WO2022055892A1 - Methods and compositions of infecting, activating, and expanding immune cells - Google Patents

Methods and compositions of infecting, activating, and expanding immune cells Download PDF

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WO2022055892A1
WO2022055892A1 PCT/US2021/049307 US2021049307W WO2022055892A1 WO 2022055892 A1 WO2022055892 A1 WO 2022055892A1 US 2021049307 W US2021049307 W US 2021049307W WO 2022055892 A1 WO2022055892 A1 WO 2022055892A1
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cell
cells
optionally
days
population
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PCT/US2021/049307
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English (en)
French (fr)
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Lei Zhang
Sunil Chada
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Cytoimmune Therapeutics, Inc.
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Priority to JP2023515125A priority Critical patent/JP2023540997A/ja
Priority to EP21867450.5A priority patent/EP4211253A1/en
Priority to CN202180055185.4A priority patent/CN116209764A/zh
Priority to AU2021341944A priority patent/AU2021341944A1/en
Publication of WO2022055892A1 publication Critical patent/WO2022055892A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • 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
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
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    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • 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)
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    • 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)
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • NK cells have great potential in tumor immunotherapy because they can kill tumor cells directly and quickly.
  • Redirecting the function of NK cells with genetic engineering (CAR-NK) is an effective strategy to overcome the multiple inhibitory receptors expressed on NK cells and to strengthen the use of targeted therapy.
  • CAR-NK genetic engineering
  • preclinical and clinical studies show that NK cells expressing a chimeric antigen receptor (CAR) may play significant anti-tumor role and it is safer than CAR-T cell therapy. See, for example, Wang et al. Int. Immunopharmacol. 2019; 74: 105695.
  • CAR-NK cell therapy still faces some challenges, such as the expansion and activation of primary NK cells in vitro, the difficulty to store and ship NK cell products and the low transduction efficiency.
  • compositions and methods efficiently preparing NK cells for therapy. This disclosure satisfies these needs and provides related advantages as well.
  • a pseudotyped gammaretroviral particle comprising a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and a modified baboon envelope glycoprotein (BaEVTR).
  • the RD114TR glycoprotein comprises, or consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a RD114 glycoprotein and a cytoplasmic domain of amphotropic murine leukemia virus (MLV-A) glycoprotein.
  • the BaEVTR glycoprotein comprises, or consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a baboon envelope glycoprotein (BaEV) and a cytoplasmic domain of MLV-A glycoprotein.
  • the RD114TR and the BaEVTR are incorporated into the envelope of the particle as membrane proteins.
  • the pseudotyped gammaretroviral particle is selected from a species of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • XMRV xenotropic murine leukemia virus-related virus
  • a method for preparing including but not limited to infecting, activating, or expanding) a population of natural killer (NK) cells.
  • the method comprises, or consists essentially of, or yet further consists of culturing a cell population comprising, or consisting essentially of, or yet further consisting of one or more of the following: an NK cell, a progenitor cell that is capable of deriving an NK cell, or a stem cell that is capable of deriving an NK cell with one or more of immune cell activators, such as NK cell activators.
  • this culturing step is repeated for once, twice, three times, or more times, with the same or different immune cell activator(s) (such as NK cell activator(s)) or a combination thereof.
  • the cell population is depleted with cells that expresses one or more of: CD3, CD4, CD8, T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population.
  • the immune cell activator(s) (such as NK cell activator(s)) is or are selected from one or more of the following: an artificial antigen presenting cell (aAPC) that expresses a tumor associated antigen (TAA) and/or a viral antigen optionally which activate and/or stimulate immune cell growth; one or more of an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the NK cell, the progenitor cell or the stem cell, thereby activating or proliferating NK cells; one or more of cytokines thereby activating or proliferating NK cells; or one or more of chemical moieties thereby activating or proliferating NK cells.
  • aAPC artificial antigen presenting cell
  • TAA tumor associated antigen
  • viral antigen optionally which activate and/or stimulate immune cell growth
  • an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the NK cell, the
  • a method for preparing a population of y6 T cells comprises, or consists essentially of, or yet further consists of culturing a cell population comprising one or more of the following: a y6 T cell, a progenitor cell that is capable of deriving a y6 T cell, or a stem cell that is capable of deriving a y6 T cell with one or more immune cell activator(s) (such as y6 T cell activators).
  • the cell population is depleted with cells that expresses one or more of: T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population.
  • this culturing step is repeated for once, twice, three times, or more times, with the same or different immune cell activator(s) (such as y6 T cell activator(s)) or a combination thereof.
  • the immune cell activator(s) (such as y6 T cell activator) is selected from one or more of: an artificial antigen presenting cell (aAPC) that expresses a tumor associated antigen (TAA) and/or a viral antigen optionally which activate and/or stimulate immune cell growth; one or more of an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the y6 T cell, the progenitor cell or the stem cell, thereby activating or proliferating y6 T cells; one or more of cytokines thereby activating or proliferating y6 T cells; or one or more of chemical moieties thereby activating or proliferating y6 T cells.
  • aAPC artificial antigen presenting cell
  • TAA tumor associated antigen
  • viral antigen optionally which activate and/or stimulate immune cell growth
  • an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the y
  • the aAPCs further express one or more of: 4-1BBL, membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (Inducible T-cell costimulator ligand, B7H2, B7RP1), MICA (MHC class I polypeptide-related sequence A), CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D super-agonist- complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc) ALT-803, or a cell surface marker mediating CD122/CD132 signaling.
  • the aAPCs further expresses mb IL-21 and 4-1BBL.
  • the cytokines are selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low-toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D super-agonist-complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL- 15RaSu-Fc; ALT-803 soluble), IL-15, IL-18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1), or MICA.
  • the cell population is cultured with any one or any two or all three of 100-500 lU/ml IL-2, 20 ng/ml IL-15, or 25 ng/mL IL-21. In some embodiments, the cell population is cultured with either or both of 50 lU/ml IL-2 and 0.5 ng/ml IL-15. In some embodiments, the cell population is cultured with 50 lU/ml IL-2.
  • any method as disclosed herein further comprises introducing a polynucleotide into the cultured cell population for expression, for example prior to and/or after one or more culturing step(s) as disclosed herein.
  • the polynucleotide encodes a CAR and/or another therapeutic protein or polypeptide, such as an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • any method as disclosed herein further comprises introducing a pseudotyped gammaretroviral particle into the cultured cell population, thereby introducing the polynucleotide as disclosed herein into the cultured cell.
  • the particle comprises RD114TR and BaEVTR as envelope proteins.
  • the pseudotyped gammaretroviral particle is selected from a species of a Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus feline sarcoma virus
  • XMRV xenotropic murine leukemia virus-related virus
  • RetroNectin to promote co-localization of a lentiviral or retroviral vector comprising a polynucleotide as disclosed herein (such as a pseudotyped gammaretroviral particle as disclosed herein) and cells to be introduced with the polynucleotide.
  • a polynucleotide as disclosed herein such as a pseudotyped gammaretroviral particle as disclosed herein
  • RetroNectin is a 63 kD fragment of recombinant human fibronectin fragment that enhances lentiviral and retroviral mediated gene transduction. It is commercially available from TaKaRa www.takarabio.com/products/gene- function/t-cell-transduction-and-culture/retronectin-reagent, last accessed on September 4, 2020.
  • a method for producing a retroviral particle comprises, consists essentially of, or yet further consists of (i) introducing a vector expressing a vector genome into a first packaging cell line suitable for packaging the vector genome into a first retroviral particle, (ii) transducing the first retroviral particle into a second packaging cell line suitable for replicating the first retroviral particle; and (iii) isolating the replicated retroviral particle.
  • the method further comprises culturing the first packaging cell line introduced with the vector.
  • the method further comprises isolating the first retroviral particle for the culture of the first packaging cell line introduced with the vector.
  • the method further comprises culturing the transduced second packaging cell line.
  • a retroviral particle produced by the method along with uses of the produced retroviral particle in producing an engineered immune cell, such as an immune cell engineered to express a chimeric antigen receptor (CAR).
  • an engineered immune cell such as an immune cell engineered to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the cell population are cultured before the introducing step for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Additionally or alternatively, the cell population are cultured before the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks, or no more than 1 month. In one embodiment, the cell population are cultured before the introducing step for about 5 days to about 10 days.
  • the cell population comprises, or consists essentially of, or yet further consists of any one or more of NK cells, y6 T cells, stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), NK cells derived from any one or more of stem cells, HSCs or iPSCs, or y6 T cells derived from any one or more of stem cells, HSCs or iPSCs.
  • the cell population is isolated from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
  • a method for inhibiting the growth of a cancer cell comprises, or consists essentially of, or yet further consists of contacting a population of CAR-expressing cells prepared by a method as disclosed herein with the cancer cell.
  • the antigen recognized by the CAR is a TAA expressed on the cancer cell.
  • the contacting step may be in vivo or in vitro.
  • a method for treating a cancer in a subject comprises, or consists essentially of, or yet further consists of administering a population of the CAR-expressing cells prepared by a method as disclosed herein.
  • the antigen recognized by the CAR is a TAA expressed by a cell of the cancer.
  • an engineered aAPC expressing an antigen and one or more of cell surface markers: 4-1BBL, membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D super-agonist- complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc) ALT-803, or a cell surface marker mediating CD122/CD132 signaling.
  • 4-1BBL membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb
  • an NK cell, a y6 T cell, or a cell population of either or both thereof prepared by a method as disclosed herein.
  • the cell and/or cell population expresses a CAR and/or another therapeutic protein or polypeptide, such as an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • composition comprising, or consisting essentially of, or yet further consisting of a cell or a population thereof as disclosed herein and a carrier, optionally a pharmaceutical acceptable carrier.
  • kits comprising, or consisting essentially of, or yet further consisting of one or more of agents suitable for use in a method as disclosed herein and an optional instruction.
  • the agents are selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting cell phenotype, an antibody for isolating or enriching or purifying immune cells, primers for detecting the polynucleotide, cytokines, and an aAPC.
  • a viral packaging system for producing a pseudotyped gammaretroviral particle, as well as a method for producing a pseudotyped gammaretroviral particle.
  • the system comprises, or consists essentially of, or yet further consists of: (a) a plasmid expressing a vector genome; (b) a packaging plasmid; and (c) one or more of envelope plasmids expressing RD114TR and BaEVTR, while the method comprises, or consists essentially of, or yet further consists of introducing the system to a packaging cell line under conditions suitable to package the pseudotyped gammaretroviral particle.
  • FIG. 1 provides a schema of gammaretroviral vector (PCIR) based therapeutic gene delivery to human primary NK cells.
  • PCIR gammaretroviral vector
  • FIGS. 2A to 2B provide determination of the growth curve and time point when the primary NK cells entered a marked proliferative state for gene delivery.
  • Time point for retroviral transduction of primary NK cells expanded was optimized.
  • the growth curve was determined along with time point when the primary NK cells entered a marked proliferative state for gene delivery.
  • the primary human NK cells used in this disclosure were peripheral blood (PB)-derived, and isolated with MACSxpress Whole Blood NK Cell Isolation Kit, human (Miltenyi Biotec, 130-098-185). The purity was determined via staining the cells with CD56 and CD3 antibody, and NK cells were defined as CD56+/CD3- population (FIG. 2A).
  • NK cells were cultured with NK MACS media (130-114-429) at presence of 50 lU/ml of human IL-2 and irradiated K562-mb21-41BBL feeders with the ratio of 1 : 1.
  • NK MACS media 130-114-429
  • K562-mb21-41BBL feeders 50 lU/ml
  • K562-mb21-41BBL feeders 50 lU/ml of human IL-2 and irradiated K562-mb21-41BBL feeders with the ratio of 1 : 1.
  • the activated NK cells at day 6-10 were used for gene delivery (FIG. 2B).
  • FIGS. 3A to 3D show efficacy of RD114TR and BaEVTR pseudotyped gamma retrovirus particles (PCIR) accomplished on RetroNectin allowing the great level of gene transfer in NK cells with high viability and yield.
  • the RetroNectin-bound virus (RBV) infection strategy was used in this disclosure and the details were illustrated in FIG. 3A.
  • FSC low-forward scatter
  • SSC high-side scatter
  • EGFR CAR gene
  • FIG. 4 provides determination of retrovirus titration based on direct infection of activated NK cells at same proliferate state with same delivery strategy.
  • PCIR gammaretrovirus vector
  • the titration determination methods were based on exactly same infection strategy for same proliferative state of activated NK cells at day 7.
  • the empty vector used was PCIR- EV-EGFP.
  • the serial dilution of pooled raw virus supernatant harvested at 48- and 72-hours post transfection were added to RetroNectin coated plate and the procedure was same as FIG. 1 and FIG. 3A.
  • the mouse source single-chain fragment variable (ScFv) for the CAR was detected using ALEXA FLUOR 1 ' 647 AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch i 15-605-006).
  • the MOI 3 was used in this study (see FIGS. 3A to 3D).
  • FIGS. 5A to 5C show that gammaretrovirus vector engineered CAR-NK outperformed lentivirus vector engineered CAR-NK with highly stable transduced gene expression in a long term culture.
  • Lentiviral engineered PCIL-EGFR-CAR-NK and EV-GFP control were enriched based on sorting GFP positive population, and retrovirus engineered PCIR-EGFR-CAR-NK was based on sorting of goat anti mouse Fab-AF647 positive cells.
  • FIGS. 6A and 6B show efficacy of highly transduction efficiency and cytotoxicity of gammaretrovirus vector PCIR/BaEVTR engineered EGFR-CAR-NK cell, using real-time cell analysis (RTCA).
  • NovoCyte 3005 flow cytometry analysis demonstrated the highly transduction efficiency for gammaretrovirus vector PCIR/BaEVTR engineered EGFR-CAR- NK from four donors. There were average 85% (CD19-PE positive) and 79.6% (Goat anti Mouse Fab-AF647 positive) transduction rates for empty vector carrying truncated CD 19 (EV-Tcdl9) and EGFR transgene separately (FIG. 6A).
  • the immune cell killing assays were performed using xCELLigence RTCA MP Bundle (ACEA Biosciences, Cat# 00380601040). These plots showed the impedance data for the experiment with 5,000 cells/well LN229, and with the addition of 500 cells/well effectors (1 : 10 E:T) from two donors. There were dynamic real time killing for engineered EGFR-CAR-NK compared to empty vector mock transduction and non-transduced NK groups with lowest live cells index (FIG. 6B).
  • FIG. 7 shows NK cell expansion of 17-24 days.
  • FIGS. 8A to 8C provide retrovirus entry receptors expression on 293 Vec-GalV and 293Vec-BaEV packaging cells, and two cell lines Jurkat T and HT1080 used for titration.
  • the baboon endogenous virus (BaEV) and feline endogenous retrovirus (RD114) use a common cell-surface receptor ASCT2 (sodiumdependent neutral amino acid transporter) for cell entry.
  • ASCT2 sodiumdependent neutral amino acid transporter
  • baboon endogenous viruses (BaEV) also uses ASCT1 as a cell entry receptor.
  • FIG. 8B provides immunostaining for ASCT1, ASCT2, and Pitl on two packaging cell line, 293 Vec-GalV and 293Vec-BaEV, and titration cell lines, Jurkat T and HT1080.
  • Histograms represent the mean fluorescence intensity (MFI) of cells stained with rabbit IgG isotype control (Invitrogen, cat#02-6102) and the corresponding secondary antibody, Alexa Fluor 647-conjugated goat anti-rabbit IgG (A-21245; Invitrogen), cells stained with anti-ASCTl (LifeSpan BioSciences, LS-C 179222) and the corresponding secondary antibody, cells stained with anti-ASCT2 (Cell Signaling Technology, 8057S) and the corresponding secondary antibody, and cells stained with anti-Pitl (ThermoFisher, PA5-98650).
  • MFI mean fluorescence intensity
  • FIG. 9 illustrates a workflow of the transduction strategy for generation of stable retrovirus virus producer-293 Vec-BaEV.
  • the moloney murine leukemia virus (Mo-MuLV)- based retroviral vector (PCIR) was used for target transgene delivery vehicle.
  • the BaEV pseudotyping 293 Vec-BaEV packaging cell line was used for vehicle production to infect cord blood derived NK cells.
  • Two packaging cell lines were used for this production: 293 Vec-GALV to produce transient gibbon ape leukemia virus (GALV)-pseudotyped supernatant and 293 Vec-BaEV to generate the final vector. Both cell lines were supplied by BioVec Pharma.
  • This supernatant was then used to transduce a BaEV pseudotyping packaging cell line 293 Vec-BaEV to generate a bulk producer.
  • this 293 Vec- BaEV bulk producer can be further sorted or single-cell cloned by limiting dilution and using a high-titer clone selected by titration of the supernatant generated from each clone. Then the supernatant can be continually produced from the high titer stable Retrovirus virus producer-293 Vec-BaEV.
  • FIGS. 10A and 10B show improved titer of raw supernatant from the stable retrovirus virus producer-293 Vec-BaEV producing a vector with large size transgenes. “Combination of Multiple Targets in one” is a unique therapeutic strategy developed by the Applicant.
  • the BaEV pseudotyping 293 Vec-BaEV packaging cell line was used for vehicle production.
  • FIG. 10 A provides that a representative retrovirus with large transgenes inserts (>11 kb) was both produced transiently and stably by 293 Vec-BaEV packaging cell.
  • a total of 3.5 million of 293 Vec-BaEV packaging cells were seeded in a 100 mm tissue culture dish, and incubated at 37 degrees for 48-72 h.
  • the raw supernatant was collected and spun at 1500 g for 5 min. Titration of the above supernatant was performed on Jurkat T cell. The serials of virus supernatant were loaded on the RetroNectin precoated non-tissue culture treated 24 well plate and brought to 500 pl with complete DMEM medium supplied with 10% heat inactivated fetal bovine serum. The plate was spun at 32 degrees, 2000g for 2 hours, and 0.1 million of Jurkat T cells were added, and spun at 1000g for 5 min.
  • Transducing Units/ml [(target cell number) X (%Positive cells)].
  • TU Transducing Units
  • RQR8 expression of a reporter gene RQR8 was detected by flow antibodies against human CD34 Antibody (QBEnd/10) (Allophycocyanin) (Novus Biologicals, LLC, #FAB7227A). As shown in FIG.
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like.
  • Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
  • “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • first and second cell line are used to distinguishing two cell lines.
  • isolated refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides, proteins, viruses and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides, proteins, viruses and/or host cells.
  • the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, virus, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, virus, or antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
  • the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species.
  • the term “engineered” or “recombinant” refers to being synthetized by human intervention.
  • the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • amplification of polynucleotides includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR).
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.
  • Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively, the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.
  • a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • ORF open reading frame
  • the term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • the gene product may refers to an mRNA generated when a gene is transcribed.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • Under transcriptional control which is also used herein as “directing expression of’, is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
  • a regulatory sequence intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed and/or replicated, and facilitates the expression and/or replication of the target polynucleotide.
  • a promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters.
  • promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • promoters include the EFl alpha promoter and the CMV promoter.
  • the EFl alpha sequence is known in the art (see, e.g., addgene.org/11154/sequences/; ncbi.nlm.nih.gov/nuccore/J04617, each last accessed on March 13, 2019, and Zheng and Baum (2014) IntT. J. Med. Sci.
  • An example is: EFl alpha promoter sequence: SEQ ID NO: 148, and optionally, an equivalent thereof.
  • An enhancer is a regulatory element that increases the expression of a target sequence.
  • a “promoter/enhancer” is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions.
  • the enhancer/promoter may be "endogenous” or “exogenous” or “heterologous.”
  • An “endogenous" enhancer/promoter is one which is naturally linked with a given gene in the genome.
  • an “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg 2+ normally found in a cell.
  • a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
  • Homology or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.
  • a polynucleotide or polynucleotide region has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
  • default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters.
  • the program is any one of: Clustal Omega accessible at www.ebi.ac.uk/Tools/msa/clustalo/, Needle (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_needle/, Stretcher (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_stretcher/, Water (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_water/, Matcher (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_matcher/, LALIGN accessible at www.ebi.ac.uk/Tools/psa/lalign/.
  • the default setting is used.
  • the polynucleotide as disclosed herein is a RNA. In some embodiments, the polynucleotide as disclosed herein is a DNA. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA.
  • an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, and/or a reverse-complement reference, optionally under conditions of high stringency.
  • an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70%, or at least 75%, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complement.
  • the equivalent must encode functional protein that optionally can be identified through one or more assays described herein.
  • the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.
  • transduce or “transduction” as it is applied to the production of an engineered cell, such as chimeric antigen receptor cells, refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, such as a viral vector or a non-viral vector.
  • a restriction enzyme is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. They are used to assist insertion of a polynucleotide, such as a gene, into plasmid vectors during gene cloning and protein production experiments.
  • plasmids for example those encoding viral vector genomes, that are commonly used for gene cloning are modified to include a short polylinker sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences.
  • MCS multiple cloning site
  • both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.
  • protein protein
  • peptide and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • antibody collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins.
  • the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10 3 M' 1 greater, at least 10 4 M' 1 greater or at least 10 5 M' 1 greater than a binding constant for other molecules in a biological sample).
  • the term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies).
  • the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains").
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions" or "CDRs".
  • framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference).
  • the Kabat database is now maintained online.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, largely adopts a P- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located (heavy chain regions labeled CDRH, such as CDRH1, CDRH2, and CDRH3, and light chain regions labeled CDRL, such as CDRL1, CDRL2 and CDRL3).
  • CDRH3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found
  • a CDRL1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • a TNT antibody will have a specific VH region and the VL region sequence unique to the TNT relevant antigen, and thus specific CDR sequences.
  • Antibodies with different specificities i.e., different combining sites for different antigens
  • have different CDRs Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
  • a fragment crystallizable (Fc) region refers to the tail region of an antibody that in some embodiments, can serve to stabilize the antibody and optionally interacts with (such as binds) an Fc receptor on an immune cell or on a platelet or that binds a complement protein.
  • a Fc mutant may be used, such as comprising one or two or all three mutations of F234A, L235A and N297Q of human IgG4 Fc region in a Fc or an equivalent thereof at positions corresponding to those of human IgG4 Fc region, such as for SEQ ID NO: 81, the corresponding positions are amino acid (aa) 16, aa 17 and aa 79 of SEQ ID NO: 81.
  • polypeptide or equivalents of each thereof can be followed by an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxyterminus.
  • An equivalent thereof comprises an polypeptide having at least 80% amino acid identity to the CAR or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complement of a polynucleotide encoding the CAR, wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water.
  • Alternative embodiments include one or more of the CDRs (e.g., CDR1, CDR2, CDR3) from the LC variable region with appropriate CDRs from other antibody CDRs. And equivalents of each thereof.
  • the CDR1 and CDR2 from the LC variable region can be combined with the CDR3 of another antibody’s LC variable region, and in some aspects, can include an additional 50 amino acids, or alternatively about 40 amino acids, or alternatively about 30 amino acids, or alternatively about 20 amino acids, or alternatively about 10 amino acids, or alternatively about 5 amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids at the carboxy-terminus.
  • the EGFR CAR is the CAR disclosed in WO 2016/164370.
  • the term “equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or fragment thereof as measured by ELISA or other suitable methods.
  • Biologically equivalent antibodies include, but are not limited to, those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody.
  • an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.
  • antibody variant intends to include antibodies produced in a species other than a mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.
  • antibody derivative is intended to encompass molecules that bind an epitope as defined above and which are modifications or derivatives of a native monoclonal antibody of this disclosure.
  • Derivatives include, but are not limited to, for example, bi specific, multi specific, heterospecific, tri specific, tetraspecific, multi specific antibodies, diabodies, chimeric, recombinant and humanized.
  • the term “specific binding” means the contact between an antibody and an antigen with a binding affinity of at least 10 -6 M.
  • antibodies bind with affinities of at least about 10 -7 M, and preferably 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10’ 12 M.
  • the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.
  • Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.
  • antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
  • antigen of a binding moiety such as an antibody, an antigen binding fragment thereof, or a CAR
  • a binding moiety such as a BCMA CAR
  • antigen of a binding moiety such as a BCMA CAR
  • TAA tumor associated antigen
  • cancer antigen cancer antigen
  • tumor antigen cancer relevant antigen
  • tumor relevant antigen are used interchangeably herein, referring to antigenic substance of a cancer or tumor cells.
  • a TAA presents on some tumor or cancer cells and also on some normal cells, optionally at a lower level.
  • a TAA only presents on a tumor or cancer cell but not on a normal cell.
  • a TAA is selected from G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), B-cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wildtype epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFR VIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD 133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransfera
  • a viral antigen refers to an antigen expressed in a virus and/or encoded by a viral genome.
  • Non-limiting example includes hemagglutinin (HA) and neuraminidase (NA) of an influenza virus, and spike protein, SI, S2, nucleocapsid envelope protein of a COVID-19.
  • antigen binding domain refers to any protein or polypeptide domain that can specifically bind to an antigen target.
  • autologous in reference to cells refers to cells that are isolated and infused back into the same subject (recipient or host). “Allogeneic” refers to non-autologous cells.
  • chimeric antigen receptor refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain.
  • the “chimeric antigen receptor (CAR)” is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor (CIR).”
  • extracellular domain capable of binding to an antigen means any oligopeptide or polypeptide that can bind to a certain antigen.
  • intracellular domain or “intracellular signaling domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
  • the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signaling domains in addition to the primary signaling domain.
  • transmembrane domain means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains.
  • a chimeric antigen receptor may optionally comprise a “hinge domain” which serves as a linker between the extracellular and transmembrane domains.
  • Non-limiting examples of such domains are provided herein, e.g.: Hinge domain: IgGl heavy chain hinge coding sequence: SEQ ID NO: 112. Additional nonlimiting example includes an IgG4 hinge region, IgD and CD8 domains, as known in the art.
  • transmembrane domain CD28 transmembrane region coding sequence: SEQ ID NO: 113
  • Intracellular domain: 4-1BB co-stimulatory signaling region coding sequence SEQ ID NO: 114
  • Intracellular domain CD3 zeta signaling region coding sequence: SEQ ID NO: 116.
  • each exemplary domain component includes other proteins that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the proteins encoded by the above disclosed nucleic acid sequences. Further, non-limiting examples of such domains are provided herein.
  • CD8 a hinge domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD8 a hinge domain sequence as shown herein.
  • the example sequences of CD8 a hinge domain for human, mouse, and other species are provided in Pinto, R.D. et al. (2006) Vet. Immunol. Immunopathol. 110:169-177.
  • the sequences associated with the CD8 a hinge domain are provided in Pinto, R.D. et al. (2006) Vet. Immunol. Immunopathol. 110: 169-177.
  • Nonlimiting examples of such include: Human CD8 alpha hinge domain: SEQ ID NO: 117; Mouse CD8 alpha hinge domain: SEQ ID NO: 118; and Cat CD8 alpha hinge domain: SEQ ID NO: 119.
  • CD8 a transmembrane domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD8 a transmembrane domain sequence as shown herein.
  • the fragment sequences associated with the amino acid positions 183 to 203 of the human T-cell surface glycoprotein CD8 alpha chain (GenBank Accession No: NP 001759.3), or the amino acid positions 197 to 217 of the mouse T-cell surface glycoprotein CD8 alpha chain (GenBank Accession No: NP 001074579.1), and the amino acid positions 190 to 210 of the rat T-cell surface glycoprotein CD8 alpha chain(GenBank Accession No: NP_ 113726.1) provide additional example sequences of the CD8 a transmembrane domain.
  • CD28 transmembrane domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, at least 90% sequence identity, or alternatively at least 95% sequence identity with the CD28 transmembrane domain sequence as shown herein.
  • GenBank Accession Nos: XM_006712862.2 and XM_009444056.1 provide additional, non-limiting, example sequences of the CD28 transmembrane domain.
  • the sequences associated with each of the listed accession numbers are provided herein.
  • the term “4- IBB costimulatory signaling region” refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the 4- IBB costimulatory signaling region sequence as shown herein.
  • Non-limiting example sequences of the 4- IBB costimulatory signaling region are provided in U.S. Publication 20130266551 Al, such as the exemplary sequence provided below: 4-1BB costimulatory signaling region: SEQ ID NO: 123.
  • CD28 costimulatory signaling region refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD28 costimulatory signaling region sequence shown herein.
  • the example sequences CD28 costimulatory signaling domain are provided in U.S. Patent No. 5,686,281; Geiger, T.L. et al., Blood 98: 2364-2371 (2001); Hornbach, A. et al., J Immunol 167: 6123- 6131 (2001); Maher, J. et al.
  • Nonlimiting examples include residues 114-220 of the below CD28 Sequence: SEQ ID NO: 124, and equivalents thereof.
  • ICOS costimulatory signaling region refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the ICOS costimulatory signaling region sequence as shown herein.
  • Non-limiting example sequences of the ICOS costimulatory signaling region are provided in U.S. Publication 2015/0017141A1 the exemplary polynucleotide sequence provided below: ICOS costimulatory signaling region coding sequence: SEQ ID NO: 125.
  • 0X40 costimulatory signaling region refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, or alternatively 90% sequence identity, or alternatively at least 95% sequence identity with the 0X40 costimulatory signaling region sequence as shown herein.
  • Nonlimiting example sequences of the 0X40 costimulatory signaling region are disclosed in U.S. Publication 2012/20148552A1, and include the exemplary sequence provided below: 0X40 costimulatory signaling region coding sequence: ID NO: 126, and equivalents thereof.
  • costimulatory signaling regions may be used, such as those of CD27, CD40, CD40L, and/or TLRs. See for example, those disclosed in, US Publication 20160340406A1.
  • CD3 zeta signaling domain refers to a specific protein fragment associated with this name and any other molecules that have analogous biological function that share at least 70%, or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity with the CD3 zeta signaling domain sequence as shown herein.
  • Non-limiting example sequences of the CD3 zeta signaling domain are provided in U.S. Publication 20130266551 Al, e.g., SEQ ID NO: 3.
  • a signal peptide refers to (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined toward the secretory pathway.
  • the signal peptide is a secretary signal.
  • a secretary signal intends a secretory signal peptide that allows the export of a protein from the cytosol into the secretory pathway. Proteins can exhibit differential levels of successful secretion and often certain signal peptides can cause lower or higher levels when partnered with specific proteins.
  • the signal peptide is a hydrophobic string of amino acids that is recognized by the signal recognition particle (SRP) in the cytosol of eukaryotic cells. After the signal peptide is produced from an mRNA-ribosome complex, the SRP binds the peptide and stops protein translation.
  • SRP signal recognition particle
  • the SRP then shuttles the mRNA/ribosome complex to the rough endoplasmic reticulum where the protein is translated into the lumen of the endoplasmic reticulum.
  • the signal peptide is then cleaved off the protein to produce either a soluble, or membrane tagged (if a transmembrane region is also present), protein in the endoplasmic reticulum.
  • a cleavable peptide which is also referred to as a cleavable linker, means a peptide that can be cleaved, for example, by an enzyme.
  • One translated polypeptide comprising such cleavable peptide can produce two final products, therefore, allowing expressing more than one polypeptides from one open reading frame.
  • cleavable peptides is a self-cleaving peptide, such as a 2A self-cleaving peptide.
  • 2A selfcleaving peptides is a class of 18-22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell.
  • the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. 2 A self-cleaving peptide-based multi -gene expression system in the silkworm Bombyx mori. Sci Rep. 2015;5: 16273. Published Nov 5, 2015.
  • T2A and 2A peptide are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising the requisite amino acids in a relatively short peptide sequence (on the order of 20 amino acids long depending on the virus of origin) containing the consensus polypeptide motif D-V/I-E-X-N-P-G-P (SEQ ID NO: 177), wherein X refers to any amino acid generally thought to be self-cleaving.
  • Detectable label “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein.
  • label or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically active isotopes, e.g., 115 Sn, 117 Sn and 119 Sn, a non-radioactive isotopes such as 13 C and 15 N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
  • N-terminal histidine tags N-His
  • magnetically active isotopes e.g., 115 Sn, 117 Sn and 119 Sn
  • a non-radioactive isotopes such as 13 C and 15 N
  • polynucleotide or protein such as an antibody so as to generate a “labeled” composition.
  • the term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequence
  • the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • the label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed
  • a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence.
  • Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal.
  • Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed).
  • Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
  • the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
  • a second agent such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
  • fluorescein rhodamine
  • tetramethylrhodamine eosin
  • erythrosin erythrosin
  • coumarin methyl-coumarins
  • pyrene Malacite green
  • stilbene Lucifer Yellow
  • Cascade BlueTM Lucifer Yellow
  • Texas Red Texas Red
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag.
  • an epitope tag including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag
  • an affinity tag including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)
  • fluorescent tag including but not limited to
  • a cell surface marker mediating CD122/CD132 signaling refers to a protein or polypeptide or another moiety expressed on a cell, such as an immune cell, and capable of activating the IL2 pathway, such as the CD122 signaling pathway and/or the CD132 signaling pathway.
  • CD122 is also known as interleukin-2 receptor subunit beta
  • CD 132 is also known as interleukin-2 receptor subunit gamma.
  • the interleukin 2 receptor which is involved in T cell-mediated immune responses, is present in 3 forms with respect to ability to bind interleukin 2.
  • the low affinity form is a monomer of the alpha subunit (also called CD25) and is not involved in signal transduction.
  • the intermediate affinity form consists of a gamma/beta subunit heterodimer, while the high affinity form consists of an alpha/beta/gamma subunit heterotrimer.
  • Both the intermediate and high affinity forms of the receptor are involved in receptor-mediated endocytosis and transduction of mitogenic signals from interleukin 2. These proteins also form one of the three subunits of the IL- 15 receptor, while CD 132 partners with other ligand-specific receptors to direct lymphocytes to respond to cytokines including IL4, IL7, IL9, and IL21. Activation of the receptor increases proliferation of CD8+ effector T cells. See for example, Noguch et al. Science. 262 (5141): 1877-80.
  • non-limiting examples of a cell surface marker mediating CD 122/CD 132 signaling includes IL2 receptor, IL4 receptor, IL7 receptor, IL9 receptor, IL 15 receptor, IL21 receptor, membrane bound IL2, membrane bound IL4, membrane bound IL7, membrane bound IL9, membrane bound IL15, or membrane bound IL21.
  • Other recombinant protein may be used, such as any extracellular domain fused to any transmembrane domain and a cytosolic domain of CD122, CD132, IL2 receptor, IL4 receptor, IL7 receptor, IL9 receptor, IL 15 receptor, or IL21 receptor.
  • interleukin refers to cytokines that was first seen to be expressed by white blood cells (leukocytes).
  • the function of the immune system depends in a large part on interleukins.
  • the majority of interleukins are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells.
  • an interleukin can be a soluble cytokine secreted out of a cell, and/or a membrane bound (mb) cytokine expressed on a cell surface.
  • mb membrane bound cytokine expressed on a cell surface.
  • a soluble form and a membrane bound form of a cytokine can be converted by one of skill in the art, such as engineering the transmembrane domain and/or signal peptide of the cytokine.
  • Interleukin-2 is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity.
  • the IL-2 is a human IL-2.
  • the IL-2 is of other species, such as a chimpanzee IL-2 having an NCBI Reference Sequence of XP_517425.1.
  • Non -limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04M122451, HGNC (6001), NCBI Entrez Gene (3558), Ensembl (ENSG00000109471), OMIM® (147680), UniProtKB/Swiss-Prot (P60568), and Open Targets atform(ENSGOOOOO 109471), each of which is incorporated by reference herein in its entirety.
  • the IL-2 comprises, or consists essentially of, or yet further consists of MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 178).
  • the IL-2 comprises, or consists essentially of, or yet further consists of amino acid (aa) 21 to aa 153 of SEQ ID NO: 178.
  • IL-2 as used herein is a wildtype IL-2 or an equivalent thereof.
  • IL-2 as used herein is a recombinant IL-2 produced by a host cell (such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris) in vitro.
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • the IL-2 equivalent stimulates the proliferation or activates the cytotoxic function of NK cells or both significantly similar to the wildtype IL
  • Assays for evaluating the proliferation and cytotoxic function of NK cells are available for one of skill in the art, such as ex vivo culturing and cell counting (see, for example Choi et al. J Immunother Cancer. 2019 Jul 5;7(1): 168), 51 chromium release assay (see, for example, Dong et al. Cancer Discov. 2019 Oct;9(10): 1422-1437. doi: 10.1158/2159-8290.CD-18-1259. Epub 2019 Jul 24), colorimetric measurement-based cytotoxicity assay (see, for example, Chava et al. J Vis Exp.
  • the IL-2 equivalent comprises, or consists essentially of, or further consists of a fragment of the wildtype IL-2, such as aa 22 to aa 153 of SEQ ID NO: 179.
  • the IL-2 equivalent comprises, or consists essentially of, or further consists of a variant of the wildtype IL-2 or a fragment thereof, such as adding an extra Methionine at the N-terminus, or having one or more following mutations: the amino acid residue aligned to aa 38 of SEQ ID NO: 178 optionally mutated to methionine, the amino acid residue aligned to aa 39 of SEQ ID NO: 178 optionally mutated to serine, the amino acid residue aligned to aa 58 of SEQ ID NO: 178 optionally mutated to alanine or lysine, the amino acid residue aligned to aa 62 of SEQ ID NO: 178 optionally mutated to lysine or isoleucine or alanine or glutamine, the amino acid residue aligned to aa 65 of SEQ ID NO: 178 optionally mutated to asparagine or glutamic acid or alanine or arginine, the amino acid residue align
  • the IL-2 equivalent comprises, or consists essentially of, or further consists of an IL-2 derivative, such as modified by glycosylation, acetylation, or phosphorylation.
  • Interleukin- 15 is a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL- 15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by virus(es). This cytokine induces the proliferation of natural killer cells. In some embodiments, the IL- 15 is a human IL-15.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04P141636, HGNC: 5977, NCBI Entrez Gene: 3600, Ensembl: ENSG00000164136, OMIM®: 600554, or UniProtKB/Swiss-Prot: P40933, each of which is incorporated by reference herein in its entirety.
  • the IL- 15 is a human IL-15 isoform 1.
  • the IL-15 comprises, or consists essentially of, or yet further consists of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 181).
  • the IL-15 comprises, or consists essentially of, or yet further consists of amino acid (aa) 30 to aa 162 of SEQ ID NO: 181.
  • the IL- 15 is a human IL-15 isoform 2.
  • the IL-15 comprises, or consists essentially of, or yet further consists of MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV TAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNI KEFLQSFVHIVQMFINTS (SEQ ID NO: 182).
  • the IL-15 is of other species, such as a Rhesus macaque IL- 15 having a UniProtKB reference ID of P48092.
  • IL-15 as used herein is a wildtype IL-15 or an equivalent thereof.
  • IL- 15 as used herein is a recombinant IL- 15 produced by a host cell (such as HEK 293 cells, or E. coli) in vitro. See, for example, human IL- 15 sold by Sigma-Aldrich (SRP6293, or SRP3077), and STEMCELLTM Technologies (78031).
  • the IL- 15 equivalent stimulates the proliferation or activates the cytotoxic function of NK cells or both significantly similar to the wildtype IL-15. Assays for evaluating the proliferation and cytotoxic function of NK cells are available for one of skill in the art, and are described herein.
  • Non-limiting examples of the IL-15 equivalents include those disclosed in US20190263877A1, US10450359B2, and US10537615B2. Additionally or alternative, the IL- 15 equivalent comprises, or consists essentially of, or further consists of an IL- 15 derivative, such as modified by glycosylation, acetylation, or phosphorylation.
  • Interleukin-21 which is also referred to herein as an IL-21 polypeptide, is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells that can destroy virally infected or cancerous cells. This cytokine induces cell division/proliferation in its target cells.
  • the IL-21 is a human IL-21.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04M122612, HGNC: 6005, NCBI Entrez Gene: 59067, Ensembl: ENSG00000138684, OMIM®: 605384, and UniProtKB/Swiss-Prot: Q9HBE4, each of which is incorporated by reference herein in its entirety.
  • the IL-21 equivalent stimulates the proliferation or sustains the viability of immune cells (such as NK cells) or both significantly similar to the wildtype IL-21.
  • Assays for evaluating cell proliferation and viability are available for one of skill in the art, such as ex vivo culturing and cell counting, or live/dead cell staining (e.g., a tetrazolium reduction assay, a resazurin reduction assay, a protease viability marker assay, an ATP assay, or a real-time assay for viable cells). See, for example, Choi et al. J Immunother Cancer. 2019 Jul 5;7(1): 168, and Riss et al.
  • CD 122 is a receptor for interleukin-2 and also termed as Interleukin 2 Receptor Subunit Beta. This beta subunit is involved in receptor mediated endocytosis and transduces the mitogenic signals of IL2. It is probably in association with IL15RA, involved in the stimulation of neutrophil phagocytosis by IL15.
  • Non-limiting exemplary sequences of this protein or the underlying gene or suitable antibodies for detection of the protein can be found under Gene Cards ID: GC22M037125, HGNC: 6009, NCBI Entrez Gene: 3560, Ensembl: ENSG00000100385, OMIM®: 146710, or UniProtKB/Swiss-Prot: P14784, each of which is incorporated by reference herein in its entirety.
  • an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence.
  • Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST.
  • the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell’s genome.
  • Plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses.
  • the gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
  • MCS multiple cloning site
  • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • the DNA viruses constitute classes I and II.
  • the RNA viruses and retroviruses make up the remaining classes.
  • Class III viruses have a double-stranded RNA genome.
  • Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA
  • Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis.
  • Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.
  • the vector is derived from or based on a wild-type virus.
  • the vector is derived from or based on a wild-type adenovirus, adeno- associated virus, or a retrovirus such as a gammaretrovirus and/or a lentivirus.
  • retrovirus include without limitation, moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), or friend murine embryonic stem cell virus (FMEV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV).
  • the viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Vector components can be manipulated to obtain desired characteristics such as target cell specificity.
  • the recombinant vectors of this disclosure may be derived from primates and nonprimates. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus
  • AIDS causative agent of human acquired immunodeficiency syndrome
  • SIV simian immunodeficiency virus
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritisencephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritisencephalitis virus
  • EIAV equine infectious anaemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • Prior art recombinant lentiviral vectors are known in the art, e.g., see US Patent Nos. 6,924,123; 7,056,699; 7,419,829 and 7,442,551, incorporated herein by reference.
  • the lentiviral vector is a self-inactivating lentiviral vector.
  • the lentiviral vector has a U3 region
  • RNA usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs
  • proteins such as a protease.
  • U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes.
  • LTRs long terminal repeats
  • the LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.
  • Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA
  • U5 is derived from the sequence unique to the 5'end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR.
  • U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • RT reverse transcriptase
  • I integrase
  • one or more of these structural genes are provided by a packing cell (also referred to herein as a host cell) producing a viral vector, instead of in the vector genome itself.
  • the vector RNA genome is expressed from a DNA construct encoding it, in a host cell.
  • the components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell.
  • the set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
  • Gammaretrovirus is a genus in the retroviridae family and may be used in the disclosure herein.
  • Example species are the moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV moloney murine leukemia virus
  • MSCV murine stem cell virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus feline sarcoma virus
  • XMRV xenotropic murine leukemia virus-related virus
  • Gammaretrovirus is a spherical, enveloped virion ranging from 80-100 nm in diameter. It contains a nucleocapsid, reverse-transcriptase, integrase, capsid,
  • the nucleocapsid is a nucleic acid protein assembly within the virus particle, it is a substructure of the virion.
  • Reversetranscriptase is the enzyme responsible for the transformation of RNA to DNA during the virion replication cycle. Integrase works with reverse transcriptase to convert RNA to DNA.
  • the genome of the gammaretrovirus is a single-stranded RNA (+) genome that is approximately 8.3 kb in size. It has a 5’ cap with a 3’ poly-A tail, and it contains two long terminal repeater regions at both the 5’ and 3’ ends. These long terminal repeat regions have the U5, R, and U3 regions as well as a polypurine tract at the 3’ end and a primer binding site at the 5’ end.
  • the typical gammaretrovirus genome contains the gag gene, pol gene, and an env gene, all of which can be omitted in a gene therapy vector.
  • the capsid is a protein shell that surrounds the genome of a virus particle, its main functions are to protect and deliver the genome to the host cell.
  • the viral envelope is the membrane that surround the viral capsid, it is a host cell derived lipid bilayer.
  • gammaretroviruses have some advantages over HIV as a lentiviral vector. Specifically, the gammaretroviral packaging system does not require the incorporation of any sequences overlapping with coding sequences of gag, pol, or accessory genes. See, for example, Tobias Maetzig et al. Viruses. 2011 Jun; 3 (6): 677-713. Epub 2011 Jun 3.
  • pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins.
  • the result is a pseudotyped virus particle, also called a pseudovirus.
  • the foreign viral envelope proteins can be used to alter host tropism or increase or decrease the stability of the virus particles.
  • Pseudotyped particles do not carry the genetic material to produce additional viral envelope proteins, so the phenotypic changes cannot be passed on to progeny viral particles.
  • the inability to produce viral envelope proteins renders the pseudovirus replication incompetent. In this way, the properties of dangerous viruses can be studied in a lower risk setting. Pseudotyping allows one to control the expression of envelope proteins.
  • VSV-G glycoprotein G from the Vesicular stomatitis virus (VSV) which mediates entry via the LDL receptor.
  • VSV-G glycoprotein G from the Vesicular stomatitis virus (VSV) which mediates entry via the LDL receptor.
  • Envelope proteins incorporated into the pseudovirus allow the virus to readily enter different cell types with the corresponding host receptor.
  • Retroviruses use specific receptors to bind and enter cells; both RD114 and BaEV envelope-containing retroviruses use the neutral amino acid (aa) transporter (ASCT2).
  • ASCT2 neutral amino acid transporter
  • ASCT2 sodium-dependent neutral amino acid transporter
  • BaEV uses two receptors, both ASCT1 and ASCT2 for cell entry.
  • ASCT1 and ASCT2 mRNAs were elevated in both IL-15- and IL- 21 -NK cells compared to freshly isolated NK and this is likely why BaEV is the preferred retroviral pseudotype for NK transduction (Colmartino et al., Front Immunol. 2019 Dec 16; 10:2873).
  • Applicant developed a retroviral producer cell line expressing both BaEV and RD114 envelopes, resulting in viruses with both envelopes on each virion, facilitating increased transduction efficiency by accessing both receptors on the target cell.
  • RD114TR modified RD114 feline endogenous retrovirus envelope glycoprotein
  • BaEVTR modified baboon envelope glycoprotein
  • the transduction efficiency of human primary lymphocytes is dependent on the type of envelope proteins used to coat retroviral vectors, and the activated NK cells highly express the receptors, neutral amino acid transporter A (SLC1 A4, which is also referred to herein as ASCT-1) and Neutral amino acid transporter B(0) (SLC1 A5, which is also referred to herein as ASCT-2) , which are baboon envelope glycoprotein (BaEV-TR) used to enter targets.
  • ASCT-2 receptor is feline endogenous retrovirus envelope glycoprotein (RD114TR) used to enter cells.
  • the term “entry receptor” as used herein means a receptor which causes membrane fusion between a virus, such as a retrovirus, and cells by binding to the virus, such as an envelope glycoprotein of the virus.
  • the retrovirus entry receptors, ASCT1, ASCT2 and Pitl are expressed on cells of the human hematopoietic system.
  • any one or any two or any three of ASCT1, ASCT2 or Pitl express on activated and proliferated NK and T cells, which greatly improve transduction efficiency of virus comprising the corresponding envelope glycoprotein.
  • ASCT1 Alanine/Serine/Cysteine/Threonine Transporter 1
  • SLC1 A4 Solute Carrier Family 1 Member 4
  • neutral amino acid transporter A In some embodiments, the ASCT1 is a human ASCT1.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC02P064988,
  • HGNC 10942, NCBI Entrez Gene: 6509, Ensembl: ENSG00000115902, OMIM®: 600229, or UniProtKB/Swiss-Prot: P43007, each of which is incorporated by reference herein in its entirety.
  • ASCT2 Sodium-Dependent Neutral Amino Acid Transporter Type 2
  • SLC1A5 Solute Carrier Family 1 Member 5
  • ASCT2 is a human ASCT2.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC19M051483, HGNC: 10943, NCBI Entrez Gene: 6510,
  • Phosphate Transporter 1 (Pitl or PiT-1 or PiTl) is also referred to as Solute Carrier Family 20 Member 1 (SLC20A1), or Sodium-Dependent Phosphate Transporter 1, or Gibbon Ape Leukemia Virus Receptor 1, or Leukemia Virus Receptor 1 Homolog. It is a retroviral receptor, causing human cells to be susceptible to infection by gibbon ape leukemia virus, simian sarcoma-associated virus, feline leukemia virus subgroup B, and 10A1 murine leukemia virus.
  • the Pitl is a human Pitl.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC02P118061 , HGNC: 10946, NCBI Entrez Gene: 6574, Ensembl: ENSG00000144136, OMIM®: 137570, or UniProtKB/Swiss-Prot: Q8WUM9, each of which is incorporated by reference herein in its entirety.
  • 4-1BBL Tumor Necrosis Factor Superfamily Member 9
  • TNFSF9 Tumor Necrosis Factor Superfamily Member 9
  • 4-1BBL polypeptide is a type 2 transmembrane glycoprotein receptor that is found on APCs (antigen presenting cells) and binds to 4-1BB (also known as CD137).
  • the 4-1BB/4- 1BBL complex belongs to the TNFR:TNF superfamily, which is expressed on activated T Lymphocytes.
  • the 4-1BBL is a human 4-1BBL.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC19P006531, HGNC: 11939, NCBI Entrez Gene: 8744, Ensembl: ENSG00000125657, OMIM®: 606182, or UniProtKB/Swiss-Prot: P41273, each of which is incorporated by reference herein in its entirety.
  • CD56 also termed as Neural Cell Adhesion Molecule 1 (NCAM)
  • NCAM Neural Cell Adhesion Molecule 1
  • CD56 is a cell adhesion protein and a member of the immunoglobulin superfamily, involved in cell-to-cell interactions as well as cell-matrix interactions during development and differentiation. It has been used as an NK cell marker. See, for example, Freud et al. Immunity. 2017 Nov 21;47(5):820-833.
  • Non-limiting exemplary sequences of this protein or the underlying gene or suitable antibodies for detection of the protein can be found under Gene Cards ID: GC11P112961, HGNC: 7656, NCBI Entrez Gene: 4684, Ensembl: ENSG00000149294, OMIM®: 116930, or UniProtKB/Swiss-Prot: P13591, each of which is incorporated by reference herein in its entirety.
  • two major NK-cell subsets can be distinguished and are characterized by the differential expression of the adhesion molecule CD56 and the low-affinity Fc receptor CD16 (FcyRIIIa). They are commonly referred to as CD56 bnght and CD56 dim NK cells.
  • CD56 dim NK cells predominate in peripheral blood whereas CD56 bnght NK cells constitute the majority of NK cells in secondary lymphoid tissues (e.g. lymph nodes) and several organ tissues (e.g. liver, uterus, and kidneys).
  • secondary lymphoid tissues e.g. lymph nodes
  • organ tissues e.g. liver, uterus, and kidneys.
  • Non-limiting exemplary sequences of proteins as referred to herein or the underlying gene or suitable antibodies for detection of the protein can be found in publically available database, such as GeneCards accessible at www.genecards.org/, or UniProtKB accessible at www.uniprot. org/uniprot.
  • RD114TR comprises, or alternatively consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a RD114 glycoprotein and a cytoplasmic domain of amphotropic murine leukemia virus (MLV-A) glycoprotein.
  • MLV-A amphotropic murine leukemia virus
  • BaEVTR comprises, or alternatively consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a baboon envelope glycoprotein (BaEV) and a cytoplasmic domain of MLV-A glycoprotein.
  • BaEV baboon envelope glycoprotein
  • MLV-A glycoprotein MLV-A glycoprotein
  • BaEVTR comprises, or alternatively consists essentially of, or yet further consists of MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVT CSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNK TYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLD TTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSN
  • a wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of MAARSTLSKPPQDKINPWKPLIVMGVLLGVGMAESPHQVFNVTWRVTNLMTGRTA NATSLLGTVQDAFPKLYFDLCDLVGEEWDPSDQEPYVGYGCKYPAGRQRTRTFDFY VCPGHTVKSGCGGPGEGYCGKWGCETTGQAYWKPTSSWDLISLKRGNTPWDTGCS KVACGPCYDLSKVSNSFQGATRGGRCNPLVLEFTDAGKKANWDGPKSWGLRLYRT GTDPITMFSLTRQVLNVGPRVPIGPNPVLPDQRLPSSPIEIVPAPQPPSPLNTSYPPSTTS TPSTSPTSPSVPQPPPGTGDRLLALVKGAYQALNLTNPDKTQECWLCLVSGPPYYEG VAVVGTYTNHSTAPANCTATSQHKLTLSEVTGQGLCMGAVPKTHQALCNTT
  • signal peptide of wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of MAARSTLSKPPQDKINPWKPLIVMGVLLGVGMA (amino acid (aa) 1 to aa 33 of SEQ ID NO: 174).
  • ectodomain domain of wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 599 of SEQ ID NO: 174.
  • transmembrane region of wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of LISTIMGPLIVLLLILLFGPCIL (aa 600 to aa 622 of SEQ ID NO: 174).
  • cytoplasmic domain of wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of NRLVQFVKDRISVVQAL (aa 623 to aa 639 of SEQ ID NO: 174).
  • cytoplasmic domain of wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of NRLVQFVKDRISVVQAL VLTQQYHQLKPLEYEP (aa 623 to aa 655 of SEQ ID NO: 174).
  • BaEV means the envelope glycoprotein of Baboon endogenous virus. Non-limiting exemplary sequences of this protein can be found at UniProtKB - Pl 0269 or NCBI Reference Sequence: YP 009109691.1, each of which is incorporated by reference herein in its entirety.
  • a wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVT CSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNK TYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLD TTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSN TSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPS YNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLA TLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGL
  • signal peptide of wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of MGFTTKIIFLYNLVLVYA (amino acid (aa) 1 to aa 18 of SEQ ID NO: 175).
  • ectodomain domain of wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 506 of SEQ ID NO: 175.
  • transmembrane region of wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of YLLPFLGPLLTLLLLLTIGPCIF (aa 507 to aa 529 of SEQ ID NO: 175).
  • cytoplasmic domain of wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of NRETAFINDKLNIIHAM (aa 530 to aa 546 of SEQ ID NO: 175). In some embodiments, cytoplasmic domain of wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of NRLTAFINDKLNIIHAMVLTQQYQVLRTDEEAQD (aa 530 to aa 563 of SEQ ID NO: 175).
  • RD114 when referring to a protein, RD114 means the envelope glycoprotein of RD114 retrovirus.
  • Non-limiting exemplary sequences of this protein can be found at GenBank: CAA61093.1, CBI Reference Sequence: YP 001497149.1, or GenBank: BAM17308.1, each of which is incorporated by reference herein in its entirety.
  • a wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of
  • signal peptide of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of MKLPTGMVILCSLIIVRA (amino acid (aa) 1 to aa 18 of SEQ ID NO: 176).
  • ectodomain domain of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 504 of SEQ ID NO: 176.
  • ectodomain domain of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 507 of SEQ ID NO: 176.
  • transmembrane region of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of FLPYLLPLLGPLLTLLLILTIGPCVF (aa 505 to aa 530 of SEQ ID NO: 176). In some embodiments, transmembrane region of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of YLLPLLGPLLTLLLILTIGPCVF (aa 508 to aa 530 of SEQ ID NO: 176).
  • cytoplasmic domain of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of SRLMAFINDRLNVVHAM (aa 531 to aa 547 of SEQ ID NO: 176). In some embodiments, cytoplasmic domain of wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of SRLMAFINDRLNVVHAMVLAQQYQALKAEEEAQD (aa 531 to aa 564 of SEQ ID NO: 176).
  • GALV means the envelope glycoprotein of Gibbon ape leukemia virus envelope glycoprotein.
  • Non-limiting exemplary sequences of this protein can be found at UniProtKB - P21415 (ENV GALV), which is incorporated by reference herein in its entirety.
  • a wild type GALV comprises, or alternatively consists essentially of, or yet further consists of MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLT WQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVR PPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLY CKEWDCETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTD KGKLSKDWITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTS LALPPPLPPREAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGA FLTLNATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTE VSGHGLC
  • a wild type GALV comprises, or alternatively consists essentially of, or yet further consists of aa 42 to aa 685 of SEQ ID NO: 183.
  • signal peptide of wild type GALV comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 41 of SEQ ID NO: 183.
  • ectodomain domain of wild type GALV comprises, or alternatively consists essentially of, or yet further consists of aa 1 to aa 489 of SEQ ID NO: 183 or aa 42 to aa 489 of SEQ ID NO: 183.
  • transmembrane region of wild type GALV comprises, or alternatively consists essentially of, or yet further consists of aa 490 to aa 670 of SEQ ID NO: 183.
  • cytoplasmic domain of wild type GALV comprises, or alternatively consists essentially of, or yet further consists of aa 671 to aa 685 of SEQ ID NO: 183.
  • Multiplicity of infection refers to the number of viral particles that are added per cell during infection.
  • RetroNectin reagent is a 63 kD fragment of recombinant human fibronectin fragment (also referred to as rFN-CH-296) that enhances the efficiency of lentiviral- and retroviral- mediated gene transduction. This is particularly important for hematopoietic cells and other hard-to-transfect cell types. Enhanced transduction is thought to result from co-localization of virus particles and target cells. This is accomplished by direct binding of viral particles to sequences in the heparin-binding domain and interaction of target-cell integrins with two other domains in rFN-CH-296.
  • RetroNectin is highly effective for cells that express integrin Integrin a4pi (VLA-4) and/or Integrin a5pi (VLA-5).
  • VLA-4-expressing cells include T cells, B cells, monocytes, NK cells, eosinophils, bone marrow monocytic cells, and lymphoid progenitors. Thymocytes, activated T cells, and mast cell express VLA-5.
  • a vector construct refers to the polynucleotide comprising the lentiviral genome or part thereof, and a therapeutic gene.
  • lentiviral mediated gene transfer or “lentiviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • a “lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.
  • Lentiviral vectors of this disclosure are based on or derived from oncoretroviruses (the sub-group of retroviruses containing MLV), and lentiviruses (the sub-group of retroviruses containing HIV). Examples include ASLV, SNV and RSV all of which have been split into packaging and vector components for lentiviral vector particle production systems.
  • the lentiviral vector particle according to the disclosure may be based on a genetically or otherwise (e.g. by specific choice of packaging cell system) altered version of a particular retrovirus.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B.
  • the AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3.
  • the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
  • That the vector particle according to the disclosure is “based on” a particular retrovirus means that the vector is derived from that particular retrovirus.
  • the genome of the vector particle comprises components from that retrovirus as a backbone.
  • the vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include gag and pol proteins derived from the particular retrovirus.
  • gag and pol proteins derived from the particular retrovirus.
  • the majority of the structural components of the vector particle will normally be derived from that retrovirus, although they may have been altered genetically or otherwise so as to provide desired useful properties.
  • certain structural components and in particular the env proteins may originate from a different virus.
  • the vector host range and cell types infected or transduced can be altered by using different env genes in the vector particle production system to give the vector particle a different specificity.
  • a cell may be a prokaryotic or a eukaryotic cell. In further embodiments, the cell is an immune cell.
  • Immuno cells includes, e.g., white blood cells (leukocytes, such as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells)) which may be derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and NKT cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • hematopoietic stem cells HSCs
  • T cells, B cells, natural killer (NK) cells, and NKT cells myeloid-derived cells
  • the immune cell is derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • the HSCs are derived from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
  • “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a “packaging cell” refers to a host cell which, by way of stable or transient transfection or transduction with heterologous nucleotide sequences, harbors a nucleic acid molecule comprising an viral helper construct, wherein the construct is capable of providing stable or transient expression of packaging functions, e.g., proteins necessary for replication and encapsidation, that can be provided in trans for production of viral particles.
  • packaging functions e.g., proteins necessary for replication and encapsidation
  • Expression of the viral helper functions can be either constitutive, or inducible, such as when the helper functions are under the control of an inducible promoter.
  • An “enriched population” of cells intends a substantially homogenous population of cells having certain defined characteristics.
  • the cells are greater than 70 %, or alternatively greater than 75 %, or alternatively greater than 80 %, or alternatively greater than 85 %, or alternatively greater than 90 %, or alternatively greater than 95 %, or alternatively greater than 98% identical in the defined characteristics.
  • the term “propagate” means to grow a cell or population of cells.
  • the term “growing” also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.
  • the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
  • Unmodified cells are sometimes referred to as “source cells” or “source stem cells”.
  • the cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, e.g., felines, canines, equines, murines, rats, simians, bovines, porcines and humans.
  • an “immature cell” refers to a cell which does not possess the desired (adult) phenotype or genotype.
  • a mature cell is a cell that is being replaced.
  • the immature cell can be subjected to techniques including physical, biological, or chemical processes which changes, initiates a change, or alters the phenotype or genotype of the cell into a “mature cell.”
  • a “mature cell” refers to a cell which possess the desired phenotype or genotype.
  • NK cell also known as natural killer cell, refers to a type of lymphocyte that originates in the bone marrow and play a critical role in the innate immune system. NK cells provide rapid immune responses against viral-infected cells, tumor cells or other stressed cell, even in the absence of antibodies and major histocompatibility complex on the cell surfaces. NK cells may either be isolated or obtained from a commercially available source.
  • Non-limiting examples of commercial NK cell lines include lines NK-92 (ATCC® CRL-2407TM), NK-92MI (ATCC® CRL-2408TM). Further examples include but are not limited to NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT.
  • Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection, or ATCC, (www.atcc.org/) and the German Collection of Microorganisms and Cell Cultures (www.dsmz.de/).
  • the potency of a cell comprises cytotoxicity of the cell, for example in killing a target cell, and/or cytokine release of the cell, such as IFN-y (also referred to herein as IFNy or IFN- gamma).
  • a naturally occurring antigen presenting cell refers to an immune cell that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes such as T cells, Classical APCs include dendritic cells, macrophages, Langerhans cell s and B cells.
  • an artificial antigen presenting cells synthetic versions of these APCs and are made by attaching the specific immune cell (such as T cell and/or NK cell) stimulating signals to various macro and micro biocompatible surfaces and/or cells. This can potentially reduce the cost while allowing control over generating large numbers of functional pathogen-specific immune cells for therapy.
  • stem cell refers to a cell that is in an undifferentiated or partially differentiated state and has the capacity for self-renewal and/or to generate differentiated progeny.
  • Self-renewal is defined as the capability of a stem cell to proliferate and give rise to more such stem cells, while maintaining its developmental potential (i.e., totipotent, pluripotent, multipotent, etc.).
  • embryo stem cell is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue.
  • Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle.
  • exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural or neuronal stem cells (NSCs).
  • the stem or progenitor cells can be embryonic stem cells or an induced pluripotent stem cell (iPSC).
  • embryonic stem cells refers to stem cells derived from tissue formed after fertilization but before the end of gestation, including pre- embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Most frequently, embryonic stem cells are pluripotent cells derived from the early embryo or blastocyst. Embryonic stem cells can be obtained directly from suitable tissue, including, but not limited to human tissue, or from established embryonic cell lines. “Embryonic-like stem cells” refer to cells that share one or more, but not all characteristics, of an embryonic stem cell.
  • “Differentiation” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, or muscle cell.
  • “Directed differentiation” refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type.
  • “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell.
  • the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell.
  • a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively.
  • Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, peri cardiogenic, or stromal.
  • the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell. “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell. Induced pluripotent stem cells are examples of dedifferentiated cells.
  • a first cell derived from a second cell refers to the first cell differentiated from the second cell. Additionally or alternatively, a first cell derived from a second cell refers to the first cell engineered from the second cell.
  • the "lineage" of a cell defines the heredity of the cell, i.e. its predecessors and progeny.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a “multi-lineage stem cell” or “multipotent stem cell” refers to a stem cell that reproduces itself and at least two further differentiated progeny cells from distinct developmental lineages.
  • the lineages can be from the same germ layer (i.e. mesoderm, ectoderm or endoderm), or from different germ layers.
  • An example of two progeny cells with distinct developmental lineages from differentiation of a multi-lineage stem cell is a myogenic cell and an adipogenic cell (both are of mesodermal origin, yet give rise to different tissues).
  • Another example is a neurogenic cell (of ectodermal origin) and adipogenic cell (of mesodermal origin).
  • a “precursor” or “progenitor cell” intends to mean cells that have a capacity to differentiate into a specific type of cell.
  • a progenitor cell may be a stem cell.
  • a progenitor cell may also be more specific than a stem cell.
  • a progenitor cell may be unipotent or multipotent. Compared to adult stem cells, a progenitor cell may be in a later stage of cell differentiation.
  • An example of progenitor cell includes, without limitation, a progenitor nerve cell.
  • a “pluripotent cell” defines a less differentiated cell that can give rise to at least two distinct (genotypically and/or phenotypically) further differentiated progeny cells.
  • a “pluripotent cell” includes an Induced Pluripotent Stem Cell (iPSC) which is an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell, that has historically been produced by inducing expression of one or more stem cell specific genes.
  • iPSC Induced Pluripotent Stem Cell
  • stem cell specific genes include, but are not limited to, the family of octamer transcription factors, i.e.
  • Oct-3/4 the family of Sox genes, i.e., Soxl, Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, i.e. Klfl, Klf2, Klf4 and Klf5; the family of Myc genes, i.e. c-myc and L-myc; the family of Nanog genes, i.e., OCT4, NANOG and REXI; or LIN28.
  • Examples of iPSCs are described in Takahashi et al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.
  • Embryoid bodies or EBs are three-dimensional (3D) aggregates of embryonic stem cells formed during culture that facilitate subsequent differentiation. When grown in suspension culture, EBs cells form small aggregates of cells surrounded by an outer layer of visceral endoderm. Upon growth and differentiation, EBs develop into cystic embryoid bodies with fluid-filled cavities and an inner layer of ectoderm-like cells.
  • An “induced pluripotent cell” intends embryonic-like cells reprogrammed to the immature phenotype from adult cells.
  • Various methods are known in the art, e.g., "A simple new way to induce pluripotency: Acid.” Nature, 29 January 2014 and available at sciencedaily. com/releases/2014/01/140129184445, last accessed on February 5, 2014 and U.S. Patent Application Publication No. 2010/0041054.
  • Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.
  • a “parthenogenetic stem cell” refers to a stem cell arising from parthenogenetic activation of an egg. Methods of creating a parthenogenetic stem cell are known in the art. See, for example, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(Suppl. 1)11911-6.
  • pluripotent gene or marker intends an expressed gene or protein that has been correlated with an immature or undifferentiated phenotype, e.g., Oct %, Sox2, Nanog, c-Myc and LIN-28. Methods to identify such are known in the art and systems to identify such are commercially available from, for example, EMD Millipore (MILLIPLEX® Map Kit).
  • phenotype refers to a description of an individual’s trait or characteristic that is measurable and that is expressed only in a subset of individuals within a population.
  • an individual’s phenotype includes the phenotype of a single cell, a substantially homogeneous population of cells, a population of differentiated cells, or a tissue comprised of a population of cells.
  • a population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.
  • the population can be purified, highly purified, substantially homogenous or heterogeneous as described herein.
  • effective period (or time) and effective conditions refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or composition to achieve its intended result, e.g., the differentiation or dedifferentiation of cells to a pre-determined cell type.
  • controllable conditions e.g., temperature, humidity for in vitro methods
  • “Substantially homogeneous” describes a population of cells in which more than about 50%, or alternatively more than about 60 %, or alternatively more than 70 %, or alternatively more than 75 %, or alternatively more than 80%, or alternatively more than 85 %, or alternatively more than 90%, or alternatively, more than 95 %, of the cells are of the same or similar phenotype. Phenotype can be determined by a pre-selected cell surface marker or other marker.
  • a cell population depleted CD3+ cells refers to the cell population comprising less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6% , less than about 0.7% , less than about 0.8% , less than about 0.9% , less than about 1.0% , less than about 1.1% , less than about 1.2% , less than about 1.3% , less than about 1.4% , less than about 1.5% , less than about 1.6% , less than about 1.7% , less than about 1.8% , less than about 1.9%, or less than about 2.0% CD3+ cells.
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or can be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • treatment include but are not limited to: preventing a disorder from occurring in a subject that may be predisposed to a disorder, but has not yet been diagnosed as having it; inhibiting a disorder, i.e., arresting its development; and/or relieving or ameliorating the symptoms of disorder.
  • treatment is the arrestment of the development of symptoms of the disease or disorder, e.g., a cancer.
  • they refer to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • the disease is cancer
  • the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
  • treatment excludes prophylaxis.
  • treatment excludes prophylaxis.
  • a therapeutic protein or polypeptide refers to a protein and/por a polypeptide suitable for a treatment, including but not limited to an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • Such therapeutic protein or polypeptide may be chose by a physician or one of skill in the art, based on the disease to be treated.
  • an antibody to an immune checkpoint receptor or a ligand thereof may be used, such as an anti-PD-1 antibody and/or an anti-PD-Ll antibody.
  • the term “disease” or “disorder” as used herein refers to a cancer, a status of being diagnosed with a cancer, a status of being suspect of having a cancer, or a status of at high risk of having a cancer.
  • a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.”
  • cancer or tumor antigen refers to an antigen known to be associated and expressed on the surface with a cancer cell or tumor cell or tissue
  • cancer or tumor targeting antibody refers to an antibody that targets such an antigen.
  • a “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant, metastatic or non-metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant , diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • inert for example, a detectable agent or label
  • active such as an adjuvant , diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose
  • a “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).
  • pharmaceutically acceptable carrier refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein).
  • biodegradable materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable).
  • a biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
  • unit dose or "dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.
  • contacting means direct or indirect binding or interaction between two or more molecules.
  • a particular example of direct interaction is binding.
  • a particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity.
  • Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.
  • administering or “delivery” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application.
  • administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • the disclosure is not limited by the route of administration, the formulation or dosing schedule.
  • administering can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the optimal route will vary with the condition and age of the recipient, and the disease being treated.
  • a “subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human.
  • the present disclosure is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents.
  • the mammals include horses, dogs, and cats.
  • the human is a fetus, an infant, a pre-pubescent subject, an adolescent, a pediatric patient, or an adult.
  • the subject is pre-symptomatic mammal or human.
  • the subject has minimal clinical symptoms of the disease.
  • the subject can be a male or a female, adult, an infant or a pediatric subject.
  • the subject is an adult.
  • the adult is an adult human, e.g., an adult human greater than 18 years of age.
  • the term “suffering” as it related to the term “treatment” refers to a patient or individual who has been diagnosed with or is predisposed to syndrome cancer or a viral infection.
  • a patient may also be referred to being “at risk of suffering” from a disease because they carry one or more genetic mutations. This patient has not yet developed characteristic disease pathology.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration.
  • one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.
  • “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response such as passive immunity; or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient.
  • a therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
  • “Separate therapy,” as used herein, includes but is not limited to surgical resection, chemotherapy, cryotherapy, radiation therapy, immunotherapy and targeted therapy. Agents that act to reduce cellular proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they are dividing are termed cell-cycle specific. These drugs include agents that act in S-phase, including topoisomerase inhibitors and antimetabolites.
  • Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication, and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
  • Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
  • folic acid antagonists e.g., methotrexate
  • pyrimidine antagonist e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine
  • purine antagonist e.g., 6-mercaptopurine and 6-thi
  • Plant alkaloids are derived from certain types of plants.
  • the vinca alkaloids are made from the periwinkle plant (Catharanthus rosea).
  • the taxanes are made from the bark of the Pacific Yew tree (taxus).
  • the vinca alkaloids and taxanes are also known as antimicrotubule agents.
  • the podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors.
  • the plant alkaloids are generally cell-cycle specific.
  • Examples of these agents include vinca alkaloids, e.g., vincristine, vinblastine and vinorelbine; taxanes, e.g., paclitaxel and docetaxel; podophyllotoxins, e.g., etoposide and tenisopide; and camptothecan analogs, e.g., irinotecan and topotecan.
  • vinca alkaloids e.g., vincristine, vinblastine and vinorelbine
  • taxanes e.g., paclitaxel and docetaxel
  • podophyllotoxins e.g., etoposide and tenisopide
  • camptothecan analogs e.g., irinotecan and topotecan.
  • Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.
  • Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art.
  • exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy and/or a dose of ultraviolet radiation at a range from at least about 5 J/m2 to not more than about 50 J/m2, usually about 10 J/m2.
  • first line or “second line” or “third line” refers to the order of treatment received by a patient.
  • First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively.
  • the National Cancer Institute defines first line therapy as “the first treatment for a disease or condition.
  • primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies.
  • First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008.
  • a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.
  • compositions and/or a method as disclosed herein were observed and exemplified in the Drawings as well as in the Experimental Methods. These advantages include but are not limited to, efficient gene delivery to an immune cell, such as NK cells, stability of the delivered genes, successful expansion of the cells, and high viability of the transduced cells, thus facilitating and improving transduced immune cells (such as CAR expressing immune cells) developing and manufacturing.
  • an immune cell such as NK cells
  • stability of the delivered genes successful expansion of the cells
  • high viability of the transduced cells thus facilitating and improving transduced immune cells (such as CAR expressing immune cells) developing and manufacturing.
  • transduced immune cells such as CAR expressing immune cells
  • Various methods may be used to assess such expression, such as fluorescence-activated Cell Sorting (FACS) or another immunostaining method utilizing an antibody or an antigen binding fragment thereof specifically recognizing and binding the CAR and/or therapeutic protein or polypeptide.
  • FACS fluorescence-activated Cell Sorting
  • a cell population prepared by a method as disclosed herein expresses a CAR and/or another therapeutic protein or polypeptide for at least about 3 days, at least about 5 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 1 months, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, at least about 150 days
  • a method as disclosed herein generates more than 1 x 10 9 cells, more than 2 x 10 9 cells, more than 3 x 10 9 cells, more than 4 x 10 9 cells, more than 5 x 10 9 cells, more than 6 x 10 9 cells, more than 7 x 10 9 cells, more than 8 x 10 9 cells, more than 9 x 10 9 cells, more than 1 x IO 10 cells, more than 2 x IO 10 cells, more than 3 x IO 10 cells, more than 4 x IO 10 cells, more than 5 x IO 10 cells, more than 6 x IO 10 cells, more than 7 x
  • a method as disclosed herein generates up to 5 x IO 10 cells, up to 6 x IO 10 cells, up to 7 x IO 10 cells, up to 8 x IO 10 cells, up to 9 x IO 10 cells, up to 1 x 10 11 cells, up to 2 x 10 11 cells, up to 3 x 10 11 cells, up to 4 x
  • a method as disclosed herein generates about 5 x IO 10 cells, about 6 x IO 10 cells, about 7 x IO 10 cells, about 8 x IO 10 cells, about 9 x IO 10 cells, about 1 x 10 11 cells, about 2 x 10 11 cells, about 3 x 10 11 cells, about 4 x 10 11 cells, about 5 x 10 11 cells, or about 6 x 10 11 cells from 1 x 10 8 cells, or an equivalent thereof.
  • Such equivalent may be calculated by one of skill in the art via dividing or multiplying both the generated cell number and the initial cell number by a same positive number.
  • viability of a cell population as disclosed herein may be at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.
  • Multiple methods of testing cell viability are available, such as an MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay or an APT measurement assay.
  • the gammaretroviral particle comprises, or alternatively consists essentially of, or yet further consists of a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR). Additionally or alternatively, the gammaretroviral particle comprises or consists essentially of, or yet further consists of a modified baboon envelope glycoprotein (BaEVTR).
  • RD114TR modified RD114 feline endogenous retrovirus envelope glycoprotein
  • BaEVTR modified baboon envelope glycoprotein
  • the RD114TR glycoprotein comprises, or alternatively consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a RD114 glycoprotein and a cytoplasmic domain of amphotropic murine leukemia virus (MLV-A) glycoprotein.
  • the BaEVTR glycoprotein comprises, or alternatively consists essentially of, or yet further consists of an ectodomain and a transmembrane domain of a baboon envelope glycoprotein (BaEV) and a cytoplasmic domain of MLV-A glycoprotein.
  • the RD114TR and the BaEVTR are incorporated into the envelope of the particle as membrane proteins.
  • a pseudotyped gammaretroviral particle as disclosed herein further comprises a vector genome encapsulated in the envelope.
  • the vector genome comprises, or alternatively consists essentially of, or yet further consists of one or more of the following flanked by two long terminal repeats (LTRs): (A) a polynucleotide encoding a chimeric antigen receptor (CAR) and/or another therapeutic protein or polypeptide; (B) a reverse-complement of (A); or (C) a polynucleotide comprising one or more of recognition sites.
  • LTRs long terminal repeats
  • the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • the recognition sites are recognized and cleaved by a restriction enzyme suitable for inserting a sequence of interest, such as either or both of (A) and (B) into the polynucleotide.
  • a vector genome further comprises one or more of the following: a 5’ LTR, a 5’ cap, a 3’ poly-A tail, and a 3’ LTR.
  • a pseudotyped gammaretroviral particle as disclosed herein further comprises either of both of a reverse transcriptase or an integrase.
  • a pseudotyped gammaretroviral particle is based on, or is derived from, or is selected from any one of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV), or the feline leukemia virus.
  • an artificial antigen presenting cell expresses one or more of a tumor associated antigen (TAA) or a viral antigen as disclosed herein or both.
  • TAA tumor associated antigen
  • the aAPC expresses one or more of the following: 4-1BBL, membrane-bound (mb) IL- 15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL- 18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D super-agonist- complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc) ALT-803, or a cell surface marker mediating CD122/CD132 signaling.
  • 4-1BBL membrane-bound (mb) IL- 15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb
  • the aAPCs further expresses mb IL-21 and 4-1BBL.
  • an aAPC as disclosed herein expresses a tumor associated antigen (TAA) and/or a viral antigen which activate and/or stimulate growth of an immune cell, such as an NK cell or a y6 T cell.
  • TAA tumor associated antigen
  • an immune cell such as an NK cell or a y6 T cell.
  • the aAPCs are engineered K562 cells.
  • the aAPCs lacks cell proliferation and/or lacks long-term survival.
  • Cell proliferation and long-term survival can be evaluated by one of skill in the art, for example via culturing and counting live cells.
  • an aAPC as disclosed herein do not substantially survive for more than about 5 days, about 7 days, about 10 days, about 14 days, about 15 days, about 21 days or about 30 days.
  • the aAPCs are irradiated to inhibit cell proliferation and/or reduce long-term survival.
  • the aAPC are irradiated at 50 Gy or more, 60 Gy or more, 70 Gy or more, 75 Gy or more, 80 Gy or more, 90 Gy or more, 100 Gy or more, 110 Gy or more, 120 Gy or more, 130 Gy or more, 140 Gy or more, 150 Gy or more, 160 Gy or more, 170 Gy or more, 180 Gy or more, 190 Gy or more, 200 Gy or more, 210 Gy or more, 220 Gy or more, 230 Gy or more, 240 Gy or more, 250 Gy or more, and/or 300 Gy or more.
  • the aAPC are irradiated at 1000 Gy or less, 900 Gy or less, 800 Gy or less, 700 Gy or less, 600 Gy or less, 500 Gy or less, 400 Gy or less, 350 Gy or less, 300 Gy or less, 250 Gy or less, 240 Gy or less, 230 Gy or less, 220 Gy or less, 210 Gy or less, 200 Gy or less, 190 Gy or less, 180 Gy or less, 170 Gy or less, 160 Gy or less, 150 Gy or less, 140 Gy or less, 130 Gy or less, 120 Gy or less, 110 Gy or less, 100 Gy or less, 90 Gy or less, 80 Gy or less, 70 Gy or less, or 60 Gy or less.
  • the aAPCs are irradiated at about 50 Gy to about 300 Gy, including but not limited to about 50 Gy to about 100 Gy, about 50 Gy to about 150 Gy, about 50 Gy to about 200 Gy , about 50 Gy to about 250 Gy, about 100 Gy to about 150 Gy, about 100 Gy to about 200 Gy, about 100 Gy to about 150 Gy, about 150 Gy to about 200 Gy, about 150 Gy to about 250 Gy, about 200 Gy to about 250 Gy.
  • the aAPCs are irradiated at about 50 Gy, about 100 Gy, about 150 Gy or about 200 Gy.
  • the irradiation is performed before culturing the aAPCs with an immune cell (such as one or more of NK cell, NKT cells and/or y6 T cells) or a cell population thereof.
  • an immune cell such as one or more of NK cell, NKT cells and/or y6 T cells
  • a method for preparing a population of natural killer (NK) cells comprises, or alternatively consists essentially of, or yet further consists of culturing a cell population comprising one or more of the following: an NK cell, a progenitor cell that is capable of deriving an NK cell, or a stem cell that is capable of deriving an NK cell with an immune cell activator (such as an NK cell activator).
  • the cell population is depleted with cells that expresses one or more of: CD3, CD4, CD8, T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population.
  • the immune cell activator(s) is selected from one or more of the following: an artificial antigen presenting cell (aAPC) that expresses a tumor associated antigen (TAA) and/or a viral antigen optionally which activate and/or stimulate immune cell growth; one or more of an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the NK cell, the progenitor cell or the stem cell, thereby activating or proliferating NK cells; one or more of cytokines thereby activating or proliferating NK cells; and/or one or more of chemical moieties thereby activating or proliferating NK cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs).
  • aAPC artificial antigen presenting cell
  • TAA tumor associated antigen
  • viral antigen optionally which activate and/or stimulate immune cell growth
  • the stimulatory receptor is one or more of the following: CD2, NKp46, CD 16, NKG2D, DNAM-1 (CD226), 2B4 (Natural killer cell receptor 2B4, CD244), NTB-A (SLAM family member 6, SLAMF6), and/or NKp46.
  • the aAPC is an aAPC as disclosed herein.
  • the aAPC is cultured with the cell population at a cell number ratio (cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population) of about 100: 1 or more, about 50:1 or more, about 20: 1 or more, about 10: 1 or more, about 9: 1 or more, about 9: 1 or more, about 8: 1 or more, about 7:1 or more, about 6: 1 or more, about 5 : 1 or more, about 4: 1 or more, about 3 : 1 or more, about 2: 1 or more, about 1 : 1 or more, about 1 :2 or more, about 1 :3 or more, about 1 :4 or more, about 1 :5 or more, about 1 :6 or more, about 1 :
  • the aAPC is cultured with the cell population at a cell number ratio (cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population) of about 100: 1 or less, about 50: 1 or less, about 20: 1 or less, about 10: 1 or less, about 9:1 or less, about 9: 1 or less, about 8: 1 or less, about 7: 1 or less, about 6: 1 or less, about 5:1 or less, about 4: 1 or less, about 3: 1 or less, about 2: 1 or less, about 1 : 1 or less, about 1 :2 or less, about 1 :3 or less, about 1 :4 or less, about 1 :5 or less, about 1 :6 or less, about 1 :7 or less, about 1 :8 or less, about 1 :9 or less, about 1 : 10 or less
  • the aAPC is cultured with the cell population at a cell number ratio (cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population) of about 10: 1 to about 1 : 10, about 5:1 to about 1 :5, about 3: 1 to about 1 :3, about 2: 1 to about 1 :2.
  • a cell number ratio cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population
  • the aAPC is cultured with the cell population at a cell number ratio (cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population) of about 10: 1, about 5: l, about 3: l, about 2: l, about 1 : 1, about 1 :2, about 1 :3, about 1 :5, or about 1 : 10.
  • a cell number ratio cell number of aAPCs: cell number of the cell population and/or cell number of aAPCs: cell number of immune cells (such as one or more of NK cell, NKT cells and/or y6 T cells) in the cell population
  • the cytokines are selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low-toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D super-agonist-complexed with IL- 15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc; ALT-803 soluble), IL-15, IL-18, IL- 21, LEC, OX40L, ICOSL (B7H2, B7RP1), or MICA.
  • the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-2.
  • the cell population is cultured with about 1 lU/mL to about 1000 lU/ml IL2.
  • the cell population is cultured with about 1 lU/mL or more, about 2 lU/mL or more, about 3 lU/mL or more, about 5 lU/mL or more, about 10 lU/mL or more, about 20 lU/mL or more, about 30 lU/mL or more, about 40 lU/mL or more, about 50 lU/mL or more, about 60 lU/mL or more, about 70 lU/mL or more, about 80 lU/mL or more, about 90 lU/mL or more, about 100 lU/mL or more, about 110 lU/mL or more, about 120 lU/mL or more, about 130 lU/mL or more, about 140 lU/mL or more, about 150 lU/mL or more, about 160 lU/mL or more, about 170 lU/mL or more, about 180 lU/mL or more,
  • the cell population is cultured with about 2000 lU/mL or less, about 1500 lU/mL or less, about 1000 lU/mL or less, about 900 lU/mL or less, about 800 lU/mL or less, about 700 lU/mL or less, about 600 lU/mL or less, about 500 lU/mL or less, about 400 lU/mL or less, about 300 lU/mL or less, about 250 lU/mL or less, about 200 lU/mL or less, about 100 lU/mL or less, about 90 lU/mL or less, about 80 lU/mL or less, about 70 lU/mL or less, about 60 lU/mL or less, about 50 lU/mL or less, about 40 lU/mL or less, about 30 lU/mL or less, about 20 lU/mL or less, about 10 lU/mL or less,
  • the cytokines comprises an IL-2.
  • the cell population is cultured with about 1 lU/mL to about 1000 lU/ml IL2, including but not limited to about 10 lU/ml to about 100 lU/ml, about 100 lU/ml to 200 lU/ml, about 200 lU/ml to about 300 lU/ml, about 300 lU/ml to about 400 lU/ml, about 400 to about 500 lU/ml, about 100 lU/ml to about 500 lU/ml IL2.
  • the cell population is cultured with about 10 lU/mL, about 20 lU/rnL, about 30 lU/rnL, about 40 lU/rnL, about 50 lU/rnL, about 60 lU/mL, about 70 lU/mL, about 80 lU/mL, about 90 lU/mL, about 100 lU/mL, about 110 lU/mL, about 120 lU/mL, about 130 lU/mL, about 140 lU/mL, about 150 lU/mL, about 160 lU/mL, about 170 lU/mL, about 180 lU/mL, about 190 lU/mL, about 200 lU/mL, about 250 lU/mL, about 300 lU/mL, about 400 lU/mL, about 500 lU/mL, about 600 lU/mL, about 700 lU/mL, about 800 lU/m
  • the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-15.
  • the cell population is cultured with about 0.1 ng/mL to about 500 ng/mL IL 15, including any ranges and/or numbers falling therein.
  • the cell population is cultured with about 1 ng/mL to about 100 ng/ml IL 15, including but not limited to about 1 ng/ml to about 10 ng/ml, about 10 ng/ml to about 20 ng/ml, about 20 ng/ml to about 30 ng/ml, about 30 ng/ml to about 40 ng/ml, about 40 ng/ml to about 50 ng/ml IL 15.
  • the cell population is cultured with about 1 ng/mL, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, or about 100 ng/ml IL15.
  • the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-21.
  • the cell population is cultured with about 0.1 ng/mL to about 500 ng/mL IL21, including any ranges and/or numbers falling therein.
  • the cell population is cultured with about 1 ng/mL to about 100 ng/ml IL21, including but not limited to about 1 ng/ml to about 10 ng/ml, about 10 ng/ml to about 20 ng/ml, about 20 ng/ml to about 30 ng/ml, about 30 ng/ml to about 40 ng/ml, about 40 ng/ml to about 50 ng/ml IL21.
  • the cell population is cultured with about 1 ng/mL, about 5 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 90 ng/ml, or about 100 ng/ml IL 21.
  • the cell population is cultured with more than one cytokines, such as a combination of the cytokines as disclosed herein.
  • the cell population is cultured with any one or any two or all three of 100-500 lU/ml IL-2, 20 ng/ml IL- 15, or 25 ng/mL IL-21.
  • the cell population is cultured with either or both of 50 lU/ml IL-2 and 0.5 ng/ml IL-15.
  • a chemical moiety may also be used as an immune cell activator (e.g., an NK activator), such as, treatment with inhibitors of ADAM17 was shown to augment NK cell ADCC by preventing shedding of the CD 16 receptor, and treatment of NK cells with nicotinamide enhances their expression of L-selectin, which is known to be essential for cellular trafficking.
  • an immune cell activator e.g., an NK activator
  • a method as disclosed herein further comprises introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide into the cultured cell population for expression.
  • the CAR specifically recognizes and binds to a tumor associated antigen (TAA) and/or a viral antigen.
  • TAA tumor associated antigen
  • the tumor associated antigen (TAA) and/or the viral antigen recognized and bound by the CAR is the one expressed by aAPC.
  • therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • a method as disclosed herein further comprises culturing the cell population with an immune cell activator (such as an NK cell activator) prior to and/or after the introducing step.
  • an immune cell activator such as an NK cell activator
  • the culturing step is repeated for once, twice, three times, or more times, with same or different immune cell activator(s) (such as NK cell activator(s)) or a combination thereof.
  • the activator(s) cultured with the cell population before and after the introducing step In further embodiments, two of the activator(s) cultured with the cell population before and after the introducing step are the same. In some embodiments, two of the activator(s) cultured with the cell population before and after the introducing step are different from each other.
  • Some embodiments of the method comprises introducing a pseudotyped gammaretroviral particle into the cultured cell population, thereby introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide as disclosed herein into the cultured cell population.
  • the pseudotyped gammaretroviral particle is disclosed herein.
  • the pseudotyped gammaretroviral particle comprises the polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide as disclosed herein and/or a reverse-complement thereof.
  • the coding polynucleotide and/or a reverse-complement thereof is flanked by two long terminal repeats (LTRs).
  • the pseudotyped gammaretroviral particle comprises an RD114TR and/or a BaEVTR as disclosed herein.
  • the pseudotyped gammaretroviral particle comprises a vector genome comprising, or alternatively consisting essentially of, or yet further consisting of a 5’ LTR, a 5’ cap, the coding polynucleotide or a reverse-complement thereof, a 3’ poly- A tail, and a 3’ LTR.
  • the components as disclosed herein are located in the vector genome from 5’ to 3’.
  • the pseudotyped gammaretroviral particle further comprises either of both of a reverse transcriptase and an integrase.
  • the pseudotyped gammaretroviral particle is based one, and/or derived from and/or is selected from any species of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • XMRV xenotropic murine leukemia virus-related virus
  • the pseudotyped gammaretroviral particle is introduced to the cultured cell population at a multiplicity of infection (MOI) of about 0.01 to about 100, including any ranges and/or numbers falling therein.
  • MOI multiplicity of infection
  • pseudotyped gammaretroviral particle is introduced to the cultured cell population at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5.
  • pseudotyped gammaretroviral particle is introduced to the cultured cell population at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10.
  • the cell population are cultured before the introducing step for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Additionally or alternatively, the cell population are cultured before the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks, or no more than 1 month. In some embodiments, the cell population are cultured before the introducing step for about 1 days to about 180 days, including any ranges and/or numbers falling therein. In some embodiments, the cell population are cultured before the introducing step for about 5 days to about 10 days, such as about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
  • the coding polynucleotide is introduced to the cell population via transducing a viral vector comprising the coding polynucleotide or reverse-complement thereof in the presence of RetroNectin.
  • the RetroNectin is coated on the inner surface of a container in which the cell population is transduced.
  • the container is a bag suitable for culturing a cell.
  • the container is a plate suitable for culturing a cell,
  • the container is a flask suitable for culturing a cell.
  • the cell population expresses either or both of Integrin a4pi (VLA-4) and Integrin a5pi (VLA-5).
  • the cell population is isolated from a biological sample (such as a blood sample) of a subject.
  • the cell population is isolated from one or more of: umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
  • the cell population comprises, or alternatively consists essentially of, or yet further consists of progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • iPSCs induced pluripotent stem cells
  • HSCs hematopoietic stem cells
  • a method as disclosed herein further comprises enriching cells expressing any one or more of CD56, CD25, CD122, CD212, CD215, CD218, CD360, TGF- PR, or IL-10R in the cell population.
  • a method as disclosed herein further comprises enriching, deriving and/or generating CD56 dim cells.
  • CD56 dim cells express CD56 surface antigen in low density and specializes in cytotoxic function.
  • a method as disclosed herein further comprises enriching, deriving and/or generating CD56 bnght cells.
  • CD56 bnght cells express CD56 surface antigen in high density and specializes in cytokine secretion.
  • the cell population comprises, or alternatively consists essentially of, or yet further consists of a peripheral blood NK cell and/or a cord blood NK cell.
  • the peripheral blood NK cell and/or the cord blood NK cell expressing any one or more of NCRs, CD56, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2D, NKG2C, and/or NKG2A.
  • the expression levels of the peripheral blood NK cell and/or the cord blood NK cell are different.
  • a method as disclosed herein further comprises enriching cells expressing any one or more of NCRs, CD56, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2D, NKG2C, and/or NKG2A in the cell population.
  • a method as disclosed herein further comprises enriching a peripheral blood NK cell and/or a cord blood NK cell in the cell population.
  • a cell population of any step and/or embodiment and/or aspect may comprise, or alternatively consist essentially of, or yet further consist of natural killer (NK) cells, or substantially purified composition thereof.
  • NK natural killer
  • a cell population of any step and/or embodiment and/or aspect may comprise, or alternatively consist essentially of, or yet further consist of one or more of the following: NK cells, progenitor cells, HSCs, iPSCs or a substantially purified population of each thereof.
  • the NK cells may comprise, or alternatively consist essentially of, or yet further consist of those derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • progenitor cells embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving NK cells.
  • a cell population of any step and/or embodiment and/or aspect is substantially free of T cells. In some embodiments, a cell population of any step and/or embodiment and/or aspect is substantially free of T regulatory cells.
  • Some embodiments of a method as disclosed herein may further comprises isolating, enriching and/or purifying a cell population.
  • a cell population of any step and/or embodiment and/or aspect is isolated, enriched or purified.
  • the cell population and the aAPCs are cultured in a cell culture media selected from StemSpan (Stemcell #09960); NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076-356), TexMACS (170-076-306 GMP medium), Cellgenix Serum-free Stem Cell Growth Medium (SCGM, #20806-0500), or ImmunoCultTM-XF Medium from Stemcell technologies in the culturing step.
  • StemSpan StemSpan
  • NK MACS® Medium Miltenyi research 30-114-429; GMP: 170-076-356
  • TexMACS 170-076-306 GMP medium
  • SCGM Cellgenix Serum-free Stem Cell Growth Medium
  • ImmunoCultTM-XF Medium from Stemcell technologies in the culturing step.
  • Some embodiments of a method as disclosed herein further comprises either or both of formulating a cell population as disclosed herein, such as those expressing a CAR in a composition; and/or cryopreserving a cell population as disclosed herein, such as those expressing a CAR in a composition.
  • Some embodiments of a method as disclosed herein further comprises either or both of: washing the cell population prior to or after one or more of the steps, and/or detecting one or more of the following prior to or during or after one or more of the steps: (i) viability of the cell population; (ii) sterility of the cell population; (iii) mycoplasma in the cell population; (iv) Human Leukocyte Antigen (HLA) type of the cell population; (v) cell numbers of the cell population; (vi) cell phenotype of the cell population; (vii) HHV6 or HHV7 or both in the cell population or a composition comprising the cell population; (viii) one or more of Human immunodeficiency virus (HIV), types 1 and 2, Human T-lymphotropic virus (HTLV), types I and II, Hepatitis B virus (HBV), Hepatitis C virus (HCV), CMV, Zika, West Nile, or Treponema pallidum in the cell population or a composition
  • the phenotype detection comprises detecting cell expression and/or expression level of one or more of the following: CD3, CD56, an antigen such as CD19, CD45, HLA, NKp46, NKG2D, NKG2A, NCRs, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, Adhesion Molecules, NKG2C, CD 107a, CAR, or cell surface markers expressed by the cell population (such as an aAPC and/or the immune cells) y6 T cell Infection and Expansion
  • a method for preparing a population of y6 T cells comprises, or alternatively consists essentially of, or yet further consists of culturing a cell population comprising one or more of the following: a y6 T cell, a progenitor cell that is capable of deriving a y6 T cell, or a stem cell that is capable of deriving a y6 T cell with an immune cell activator(s) (such as a y6 T cell activator).
  • the cell population is depleted with cells that expresses one or more of: T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population.
  • TCR T cell receptor
  • the immune cell activator(s) (such as y6 T cell activator) is selected from one or more of the following: an artificial antigen presenting cell (aAPC) that expresses a tumor associated antigen (TAA) and/or a viral antigen optionally which activate and/or stimulate immune cell growth, one or more of an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the y6 T cell, the progenitor cell or the stem cell, thereby activating or proliferating y6 T cells, one or more of cytokines thereby activating or proliferating y6 T cells, or one or more of chemical moieties thereby activating or proliferating y6 T cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs).
  • aAPC artificial antigen presenting cell
  • TAA tumor associated antigen
  • viral antigen optionally which
  • Some embodiments of a method as disclosed herein further comprises introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide into the cultured cell population for expression.
  • the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • the CAR specifically recognizes and binds to a tumor associated antigen (TAA) and/or a viral antigen.
  • TAA tumor associated antigen
  • the viral antigen recognized and bound by the CAR is the one expressed on the aAPC.
  • Some embodiments of a method as disclosed herein further comprises culturing the cell population after the introducing step with an immune cell activator (such as a y6 T cell activator) or a combination thereof.
  • an immune cell activator such as a y6 T cell activator
  • the culturing step is repeated for once, twice, three times, or more times with the same or different activator(s).
  • a cell population of any step and/or embodiment and/or aspect may comprise, or alternatively consist essentially of, or yet further consist of one or more of the following: y6 T cells or substantially purified composition thereof.
  • a cell population of any step and/or embodiment and/or aspect (such as a depleted cell population as disclosed herein) may comprise, or alternatively consist essentially of, or yet further consist of one or more of the following: y6 T cells, HSCs, iPSCs or a substantially purified population of each thereof.
  • one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving y6 T cells.
  • the y6 T cells comprise, or alternatively consist essentially of, or yet further consist of those derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • the cell population is substantially free of cells expressing TCR a chain or TCR P chain.
  • the tumor associated antigen is selected from one or more of the following or a fragment of each thereof: G Protein- Coupled Receptor Class C Group 5 Member D (GPRC5D), B-cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wildtype epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD 123, CD 19, carcinoembryonic antigen (CEA), CD 133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kina
  • GPC5D G Protein- Coupled Receptor Class
  • a CAR as disclosed herein comprises, or alternatively consists essentially of, or yet further consists of (1) an antigen binding domain of an antibody that specifically recognizes and binds to the antigen; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular domain comprising a signaling domain.
  • the CAR further comprises a signal peptide.
  • an antigen binding domain of the CAR specifically recognizes and binds to one or more of the following: G Protein- Coupled Receptor Class C Group 5 Member D (GPRC5D), B-cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wildtype epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD 123, CD 19, carcinoembryonic antigen (CEA), CD 133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine
  • an antigen binding domain of the CAR comprises six CDRs of an antibody that specifically recognizes and binds to one or more of the following: G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), B-cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wildtype epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL- 1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TA), G Protein-Coupled Receptor Class C Group 5 Member
  • an antigen binding domain of the CAR comprises six CDRs of an antibody or a fragment of each thereof selected from: an anti-EGFRwt and anti-EGFRVIII antibody, an anti-FLT3 antibody, an anti-BCMA antibody and/or an anti -C SI antibody.
  • the hinge domain comprises a CD8 a hinge domain.
  • the transmembrane domain comprises a CD8a transmembrane domain.
  • the intracellular domain further comprises (1) one or two or more costimulatory signaling regions, or (2) IL2RP or a fragment thereof comprising an JAK-STAT activation domain, or both (1) and (2).
  • the costimulatory signaling region comprises a CD28 costimulatory signaling region or a 4- IBB costimulatory signaling region or both.
  • the signaling domain comprises, or alternatively consists essentially of, or yet further consists of a CD3 zeta signaling domain.
  • the introduced polynucleotide further expresses one or more of cytokines and/or antibodies, or wherein the method further comprising introducing another polynucleotide expressing one or more of cytokines and/or antibodies into the depleted cell population.
  • the cytokine expressed by the polynucleotide is selected from one or more of the following: B7.1(soluble or membrane bound), CCL19(soluble or membrane bound), CCL21 (soluble or membrane bound), CD40L(soluble or membrane bound), CD137L(soluble or membrane bound), GITRL(soluble or membrane bound), GM- CSF(soluble or membrane bound), IL-12(soluble or membrane bound), IL-2(soluble or membrane bound), low-toxicity IL-2(soluble or membrane bound), IL-2 analogs that lack cd25 binding (soluble or membrane bound), IL-15-N72D super-agonist along with a IL- 15RaSushi-Fc fusion protein (soluble or membrane bound), IL-15 (soluble or membrane bound), IL- 18 (soluble or membrane bound), IL-21 (soluble or membrane bound), LEC (soluble or membrane bound), OX40L (soluble or membrane bound), IL-7 (soluble or membrane bound), ICOSL (B7H2, B7RP1, soluble or membrane bound), or
  • the antibody expressed by the polynucleotide is a monospecific antibody, or a bispecific antibody, or a multiple specific antibody. Additionally or alternatively, the antibody expressed by the polynucleotide is an immune cell activator, such as an NK cell activator.
  • the prepared CAR-expressing cell population is suitable to inhibit the growth of a cancer cell and wherein the antigen is a tumor associated antigen (TAA) expressed by the cancer cell.
  • TAA tumor associated antigen
  • the polynucleotide encoding a CAR and/or a therapeutic protein or polypeptide (which is also referred to herein as a coding polynucleotide) further encodes a signal peptide. Additionally or alternatively, the polynucleotide encoding a CAR and/or a therapeutic protein or polypeptide further comprises a suicide gene.
  • the suicide gene product is selected from one or more of: HSV-TK (Herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, carboxylesterase, cytochrome P450 or PNP (Purine nucleoside phosphorylase), truncated EGFR, or inducible caspase (“iCasp”).
  • HSV-TK Herpes simplex virus thymidine kinase
  • cytosine deaminase cytosine deaminase
  • nitroreductase carboxylesterase
  • cytochrome P450 or PNP Purine nucleoside phosphorylase
  • truncated EGFR or inducible caspase (“iCasp”).
  • the coding polynucleotide further comprises a regulatory sequence directing expression of the suicide gene.
  • the regulatory sequence is inducible.
  • the coding polynucleotide further comprises a regulator sequence directing the expression of the CAR or the therapeutic protein or polypeptide. In further embodiments, the regulator sequence directing expression of the CAR or the therapeutic protein is inducible or constitutively active.
  • the coding polynucleotide is introduced to the cell population via a vector.
  • the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is a plasmid.
  • the viral vector is selected form a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno- associated viral vector or Herpes viral vector.
  • the viral backbone contains essential nucleic acids or sequences for integration of the coding polynucleotide into a target cell’s genome.
  • the essential nucleic acids necessary for integration to the genome of the target cell include at the 5’ and 3’ ends the minimal LTR regions required for integration of the vector.
  • This disclosure also provides a vector comprising, or alternatively consisting essentially of, or yet further consisting of a polynucleotide (such as coding polynucleotide) as disclosed herein, optionally inserted into a viral backbone.
  • the vector is selected for expression in prokaryotic or eukaryotic cells.
  • the vector comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide as described herein, encoding the modified protein.
  • the vector comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide as described herein, permitting replication of the polynucleotide.
  • the vector further comprises a regulatory sequence operatively linked to the polynucleotide and directing the replication of the polynucleotide.
  • the regulatory sequence comprises, or alternatively consists essentially of, or yet further consists of one or more of: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).
  • WP Woodchuck Hepatitis Virus
  • a coding sequence is introduced into a target cell via a pseudotyped gammaretroviral particle as disclosed herein.
  • the disclosure also provides a viral packaging system comprising: a vector as described herein, optionally wherein the backbone is derived from a virus; a packaging plasmid; and an envelope plasmid.
  • the packaging plasmid contains polynucleotides encoding the nucleoside, matrix proteins, capsids, and other components necessary for packaging a vector genome into a viral particle.
  • Packaging plasmids are described in the patent literature, e.g., U.S. Patent Nos. 7,262,049; 6,995,258; 7,252,991 and 5,710,037, incorporated herein by reference.
  • the system may also contain a plasmid encoding a pseudotyped envelope protein provided by an envelope plasmid.
  • Pseudotyped viral vectors consist of vector particles bearing glycoproteins derived from other enveloped viruses or alternatively containing functional portions. See, for example U.S. Patent No. 7,262,049, incorporated herein by reference.
  • the envelope plasmid encodes an envelope protein optionally not causing the viral particle to non-specifically bind to a cell or population of cells.
  • the specificity of the viral particle may be conferred by a protein or polypeptide, such as an antibody binding domain, that is inserted into the particle envelope.
  • suitable envelope proteins include, but are not limited to those containing the VSVG or RD114 domains.
  • envelope proteins used herein include, but are not limited to RD114TR and/or BaEVTR.
  • the packaging cell line is the HEK-293 cell line.
  • suitable cell lines are known in the art, for example, described in the patent literature within U.S. Patent Nos. 7,070,994; 6,995,919; 6,475,786; 6,372,502; 6,365,150 and 5,591,624, each incorporated herein by reference.
  • the packaging cell line is selected from or is derived from one or more of: 293Vec-Galv, 293Vec-Ampho, 293Vec-RD114, or 293 Vec-BaEV. See, for example, Ghani et al., Gene Ther.
  • This disclosure further provides a method for producing a viral particle as disclosed herein comprising, or alternatively consisting essentially of, or yet further consisting of, transducing a packaging cell line with a viral system as described above, under conditions suitable to package the viral vector. Such conditions are known in the art and briefly described herein.
  • the viral particle can be isolated from the cell supernatant, using methods known to those of skill in the art, e.g., centrifugation. Such isolated particles are further provided by this disclosure.
  • This disclosure further provides the isolated viral particle produced by this method.
  • the viral particle comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide as disclosed herein.
  • This disclosure also provides methods to prepare a viral particle (e.g., a gammaretroviral particle) comprising a polynucleotide as disclosed herein, such as a coding polynucleotide as disclosed herein by transducing a packaging cell line, as described herein with the vector, the envelope plasmid and the packaging plasmid under conditions that facilitate packaging of the vector into the envelope particle.
  • a viral particle e.g., a gammaretroviral particle
  • the viral particle is a pseudotyped viral particle.
  • the particles are separated from the cellular supernatant and conjugated to an antibody for cell-specific targeting.
  • a method for producing a retroviral particle comprises, consists essentially of, or yet further consists of (i) introducing a vector expressing a vector genome into a first packaging cell line suitable for packaging the vector genome into a first retroviral particle, (ii) transducing the first retroviral particle into a second packaging cell line suitable for replicating the first retroviral particle; and (iii) isolating the replicated retroviral particle.
  • the vector is a non-viral vector. In further embodiments, the vector is a plasmid.
  • the method further comprises culturing the first packaging cell line introduced with the vector. In further embodiments, the method further comprises isolating the first retroviral particle from the culture of the first packaging cell line introduced with the vector, such as from the supernatant.
  • the method further comprises culturing the transduced second packaging cell line.
  • a vector is produced using two steps:
  • Step 1 a plasmid (that expresses a retroviral vector genome) transfects 293 Vec- GALV cells to produce a retroviral vector.
  • a retroviral vector is enveloped with a lipid membrane comprising GALV.
  • the produced retroviral vector is also referred to as a transient vector and was tested in FIG. 10.
  • Step 2 The produced retroviral vector transduces 293 Vec-BaEV cells to replicate the retroviral vector.
  • the replicated retroviral vector has the same vector genome with the parent one, but is enveloped with a lipid membrane comprising BaEV.
  • the replicated retroviral vector here is also referred to as a stable vector and was tested in FIG. 10.
  • T cells (transduced with the RQR8-encoded retroviral vector genome) express RQR8 at a higher level using the stable vector compared to the transient vector.
  • these stable virus producer cells also referred to as producers
  • the protein product encoded by the transgene is non-toxic. Therefore, without wishing to be bound by the theory, the producer cells are just like the parental packaging cell line, which can be continually cultured in vitro for as many generations as they are in log growth phase. In some embodiments, the producers can be kept in culture in vitro for no more than 30 generations.
  • the cells are cultured for at least about 1 days, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Additionally or alternatively, the cells are cultured for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks, or no more than 1 month. In some embodiments, the cells are cultured for about 1 days to about 180 days, including any ranges and/or numbers falling therein. In some embodiments, the cells are cultured for about 5 days to about 10 days, such as about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
  • the first packaging cell line and the second packaging cell line expresses components required for packaging a retroviral particle.
  • the component required for packaging a retroviral particle is selected from one or more of: a retroviral gag, a retroviral pol, a retroviral env, a fragment of each thereof, or any combination thereof.
  • the second packaging cell line comprises a retroviral envelope protein in the cell membrane but does not comprises the entry receptor of the retroviral envelope protein in the cell membrane.
  • the cell stably produce the retroviral particle.
  • the retroviral envelope protein is BaEV and its entry receptor is ASCT1 or ASCT2. In some embodiments, the retroviral envelope protein is RD114, and its entry receptor is ASCT2. In some embodiments, the retroviral envelope protein is GALV and its entry receptor is Pit 1.
  • the first packaging cell line comprises an entry receptor of a retroviral envelope protein of the first retroviral particle. In other embodiments, the first packaging cell line does comprise an entry receptor of a retroviral envelope protein of the first retroviral particle.
  • the vector is introduced to the first packaging cell line at a multiplicity of infection (MOI) of about 0.01 to about 100, including any ranges and/or numbers falling therein.
  • MOI multiplicity of infection
  • the vector is introduced to the first packaging cell line at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5.
  • the vector is introduced to the first packaging cell line at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10.
  • the first retroviral particle is introduced (such as transduced) to the second packaging cell line at a multiplicity of infection (MOI) of about 0.01 to about 100, including any ranges and/or numbers falling therein.
  • MOI multiplicity of infection
  • the first retroviral particle is introduced (such as transduced) to the second packaging cell line at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5.
  • the first retroviral particle is introduced (such as transduced) to the second packaging cell line at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10.
  • a method for producing an engineered immune cell such as a CAR expressing immune cell.
  • the method comprises, or consists essentially of, or yet further consists of introducing (such as transducing) the retroviral particle produced using a method as disclosed herein into an immune cell or a precursor cell thereof.
  • the genetic information of the viral vector particle (which is also referred to herein as a vector genome or a viral genome) is RNA which comprises, or alternatively consists essentially of, or yet further consists of, on the 5’ and 3’ ends, the minimal LTR regions required for integration of the vector, and a polynucleotide as disclosed herein between the two LTR regions.
  • between the two LTR regions further comprises an encapsidation signal (a psi region) which is required for packaging of the vector RNA into the particle.
  • a psi region is followed by a Rev- Responsive Element (RRE) and a central polypurine tract sequence (cPPT) that enhance vector production by transporting the full-length vector transcript out of the nucleus for efficient packaging into the vector particle.
  • RRE Rev- Responsive Element
  • cPPT central polypurine tract sequence
  • the listed genetic elements are transcribed into a full-length RNA molecule which is packaged into a vector particle and contains all of the genetic information that will be integrated into the transduced cells.
  • the full-length RNA transcript is packaged inside the capsid of the vector particle that contains the nucleocapsid, capsid, and matrix proteins which are generated from the packaging plasmid.
  • the reverse transcriptase polymerase which is generated from the packaging plasmid is also located within the capsid with the RNA transcript.
  • the capsid encases and protects the full- length RNA transcript.
  • cells of a packaging cell line such as HEK-293T cells are plated at 75% confluence in complete DMEM media 24 hours prior to transfection. After at least 24 hours post-plating of cells, the transfection mixture is prepared. Three milliliters of serum free media are incubated with 150 pl of the lipofection reagent for 20 minutes at room temperature. The plasmids are then added to the media/lipofection reagent mixture at a certain ratio (packaging plasmid: viral vector plasmid: envelope plasmid) and incubated for 30 minutes.
  • the media/lipofection reagent/DNA mixture is then added to the HEK-293T cells and left overnight for the transfection to occur. The next day, the transfection media is removed and fresh complete DMEM is added. Seventy-two hours later, the cell culture supernatant can be collected and concentrated by ultracentrifugation at 20,000 rpm for 1.5 hours. [0303] Once the vector particle buds from the packaging cells and is released into the supernatant, this vector particle can be isolated and/or purified by an antibody specifically recognizes or binds the particle and/or by having a conjugated antibody on the envelope of the particle as defined herein.
  • a viral packaging system for producing a pseudotyped gammaretroviral particle, comprising: (a) a plasmid expressing a vector genome; (b) a packaging plasmid; and (c) one or more of envelope plasmids expressing RD114TR, and BaEVTR.
  • the packaging system further comprises a packaging cell line.
  • the packaging cell line is a 293T cell line.
  • the vector genome comprises one or more of the following flanked by two long terminal repeats (LTRs): (A) a polynucleotide encoding a chimeric antigen receptor (CAR) and/or another therapeutic protein or polypeptide, (B) a reversecomplement of (A), or (C) a polynucleotide comprising one or more of recognition sites recognized by a restriction enzyme suitable for inserting a coding sequence or a reversecomplement thereof into the polynucleotide.
  • the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor.
  • the vector genome further comprises one or more of the following: a 5’ LTR, a 5’ cap, a 3’ poly -A tail, and a 3’ LTR.
  • the coding polypeptide is located between the 5’ cap and the3’ poly-A tail.
  • the pseudotyped gammaretroviral particle is based on and/or derived from and/or selected from any species of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • XMRV xenotropic murine leukemia virus-related virus
  • a method for producing a pseudotyped gammaretroviral particle comprises, or alternatively consists essentially of, or yet further consists of transducing a packaging cell line with a packaging system as disclosed herein under conditions suitable to package the pseudotyped gammaretroviral particle.
  • the packaging cell line is a 293T cell line.
  • the cell line is transduced with plasmids of (a), (b) and (c), i.e., (viral vector plasmid: packaging plasmid: envelope plasmid) at a ratio of about 5:5: 1, 5:4: 1, 5:3:1, 5:2:1, 5:1:1, 4:5:1, 3:5:1, 2:5:1, 1:5:1; 4:4:1, 4:3:1, 4:2:1, 4:1:1, 3:4:1, 2:4:1, 1:4:1; 3:3:1, 3:2:1, 3:1:1, 2:3:1, 1:3:1; 2:2:1, 2:1:1, 1:2:1; 1.5:15:1, 15:1:1, 1:15:1; or 1:1:1.
  • viral vector plasmid packaging plasmid: envelope plasmid
  • the packaging system comprises at least two envelope plasmids, one expressing RD114TR and the other expressing BaEVTR.
  • the RD114TR expressing plasmid and the BaEVTR expressing plasmid are transduced at a ratio of 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, or 1:3.
  • the cell line is transduced with the plasmid of (a), the plasmid of (b), the RD114TR expressing plasmid, and the BaEVTR expressing plasmid at a ratio of 3 :3 : 1 : 1, 2:2: 1 : 1, 1.5:1.5:1:1, 1 : 1 : 1 : 1, 5 :5: 1 : 1, 5:4:1:1, 5:3:1:1, 5:2:1:1, 5:1:1:1, 4:5:1:1, 3:5:1:1, 2:5:1:1, 1:5:1:1; 4:4:1:1, 4:3:1:1, 4:2:1:1, 4:1:1:1, 3:4:1:1, 2:4:1:1, 1:4:1:1; 3:3:1:1, 3:2:1: 1, 3:1:1:1, 2:3:1:1, 1:3:1:1; 2:2:1:1, 2:1:1:1, 1:2
  • a cell population as disclosed herein and/or a progeny thereof is provided.
  • the cell population is clonal.
  • the cell population is isolated and/or enriched and/or engineered.
  • an immune cell such as an NK cell and/or a y6 T cell
  • a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier.
  • the immune cell or population thereof is isolated and/or enriched and/or engineered.
  • the cell as disclosed herein are derived or differentiated from a stem cell.
  • the derived and/or differentiated cell and/or the cell population thereof comprise, or consist essentially of, or yet further consist of an immune cell.
  • the immune cell is selected from a B-cell, T-cell, Natural Killer (NK) cell, Natural Killer T (NKT) cell, dendritic cell, a cell of the myeloid lineage, and/or a neutrophil.
  • the T cell does not express CD3, i.e. is a CD3- T cell.
  • the T cell does not express CD4, i.e. is a CD4- T cell.
  • the T cell does not expresses CD8, i.e. is a CD8- T cell. In some embodiments, the T cell does not express any one or more of: T cell receptor (TCR) a chain, TCR P chain, or aPTCR. In some embodiments, the T cell expresses TCR y chain. Additionally or alternatively, the T cell expresses TCR 6 chain. In some embodiments, the T cell is a y6 T cell. In some instances, the isolated cell or an enriched population of immune cells comprise, or consist essentially of, or yet further consist of, a monocyte, a macrophage, and/or a microglia.
  • the cell population comprises substantially an immune cell, optionally derived from a stem cell such as a HSC, and/or an induced pluripotent stem cell (iPSC).
  • a stem cell such as a HSC
  • iPSC induced pluripotent stem cell
  • the cell population comprises substantially a stem cell, such as HSCs, and/or an iPSC, optionally deriving to an immune cell.
  • the cell population is substantially homogenous, for example, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92% , or at least about 93% , or at least about 94% , or at least about 95% , or at least about 96% , or at least about 97% , or at least about 98% , or at least about 99% of the cells in the population are the same.
  • an NK cell or a population thereof produced or prepared by a method as disclosed herein.
  • a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier.
  • a y6 T cell or a population thereof produced or prepared by a method as disclosed herein.
  • a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier.
  • the cell and/or a population thereof, and/or the composition as disclosed herein comprise less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6% , less than about 0.7% , less than about 0.8% , less than about 0.9% , less than about 1.0% , less than about 1.1% , less than about 1.2% , less than about 1.3% , less than about 1.4% , less than about 1.5% , less than about 1.6% , less than about 1.7% , less than about 1.8% , less than about 1.9%, or less than about 2.0% aAPCs.
  • the cell and/or a population thereof, and/or the composition as disclosed herein are no less than 30%, no less than 40%, no less than 50%, no less than 55%, no less than 60%, no less than 65%, no less than 70%, no less than 75%, no less than 80%, no less than 85%, no less than 90%, or no less than 95% viable;
  • the cell and/or a population thereof, and/or the composition as disclosed herein are sterile.
  • the cell and/or a population thereof, and/or the composition as disclosed herein comprises less than a threshold pyrogenic dose (TPD) of endotoxin, which represents a statistical assessment of the levels of endotoxin activity that it takes to induce a fever in a test subject (such as a rabbit, see, for example, Wachtel and Tsuji, 1976; Dabbah, et al, 1980).
  • TPD pyrogenic dose
  • TPD may be determined based on administration routes.
  • the TPD is 5 EUKg -1 h -1 endotoxin, such as for intravenous or intramuscular administration.
  • the TPD is 0.2 EUKg -1 h -1 endotoxin, such as for intrathecal administration.
  • the cell and/or a population thereof, and/or the composition as disclosed are negative for one or more of: mycoplasma, Adventitious viruses, or HHV (such as HHV6 and/or HHV7).
  • mycoplasma such as HHV6 and/or HHV7.
  • HHV such as HHV6 and/or HHV7.
  • the cell population and/or the composition as disclosed herein comprise more than about 25%, more than about 30%, more than 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, or more than about 95% cells expressing the CAR among the total cells.
  • the cell population and/or the composition as disclosed herein comprise about 25%, about 30%, 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% cells expressing the CAR among the total cells.
  • the cell population and/or the composition as disclosed herein comprise more than about 25%, more than about 30%, more than 40%, more than about 45%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 99% cells expressing CD56 among the total cells.
  • the cell population and/or the composition as disclosed herein comprise about 25%, about 30%, 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% cells expressing CD56 among the total cells.
  • the cell population and/or the composition as disclosed herein comprise less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6% , less than about 0.7% , less than about 0.8% , less than about 0.9% , less than about 1.0% , less than about 1.1% , less than about
  • the cell and/or a population thereof, and/or the composition as disclosed herein comprises less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8, less than about 9, or less than about 10 copies of the polynucleotide per cell.
  • the cell and/or a population thereof, and/or the composition as disclosed herein lack proliferation in absence of IL-2 and/or IL-21.
  • the cell and/or a population thereof, and/or the composition as disclosed herein have a potent ability (i.e., potency), for example, at s more than 1-fold, more than 2-fold, more than 3-fold, more than 4-fold, more than 10-fold increase, more than 15- fold, more than 20-fold or higher level compared to that of an immune cell not prepared by any culturing step and/or introducing step as disclosed herein.
  • potency may be measured as secretion of IFNy and/or other proinflammaotry cytokines and/or expression of CD107 (such as CD107a). See, for example, Orange et al. J Exp Med. 1995 Oct 1; 182(4): 1045-56; and McElroy et al. J Immunol Methods. 2007 Dec l;328(I-2):45-52.
  • the cell and/or a population thereof, and/or the composition as disclosed herein express one or more of an antigen such as CD19, NKp46, or NKG2D.
  • these cells, cell populations, and/or compositions are useful to treat and/or prevent a cancer in a subject in need thereof or to test new therapies.
  • the cell and/or a population thereof, and/or the composition as disclosed herein, such as those administered to a subject in need thereof comprises, or alternatively consists essentially of, or yet further consists of more than 1 x 10 6 cells per kilogram of body weight of the subject to be treated, including but not limited to more than 2 x 10 6 cells per kilogram, more than 3 x 10 6 cells per kilogram, more than 4 x 10 6 cells per kilogram, more than 5 x 10 6 cells per kilogram, more than 6 x 10 6 cells per kilogram, more than 7 x 10 6 cells per kilogram, more than 8 x 10 6 cells per kilogram, more than 9 x 10 6 cells per kilogram, more than 1 x 10 7 cells per kilogram, more than 2 x 10 7 cells per kilogram, more than 3 x 10 7 cells per kilogram, more than 4 x 10 7 cells per kilogram, more than 5 x 10 7 cells per kilogram, more than 6 x 10 7 cells per kilogram, more than 7 x 10 7 cells per kilogram, more than 8 x 10 7
  • a method for inhibiting the growth of a cancer cell comprises, or alternatively consists essentially of, or yet further consists of contacting, for example an effective amount of, a population of CAR-expressing cells prepared by a method as disclosed herein with the cancer cell.
  • the antigen recognized by the CAR is a tumor associated antigen (TAA) expressed by the cancer cell.
  • TAA tumor associated antigen
  • the contacting is in vivo or in vitro.
  • a method for treating a cancer in a subject comprises, or alternatively consists essentially of, or yet further consists of administering, for example an effective amount of, a population of the CAR-expressing immune cells prepared by a method as disclosed herein to the subject.
  • the antigen recognized by the CAR is a TAA expressed by a cell of the cancer.
  • the administration is a first line therapy, a second line therapy, a third line therapy, or a fourth line therapy.
  • the cell population comprises, or alternatively consists essentially of, or yet further consists of NK cells. Additionally or alternatively, the cell population comprises less than or equal to about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0% CD3+ cells.
  • the subject is administered with less than 1 x 10 2 , 2 x 10 2 , 3 x
  • the cancer cell is selected from cancer cells of the: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), al
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • bronchogenic carcinoma squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinom
  • the cancer cell is a solid tumor cell. In other embodiments, the cancer cell is not a cell of a solid tumor. In further embodiments, the cancer cell is a leukemia cancer cell. In some embodiments, the cancer cell is a primary cancer cell or a metastatic cancer cell. In some embodiments, the cancer cell is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma.
  • Additional effective therapies can be combined with this disclosure and/or added as necessary.
  • Some embodiments of a method as disclosed herein further comprises contacting the cell with or administering a separate therapy.
  • the separate therapy comprises, or alternatively consists essentially of, or yet further consists of surgical resection, chemotherapy, radiation therapy, immunotherapy and targeted therapy.
  • the separate therapy is a first line therapy, a second line therapy, a third line therapy, or a fourth line therapy.
  • an “effective amount” is delivered, that is it is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration.
  • Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration.
  • administration shall include without limitation, local or systemic administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), intracranial, or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant
  • the disclosure is not limited by the route of administration, the formulation or dosing schedule.
  • the administration is performed locally, such as to the bone marrow or in the brain.
  • the administration is performed systemically.
  • the administration is an infusion, for example over about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, or about 1 day.
  • kits comprising, or alternatively consisting essentially of, or yet further consisting of one or more of agents suitable for use in a method as disclosed herein and an optional instruction.
  • the agents are selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting cell phenotype, an antibody for isolating or enriching or purifying immune cells, primers for detecting the polynucleotide, cytokines, and an aAPC as disclosed herein.
  • PCIR Gamma Retroviral vector
  • PCIR Gamma Retroviral vector
  • CARs chimeric antigen receptors
  • Lentivirus and Retrovirus vector mediated gene delivery have been proven a safety way for genetic engineering.
  • the conventional lentiviral vectors mediated transduction is inefficient for NK cells due to transgene expression dramatically reduced during CAR-NK expansion. See, for example, FIGS. 5A and 5C. Therefore, much endeavors Applicant put aimed to find an alternative gene delivery method to overcome the inefficiency of Lentivirus vector.
  • the primary human NK cells used in this disclosure were peripheral blood (PB)-derived, and isolated with MACSxpress Whole Blood NK Cell Isolation Kit, human (Miltenyi Biotec ,130-098-185). The purity was determined via staining the cells with CD56 and CD3 antibody (FIG. 2A), and NK cells were defined as CD56+/CD3- population.
  • NK cells were cultured with NK MACS media (130-114-429) at presence of 50 lU/ml of human IL-2 and Irradiated K562-mb21-41BBL feeders with the ratio of 1 : 1.
  • PCIR gammaretrovirus plasmids
  • PEQ-PEM3(-E) All the gammaretrovirus plasmids (PCIR) were packaged with PEQ-PEM3(-E) and enveloped with BaEVTR or RD114TR plasmids at 1.5: 1.5: 1 ratio and transiently transfected into 293T cells (ATCC CRL-3216) via lipofectamine 3000 reagent (InvitrogenTM L3000075) or PEI MAX 40K (Polysciences, Inc, Cat #24765).
  • BaEVTR plasmids are practically benefit for the larger inserts delivery to activated NK cells who expressed two virus entry receptors ASCT-1 and ASCT-2 compared to ASCT-2 recognized by RD114TR.
  • retronectin reagent TakaRa, T100A/B coated non-tissue culture plate
  • Applicant performed directly titration determination with infection of above retrovirus particles into activated NK cells (cultured 7 days) and the MOI 3 was used for CAR-NK generation.
  • the primary NK cells entered a marked proliferative state, therefore the activated NK cell at day 6-10 were used for gene delivery (FIG. 2B).
  • Retrovirus titration based on direct infection of activated NK cells at same proliferate state with same deliver strategy is provided in FIG. 4.
  • Applicant optimized the time point for retroviral transduction of primary NK cells expanded. Due to the ability of gamma-retrovirus to efficiently infect dividing cells, Applicant determined the Growth Curve and time point when the primary NK cells entered a marked proliferative state for gene delivery.
  • RetroNectin reagent has proven can enhance retroviral-mediated gene transduction by aiding the co-localization of target cells and virions. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptor VLA-5 and VLA-4 with the fibronectin C-domain and CS-1 site, respectively. By facilitating proximity, RetroNectin reagent can enhance retroviral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and/or VLA-5.
  • Applicant determined the retrovirus titration based on direct infection of activated NK cells at same proliferate state with same delivery strategy, aimed to avoid overestimating or underestimating the titration calculated from indirect measurement method based on different cell types.
  • RetroNectin The findings as disclosed herein present the use of RD114-TR or BaEV-TR pseudotyped gamma retroviral particles in combination with RetroNectin is a successful strategy to deliver therapeutic genes to human primary NK cells with high transduction efficiency, sustained transgene expression, and great cell viability and expansion rates.
  • NK cells were collected from umbilical cord blood of healthy newborns. Selection of best NK cell candidates were performed as disclosed. NK cell were then expanded using the feeder cells (such as an aAPCs as disclosed herein). The expanded NK cells undergo transduction to express CAR and/or other components, such as suicide switch, antibody and cytokine. Continued ex vivo expansion was also driven by exogenous IL-2. The final cell product was then harvested, cryopreserved, and thawed with high viability for off-the-shelf administration to patients.
  • the feeder cells such as an aAPCs as disclosed herein.
  • the expanded NK cells undergo transduction to express CAR and/or other components, such as suicide switch, antibody and cytokine. Continued ex vivo expansion was also driven by exogenous IL-2.
  • the final cell product was then harvested, cryopreserved, and thawed with high viability for off-the-shelf administration to patients.
  • such methods allow to select the best umbilical cord blood product for NK cell expansion.
  • Co-culture with the aAPCs as disclosed herein achieves massive NK cell expansion.
  • the production method drastically reduces quantity of requisite retrovirus and results in 20% reduction in time for NK cell expansion.
  • High transduction efficiency of expressing CAR is achieved.
  • Cell therapies process development studies
  • NK growth and/or amplification including, but not limited to, StemSpan (Stemcell #09960), NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076-356), TexMACS (170-076-306 GMP medium), and Cellgenix Serum-free Stem Cell Growth Medium (SCGM, #20806-0500).
  • StemSpan Stemcell #09960
  • NK MACS® Medium Miltenyi research 30-114-429; GMP: 170-076-356
  • TexMACS 170-076-306 GMP medium
  • SCGM Cellgenix Serum-free Stem Cell Growth Medium
  • Cytokine additives are compared in order to optimize yield, such as one or more of the following: 100-500 U/ml IL-2, 20 ng/ml IL- 15, 25 ng/mL IL-21, or any combination thereof.
  • NK cell enrichment was performed. The following commercially available kits are tested and compared: Stemcell NK depletion EasySep NK Isolation kit (17955RF), RosetteSep NK enrichment Cocktail (#15065) with Miltenyi NK isolation kit (130-092-657) and CD56 + NK Cell Isolation Kit (130-092-660).
  • kits are tested for reducing CD3+ cells to ⁇ 0.3% (i.e., less than 0.3% of the depleted cell population.
  • Those kits include, e.g., Miltenti 130-096-535.
  • K562-engineered cells such as those expressing membrane-bound IL- 15 (sushi domain) and/or IL-21 were generated.
  • Master cell bank (MCB) and/or working cell bank (WCB) of the engineered cells are developed according to the Current Good Manufacturing Practice.
  • release testing of K562 MCB and WCB banks are completed, including but not limited to testing one or more of the following: sterility, endotoxin, mycoplasma, adventitious virus testing (in vitro and in vivo), PCR-based virus testing, isoenzyme assay, karyotyping, S + L" etc.
  • stability of K562 marker co-expression is determined, e.g., CD64/FcyRI, CD86/B7-2, CD137L/4-1BBL, truncated CD19 and membrane-bound IL- 21 over 4-week culture time and after irradiation (21 days culture). Irradiation of K562 cells is tested to ensure that irradiator is qualified/ validated.
  • Kinetics of growth and/or viability are tested after plating post-irradiation day 1-14 at 50, 100, 150 Gy irradiation.
  • Expansion of cord blood derived NK cells is tested using irradiated K562 cells at various ratios ofNK: K562, e.g., 1 :2; 1 : 1; 1 :3, 1 :5 ratios.
  • Transduction efficiency is compared using AFC bags coated with RetroNectin compared to RetroNectin-coated plates. [0367] The process is then scaled up, for example via comparing G-Rex, Xuri, Bioflow and Xcellerex bioreactor systems.
  • CD56+ cells More than 1 x 10 11 CD56+ cells were generated starting from 1 x 10 8 CD56+ cells. Test was performed initially using small cultures to define growth and amplification parameters and confirm functionality. Without wishing to be bound be the theory, cells lose potency with high level amplification, thus the balance between cell amplification and function is a key consideration for scale-up.
  • Antibodies/ reagents used include, but are not limited to: Anti-CD56 Brilliant Violet 605 (BioLegend, San Diego, CA), anti-CD3 APC-H7, granzyme B-PE-CF594, CCR4- BV421, CXCR3-PerCP Cy5.5, NKp46-BV711 (BD Biosciences, San Jose CA), CD57-PerCP (Bioss Woburn MA), anti-CD16 Brilliant Violet 650, and anti-CD19 PE (Miltenyi Biotec Inc ), CD44-BV785, CXCR4-BV605, 2B4-PE, NKG2D-PE, DNAM-FITC, TbetBV711, CD16-BV650, CX3CR1-PE-Cy7, CD62L-PE-Cy7, CXCR1-PE, CCR7-FITC, PD-1-BV421, NKp30-Biotin (from Biolegend), NKp44-PerCP-eflour 710,
  • K562 Testing are performed, including but not limited to, creating bank of clonal K562-gene modified cells (if cells are not clonal, then re-cloning); testing bank for viability, sterility, mycoplasma, endotoxin, HLA, and/or genetically modified surface markers (e.g., mb IL-15, mb IL-21, CD64, CD86, CD137L etc.); developing a Certificate of Analysis for K562-gene modified cells; ensuring irradiator is calibrated and dose administration is validated, and testing irradiation of cells at 100 Gy, 200 Gy and test for proliferation/ survival over 14 days Table 2 Exemplified phenotype analysis
  • Donor eligibility screening and testing for allogeneic products were performed, including but not limited to testing one or more of the following: Human immunodeficiency virus (HIV), types 1 and 2; Human T-lymphotropic virus (HTLV), types I and II; Hepatitis B virus (HBV); Hepatitis C virus (HCV); CMV, Zika, West Nile; Treponema pallidum; HHV6/ HHV7.
  • HSV Human immunodeficiency virus
  • HBV Human T-lymphotropic virus
  • HBV Hepatitis B virus
  • HCV Hepatitis C virus
  • CMV Zika, West Nile
  • Treponema pallidum HHV6/ HHV7.
  • Cord blood contains approximately 5 x 10 8 total nucleated cells (TNCs), of which 30% are NK cells. Thus, each cord blood unit (60 mL) contains about 1 ⁇ 2 x 10 8 NK cells.
  • NK purity was tested. Without wishing to be bound by the theory, it is critical to reduce T cells from NK product since patients develop acute graft versus host disease ( GvHD > grade II when receiving >0.5 x 10 5 T cells/kg body weight and NK cell doses are well tolerated when T-cell contamination is 0.03 x 10 5 T cells/kg body weight, therefore define acceptance criterion of ⁇ 0.3% CD3+ cells. See, for example, Stern M, Passweg JR, Meyer-Monard S, et al. Preemptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective Phase II study in two centers. Bone Marrow Transplant 2013;48:433-438.
  • CAR+ and CAR- target cells are tested with CAR- NK cells using ELISA for IFN-gamma, IL-15 and TNF-alpha; and/or assays for cytotoxic activity (such as killing assay, degranulation assay, such as FACS for CD 107a degranulation assay, and FACS for intracellular cytokines IFN-gamma and/or TNF-alpha).
  • assays for cytotoxic activity such as killing assay, degranulation assay, such as FACS for CD 107a degranulation assay, and FACS for intracellular cytokines IFN-gamma and/or TNF-alpha.
  • Retrovirus entry receptors expression was evaluated on 293 Vec-GalV and 293 Vec- BaEV packaging cells, and two cell lines Jurkat T and HT1080 used for titration.
  • the baboon endogenous virus (BaEV) and feline endogenous retrovirus (RD114) use a common cell-surface receptor ASCT2 (sodium-dependent neutral amino acid transporter) for cell entry.
  • ASCT2 sodium-dependent neutral amino acid transporter
  • BaEV also uses ASCT1 as a cell entry receptor.
  • GLV Gibbon ape leukemia virus
  • itl Sodium-Dependent Phosphate Transporters
  • a workflow of transduction strategy for generation of stable retrovirus virus producer-293 VEC-BaEV is provided herein.
  • moloney murine leukemia virus Mo-MuLV
  • PCJR retroviral vector
  • BaEV pseudotyping 293 Vec-BaEV packaging cell line was used for vehicle production to infect Cord blood derived NK cells.
  • Two packaging cell lines were used for this production: 293 Vec-GALV to produce transient gibbon ape leukemia virus (GALV)-pseudotyped supernatant and 293 Vec-BaEV to generate the final vector. Both cell lines were supplied by BioVec Pharma.
  • GALV-pseudotyped retroviral supernatant was generated by transient transfection of the packaging cell line 293 Vec-GALV with the reagent PEI MAX 40K (Polysciences, Inc, Cat #24765). Briefly, on the day before transfection, 0.6 million of 293 Vec-GALV was seeded onto a well of 6-well plate in 3 ml of complete DMEM medium supplied with 10% heat inactivated fetal bovine serum. On the transfection day, the 293 Vec- GALV should be 80% confluence.
  • a total of 2.5 pg of a single Mo-MuLV-based retroviral vector containing a target transgenes was diluted in 150 pl of Opti-MEMTM Reduced-Serum Medium. Meanwhile, a total of 10 pg/lOpl of PEI MAX 40K was also diluted in 150 pl of Opti-MEMTM Reduced-Serum Medium. The ratio of plasmid/DNA and PEI MAX 40K is 1 :4. Then the diluted DNA was added to PEI 40K, mixed well, and incubated for 12 minutes in a hood at room temperature. After the incubation, the 300 pl of DNA-PEI MAX 40K complex was added gently drop by drop to cells in 3 ml culture.
  • This supernatant was then used to transduce a BaEV pseudotyping packaging cell line 293Vec-BaEV to generate a bulk producer.
  • this 293Vec- BaEV bulk producer can be further single-cell cloned by limiting dilution and screening for a high-titer clone selected by titration of the supernatant generated from each clone.
  • the supernatant can be continually produced from the high titer stable retrovirus virus producer-293 VEC-BaEV.
  • the serials of virus supernatant were loaded on the RetroNectin precoated non tissue culture treated 24 well plate and brought to 500 pl with complete DMEM medium supplied with 10% heat inactivated fetal bovine serum.
  • the plate was spun down at 32 degrees, 2000g for 2 hours, and 0.1 million of Jurkat T cells were added, and spun at 1000g for 5 min.
  • a reporter gene RQR8 was detected by flow antibodies against human CD34 Antibody (QBEnd/10) (Allophycocyanin) (Novus Biologicals, LLC, #FAB7227A).
  • QBEnd/10 Allophycocyanin
  • IgGl hinge domain LEPKSCDKTHTCPPCPDPKGT (SEQ ID NO: 1)
  • CD28 transmembrane and cytoplasmic domain [0391] CD28 transmembrane and cytoplasmic domain:
  • CD3 zeta signaling domain [0392] CD3 zeta signaling domain:
  • IL 2 signal peptide MYRMQLLSCIALSLALVTNS (SEQ ID NO: 4),
  • IgGl signal peptide MGWSSIILFLVATATGVH (SEQ ID NO: 5)
  • CDRL1 :SGSSSNIGNNAVN SEQ ID NO: 6
  • CDRL2 YDDLLPS (SEQ ID NO: 7)
  • CDRL3 AAWDDSLNGPV (SEQ ID NO: 8)
  • CDRH2 RYDGSN (SEQ ID NO: 10)
  • Anti-NKG2D light chain variable region [0396] Anti-NKG2D light chain variable region:
  • Anti-NKG2D heavy chain variable region [0397] Anti-NKG2D heavy chain variable region:
  • Peptide linker GGGGSGGGGSGGGGS (SEQ ID NO: 14)
  • human IgG4 Fc region GGGGSGGGGSGGGGS (SEQ ID NO: 14)
  • human IgG4 Fc region equivalent having F234A, L235A and N297Q mutations (i.e., mutations at aa 16, aa 17 and aa 79 of SEQ ID NO: 81, respectively)
  • HMA Human muscle aldolase
  • Detectable marker YPYDVPDYA (SEQ ID NO: 84)
  • T2A peptide HVGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 85)
  • ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAA ACAGT SEQ ID NO: 87
  • a nucleotide sequence encoding a signaling domain [0408]
  • a nucleotide sequence encoding a hinge domain [0411]
  • AAGGTACC (SEQ ID NO: 109)
  • peptide linker (glycine-serine)n wherein n is an integer from 1 to 6 (SEQ ID NO: 110)
  • Hinge domain IgGl heavy chain hinge coding sequence:
  • CD28 transmembrane region coding sequence [0415]
  • 4-1BB co-stimulatory signaling region coding sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA
  • CD28 co-stimulatory signaling region coding sequence [0417]
  • CD3 zeta signaling region coding sequence [0418]
  • Human CD8 alpha hinge domain PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 117).
  • Mouse CD8 alpha hinge domain KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 118).
  • Cat CD8 alpha hinge domain PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY (SEQ ID NO: 119).
  • Human CD8 alpha transmembrane domain IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 120).
  • Mouse CD8 alpha transmembrane domain IWAPLAGICVALLLSLIITLI (SEQ ID NO: 121).
  • Rat CD8 alpha transmembrane domain IWAPLAGICAVLLLSLVITLI (SEQ ID NO: 122).
  • 4- IBB costimulatory signaling region KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 123)
  • ICOS costimulatory signaling region coding sequence acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga gcagtgaaca cagccaaaa atccagactc acagatgtga cccta (SEQ ID NO: 125)
  • Linker GGGGS (SEQ ID NO: 134).
  • n may be an integer of 1 (SEQ ID NO: 134), or 2 (SEQ ID NO: 135), or 3 (SEQ ID NO: 14), or 4 (SEQ ID NO: 136), or 5 (SEQ ID NO: 137), or 6 (SEQ ID NO: 138), or 7 (SEQ ID NO: 139), or 8 (SEQ ID NO: 140), or 9 (SEQ ID NO: 141), or 10 (SEQ ID NO: 142), or 11(SEQ ID NO: 143), or 12(SEQ ID NO: 144), or 13 (SEQ ID NO: 145), or 14 (SEQ ID NO: 146), or 15(SEQ ID NO: 147), or more.
  • Embodiment 1 A pseudotyped gammaretroviral particle comprising a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and a modified baboon envelope glycoprotein (BaEVTR), wherein: a. the RD114TR glycoprotein comprises an ectodomain and a transmembrane domain of a RD114 glycoprotein and a cytoplasmic domain of amphotropic murine leukemia virus (MLV-A) glycoprotein; and b. wherein the BaEVTR glycoprotein comprises an ectodomain and a transmembrane domain of a baboon envelope glycoprotein (BaEV) and a cytoplasmic domain of MLV-A glycoprotein.
  • RD114TR modified RD114 feline endogenous retrovirus envelope glycoprotein
  • BaEVTR modified baboon envelope glycoprotein
  • Embodiment 2 The pseudotyped gammaretroviral particle of embodiment 1, wherein the RD114TR and the BaEVTR are incorporated into the envelope of the gammaretroviral particle as membrane proteins.
  • Embodiment 3 The pseudotyped gammaretroviral particle of embodiment 1 or 2, further comprising a vector genome encapsulated in the envelope, wherein the vector genome comprises one or more of the following flanked by two long terminal repeats (LTRs):
  • LTRs long terminal repeats
  • A a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein or polypeptide optionally selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor,
  • CAR chimeric antigen receptor
  • (C) a polynucleotide comprising one or more of recognition sites that optionally are recognized and cleaved by a restriction enzyme suitable for inserting a CAR coding sequence or a reverse-complement thereof into the polynucleotide.
  • Embodiment 4 The pseudotyped gammaretroviral particle of any one of embodiments 1 to 3, wherein the vector genome further comprises one or more of the following: a 5’ LTR, a 5’ cap, a 3’ poly-A tail, and a 3’ LTR.
  • Embodiment 5. The pseudotyped gammaretroviral particle of any one of embodiments 1 to 4, further comprising either of both of a reverse transcriptase or an integrase.
  • Embodiment 6 The pseudotyped gammaretroviral particle of any one of embodiments 1 to 5, which is selected from any one of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • XMRV xenotropic murine leukemia virus-related virus
  • Embodiment 7 A method for preparing a population of natural killer (NK) cells, comprising culturing a cell population comprising one or more of the following: an NK cell, a progenitor cell that is capable of deriving an NK cell, or a stem cell that is capable of deriving an NK cell with an immune cell activator (such as an NK cell activator), wherein the cell population is depleted with cells that expresses one or more of: CD3, CD4, CD8, T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population, and wherein the immune cell activator is selected from one or more of the following (i) to (iv):
  • an artificial antigen presenting cell that expresses a tumor associated antigen (TAA) or a viral antigen optionally which activate and/or stimulate immune cell growth,
  • an antibody or an antigen binding fragment thereof which specifically recognizes and binds to a stimulatory receptor on one or more of the NK cell, the progenitor cell or the stem cell, thereby activating or proliferating NK cells, optionally wherein the antibody is selected from one or more of an anti-CD2 antibody, an anti-CD16 antibody, an anti-NKG2D antibody, an anti-DNAM-1 antibody, an anti-2B4 antibody, an anti-NTB-A antibody, or an anti-NKp46 (natural cytotoxicity receptor 1 (NCR1)) antibody,
  • one or more of chemical moieties thereby activating or proliferating NK cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs).
  • Embodiment 8 The method of embodiment 7, further comprising introducing a polynucleotide encoding a CAR or another therapeutic protein or polypeptide optionally selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor into the cultured cell population for expression, wherein the CAR specifically recognizes and binds to the tumor associated antigen (TAA) or a viral antigen.
  • TAA tumor associated antigen
  • Embodiment 9 The method of embodiment 8, further comprising culturing the cell population with an immune cell activator after the introducing step of embodiment 8, wherein the immune cell activator is selected from one or more of the following (i) to (iv):
  • An artificial antigen presenting cell that expresses a tumor associated antigen (TAA) or a viral antigen optionally which activate and/or stimulate immune cell growth,
  • one or more of chemical moieties thereby activating or proliferating NK cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs).
  • the culturing step is repeated for once, twice, three times, or more times, with same or different immune cell activator(s) or a combination thereof.
  • Embodiment 10 The method of any one of embodiments 7 to 9, wherein the aAPCs further express one or more of: 4-1BBL, membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D superagonist-complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc) ALT- 803, or a cell surface marker mediating CD122/CD132 signaling.
  • 4-1BBL membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40
  • Embodiment 11 The method of any one of embodiments 7 to 10, wherein the aAPCs further expresses mb IL-21 and 4-1BBL.
  • Embodiment 12 The method of any one of embodiments 7 to 11, wherein the aAPCs are engineered K562 cells.
  • Embodiment 13 The method of any one of embodiments 7 to 12, wherein the aAPCs are irradiated, thereby lacking cell proliferation or lacking long-term survival.
  • Embodiment 14 The method of embodiment 13, wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy.
  • Embodiment 15 The method of any one of clams 7 to 14, wherein the aAPC is cultured with the cell population at a cell number ratio of about 10:1, about 5: 1, about 3: 1, about 2:1, about 1 : 1, about 1:2, about 1 :3, about 1 :5, or about 1 : 10.
  • Embodiment 16 The method of any one of embodiments 7 to 15, wherein the cytokines are selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low-toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D super-agonist-complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL- 15RaSu-Fc; ALT-803 soluble), IL-15, IL-18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1), or MICA.
  • the cytokines are selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low-toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL
  • Embodiment 17 The method of embodiment 16, wherein the cell population is cultured with any one or any two or all three of 100-500 lU/ml IL-2, 20 ng/ml IL- 15, or 25 ng/mL IL-21.
  • Embodiment 18 The method of embodiment 16, wherein the cell population is cultured with either or both of 50 lU/ml IL-2 and 0.5 ng/ml IL-15.
  • Embodiment 19 The method of embodiment 16, wherein the cell population is cultured with 50 lU/ml IL-2.
  • Embodiment 20 The method of any one of embodiments 9 to 19, wherein the activator cultured with the cell population before and after the introducing step is the same.
  • Embodiment 21 The method of any one of embodiments 9 to 19, wherein the activator cultured with the cell population before and after the introducing step is different with each other.
  • Embodiment 22 The method of any one of embodiments 8 to 21, comprising introducing a pseudotyped gammaretroviral particle into the cultured cell population, thereby introducing the CAR coding polynucleotide into the cultured cell, wherein the particle comprises the CAR coding polynucleotide or reverse-complement thereof flanked by two long terminal repeats (LTRs), an RD114TR, and a BaEVTR.
  • LTRs long terminal repeats
  • RD114TR RD114TR
  • BaEVTR BaEVTR
  • the pseudotyped gammaretroviral particle comprises a vector genome comprising a 5’ LTR, a 5’ cap, the CAR coding polynucleotide or a reverse-complement thereof, a 3’ poly-A tail, and a 3’ LTR.
  • Embodiment 24 The method of embodiment 22 or 23, wherein the pseudotyped gammaretroviral particle further comprises either of both of a reverse transcriptase and an integrase.
  • Embodiment 25 The method of any one of embodiments 22 to 24, wherein the pseudotyped gammaretroviral particle is selected from any species of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • XMRV xenotropic murine leukemia virus-related virus
  • Embodiment 26 The method of any one of embodiments 22 to 25, wherein the pseudotyped gammaretroviral particle is introduced to the cultured cell population at a multiplicity of infection (MOI) of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10.
  • MOI multiplicity of infection
  • Embodiment 27 The method of any one of embodiments 8 to 26, wherein the cell population are cultured before the introducing step for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days.
  • Embodiment 28 The method of any one of embodiments 8 to 27, wherein the cell population are cultured before the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks, or no more than 1 month.
  • Embodiment 29 The method of any one of embodiments 8 to 27, wherein the cell population are cultured before the introducing step for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
  • Embodiment 30 The method of any one of embodiments 8 to 29, wherein the CAR coding polynucleotide is introduced to the cell population via transducing a viral vector comprising the CAR coding polynucleotide or reverse-complement thereof in the presence of RetroNectin.
  • Embodiment 31 The method of embodiment 30, wherein the RetroNectin is coated on the inner surface of a container in which the cell population is transduced.
  • Embodiment 32 The method of embodiment 30 or 31, wherein the cell population expresses either or both of Integrin a4pi (VLA-4) and Integrin a5pi (VLA-5).
  • Embodiment 33 The method of any one of embodiments 7 to 32, wherein the cell population is isolated from a blood sample of a subject.
  • Embodiment 34 The method of any one of embodiments 7 to 33, wherein the cell population is isolated from one or more of: umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
  • Embodiment 35 The method of any one of embodiments 7 to 34, wherein the cell population comprises progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • the cell population comprises progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • the cell population comprises progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (
  • Embodiment 36 The method of any one of embodiments 7 to 35, further comprising enriching cells expressing any one or more of CD56, CD25, CD122, CD212, CD215, CD218, CD360,TGF-PR, or IL-10R in the cell population, and optionally further comprising enriching either or both CD56 dim cells and CD56 bnght cells.
  • Embodiment 37 The method of any one of embodiments 7 to 36, wherein any cell population comprises natural killer (NK) cells, or substantially purified composition thereof.
  • NK natural killer
  • Embodiment 38 The method of any one of embodiments 7 to 37, wherein the depleted cell population comprises one or more of the following: NK cells, progenitor cells, HSCs, iPSCs or a substantially purified population of each thereof.
  • Embodiment 39 The method of embodiment 37 or 38, wherein the NK cells comprise those derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • Embodiment 40 The method of embodiment 38, wherein one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving NK cells.
  • Embodiment 41 The method of any one of embodiments 7 to 40, wherein any cell population is substantially free of T cells.
  • Embodiment 42 The method of any one of embodiments 7 to 41, wherein any cell population is substantially free of T regulatory cells.
  • Embodiment 43 The method of any one of embodiments 7 to 42, wherein any cell population is isolated, enriched or purified.
  • Embodiment 44 The method of any one of embodiments 7 to 43, wherein the cell population and the aAPCs are cultured in a cell culture media selected from StemSpan (Stemcell #09960); NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076- 356), TexMACS (170-076-306 GMP medium), Cellgenix Serum-free Stem Cell Growth Medium (SCGM, #20806-0500), or ImmunoCultTM-XF Medium from Stemcell technologies in the culturing step.
  • a cell culture media selected from StemSpan (Stemcell #09960); NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076- 356), TexMACS (170-076-306 GMP medium), Cellgenix Serum-free Stem Cell Growth Medium (SCGM, #20806-0500), or ImmunoCultTM-XF Medium from Stemcell technologies in the culturing step.
  • Embodiment 45 The method of any one of embodiments 7 to 44, further comprising either or both of formulating the CAR-expressing population in a composition; and cry opreserving the CAR-expressing population.
  • Embodiment 46 The method of any one of embodiments 7 to 45, generating more than 1 x 10 11 cells from 1 x 10 8 cells .
  • Embodiment 47 The method of any one of embodiments 7 to 46, further comprising either or both of: washing the cell population prior to or after one or more of the steps, and detecting one or more of the following prior to or during or after one or more of the steps:
  • HIV Human immunodeficiency virus
  • types 1 and 2 Human T-lymphotropic virus
  • HBV Human T-lymphotropic virus
  • types I and II Hepatitis B virus
  • HCV Hepatitis C virus
  • CMV Zika, West Nile, or Treponema pallidum
  • Embodiment 48 The method of embodiment 47, wherein the phenotype detection comprises detecting cell expression or expression level of one or more of the following: CD3, CD56, an antigen such as CD19, CD45, HLA, NKp46, NKG2D, NKG2A, NCRs, DNAM-1, CD 16, IL-2R, CXCR4, KIRS, CD8, CD57, Adhesion Molecules, NKG2C, CD 107a, CAR, or cell surface markers expressed by the cell population.
  • Embodiment 49 The method of embodiment 48, wherein the phenotype detection comprises detecting cell expression level of CD56 by the cell population.
  • Embodiment 50 The method of any one of embodiments 8 to 49, wherein an antigen binding domain of the CAR specifically recognizes and binds to one or more of the following:
  • G Protein-Coupled Receptor Class C Group 5 Member D GPRC5D
  • B-cell maturation antigen BCMA
  • SLAMF7 SLAMF7
  • EGFR wildtype epidermal growth factor receptor
  • EGFRwt wildtype epidermal growth factor receptor
  • EGFR VIII epidermal growth factor receptor variant III
  • FLT3 CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL- 1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferas
  • G Protein-Coupled Receptor Class C Group 5 Member D GPRC5D
  • B-cell maturation antigen BCMA
  • SLAMF7 SLAMF7
  • EGFR wildtype epidermal growth factor receptor
  • EGFRwt wildtype epidermal growth factor receptor
  • EGFR VIII epidermal growth factor receptor variant III
  • FLT3 CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL- 1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferas
  • Embodiment 52 The method of any one of embodiments 8 to 51, wherein the
  • CAR comprises (1) an antigen binding domain of an antibody that specifically recognizes and binds to the antigen; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular domain comprising a signaling domain.
  • Embodiment 53 The method of any one of embodiments 8 to 52, wherein the CAR comprises (1) an antigen binding domain of an antibody that specifically recognizes and binds to the antigen; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular domain comprising a signaling domain and a costimulatory domain.
  • Embodiment 54 The method of any one of embodiments 8 to 53, wherein the CAR further comprises a signal peptide.
  • Embodiment 55 The method of embodiment 53 or 54, wherein the hinge domain comprises a CD8 a hinge domain.
  • Embodiment 56 The method of any one of embodiments 53 to 55, wherein the transmembrane domain comprises a CD8a transmembrane domain.
  • Embodiment 57 The method of any one of embodiments 53 to 56, wherein the intracellular domain further comprises (1) one or two or more costimulatory signaling regions.
  • Embodiment 58 The method of embodiment 57, wherein the costimulatory signaling region comprises a CD28 costimulatory signaling region or a 4- IBB costimulatory signaling region or both.
  • Embodiment 59 The method of any one of embodiments 53 to 58, wherein the signaling domain comprises a CD3 zeta signaling domain.
  • Embodiment 60 The method of any one of embodiments 8 to 59, wherein the introduced polynucleotide further expresses one or more of cytokines and antibodies, or wherein the method further comprising introducing another polynucleotide expressing one or more of cytokines and antibodies into the depleted cell population.
  • Embodiment 61 The method of embodiment 60, wherein the cytokine expressed by the polynucleotide is selected from one or more of the following: B7.1(soluble or membrane bound), CCL19(soluble or membrane bound), CCL21 (soluble or membrane bound), CD40L(soluble or membrane bound), CD137L(soluble or membrane bound), GITRL(soluble or membrane bound), GM-CSF(soluble or membrane bound), IL-12(soluble or membrane bound), IL-2(soluble or membrane bound), low-toxicity IL-2(soluble or membrane bound), IL-2 analogs that lack cd25 binding (soluble or membrane bound), IL-15- N72D super-agonist along with a IL-15RaSushi-Fc fusion protein (soluble or membrane bound), IL- 15 (soluble or membrane bound), IL- 18 (soluble or membrane bound), IL-21 (soluble or membrane bound), LEC (soluble or membrane bound), OX40L (soluble or membrane bound), IL-7 (soluble or membrane bound), ICOSL (B7H2, B
  • Embodiment 62 The method of embodiment 60 or 61, wherein the antibody expressed by the polynucleotide is a monospecific antibody, or a bispecific antibody, or a multiple specific antibody.
  • Embodiment 63 The method of any one of embodiments 60 to 62, wherein the antibody expressed by the polynucleotides an immune cell activator.
  • Embodiment 64 The method of any one of embodiments 8 to 63, wherein the polynucleotide further encodes a signal peptide.
  • Embodiment 65 The method of any one of embodiments 8 to 64, wherein the polynucleotide further comprises a suicide gene.
  • Embodiment 66 The method of embodiment 65, wherein the suicide gene product is selected from one or more of: HSV-TK (Herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, carboxylesterase, cytochrome P450 or PNP (Purine nucleoside phosphorylase), truncated EGFR, or inducible caspase (“iCasp”).
  • HSV-TK Herpes simplex virus thymidine kinase
  • cytosine deaminase cytosine deaminase
  • nitroreductase carboxylesterase
  • cytochrome P450 or PNP Purine nucleoside phosphorylase
  • truncated EGFR or inducible caspase (“iCasp”).
  • Embodiment 67 The method of embodiment 65 or 66, wherein the polynucleotide further comprises a regulatory sequence directing expression of the suicide gene and wherein the regulatory sequence is inducible.
  • Embodiment 68 The method of any one of embodiments 8 to 67, wherein the polynucleotide further comprises a regulator sequence directing the expression of the CAR or the therapeutic protein.
  • Embodiment 69 The method of embodiment 68, wherein the regulator sequence directing expression of the CAR or the therapeutic protein is inducible or constitutively active.
  • Embodiment 70 The method of any one of embodiments 8 to 69, wherein the polynucleotide is introduced to the cell population via a vector.
  • Embodiment 71 The method of embodiment 70, wherein the vector is a viral vector or a non-viral vector.
  • Embodiment 72 The method of embodiment 71, wherein the non-viral vector is a plasmid.
  • Embodiment 73 The method of embodiment 71, wherein the viral vector is selected form a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or Herpes viral vector.
  • Embodiment 74 The method of any one of embodiments 8 to 73, wherein the prepared CAR-expressing cell population is suitable to inhibit the growth of a cancer cell and wherein the antigen is a tumor associated antigen (TAA) expressed by the cancer cell.
  • TAA tumor associated antigen
  • Embodiment 75 A method for inhibiting the growth of a cancer cell, comprising contacting a population of CAR-expressing cells prepared by a method of any one of embodiments 8 to 74 with the cancer cell, wherein the antigen recognized by the CAR is a TAA expressed by the cancer cell.
  • Embodiment 76 A method for treating a cancer in a subject, comprising administering a population of the CAR-expressing immune cells prepared by a method of any one of embodiments 8 to 74 to the subject, wherein the antigen recognized by the CAR is a TAA expressed by a cell of the cancer.
  • Embodiment 77 The method of embodiment 76, wherein the cell population comprises NK cells and less than or equal to 0.3% CD3+ cells.
  • Embodiment 78 The method of embodiment 76 or 77, wherein the subject is administered with less than 3 x 10 3 T cells per kilogram of body weight.
  • Embodiment 79 The method of any one of embodiments 74 to 78, wherein the cancer cell is selected from cancer cells of the: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lympho
  • SCLC
  • Embodiment 80 The method of any one of embodiments 74 to 79, wherein the cancer cell is a solid tumor cell.
  • Embodiment 8E The method of any one of embodiments 74 to 79, wherein the cancer cell is not a cell of a solid tumor, optionally wherein the cancer cell is a leukemia cancer cell.
  • Embodiment 82 The method of any one of embodiments 74 to 81, wherein the cancer cell is a primary cancer cell or a metastatic cancer cell.
  • Embodiment 83 The method of any one of embodiments 74 to 82, wherein the cancer cell is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma.
  • Embodiment 84 The method of any one of embodiments 75 and 79 to 83, wherein the contacting is in vivo or in vitro.
  • Embodiment 85 The method of any one of embodiments 74 to 84, further comprising contacting the cell with or administering a separate therapy.
  • Embodiment 86 The method of embodiment 85, wherein the separate therapy comprises surgical resection, chemotherapy, radiation therapy, immunotherapy and targeted therapy.
  • Embodiment 87 The method of embodiment 85 or 86, wherein the separate therapy is a first line therapy, a second line therapy, a third line therapy, or a fourth line therapy.
  • Embodiment 88 An NK cell or a population thereof produced or prepared by the method of any one of embodiments 7 to 74.
  • Embodiment 89 A composition comprising the cell or a population thereof of embodiment 88 and a carrier, optionally a pharmaceutical acceptable carrier.
  • Embodiment 90 The cell or a population thereof of embodiment 88, or the composition of embodiment 89, comprising less than 0.5% aAPCs.
  • Embodiment 91 The cell or a population thereof of embodiment 88 or 90, or the composition of embodiment 89 or 90, comprising less than 0.1%, or less than 0.2%, or less than 0.3%, less than 0.4%, or less than 0.5% aAPCs.
  • Embodiment 92 The cell or a population thereof of any one of embodiments 88 and 90-91 or the composition of any one of embodiments 89 to 91, having one or more of the following properties:
  • (E) comprising more than 1 x 10 6 cells per kilogram of body weight of a subject to be treated;
  • Embodiment 93 The cell or a population thereof of embodiment 92 or the composition of embodiment 92 having one or both of the following properties:
  • (E’) comprising more than 1 x 10 7 cells per kilogram of body weight of a subject to be treated;
  • Embodiment 94 An engineered aAPC expressing an antigen and one or more of cell surface markers: 4-1BBL, membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D super-agonist-complexed with IL-15RaSushi-Fc fusion protein (IL-15SA/IL-15RaSu-Fc) ALT-803, or a cell surface marker mediating CD122/CD132 signaling.
  • 4-1BBL membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD
  • Embodiment 95 The aAPC of embodiment 94, wherein the aAPCs are engineered K562 cells.
  • Embodiment 96 The aAPC of embodiment 94 or 95, wherein the aAPCs are irradiated, thereby lacking cell proliferation or long-term survival or both.
  • Embodiment 97 The aAPC of embodiment 96, wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy.
  • Embodiment 98 The aAPC of any one of embodiments 94 to 97, wherein the aAPCs do not substantially survive for more than 14 days.
  • Embodiment 99 A kit comprising one or more of agents suitable for use in the method of any one of embodiments 7 to 87 and an optional instruction.
  • Embodiment 100 The kit of embodiment 99, wherein the agents are selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting cell phenotype, an antibody for isolating or enriching or purifying immune cells, primers for detecting the polynucleotide, cytokines, and an aAPC.
  • the agents are selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting cell phenotype, an antibody for isolating or enriching or purifying immune cells, primers for detecting the polynucleotide, cytokines, and an aAPC.
  • Embodiment 101 A method for preparing a population of y6 T cells, comprising culturing a cell population comprising one or more of the following: a y6 T cell, a progenitor cell that is capable of deriving a y6 T cell, or a stem cell that is capable of deriving a y6 T cell with an immune cell activator (such as a y6 T cell activator), wherein the cell population is depleted with cells that expresses one or more of: T cell receptor (TCR) a chain, TCR P chain, or aPTCR in a cell population, and wherein the activator is selected from one or more of the following (i) to (iv):
  • TCR T cell receptor
  • an artificial antigen presenting cell that expresses a tumor associated antigen (TAA) or a viral antigen optionally which activate and/or stimulate immune cell growth,
  • one or more of chemical moieties thereby activating or proliferating y6 T cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs).
  • Embodiment 102 The method of embodiment 101, further comprising introducing a polynucleotide encoding a CAR or another therapeutic protein optionally selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor into the cultured cell population for expression, wherein the CAR specifically recognizes and binds to the tumor associated antigen (TAA) or the viral antigen.
  • TAA tumor associated antigen
  • Embodiment 103 The method of embodiment 102, further comprising culturing the cell population after the introducing step of embodiment 102 with an immune cell activator (such as a y6 T cell activator), wherein the activator is selected from one or more of the following (i) to (iv): (i) an artificial antigen presenting cell (aAPC) that expresses a tumor associated antigen (TAA) or a viral antigen optionally which activate and/or stimulate immune cell growth,
  • an immune cell activator such as a y6 T cell activator
  • one or more of chemical moieties thereby activating or proliferating y6 T cells optionally selected from a mTOR inhibitor, a PI3K inhibitor or a STING-activating cyclic dinucleotides (CDNs);
  • the culturing step is repeated for once, twice, three times, or more times with the same or different activator(s).
  • Embodiment 104 The method of any one of embodiments 101 to 103, wherein the cell population comprises one or more of the following: y6 T cells or substantially purified composition thereof.
  • Embodiment 105 The method of any one of embodiments 101 to 104, wherein the depleted cell population comprises one or more of the following: y6 T cells, HSCs, iPSCs or a substantially purified population of each thereof.
  • Embodiment 106 The method of embodiment 105, wherein one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving y6 T cells.
  • Embodiment 107 The method of any one of embodiments 101 to 106, wherein the y6 T cells comprise those derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • progenitor cells embryonic stem cells, embryonic stem cell derived cells, embryonic germ cells, embryonic germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells.
  • Embodiment 108 The method of any one of embodiments 101 to 107, wherein the cell population is substantially free of cells expressing TCR a chain or TCR P chain.
  • Embodiment 109 A viral packaging system for producing a pseudotyped gammaretroviral particle, comprising: (a) a plasmid expressing a vector genome; (b) a packaging plasmid; and (c) one or more of envelope plasmids expressing RD114TR, and BaEVTR.
  • Embodiment 110 The viral packaging system of embodiment 109, wherein the vector genome comprises one or more of the following flanked by two long terminal repeats (LTRs):
  • A a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein optionally selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor,
  • CAR chimeric antigen receptor
  • (C) a polynucleotide comprising one or more of recognition sites recognized by a restriction enzyme suitable for inserting a CAR coding sequence or a reverse-complement thereof into the polynucleotide.
  • Embodiment 111 The viral packaging system of embodiment 109 or 110, wherein the vector genome further comprises one or more of the following: a 5’ LTR, a 5’ cap, a 3’ poly-A tail, and a 3’ LTR.
  • Embodiment 112. The viral packaging system of any one of embodiments 109 to
  • the pseudotyped gammaretroviral particle is selected from any species of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus xenotropic MuLB-related virus
  • feline sarcoma virus xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • Embodiment 113 The viral packaging system of any one of embodiments 109 to
  • Embodiment 114 The viral packaging system of embodiment 113, wherein the packaging cell line is a 293T cell line.
  • Embodiment 115 A method for producing a pseudotyped gammaretroviral particle, comprising transducing a packaging cell line with the system of any one of embodiments 109 to 112 under conditions suitable to package the pseudotyped gammaretroviral particle.
  • Embodiment 116 The method of embodiment 115, wherein the packaging cell line is a 293T cell line.
  • Embodiment 117 The method of embodiment 115 or 116, wherein the cell line is transduced with plasmids of (a), (b) and (c) at a ratio of 1.5: 1.5: 1.
  • Embodiment 118 The method of any one of embodiments 115 to 116, wherein the packaging system comprises at least two envelope plasmids, one expressing RD114TR and the other expressing BaEVTR.
  • Embodiment 119 The method of embodiment 118, wherein the cell line is transduced with the plasmid of (a), the plasmid of (b), the RD114TR expressing plasmid, and the BaEVTR expressing plasmid at a ratio of 1.5: 1.5: 1 : 1.

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