; 52,146 cells vs 2775 cells).
  • Culture in 1L-4/IL-7 expanded 14.0-fold more CD3+ cells on average compared with no cytokine controls (FIG. 18A; 38,838 vs 2755 cells).
  • Viability of CD3+ cells on average was not significantly different between wells containing IL-I5/IL-6 or IL-4/IL-7, averaging 90% and 89%, respectively.
  • NK cells were present in cultures expanded with IL-15 + IL-6, averaging 3878 cells, which was 6.2% of total cells recovered (FIG. 18A). Less than 300 NK cells were recovered on average from wells containing IL-4/IL-7, representing 0.6% of total cells recovered.
  • CD4+ and CD8+ cells were also not significantly different comparing culture in IL-I5/IL-6 and IL-4/IL-7 (data not shown; CD4 viability p ::: 0.4381; CD8 viability p ::: 0.1033), indicating the different cy tokine combinations were stimulating outgrowth of CD4+ vs CD8+ cells rather than preserving the selective survival of individual subsets.
  • This substantial difference in the ratio of helper vs. cytotoxic cells within the final cell product demonstrates a potential avenue for purposeful skewing of cell therapy products which was previously unrecognized.
  • Both culture conditions expanded CD3+ T cells which produced IFNy in response to CMV peptide re-stimulation.
  • An average of 24% of CD8+ T cells produced IFNy in response to antigenic pep tide in wells cultured in the highest concentration of IL- 15+IL-6, while approximately 30% of CD8+ T cells cultured in the highest concentration of IL-4+IL-7 recognized CMV peptide (FIG. 18C).
  • Over 30% of ( 1)4 T cells also recognized CMV peptide when cultured in IL-I5+IL- 6, as compared with under 20% of CD4+ T cells cultured in the highest concentration of IL-4+IL-7.
  • T cells produced as cellular therapy products are e ffector memory in phenotype.
  • surface expression of markers associated with T cell memory in addition to intracellular cytokine staining wns measured as part of the comprehensive panel. Cells were stained with antibodies specific for CCR7 and CD45RO as primary' indicators of memory', along with CD95, CD28, and CD45RA to investigate how the different cytokine combinations affected the evolution of memory in the products.
  • Pre-culture naive/stem cell memory cells comprised 32% of CD3+ T cells on average, with effector memory' cells comprising the next highest fraction with 30.4% of cells on average.
  • Central memory' cells were 22% of the CD3+ population on average, and terminal effectors were the smallest fraction of CD3+ T cells on average (15.4%).
  • Further analysis of CD4+ and CD8+ subsets identified helper and cytotoxic specific staining patterns as shown in one representative sample. For this sample, 44% of CD4+ T cells displayed a naive/stem cell memory phenotype (CCR7+ CD45RO-) compared with 13% of CD8+ T cells.
  • CD4+ T cells were also central memory (37%; CCR7+ CD45RO+) than effector memory (18%; CCR7- CD45RO+).
  • CD8+ T ceils reversed this pattern, with 27% of cells displaying an effector memory' phenotype compared with 9% displaying central memory' phenotype.
  • less than 1% of CD4+ T cells were terminal effectors (CCR7- CD45RO-), while 51% of CD8+ T cells were terminal effectors.
  • the memory' phenotype of antigen specific cells and antigen n on-responsive cells within the same well was determined by IFNy production in response to peptide.
  • CD3+ IFNy+ cells were also predominantly effector memory in phenotype, while a small but recurrent fraction of naive cells were identified within the antigen non-reactive fraction (IFN-y negative) in cells cultured with IL-4 and IL-7 (4.2%) which was lower than when cells were grown in IL-15 and IL-6 (1.7%).
  • IFN-y negative antigen non-reactive fraction
  • Cells grown in IL- 15/IL-6 produced an average of 646 spots per 100,000 added cells after re-stimulation with 1E1 and pp65 peptide, while cells cultured in IL-4 and IL-7 produced an average of 621 spots per 100,000 added cells in response to CMV peptide restimulation.
  • the CMV response was also antigen-specific, as cells produced less than 10 spots on average in response to either actin or no peptide controls per 100,000 added cells.
  • IL-15/IL-7 to maximize specificity (CD4+ and CD8+) and cell expansion compared to IL-4/ IL-7 in Grex expansion.
  • IL-15 and IL-7 Expanded in microexpansion format by initially choosing 2 lines with prior CD4+ specificity to membrane and spike proteins, then choosing 3 lines with some faint CD8+- specificity on prior microexpansion. Flow cytometry was performed on Day 10 by comparing to IL-4/7 (standard condition). IL-15/7 showed preserved total CD3+ cell counts. Mildly decreased total CD3+ cell counts were seen in IL-15, IL-15/4, and IL-15/6.
  • 1L-15/4 and IL-15/7 showed preserved CD4+ cell counts. Decreased CD4+ cell counts were seen in IL-15 and IL-15/6. Increased CD8+ cell counts were seen in IL-15/6 and IL-15/7. Regarding specificity on flow' cytometry for CD4+, IL-15/7 appeared to maximize CD4+ specificity comparing the other combinations. Regarding specificity on flow cytometry for CD8+, IL- 15 and IL-15/6 show'ed highest CD8+ specificity. IL-15/7 showed high specificity' in some lines comparing to IL-4/7. Grex expansion was used to validate cell expansion and maximization of specificity of CD4+ and CD8+ by comparing IL-15/7 and IL-4/7.
  • FIG. 26A-C show ICS (CD4+ and CD8+) and ELISpot specificities.
  • FIG. 26A shows significantly increased CD4+ specificity using IL-15/7 compared to IL-4/7 in Grex validation.
  • FIG. 26B shows increased CD8+ specificity using IL-15/7 compared to IL-4/7 in Grex validation.
  • FIG. 26C shows increased specificity using IL-15/7 compared to IL-4/7 in Grex -ELISpot.
  • IL-15/7 showed increased CD4+ and CD8+ specificity compared to IL -4/7.
  • IL-7 was responsible for the growth of VSTs, while IL-4 could not support memory' T cell expansion without addition of IL-15 or IL-7.
  • This requirement for memory T cell expansion via stimulation through the IL- 15 receptor or IL-7 receptor is not unexpected as both receptors share homology with IL.2 via utilizing the common y- chain and its associated Jak/STAT signaling proteins.
  • the IL- 15 receptor is a heterotrimer composed of the IL2RP, the common y-chain, and the specific IL-I5Ra, while the IL-7 receptor is a heterodimer consisting of the common y-chain with IL-7Ra/CD127, Recombinant IL-7 had been previously used clinically to expand T cell subsets in cases of lymphopenia, and was included with IL-4 for its pro-survival benefits for T cells. This previous investigation suggested inclusion of IL- 15 was inferior to IL-4/IL-7 because of a lack of CD4+ T cell expansion and excessive CD56+ NK cell growth (37.7%).
  • IL-4 had been described to support T cell survival by inhibiting degradation of anti-apoptotic factors Bcl2 and Bcl-xL, while IL-4 induced proliferation w ? as more limited to naive cells.
  • Tire data also demonstrated that addition of IL-6 to IL- 15 improved the total CD3+- and CD8+- expansion, while showing no significant difference for effector function compared with IL -15 alone.
  • Initial experiments in knockout mice previously demonstrated that IL-6 w ? as not required for survival of naive cells.
  • signaling via IL-6 has been described to reduce the threshold for TCR signaling in CD8+ T cells, which aligns with the data and would be ideal for promoting memory T cell expansion in response to peptide re-stimulation.
  • VST products grown in either 96 well plates or G-rex vessels were CCR7- CD45RO+ CD45RA- CD62L-, nominally effector memory, when cultured with either IL-4/IL-7 or IL-15/IL-6.
  • Cytokine combinations which produced a substantial frequency of either central memory cells, or stem cell memory cells were not identified.
  • Culture in IL-4 promoted survival of a naive CD4+ population winch was absent in cultures with IL-15/IL-6. While transfer of less differentiated T cells has been indicated to be more favorable for long term reconstitution for cellular therapy against tumors, naive, non-antigen reactive cells would not be expected to contribute to the antiviral response in the near term.
  • a plate-based flow cytometric assay can effectively measure growth, function, and phenotype in a high throughput fashion for process development.
  • This assay was demonstrated effective for screening 90 different cytokine combinations, rapidly identifying IL-15/IL-6 as equivalent to current GMP culture conditions using IL-4/IL-7.
  • culture in IL-15/IL-6 provides an advantage when expanding CD8+ T cells, while culture in IL-4/IL-7 would be advantageous for expansion favoring CD4+ T cells.
  • the modular nature of the assay can promote future investigations.
  • T-cell responses to SARS-CoV-2 have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals.
  • SARS-CoV-2-specific T-cells can be expanded from convalescent donors, and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleoprotein.
  • VSTs virus-specific T cells
  • PBMCs Peripheral blood mononuclear cells
  • Evaluated T cell products included SARS-CoV-2 specific T cells (CSTs), manufactured from PBMCs of seropositive and seronegative volunteers. VSTs were produced using a rapid expansion protocol previously described. Briefly, PBMCs w'ere pulsed simultaneously with overlapping peptide pools encompassing viral structural proteins (1 pg /15 x 10 6 PBMCs) for 30 minutes at 37°C. Peptide libraries of 15-mers with 11 amino acid overlaps encompassing the spike, membrane, nucleoprotein, and envelope proteins w-ere generated (A&A peptide, San Diego, CA, USA) from the SARS-CoV-2 reference sequence (NC_045512,2).
  • IL-4 400 lU/ml; R&D Systems, Minneapolis, MN
  • IL-7 10 ng/ml; R&D Systems
  • CTL media consisting of 45% RPMI (GE Healthcare, Logan, UT), 45% Click’s medium (Irvine Scientific, Santa Ana, CA), 10% fetal bovine serum, and supplemented with 2 mM GIutaMax (Gibco, Grand Island, NY) according to our Good Manufacturing Practice (GMP)- compliant standard operating procedures (SOPs).
  • Cytokines were replenished on day 7. On day 10, cells were harvested and evaluated for antigen specificity and functionality.
  • a subset of samples were re-stimulated with autologous PBMCs that were pulsed with the viral peptide libraries, irradiated at 75 grey, and co-cultured with the CSTs at a ratio of 1:4 (CSTs to PBMCs). These restimulated cells were incubated in IL-4 (400 lU/ml) and IL-7 (lOng/ml), with cytokines replenished at day 17, and harvested at day 21 for further testing.
  • IL-4 400 lU/ml
  • IL-7 lOng/ml
  • T-cells producing IFN-y in response to this stimulation were enriched using the IFN-y secretion detection and enrichment kit (Miltenyi cat #130-054-201) in accordance with the manufacturer’s instructions. These T-cells were plated at a series of dilutions in 96- well plates with irradiated feeder medium (RPMI 1640 supplemented with 10% FBS, L -glutamine, and PenStrep [R-10] with 1 x 10 6 cells/ml 5,000 rad irradiated PBMC + 50U/'ml IL-2 + lOng/ml IL-15 + O.lug/ml each of anti-CD3 (Ultra- LEAF purified Anti-human CD3 antibody clone OKT3, Biolegend, cat 317325) and anti-CD28 (Ultra-LEAF purified Anti-human CD28 antibody clone 28.2, Biolegend, cat 302933).
  • RPMI 1640 supplemented with 10% FBS, L
  • colonies were selected from the lowest dilution plates with positive wells ( ⁇ l/3 of wells positive) and screened for responsiveness to Membrane or Spike peptide pools by intracellular cytokine staining for IFN-gamma, TNF-alpha, and degranulation by CD 107a staining.
  • Membrane and Spike-specific T-cell clones were expanded bi-weekly with irradiated feeder medium.
  • T-cells Antigen specificity of T-cells was measured by IFN-y ELISpot (Millipore, Burlington, MA). T-cells were plated at 1 x 10 5 /well with no peptide, actin (control), or each of the individual SARS-CoV-2 pepmixes (200 ng/peptide/well). Plates were sent for IFN-y spots forming cells (SFC) counting (Zellnet Consulting, Fort Lee, NJ).
  • SFC spot forming cells
  • VSTs were stained with fluorophore-conjugated antibodies against CD4, CD8, TCR ⁇ , TCRy ⁇ , CXCR3, CXCR5, CCR6, CD127, CD25, and CD56 (Miltenyi Biotec, Bergisch Gladbach, Germany; BioLegend, San Diego, CA). Ah samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA).
  • Intracellular cytokine staining w'as performed as follows: 1 x 10 6 VSTs w'ere plated in a 96-well plate and stimulated with pooled pepmixes or individual peptides (200 ng/peptide/well) or actin (control) in the presence of brefeldin A (Golgiplug; BD Biosciences, San Jose, CA) and CD28/CD49d (BD Biosciences) for 6 hours. T-cells were fixed, made permeable with Cytofix/Cytoperm solution (BD Biosciences) and stained with IFN-y and TNF-a, and IL-2 antibodies (Miltenyi Biotec).
  • Cells were fixed, permeabilized using BD Cytofix/Cytoperm solution, and stained with anti-IFN-y Brilliant Violet 421 clone 4S.B3, anti-TNF-77a PerCP-Cyanine5.5 clone Mabll (both from Biolegend). Cells were analyzed on an Attune NxT flow cytometer. Data was analyzed with FlowJo X (FlowJo LLC, Ashland, OR).
  • CSTs were tested for specificity to minipools containing 8-24 peptides spanning the SARS- CoV2 antigens by IFN-y ELISpot. Cross-reactive pools were analyzed and individual peptides were tested to confinn epitope specificity. In silico predictions of MHC restrictions was performed using MARIA (hypertex transfer protocol ://maria. stanford.edu) and NetMHCIIPan (http://www.cbs.dtu.dk/services/NetMHCIIpan-4.0/).
  • CSTs were plated at 1 x 10 5 well with partially HLA-matched phytohemagglutinin (PHA)-treated lymphoblasts (PHA-blasts, 25 Gy irradiated) either alone or pulsed with peptide (1 ug/ml) and tested via IFN-y ELISpot.
  • PHA phytohemagglutinin
  • Cutoff limits for determining positive antibodies in the SARS-CoV-2-infected samples were based on the mean plus three standard deviations of the serum values derived from uninfected blood donor controls or by receiver operator characteristics (ROC) analysis. For some of the data percentages for categorical variables, mean and range, geometric mean plus 95% CI were used to describe the data. Wilcoxon signed rank were used for statistical analysis.
  • CSTs were plated at 1 x lOVwell in 96-well plates, stimulated with pooled pepmixes (200 ng/peptide/well) or control actin peptide, and incubated 48 hours. Supernatants were harvested and the cytokine profile analysis was performed using the Bio-plex Pro Human 17-plex Cytokine Assay kit (Bio-Rad, Hercules, CA), and read on a MAGPIX system (Luminex, Austin, TX).
  • Phytohemagglutinin blasts were labeled with Chromium-51 (Perkin Elmer, Waltham, MA, USA) at 10 ⁇ Ci per 5 x 10 5 cells.
  • CST were co ⁇ piated with 51 Cr-labeled, MHC -mismatched irradiated PHA blasts at effector: target ratios between 40:1 and 5: 1, and incubated at 37°C for 4 hours.
  • Maximal release was evaluated by lysis of 51 Cr-labeled targets with Triton-X-100. Supernatants were transferred to lumiplates and read on a MicroBeta2 plate reader (Perkin Elmer). Specific lysis was calculated as follows: (Experimental Counts per minute[CPM] - Background CPM) / (Maximal CPM - Background CPM).
  • Non-amplified responses to SARS- CoV-2 viral antigens were detectable from PBMCs via IFN-y ELISpot in only 2 of 46 patients and none of 15 controls suggesting that the frequency of the SARS-CoV-2 response is relatively low, consistent with T cell immune responses observed against other respiratory viruses (e.g. adenovirus).
  • Post-expansion T cells were predominantly CD4 + , with central memory' and effector memory' subsets.
  • the predominant CD4+ T cell population was CXCR3 + CCR6" (mean 42.3% of CD4+ T-cells) consistent with a Till population, with minor populations expressing CXCR57CXCR3" (mean 12.9.5% of CD4+ T-cells) and CD1277CD25 + (mean 15.18% of CD4+ T- ceils). These ratios were proportionate to rapidly expanded Virus-specific T cells targeting cytomegalovirus, Epstein-Barr vims, and adenovirus. Responses to spike and membrane proteins were confirmed to be predominantly CD4+ restricted in 11/11 tested patients (FIG.
  • CSTs Following re-stimulation with viral structural proteins, CSTs produced multiple cytokines, with significant production of IL- 1 p, IL-2, IL-4, IL-6, IL-7, IL-12, G-CSF, IFN-y, and TNFa. CSTs expanded to 18 days following a second stimulation showed a similar pattern of cytokine production, which was not statistically different from the cytokine profile following the first stimulation.
  • CSTs were tested against peptides corresponding to variant epitopes in circulating SARS-CoV-2 genotypes, and from the HKU1 and OC43 coronaviruses.
  • CSTs from seropositive donors recognize a broader array of viral antigens than CSTs derived from donors who lack detectable humoral responses.
  • 25 had demonstrable antibody and T-cell responses to SARS-CoV-2.
  • Seven convalescent donors had no detectable T-cell or antibody responses.
  • Seven donors had antibody responses without detectable T-cell responses, and 6 donors had T-cell responses without accompanying antibody responses, as has been observed with other infections such as EBV and HSV.
  • Subject 4 had mild GI disease, fever, and shortness of breath, and developed a CD4 + T-cell response to Spike protein (which was not detectable preillness), but no detectable antibody response to Spike or Nucleocapsid.
  • SARS-CoV-2 immune (humoral and adaptive) responses were absent in the pre-pandemic sample, and post infection (after being confirmed to be PCR+ for SARS-CoV-2), a robust T cell response to spike protein was demonstrated, though this individual did not have an antibody response to Spike, Subject 46 had mild respiratory symptoms, anosmia, and GI symptoms, and developed a CD4+ T-cell response, as well as antibody response to both Spike and Nucleocapsid, both of which were absent two months prior to his illness.
  • CSTs recognize multiple Immunodominant epitopes in membrane, nucleocapsld, and spike proteins. Epitope mapping of the membrane protein yielded multiple epitopes at the C ⁇ terminal domain (FIG. 23A). Two epitopes at AA 144-163, were recognized by 8 donors, and were exclusively CD4-restricted (FIG. 24A). Using in silico analysis, the predicted HLA restrictions of these responses were HLA-DRB1*11 and DRB4*01 (Table 9). Similarly, epitopes at AA 173-192 were recognized by 6 donors, and were also confirmed to be CD4-restncted (FIG. 24B).
  • epitopes contained within the peptides described in Table 9 and the other tables herein may be employed in the methods for culturing T cells disclosed herein or for treatment or prevention of disease using T cells recognizing these epitopes.
  • these peptide epitopes are expressed or complexed with HLA molecules capable of presenting the epitopes to the cellular immune system such as the HLA molecules disclosed by the Tables herein.
  • epitopes contained within the peptides described in Table 10 and the other tables herein may be employed in the methods for culturing T cells disclosed herein or for treatment or prevention of disease using T cells recognizing these epitopes.
  • these peptide epitopes are expressed or complexed with HLA molecules capable of presenting the epitopes to the cellular immune system such as the HLA molecules disclosed by the Tables herein
  • ex vivo expanded CSTs may be easily generated from convalescent patients, following recoveiy from COVID-19, and recognize multiple immunodominant epitopes within membrane protein, which represent class II restricted T-cell epitope "‘hot spots”.
  • Cross-reactivity with corresponding epitopes from circulating coronaviruses further suggest that T-cell recognition of these domains is common.
  • Membrane, spike, and nucleoprotein showed a clear hierarchy of immunodominance, and were associated with significant increases in IFN-y/TNF-a producing CD4* T-cell populations.
  • T-cell populations in combating SARS-CoV-2 remains limited, decreases in activated T-cell populations have been shown to correlate with patient acuity scores. Furthermore, the importance of polyfunctional CD4 T-cell responses are well-documented for antiviral immunity against other respiratory viruses. Moreover, the efficacy of adoptive, predominantly MHC class Il-restricted T-cell therapies targeting adenovirus in immunocompromised patients is a prime example of the potency of T cell therapies for clearance of respiratory' viruses in the immune compromised host. Though T-cell immunotherapy targeting RNA viruses has not been attempted, the concept is supported by prior murine RSV studies.
  • CSTs derived from an HSCT donor may be an effective preventative therapy especially for patients undergoing BMT. Further, for patients who lack a. donor with immunity to COVID-19, the administration of partially-HLA matched third-party CSTs may be a consideration as an “on-demand: treatment of COVID-19 early in the course of infection to prevent invasive disease, with the goal to reduce the length and severity of illness.
  • HLA-DRB 1*11:01 Confirmation of HLA-restriction for Membrane peptide 37 was confirmed to be mediated by HLA-DRB 1*11:01, and in silico analysis suggested restriction of these epitopes through HLA-DR11, DR7, DQ3, and DQ7, which are present in roughly 50% of the population. This information is therefore highly useful for the manufacture of a CST bank for clinical use.
  • CSTs with specificity for one or more viral antigens could be successfully produced from 58% of the evaluated convalescent donors, and an association was detected between SARS-CoV-2 seropositivity and T-cell responses to non-Spike antigens. It is plausible that T- follicular helper cells play a role in this association, and a population of CXCR5 + CD4 + T-cells were noted in expanded CSTs. Interestingly, not all convalescent donors had detectable humoral and cellular responses, and many incongruous responses were noted.
  • biological samples may be obtained from subjects recognizing homologous or similar epitopes from other coronaviruses, such as those described above, and used to produce T ceils recognizing S ARS-CoV-2 or other related coronaviruses.
  • T-cell and humoral responses measured here represent an effective adaptive immune response to SARS-CoV -2 which can be effectively harnessed (especially from BMT donors) for the manufacture of CST products for clinical use.
  • Embodiments of this technology include, but are not limited to the following.
  • Embodiment 1 A method of culturing a composition comprising a population of one or a plurality of CD4+ and/or CD8+ T cells, said method comprising, consisting essentially of, or consisting of:
  • Embodiment 3 The method of embodiment 1 or 2, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is from about 2 to about 10 days.
  • Embodiment 4 The method of any of embodiments 1 through 3, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality' of CD4+ and/or CD8+ T cells is from about 3 to about 7 days.
  • Embodiment 5 The method of any of embodiments 1 through 4 ? wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 5% of CD8+ or CD4+ T cells produce either interferongamma or TNF-alpha.
  • Embodiment 6 The method of any of embodiments 1 through 5, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality' of CD4+ and/or CD8+ T cells is a time period in which no less than about 10% of CD8+ or CD4+ T cells produce either interferon-gamma or TNF-alpha.
  • Embodiment 7 The method of any of embodiments 1 through 6. wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 15% of CD8+ or CD4+ T cells produce either interferon-gamma or TNF-alpha.
  • Embodiment 8 The method of any of embodiments 1 through 7, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 20% of CD8+ or CD4+ T cells produce interferon- gamma or TNF-alpha.
  • Embodiment 9 The method of any of embodiments 1 through 8, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ and/or CD8+ T cells is a time period in which no less than about 22% of CD8+ or CD4+ T cells produce either interferon-gamma or TNF-alpha, Embodiment 10.
  • PBMCs peripheral blood mononuclear cells
  • Embodiment 11 The method of any of embodiments 1 through 9, further comprising a step of isolating PBMCs from a subject and culturing the PBMCs in vitro prior to performing step (a).
  • Embodiment 12 The method of any of embodiments 1 through 11, wherein the one or plurality of T cells are isolated from:
  • Embodiment 13 The method of any of embodiments 1 through 12, wherein the T cells are obtained through an ex vivo expansion of a single population of T cells.
  • Embodiment 14 The method of any of embodiments 1 through 12, wherein the T cells are obtained through an ex vivo expansion of separate T cell subpopulations, wherein each T cell subpopulation is specific for a single viral antigen.
  • Embodiment 15 The method of any of embodiments 1 through 14, wherein the one or plurality of T cells are obtained from an allogeneic source.
  • Embodiment 16 The method of any of embodiments 1 through 14, wherein the one or plurality' of T cells are obtained from an autologous source.
  • Embodiment 17 The method of any of embodiments 1 through 16, wherein the composition further comprises NKT cells and yd T cells.
  • Embodiment 18 The method of any of embodiments 1 through 17, wherein the at least two peptides are:
  • Embodiment 19 The method of any of embodiments 1 through 18, wherein the method is performed in a closed tissue culture system over at least 1, 2, 3, 4 or 5 days.
  • Embodiment 20 The method o f any of embodiments 1 through 19, wherein the method is performed in a closed tissue culture system over from about 5, 6, 7, 8, 9 to about 10 days.
  • Embodiment 21 The method of any of embodiments 1 through 20, wherein the one or plurality of T cells are naive cells prior to step (a) and the one or plurality of T cells are CCR7+ CD45RO+ after performing step (a).
  • Embodiment 22 A method of expanding a population of CD8+ memory effector T cells in a composition of cultured cells, said method comprising, consisting essentially of, or consisting of:
  • Embodiment 23 The method of embodiment 22 further comprising:
  • Embodiment 24 The method of embodiment 22 or 23, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD8+ memory effector T ceils is a time period sufficient to cause an increase of expression of CCR7 or CD45RO on the T cells after performing step (a).
  • Embodiment 25 The method of any of embodiments 22 through 24, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD8+ memory' effector T cells is a time period sufficient to cause an increase of expression of CCR7 and CD45RO on the T cells after performing step (a).
  • Embodiment 26 The method of any of embodiments 22 through 25, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality’ of CD8+ memory effector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 2% of the CD8+ T cells.
  • Embodiment 2.7 The method of any of embodiments 2.2 through 26, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD8+ memory effector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 5% of the CD8+ T cells.
  • Embodiment 28 The method of any of embodiments 22 through 27, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD8+ memory' effector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 10% of the CD8+ T cells.
  • Embodiment 29 The method of any of embodiments 22 through 28, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD8+ memory effector T cells is a timer period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than 15% of the CD8+ T cells.
  • Embodiment 30 The method of any of embodiments 22 through 29, wherein the CD8+ T cells secrete IFN-gamma or TNF-alpha,
  • Embodiment 31 The method of any of embodiments 22 through 30, wherein the CD8+ I' cells secrete IFN-gamma and TNF-alpha.
  • Embodiment. 32 The method of any of embodiments 22 through 31, wherein the CD8+ T cells are CD3+ and secrete IFN-gamma or TNF-alpha.
  • Embodiment 33 The method of any of embodiments 22 through 32, wherein the method further comprises stimulating proliferation of a subpopulation of CD4+ T cells that secrete IFN- gamma or TNF-alpha by exposing the cells simultaneously or sequentially with a polypeptide or nucleic acid encoding a polypeptide of IL-7, or functional fragments or variants thereof, and a polypeptide or nucleic acid encoding a polypeptide of IL-4, or functional fragments or variants thereof.
  • Embodiment 34 A method of expanding a population of CD4+ memory effector T cells in a composition of cultured lymphocytes, said method comprising, consisting essentially of or consisting of:
  • Embodiment 35 The method of embodiment 34 further comprising:
  • Embodiment 36 The method of embodiment 34 or 35, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4 ⁇ memory effector T cells is a time period sufficient to cause an increase of expression of CCR7 or CD45RO on the T cells after performing step (a).
  • Embodiment 37 The method of any of embodiments 34 through 36, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ memory effector T cells is a time period sufficient to cause an increase of expression of CCR7 and CD45RO on the T ceils after performing step (a).
  • Embodiment 38 The method of any of embodiments 34 through 37, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ memory effector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 2% of the CD4+ T cells.
  • Embodiment 39 The method of any of embodiments 34 through 38, wherein the time period sufficient to stimulate growth or proliferation of the one or plurality of CD4+ memory- effector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 5% of the CD4+ T cells.
  • Embodiment 40 The method of any of embodiments 34 through 39, wherein the time period sufficient to stimulate grow th or proliferation of the one or plurality of CD4+ memoryeffector T cells is a time period sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than about 10% of the CD4+ T cells.
  • Embodiment 41 The method of any of embodiments 34 through 40, wherein the time period sufficient to stimulate growfh or proliferation of the one or plurality- of CD4+ memoryeffector T cells is sufficient to cause expression of CCR7 and CD45RO after performing step (a) in no less than 15% of the CD4+ T cells.
  • Embodiment 42 The method of any of embodiments 34 through 41, wherein the CD4+ T cells secrete IFN-gamma or TNF-alpha.
  • Embodiment 43 The method of any of embodiments 34 through 42, wherein the ( 1)4 T cells secrete IFN-gamma and TNF-alpha.
  • Embodiment 44 The method of any of embodiments 34 through 43, wherein the CD4 ⁇ T cells are CD3+ and secrete IFN-gamma or TNF-alpha.
  • Embodiment 45 The method of any of embodiments 34 through 44, w-herein the method further comprises stimulating proliferation of a subpopulation of CD8+ T cells that secrete IFN- gamma or TNF-alpha by exposing the cells simultaneously or sequentially with a polypeptide or nucleic acid encoding a polypeptide of IL-15, or functional fragments or variants thereof, and a polypeptide or nucleic acid encoding a polypeptide of IL-6, or functional fragments or variants thereof.
  • Embodiment 46 A method of generating, culturing and/or manufacturing CD8+ and/or CD4+ effector memory cells, sard method comprising:
  • Embodiment 47 The method of embodiment 46 further comprising:
  • Embodiment 48 The method of embodiment 46 or 47 further comprising:
  • Embodiment 49 The method of any of embodiments 46 through 48, wherein the one or plurality of lymphocytes are from naive T cells from a subject.
  • Embodiment 50 The method of any of embodiments 46 through 48 further comprising isolating PBMCs from a subject.
  • Embodiment 51 The method of embodiment 50, wherein the step of isolating PBMCs comprising placing a whole blood sample from the subject through an apheresis unit.
  • Embodiment 52 The method of any of embodiments 46 through 51, further comprising a step of aliquoting PBMCs into a cell culture unit comprising a plurality' of vessels prior to exposing the cells to the cytokines, wherein each of the vessels comprises a volume defined by at least one base surface and at least one sidewall.
  • Embodiment 53 A T cell composition comprising from about 2.% to about 23% of CD4+ and/or CD8+ T cells manufactured by any of the methods of embodiments 1 through 21 and embodiments 69 through 72 or ( 1)4 and/or CD8+- memory effector T cells manufactured by any of methods of embodiments 22 through 52 and embodiments 73 through 84; preferably wherein said CD4+ and/orCD8+ memory effector T cells recognize, or are contacted with, a peptide epitope of SARS-CoV -2.
  • Embodiment 54 An isolated composition comprising lymphocytes, wherein the lymphocytes comprise:
  • Embodiment 55 A tissue culture system comprising a composition comprising lymphocytes and tissue culture media, wherein the lymphocytes comprise:
  • Embodiment 56 The tissue culture system of embodiment 55, further comprising NKT cells.
  • Embodiment. 57 The tissue culture system of embodiment. 55 or 56, wherein the system further comprises at least one cell culture surface area onto which the lymphocytes adhere, a tissue culture media reservoir in closed fluid communication with the at least one cell culture surface area by one or more media lines and a pump in operable connection to the reservoir and configured to create pressure in the media lines sufficient to generate a rate of flow of tissue culture media over the cell culture surface area.
  • Embodiment 58 A method of inducing an antigen-specific immune response against a viral antigen, the method comprising:
  • Embodiment 59 The method of embodiment 58 further comprising:
  • Embodiment 61 The method of embodiment 60, wherein the viral antigens are:
  • peptides comprising at least about 70%, 75%, 80%. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to any one or combination of sequences provided in FIG, 9, or any functional fragment or variant thereof;
  • nucleic acids encoding an ammo acid sequence that comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to any one or combination of sequences provided in FIG. 9, or any functional fragment or variant thereof; or
  • Embodiment 62 The method of any of embodiments 58 through 61 , wherein the viral antigens are selected from one or a combination of EBV, HHV, CMV, BKV, or HPIV.
  • Embodiment 63 The method of embodiment 60, wherein the viral antigens are:
  • Embodiment 64 A method of selectively growing CD8+ and/or CD4+ memory' effector T cells from a. cell composition comprising naive T cells, said method comprising, consisting essentially of, or consisting of:
  • Embodiment 65 The method of embodiment 64, wherein, if growth and proliferation of a population of CD8+ memory' effector T cells is stimulated, the step of contacting one or plurality of lymphocytes comprises: contacting one or plurality' of lymphocytes comprising the naive I' cells with at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, or IL-6, or functional fragments or variants thereof; or one or a plurality of vectors that encode at least two peptides that comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to IL-18, IL-15, or IL-6, or functional fragments or variants thereof,
  • Embodiment 66 The method of embodiment 64 or 65 further comprising a step of contacting the one or plurality-- of lymphocytes or the one or plurality- of CD8+ and/or CD4+ memory effector T cells with one or a plurality of viral antigens.
  • Embodiment 67 The method of embodiment 64 or 65 further comprising a step of contacting the one or plurality- of lymphocytes or the one or plurality of CD8+ and/or C D 4 memory effector T cells with one or a plurality of viral antigens for a time period sufficient to induce an antigen -specific immune response against the one or plurality of viral antigens.
  • Embodiment 68 The method of either of embodiments 64 or 65 further comprising a step of contacting the one or plurality-- of lymphocytes or the one or plurality- of CD8+ and/or CD4+ memory effector T cells with one or a plurality of viral antigens for a time period sufficient to induce an antigen-specific immune response against the one or plurality of viral antigens as measured by the number of CD3+ T cells that secrete mterfer on-gamma after exposure to the one or plurality- of viral antigens.
  • Embodiment 69 The method of any of embodiments 1 through 21, wherein the at least two peptides comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity- to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • Embodiment 70 The method of any of embodiments 1 through 21 and embodiment 69, wherein the one or plurality of CI)4+ and/or CD8+ T cells are specific for one or a plurality of viral antigens from Coronaviridae.
  • Embodiment 71 The method of embodiment 70, wherein the one or plurality of viral antigens is a SARS-CoV-2 antigen.
  • Embodiment 73 The method of any of embodiments 22 through 33, wherein the at least two peptides comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: I, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • Embodiment 74 The method of any of embodiments 22 through 33 and embodiment 73, wherein the one or plurality of CD4+ and/or CD8+ T cells are specific for one or a plurality of viral antigens from Coronaviridae .
  • Embodiment 75 The method of embodiment 74, wherein the viral antigen is a SARS-CoV- 2 antigen.
  • Embodiment 76 The method of embodiment 74 or 75, wherein the one or plurality of viral antigens comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • Embodiment 77 Die method of any of embodiments 34 through 45, wherein the at least two peptides comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: I, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • Embodiment 78 The method of any of embodiments 34 through 45 and embodiment 77, wherein the one or plurality of CD4+ and/or CD8+ T cells are specific for one or a plurality of Coronaviridae viral antigens.
  • Embodiment 79 The method of embodiment 78, wherein the one or plurality of antigens are SARS-CoV-2 antigens.
  • Embodiment 80 The method of embodiment 78 or 79, wherein the one or plurality of viral antigens comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • Embodiment 81 Die method of any of embodiments 46 through 52, wherein the at least two peptides comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • Embodiment 82 The method of any of embodiments 46 through 52 and embodiment 81, wherein the one or plurality of ⁇ 1)4 and/or CD8+ T cells are specific for one or a plurality of viral antigens from a coronavirus.
  • Embodiment 83 The method of embodiment 82, wherein the coronavirus is SARS-CoV-2.
  • Embodiment 84 The method of embodiment 82 or 83, wherein the one or plurality of viral antigens comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • Embodiment 85 nA method of generating, culturing and/or manufacturing one or a plurality of CD4+ and/or CD8+ T cells specific to one or a plurality' of viral antigens, the method comprising, consisting essentially of, or consisting of:
  • Embodiment 86 The method of embodiment 85, wherein the one or plurality of viral antigens are from a virus of the family Coronaviridae.
  • Embodiment 87 The method of embodiment 85 or 86, wherein the one or plurality of viral antigens are from a coronavirus.
  • Embodiment 88 The method of any of embodiments 85 through 87, wherein the one or plurality of viral antigens are from SARS-CoV-2.
  • Embodiment 89 The method of any of embodiments 85 through 88, wherein the one or plurality of viral antigens comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, and/or SEQ ID NO: 14, or functional fragments or variants thereof.
  • Embodiment 90 The method of any of embodiments 85 through 89, wherein:
  • the cytokine IL-15 comprises at ieast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 1;
  • the cy tokine IL-7 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 3;
  • the cytokine IL-6 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 5:
  • the cytokine IL-4 comprises at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 7; and/or
  • the cytokine IL-18 comprises at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ⁇ 100%, or 100% sequence identity to SEQ ID NO: 9.
  • Embodiment 91 The method of any of embodiments 85 through 90, wherein the time period sufficient to stimulate growth and proliferation of the one or plurality CD4+ and/or CD8+ T ceils and/or the time period sufficient to activate the one or plurality of CD4+ and/or CD8+ T cells against a cell expressing the one or plurality of viral antigens is from about 2 to about 10 days.
  • Embodiment 92 The method of any of embodiments 85 through 91, wherein the time period sufficient to stimulate growth and proliferation of the one or plurality of CD4+ and/or ⁇ 1)8 T cells and/or the time period sufficient to activate the one or plurality of CD4+ and/or CD8+ T cells against a cell expressing the one or plurality of viral antigens is from about 3 to about 7 days.
  • Embodiment 93 The method of any of embodiments 85 through 92, wherein the at least two cytokines are:
  • Embodiment 94 The method of any of embodiments 8.5 through 93, wherein no less than about 2% of the one or plurality of CD4 ⁇ and/or CD8+ T cells are CCR7+ CD45RO+.
  • Embodiment 95 The method of any of embodiments 85 through 94, wherein no less than about 5% of the one or a plurality of CD4+ and/or CD8+ T cells are CCR7+ CD45RO+.
  • Embodiment 96 Hie method of any of embodiments 85 through 95, wherein no less than about 10% of the one or plurality of CD4+ and/or CD8+ T cells are CCR7+ CD45RO+.
  • Embodiment 97 the method of any of embodiments 85 through 96, wherein no less than about 1, 2, 5, 10 or 15% of the one or plurality' of CD4+ and/or CD8+ T cells are CCR7 * CD45RO+.
  • Embodiment 98 The method of any of embodiments 85 through 97, wherein the one or plurality of CD4+ and/or CD8+ T cells secrete IFN-gamma or TNF-alpha.
  • Embodiment 99 A T cell composition comprising from about 2%, 5%, 10%, 15%, 20% to about 23% of CD4+ and/or CD8+ T cells manufactured by any of the methods of embodiments 85 through 98.
  • Embodiment 100 A pharmaceutical composition comprising, consisting essentially of, or consisting of:
  • Embodiment 101 A method of preventing and/or treating viral infection in a subject in need thereof, said method comprising, consisting essentially of, or consisting of administering a pharmaceutically effective amount of the one or plurality of CD4+ and/or CD8+ T cells generated by any of the methods of embodiments 85 through 98, the T cell composition of embodiment 99, or the pharmaceutical composition of embodiment 100 to the subject in need thereof.
  • Embodiment 102 The method of embodiment 101, wherein the viral infection is a Corona viridae infect! on .
  • Embodiment 103 The method of embodiment 101 or 102, wherein the viral infection is coronavirus infection.
  • Embodiment 104 The method of any of embodiments 101 through 103, wherein the viral infection is a SARS-CoV-2 of COVID-19 infection.
  • Embodiment 105 The method of embodiment 84, wherein the one or plurality' of viral antigens comprise one or plurality of viral epitopes chosen from Table A, Table B, and/or Table C.
  • Embodiment 106 A T cell composition comprising T cells manufactured by the method of embodiment 105.
  • Embodiment 107 The method of embodiment 89, wherein the one or plurality of viral antigens comprise one or plurality' of viral epitopes chosen from Table A, Table B, and/or Table C.
  • Embodiment 108. A T cell composition comprising T cells manufactured by the method of embodiment 107.
  • CMV cytomegalovirus
  • SARS-CoV-2 T-cell immunity is directed against the spike, membrane, and nucleocapsid protein and associated with COVID 19 severity, medRxiv (2020).

Abstract

La divulgation concerne des procédés de culture et d'expansion de lymphocytes T CD4+ et/ou CD8+ en culture. Dans certains modes de réalisation, les procédés comprennent l'expansion, la prolifération et le stockage de lymphocytes dans une culture tissulaire en exposant des lymphocytes à une combinaison de cytokines et/ou d'acides nucléiques exprimant des cytokines (ou des fragments fonctionnels ou des variants correspondants). L'invention concerne en outre des procédés de génération et de fabrication de lymphocytes T CD4+ et/ou CD8+ qui sont spécifiques à un ou plusieurs antigènes viraux.
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