AU2021341944A1 - Methods and compositions of infecting, activating, and expanding immune cells - Google Patents

Abstract

Provided are methods and compositions for effectively infect, activate and expanded immune cells, such as natural killer (NK) cells and γδ T cells.

Description

METHODS AND COMPOSITIONS OF INFECTING, ACTIVATING,

AND EXPANDING IMMUNE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 63/075,651, filed September 8, 2020; and 63/075,747, filed September 8, 2020, the contents of each of which are hereby incorporated by reference into this application in their entireties.

BACKGROUND

[0002] 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. At present, 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.

[0003] Nevertheless, 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. Thus, what remain needed are compositions and methods efficiently preparing NK cells for therapy. This disclosure satisfies these needs and provides related advantages as well.

SUMMARY OF THE DISCLOSURE

[0004] In one aspect, provided herein is a pseudotyped gammaretroviral particle comprising a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and a modified baboon envelope glycoprotein (BaEVTR). In some embodiments, 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. Additionally or alternatively, 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. In some embodiments, the RD114TR and the BaEVTR are incorporated into the envelope of the particle as membrane proteins. In some embodiments, 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.

[0005] In another aspect, provided is 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. In some embodiments, 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. In some embodiments, 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.

[0006] In some embodiments, 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.

[0007] In one aspect, provided is a method for preparing a population of y6 T cells, The method 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). In some embodiments, 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. In some embodiments, 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.

[0008] In some embodiments, 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.

[0009] In some embodiments that may relate to any aspect of the disclosure herein, 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. In one embodiment, the aAPCs further expresses mb IL-21 and 4-1BBL.

[0010] In some embodiments that may relate to any aspect of the disclosure herein, 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. In some embodiments, 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.

[0011] Some embodiments of 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. In some embodiments, 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.

[0012] Some embodiments of 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. In some embodiments, the particle comprises RD114TR and BaEVTR as envelope proteins. In some embodiments, 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.

[0013] Some embodiments of any method as disclosed herein further comprises using 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. One example is coating RetroNectin on the inner surface of a container in which the cell population is introduced with a polynucleotide 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.

[0014] Additionally provided is a method for producing a retroviral particle (such as a gammaretroviral particle). The method 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. In some embodiments, 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 for the culture of the first packaging cell line introduced with the vector. Additionally or alternatively, the method further comprises culturing the transduced second packaging cell line. Accordingly provided is 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).

[0015] In some embodiments that may relate to any aspect of the disclosure herein, 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.

[0016] In some embodiments that may relate to any aspect of the disclosure herein, 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. Additionally or alternatively, the cell population is isolated from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.

[0017] In one aspect, provided is a method for inhibiting the growth of a cancer cell. The method 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. In some embodiments, the antigen recognized by the CAR is a TAA expressed on the cancer cell. The contacting step may be in vivo or in vitro.

[0018] In another aspect, provided is a method for treating a cancer in a subject. The method 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. In some embodiments, the antigen recognized by the CAR is a TAA expressed by a cell of the cancer.

[0019] In yet another aspect, provided is 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.

[0020] In a further aspect, provided are an NK cell, a y6 T cell, or a cell population of either or both thereof prepared by a method as disclosed herein. In some embodiments, 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.

[0021] In one aspect, provided is a 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.

[0022] In another aspect, provided is a kit 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. In some embodiments, 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.

[0023] In yet another aspect, provided is 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.

[0024] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 provides a schema of gammaretroviral vector (PCIR) based therapeutic gene delivery to human primary NK cells.

[0026] 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. Clearly, after isolation and culture for 5 days, the primary NK cells entered a marked proliferative state, therefore the activated NK cells at day 6-10 were used for gene delivery (FIG. 2B).

[0027] 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. There were distinct high viable NK cells ranging from 73.51% - 82.81% (a rough viable cell gate can be drawn by excluding low-forward scatter (FSC) and high-side scatter (SSC) events) after infection (FIG. 3B), EGFR CAR gene (Fab-AF647 positive) transduction efficiency ranging from 65.11% - 72.75% among three donors (FIG. 3C) and yields with great proliferation state of fold 25, 34 and 45 in three donors compared with initial infection NK numbers at post infection day 8 (FIG. 3D).

[0028] FIG. 4 provides determination of retrovirus titration based on direct infection of activated NK cells at same proliferate state with same delivery strategy. In this disclosure for developing gammaretrovirus vector (PCIR) based therapeutic gene delivery to human primary NK cells, the titration determination methods were based on exactly same infection strategy for same proliferative state of activated NK cells at day 7. For this PCIR-EGFR- CAR-NK delivery assay demonstrated in FIGS. 3A to 3D, 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 FLUOR¹' 647 AffiniPure Goat Anti-Mouse IgG, F(ab')2 fragment specific (Jackson ImmunoResearch i 15-605-006). Transducing Units/mL were calculated using formula: TU/mL = (Number of cells transduced x Percent fluorescent x Dilution Factor) / (Transduction Volume in mL). For a more accurate titer, the average of multiple dilutions were taken. The MOI 3 was used in this study (see FIGS. 3A to 3D).

[0029] 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. Both flow cytometry and fluorescence microscopy imaging were used for evaluating the PCIL-EGFR-CAR-NK transduction rate and expression at post sorting day 10, and the GFP positive cells were significantly reduced from around 100% to 39% for both PCIL-EGFR- CAR-NK and PCIL-GFP empty vector control (FIGS. 5A and 5C). However, flow analysis demonstrated that there were highly stable transduced gene expression at post sorting day 10, day 14 and day 25 for PCIR-EGFR-CAR-NK (FIGS. 5B and 5C).

[0030] 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).

[0031] FIG. 7 shows NK cell expansion of 17-24 days. [0001] 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. As shown in FIG. 8A, Among the retroviruses, 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. In addition to ASCT2, baboon endogenous viruses (BaEV) also uses ASCT1 as a cell entry receptor. Gibbon ape leukemia virus (GALV) uses sodium-dependent phosphate transporters (Pitl ) as its 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). FIG. 8C shows that both cell lines (Jurkat T and HT1080) can be used for titration of the three retrovirus envelop proteins (BaEV, RD114 and GALV) due to their ubiquitous expression of the corresponding entry receptors. For the 293 based packaging cell lines, 293 Vec GALV and 293 Vec BaEV, only the GALV-pseudotyped retrovirus can infect themselves using entry receptor Pitl. For cell type, three bars are plotted in FIG. 8C: the left one represents expression of ASCT1, the middle one represents expression of ASCT2, and the right one represents expression of Pitl.

[0032] 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. For high-titer clone generation, 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.

[0033] 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. When the cells reached 90-100 % confluence, 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. The plate was incubated at 37 degrees for 48h, then a flow analysis was performed for determination of the titer (transducing Units/ml) according to the following formula: Transducing Units (TU)/mL = [(target cell number) X (%Positive cells)]. In this example, 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. 10B, there is an improved titer of raw supernatant from stable retrovirus virus producer-293 Vec-BaEV (TU=7.2E5) for a vector with a large-size transgenes compared to the virus produced transiently (TU=2.2E5).

DETAILED DESCRIPTION

Definitions

[0034] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and/or separating the subject matter described. [0035] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0036] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3^rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5^th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);

Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0037] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0038] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.

[0039] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0040] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0041] As used herein, the term “comprising” or “comprises” is intended to mean that the 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.

[0042] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0043] As used herein, “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”). [0044] “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%.

[0045] In some embodiments, the terms “first” “second” “third” “fourth” or similar in a component name are used to distinguish and identify more than one components sharing certain identity in their names. For example, “first cell line” and “second cell line” are used to distinguishing two cell lines.

[0046] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “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. The term “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. In other embodiments, 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. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, 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.

[0047] In some embodiments, 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. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention. As used herein, 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.

[0048] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer 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. The following are non-limiting examples of 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. If present, 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.

[0049] 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. Thus, the term “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.

[0050] The expression “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). In general, 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. [0051] Reagents and hardware for conducting PCR are commercially available. 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.

[0052] 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.

[0053] The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “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.

[0054] 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. In some embodiments, the gene product may refers to an mRNA generated when a gene is transcribed.

[0055] The term “encode” as it is applied to polynucleotides 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.

[0056] “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. [0057] The term “a regulatory sequence” “an expression control element” or “promoter” as used herein, 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.

[0058] The term “promoter” as used herein 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. Non-limiting examples of 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.

11(5):404-408). The CMV promoter sequence is known in the art (see, e.g., snapgene.com/resources/plasmid- files/?set=basic_cloning_vectors&plasmid=CMV_promoter, last accessed on March 13, 2019 and Zheng and Baum (2014), supra.). An example is: EFl alpha promoter sequence: SEQ ID NO: 148, and optionally, an equivalent thereof.

[0059] 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.

[0060] “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.

[0061] 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 Mg²⁺ normally found in a cell.

[0062] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary.” 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” (the degree that one polynucleotide is complementary with another) 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.

[0063] “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. [0064] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide 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. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: www.ncbi.nlm.nih.gov/cgi-bin/BLAST. In another embodiment, 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/. In further embodiments, the default setting is used.

[0065] In some embodiments, 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.

[0066] In some embodiments, 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.

[0067] Additionally or alternatively, 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. In one aspect, the equivalent must encode functional protein that optionally can be identified through one or more assays described herein. In addition or alternatively, the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.

[0068] The term “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.

[0069] As used herein, 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. For optimal use, 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. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA, since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, 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.

[0070] The term “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. As used herein the term “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. [0071] As used herein, the term “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. Unless specifically noted otherwise, 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 10³ M'¹ greater, at least 10⁴ M'¹ greater or at least 10⁵ 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). See also, Pierce Catalog and Handbook, 1994- 1995 (Pierce Chemical Co., Rockford, Ill.); Owen et al., Kuby Immunology, 7^th Ed., W.H. Freeman & Co., 2013; Murphy, Janeway’s Immunobiology, 8^th Ed., Garland Science, 2014; Male et al., Immunology (Roitt), 8^th Ed., Saunders, 2012; Parham, The Immune System, 4^th Ed., Garland Science, 2014.

[0072] As used herein, 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.

[0073] In terms of antibody structure, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, 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". The extent of the 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. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0074] 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). Thus, a CDRH3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found, whereas a CDRL1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. For example, 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).

[0075] As used herein, 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. In some embodiments, 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. [0076] The 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.

[0077] 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.

[0078] 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. Accordingly, and as an example, 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. In another aspect, the EGFR CAR is the CAR disclosed in WO 2016/164370.

[0079] In one aspect, 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.

[0080] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, antibody, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, 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. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0081] The term “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.

[0082] The term “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.

[0083] As used herein, the term “specific binding” means the contact between an antibody and an antigen with a binding affinity of at least 10^-6M. In certain aspects, antibodies bind with affinities of at least about 10^-7M, and preferably 10^-8M, 10^-9M, 10^-10M, 10^-11 M, or 10’¹²M.

[0084] As used herein, 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. Common categories of 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.

[0085] In some embodiments, antigen of a binding moiety, such as an antibody, an antigen binding fragment thereof, or a CAR, may be provided herein in a format of “antigen” followed by the binding moiety (such as a BCMA CAR), or having “anti-” before the antigen and the binding moiety after the antigen (such as an anti-BCMA antibody), or the binding moiety followed by “to” or “directed to” and then the antigen (such as an antibody to CS1).

[0086] As used herein, the terms tumor associated antigen (TAA), cancer antigen, tumor antigen, cancer relevant antigen, and tumor relevant antigen are used interchangeably herein, referring to antigenic substance of a cancer or tumor cells. In some embodiments, a TAA presents on some tumor or cancer cells and also on some normal cells, optionally at a lower level. In some embodiments, a TAA only presents on a tumor or cancer cell but not on a normal cell. In some embodiments, 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 phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT, IL-13Ra2, IL-1 IRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1.

[0087] As used herein, 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.

[0088] As used herein, the term “antigen binding domain” refers to any protein or polypeptide domain that can specifically bind to an antigen target.

[0089] As used herein, the term “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.

[0090] The term “chimeric antigen receptor” (CAR), as used herein, 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).” The “extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen. The “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. In certain embodiments, 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. The “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. Additional exemplified sequences are provided in the sequence listing, such as: 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: CD28 co-stimulatory signaling region coding sequence: SEQ ID NO: 115; and Intracellular domain: CD3 zeta signaling region coding sequence: SEQ ID NO: 116.

[0091] Further embodiments of each exemplary domain component include 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.

[0092] As used herein, the term “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.

[0093] As used herein, the term “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. The sequences associated with each of the listed accession numbers are provided as follows: Human CD8 alpha transmembrane domain: SEQ ID NO: 120; Mouse CD8 alpha transmembrane domain: SEQ ID NO: 121; and Rat CD8 alpha transmembrane domain: SEQ ID NO: 122.

[0094] As used herein, the term “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. The fragment sequences associated with the 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.

[0095] As used 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.

[0096] As used herein, the term “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. Nat Biotechnol 20: 70-75 (2002); Haynes, N.M. et al., J Immunol 169: 5780-5786 (2002); Haynes, N.M. et al., Blood 100: 3155-3163 (2002). Nonlimiting examples include residues 114-220 of the below CD28 Sequence: SEQ ID NO: 124, and equivalents thereof.

[0097] As used herein, the term “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.

[0098] As used herein, the term “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.

[0099] Other 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.

[0100] As used herein, the term “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.

[0101] A signal peptide, as used herein, 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. In one embodiment, the signal peptide is a secretary signal.

[0102] 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. In eukaryotes, 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. 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. These are known in the art, and commercially available from vendors, e.g., Oxford Genetics.

[0103] As used herein, 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. One example of 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. In some embodiments, 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.

[0104] As used herein, the terms “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. [0105] “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.

[0106] As used herein, the term “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., ¹¹⁵Sn, ¹¹⁷Sn and ¹¹⁹Sn, a non-radioactive isotopes such as ¹³C and ¹⁵N, 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 sequences, such as green fluorescent protein (GFP) and the like. 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. As such, 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, whereas 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. In luminescence or fluorescence assays, 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. Examples of 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. [0107] As used herein, 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.

[0108] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0109] In another aspect, 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.

[0110] As used herein, 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.

[OHl] As used herein, 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, while 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. Therefore, 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.

[0112] As used herein, interleukin (IL) 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. As used herein, an interleukin can be a soluble cytokine secreted out of a cell, and/or a membrane bound (mb) 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.

[0113] Interleukin-2 (IL-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. In some embodiments, the IL-2 is a human IL-2. In some embodiments, 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.

[0114] Accordingly, in some embodiments, the IL-2 comprises, or consists essentially of, or yet further consists of MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 178). In some embodiments, 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.

[0115] In some embodiments, IL-2 as used herein is a wildtype IL-2 or an equivalent thereof. In some embodiments, 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. See, for example, human IL-2 sold by Sigma-Aldrich (SRP3085, SRP6170, 17908, 12644, H7041, or 117002), and STEMCELL™ Technologies (78036). In further embodiments, the IL-2 equivalent stimulates the proliferation or activates the cytotoxic function of NK cells or both significantly similar to the wildtype IL-2. 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), ⁵¹chromium 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. 2020 Feb 22;(156): 10.3791/60714), or a flow cytometry based cytotoxicity assay (see, for example, Kim et al. Front Immunol. 2020 Aug 14; 11 : 1851). In some embodiments, 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. Additionally or alternatively, 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 aligned to aa 82 of SEQ ID NO: 178 optionally mutated to leucine or alanine, the amino acid residue aligned to aa 88 of SEQ ID NO: 178 optionally mutated to valine, the amino acid residue aligned to aa 145 of SEQ ID NO: 178 optionally mutated to serine or alanine, or any combination thereof. Non-limiting examples include Aldesleukin consisting of

PTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF LNRWITFSQSIISTLT (SEQ ID NO: 179), Teceleukin consisting of MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHL QCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT (SEQ ID NO: 180), Neoleukin 2/15 (see, for example, Silva et al. Nature. 2019 Jan;565(7738): 186-191), as well as those disclosed in US9206243B2 or US8012465B2. Additionally or alternative, 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.

[0116] Interleukin- 15 (IL- 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. In some embodiments, the IL- 15 is a human IL-15 isoform 1. Accordingly, in some embodiments, the IL-15 comprises, or consists essentially of, or yet further consists of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 181). In some embodiments, 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. In some embodiments, the IL- 15 is a human IL-15 isoform 2. Accordingly, in some embodiments, the IL-15 comprises, or consists essentially of, or yet further consists of MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV TAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNI KEFLQSFVHIVQMFINTS (SEQ ID NO: 182). In some embodiments, the IL-15 is of other species, such as a Rhesus macaque IL- 15 having a UniProtKB reference ID of P48092.

[0117] In some embodiments, IL-15 as used herein is a wildtype IL-15 or an equivalent thereof. In some embodiments, 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 STEMCELL™ Technologies (78031). In further embodiments, 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.

[0118] Interleukin-21 (IL-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. In some embodiments, 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.

[0119] In further embodiments, 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. Cell Viability Assays. 2013 May 1. In: Markossian et al., editors. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-, accessible from www.ncbi.nlm.nih.gov/books/NBK144065/ on May 14, 2021.

[0120] 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.

[0121] As used herein, 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.

[0122] In some embodiments, 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.

[0123] A “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. 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.

[0124] 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. As is known to those of skill in the art, there are 6 classes of viruses. 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.

[0125] In several embodiments, the vector is derived from or based on a wild-type virus. In further embodiments, 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. Examples of 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. [0126] 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). 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). 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. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. In further embodiments, the lentiviral vector has a U3 region lacking a TATA box. Additionally or alternatively, the lentiviral vector has a U3 region lacking one or more of transcription factor binding site(s).

[0127] A retrovirus such as a gammaretrovirus and/or a lentivirus comprises (a) envelope comprising lipids and glycoprotein, (b) a vector genome, which is a RNA (usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs) derived to the target cell, (c) a capsid, and (d) 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. These genes are flanked at both ends by regions called long terminal repeats (LTRs). 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, and 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. For the viral genome, and 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.

[0128] With regard to the structural genes gag, pol and env themselves, 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. In some embodiments, 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.

[0129] For the production of viral vector particles, 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.

[0130] 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. Gammaretrovirus is a spherical, enveloped virion ranging from 80-100 nm in diameter. It contains a nucleocapsid, reverse-transcriptase, integrase, capsid, protease, envelope and surface units. 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. As a potential vector for gene therapy, 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.

[0131] As used herein, 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. With this method, 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. In some cases, 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. A frequently used protein is the glycoprotein G (VSV-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.

[0132] Retroviruses use specific receptors to bind and enter cells; both RD114 and BaEV envelope-containing retroviruses use the neutral amino acid (aa) transporter (ASCT2). RD114-pseudotype viral vectors use only the sodium-dependent neutral amino acid transporter (ASCT2) as their entry receptor whereas 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). Here, 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.

[0133] As disclosed herein, two recombinant envelope proteins foreign to a gammaretrovirus are used, which are a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and a modified baboon envelope glycoprotein (BaEVTR). 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.

[0134] 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. In some embodiments, the retrovirus entry receptors, ASCT1, ASCT2 and Pitl, are expressed on cells of the human hematopoietic system. In further embodiments, 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.

[0135] Alanine/Serine/Cysteine/Threonine Transporter 1 (ASCT1) is also referred to as Solute Carrier Family 1 Member 4 (SLC1 A4) or 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.

[0136] Sodium-Dependent Neutral Amino Acid Transporter Type 2 (ASCT2) is also referred to as Solute Carrier Family 1 Member 5 (SLC1A5) or Neutral Amino Acid Transporter B(0). It is a RD114/Simian Type D Retrovirus Receptor, a Baboon M7 Virus Receptor, and a RD114 Virus Receptor. In some embodiments, the 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,

Ensembl: ENSG00000105281, OMIM®: 109190, or UniProtKB/Swiss-Prot: Q15758, each of which is incorporated by reference herein in its entirety.

[0137] 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. In some embodiments, 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.

[0138] The terms “4-1BBL,” “Tumor Necrosis Factor Superfamily Member 9,” “TNFSF9,” or “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. In some embodiments, 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.

[0139] CD56, also termed as Neural Cell Adhesion Molecule 1 (NCAM), 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. In humans, 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). [0140] 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.

[0141] In some embodiments, 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. See for example, Sandrin et al. Blood (2002) 100 (3): 823-832. In some further embodiments, RD114TR comprises, or alternatively consists essentially of, or yet further consists of

MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTC PGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINK TYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDT KRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNF SLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSP FIND TEQIDLGAVTFTNCTSVANVS SPEC ALNGSVFLCGNNMAYTYLPQNWTRLCVQ ASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQ YTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEK CCFYANKSGIVRNKIRTLQEELQKRRESLATNPLWTGLQGFLPYLLPLLGPLLTLLLIL TIGPCVFNRLVQFVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 172) or an equivalent thereof.

[0142] In some embodiments, 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. See, for example, Girard-Gagnepain, Blood (2014) 124 (8): 1221-1231. In further embodiments, BaEVTR comprises, or alternatively consists essentially of, or yet further consists of MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVT CSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNK TYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLD TTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSN

TSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPS YNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLA TLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYT KLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCF YVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLPYLLPFLGPLLTLLLLLTIG PCIFNRLVQ FVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 173) or an equivalent thereof.

[0143] In some embodiments, a wild type MLV-A comprises, or alternatively consists essentially of, or yet further consists of MAARSTLSKPPQDKINPWKPLIVMGVLLGVGMAESPHQVFNVTWRVTNLMTGRTA NATSLLGTVQDAFPKLYFDLCDLVGEEWDPSDQEPYVGYGCKYPAGRQRTRTFDFY VCPGHTVKSGCGGPGEGYCGKWGCETTGQAYWKPTSSWDLISLKRGNTPWDTGCS KVACGPCYDLSKVSNSFQGATRGGRCNPLVLEFTDAGKKANWDGPKSWGLRLYRT GTDPITMFSLTRQVLNVGPRVPIGPNPVLPDQRLPSSPIEIVPAPQPPSPLNTSYPPSTTS TPSTSPTSPSVPQPPPGTGDRLLALVKGAYQALNLTNPDKTQECWLCLVSGPPYYEG VAVVGTYTNHSTAPANCTATSQHKLTLSEVTGQGLCMGAVPKTHQALCNTTQSAG SGSYYLAAPAGTMWACSTGLTPCLSTTVLNLTTDYCVLVELWPRVIYHSPDYMYGQ LEQRTKYKREPVSLTLALLLGGLTMGGIAAGIGTGTTALIKTQQFEQLHAAIQTDLNE VEKSITNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYADHTGLVRDS MAKLRERLNQRQKLFETGQGWFEGLFNRSPWFTTLISTIMGPLIVLLLILLFGPCILNR LVQFVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 174). In further embodiments, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). [0144] In some embodiments, when referring to a protein, 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. In some embodiments, a wild type BaEV comprises, or alternatively consists essentially of, or yet further consists of MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVT CSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNK TYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLD TTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSN TSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPS YNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLA TLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYT KLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCF YVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLPYLLPFLGPLLTLLLLLTIG PCIFNRLTAFINDKLNIIHAMVLTQQYQVLRTDEEAQD (SEQ ID NO: 175). In further embodiments, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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).

[0145] In some embodiments, 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. In some embodiments, a wild type RD114 comprises, or alternatively consists essentially of, or yet further consists of

MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTC PGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINK TYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDT KRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNF SLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSP FIND TEQIDLGAVTFTNCTSVANVS SPEC ALNGSVFLCGNNMAYTYLPQNWTRLCVQ ASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQ YTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEK CCFYANKSGIVRNKIRTLQEELQKRRESLATNPLWTGLQGFLPYLLPLLGPLLTLLLIL TIGPCVFSRLMAFINDRLNVVHAMVLAQQYQALKAEEEAQD (SEQ ID NO: 176). In further embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, 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).

[0146] In some embodiments, when referring to a protein, 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. In some embodiments, a wild type GALV comprises, or alternatively consists essentially of, or yet further consists of MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLT WQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVR PPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLY CKEWDCETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTD KGKLSKDWITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTS LALPPPLPPREAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGA FLTLNATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTE VSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVF NQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGT GSTALIKGPIDLQQGLTSLQIAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLF LKEGGLCAALKEECCFYIDHSGAVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNN SPWFTTLLSTIAGPLLLLLLLLILGPCIINKLVQFINDRISAVKILVLRQKYQALENEGN L (SEQ ID NO: 183). In some embodiments, 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. In further embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0147] As used herein, Multiplicity of infection (MOI) refers to the number of viral particles that are added per cell during infection.

[0148] 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.

[0149] In aspects where gene transfer is mediated by a lentiviral vector, a vector construct refers to the polynucleotide comprising the lentiviral genome or part thereof, and a therapeutic gene. As used herein, “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. As used herein, 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.

[0150] 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.

[0151] The term “adeno-associated virus” or “AAV” as used herein 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. In one embodiment, 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.

[0152] 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. Thus, 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. However, 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.

[0153] As used herein, a cell may be a prokaryotic or a eukaryotic cell. In further embodiments, the cell is an immune cell.

[0154] As used herein, “Immune 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). In some embodiments, 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. In some embodiments, the HSCs are derived from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.

[0155] “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.

[0156] 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. 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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. [0161] In one embodiment, an “immature cell” refers to a cell which does not possess the desired (adult) phenotype or genotype. For example, in one embodiment, 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.

[0162] As used herein, the term “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-2407™), NK-92MI (ATCC® CRL-2408™). 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/).

[0163] As used herein, the potency of a cell (such as an NK cell, an NKT cell, a cytotoxic T cell, a or y6 T 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).

[0164] A naturally occurring antigen presenting cell (APC) 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. As used herein, an artificial antigen presenting cells (aAPCs) 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. [0165] The term "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.). The term "somatic 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). In some embodiments, the stem or progenitor cells can be embryonic stem cells or an induced pluripotent stem cell (iPSC). As used herein, "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.

[0166] “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. As used herein, the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell. As used herein “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.

[0167] As used herein, 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.

[0168] In some embodiments, 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.

[0169] As used herein, 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.

[0170] 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).

[0171] 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.

[0172] As used herein, 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. In another aspect, 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. Such 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.

[0173] “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.

[0174] 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.

[0175] 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.

[0176] As used herein, the term “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).

[0177] The term “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. In one aspect of the disclosure, 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.

[0178] In some embodiments, 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.

[0179] The terms 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.

[0180] “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.

[0181] As used herein, the term “depleting” refers to substantially lacking. For example, 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.

[0182] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0183] As used herein, the terms "treating," "treatment" and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect. In some embodiments, 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. Examples of “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. In one aspect, treatment is the arrestment of the development of symptoms of the disease or disorder, e.g., a cancer. In some embodiments, 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. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, 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. When 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. In one aspect, treatment excludes prophylaxis. In one aspect, treatment excludes prophylaxis.

[0184] As used herein, 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. For example, for treating a cancer, 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.

[0185] In one embodiment, 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.

[0186] As used herein, 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.” The term “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, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen.

[0187] 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.

[0188] A “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.

[0189] 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. Representative 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.

[0190] 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.

[0191] As used herein, 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. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton). The term pharmaceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, 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). These semi-solid and solid 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.

[0192] “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.

[0193] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "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, both according to number of treatments and unit dose, 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. Upon formulation, 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.

[0194] As used herein, the term “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.

[0195] “Administration” 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. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application.

[0196] The term 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.

[0197] “Administration” 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.

[0198] 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.

[0199] 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. Besides being useful for human treatment, the present disclosure is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present disclosure, the human is a fetus, an infant, a pre-pubescent subject, an adolescent, a pediatric patient, or an adult. In one aspect, the subject is pre-symptomatic mammal or human. In another aspect, 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. In an additional aspect, the subject is an adult. In some instances, the adult is an adult human, e.g., an adult human greater than 18 years of age.

[0200] 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.

[0201] 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. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, 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. [0202] “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.

[0203] “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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.

[0209] 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.

[0210] The phrase “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. In patients with cancer, 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. Typically, 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.

MODES FOR CARRYING OUT THE DISCLOSURE

[0211] Several advantages of a composition 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. [0212] In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, 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%, at least about 95%, or at least about 99% cells among the cell population prepared by a method as disclosed herein expresses a CAR and/or another therapeutic protein or polypeptide. 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.

[0213] In some embodiments, 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, at least about 180 days or longer, for example in a cell culture. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, 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%, at least about 95%, or at least about 99% cells among the 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, at least about 180 days or longer, for example in a cell culture.

[0214] In some embodiments, a method as disclosed herein generates more than 1 x 10⁹ cells, more than 2 x 10⁹ cells, more than 3 x 10⁹ cells, more than 4 x 10⁹ cells, more than 5 x 10⁹ cells, more than 6 x 10⁹ cells, more than 7 x 10⁹ cells, more than 8 x 10⁹ cells, more than 9 x 10⁹ cells, more than 1 x IO¹⁰ cells, more than 2 x IO¹⁰ cells, more than 3 x IO¹⁰ cells, more than 4 x IO¹⁰ cells, more than 5 x IO¹⁰ cells, more than 6 x IO¹⁰ cells, more than 7 x

10¹⁰ cells, more than 8 x IO¹⁰ cells, more than 9 x IO¹⁰ cells, more than 1 x 10¹¹ cells, more than 2 x 10¹¹ cells, more than 3 x 10¹¹ cells, more than 4 x 10¹¹ cells, more than 5 x 10¹¹ cells, more than 6 x 10¹¹ cells, more than 7 x 10¹¹ cells, more than 8 x 10¹¹ cells, more than 9 x 10¹¹ cells, more than 1 x 10¹² cells, more than 2 x 10¹² cells, more than 3 x 10¹² cells, more than 4 x 10¹² cells, more than 5 x 10¹² cells, more than 6 x 10¹² cells, more than 7 x 10¹² cells, more than 8 x 10¹² cells, more than 9 x 10¹² cells, or more than 1 x 10¹³ cells, from 1 x 10⁸ cells, or an equivalent thereof. Additionally or alternatively, a method as disclosed herein generates up to 5 x IO¹⁰ cells, up to 6 x IO¹⁰ cells, up to 7 x IO¹⁰ cells, up to 8 x IO¹⁰ cells, up to 9 x IO¹⁰ cells, up to 1 x 10¹¹ cells, up to 2 x 10¹¹ cells, up to 3 x 10¹¹ cells, up to 4 x

10¹¹ cells, up to 5 x 10¹¹ cells, up to 6 x 10¹¹ cells, up to 7 x 10¹¹ cells, up to 8 x 10¹¹ cells, up to 9 x 10¹¹ cells, up to 1 x 10¹² cells, up to 2 x 10¹² cells, up to 3 x 10¹² cells, up to 4 x 10¹² cells, up to 5 x 10¹² cells, up to 6 x 10¹² cells, up to 7 x 10¹² cells, up to 8 x 10¹² cells, up to 9 x 10¹² cells, or up to 1 x 10¹³ cells from 1 x 10⁸ cells, or an equivalent thereof. In some embodiments, a method as disclosed herein generates about 5 x IO¹⁰ cells, about 6 x IO¹⁰ cells, about 7 x IO¹⁰ cells, about 8 x IO¹⁰ cells, about 9 x IO¹⁰ cells, about 1 x 10¹¹ cells, about 2 x 10¹¹ cells, about 3 x 10¹¹ cells, about 4 x 10¹¹ cells, about 5 x 10¹¹ cells, or about 6 x 10¹¹ cells from 1 x 10⁸ 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.

[0215] In some embodiments, 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.

Pseudotyped Gammaretroviral Particles

[0216] In one aspect, provided is a pseudotyped gammaretroviral particle. In some embodiments, 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). In further embodiments, 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. In yet further embodiments, 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. In some embodiments, the RD114TR and the BaEVTR are incorporated into the envelope of the particle as membrane proteins.

[0217] In some embodiments, a pseudotyped gammaretroviral particle as disclosed herein further comprises a vector genome encapsulated in the envelope. In further embodiments, 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. In some embodiments, the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor. In some embodiments, 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.

[0218] In some embodiments, 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.

[0219] In some embodiments, a pseudotyped gammaretroviral particle as disclosed herein further comprises either of both of a reverse transcriptase or an integrase. [0220] In some embodiments, 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.

Artificial Antigen Presenting Cells (aAPCs)

[0221] In another aspect, provided herein is an artificial antigen presenting cell (aAPC). In some embodiments, the aAPC expresses one or more of a tumor associated antigen (TAA) or a viral antigen as disclosed herein or both. In further embodiments, 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. In one embodiment, the aAPCs further expresses mb IL-21 and 4-1BBL. In some embodiments, 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.

[0222] In some embodiments, the aAPCs are engineered K562 cells.

[0223] In some embodiments, 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. In some embodiments, 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.

[0224] In some embodiments, the aAPCs are irradiated to inhibit cell proliferation and/or reduce long-term survival. In further embodiments, 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. Additionally or alternatively, 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. In some embodiments, 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. In further embodiments, the aAPCs are irradiated at about 50 Gy, about 100 Gy, about 150 Gy or about 200 Gy. In some embodiments, 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.

NK Infection and Expansion

[0225] In a further aspect, provided is a method for preparing a population of natural killer (NK) cells. The method 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). In some embodiments, 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.

[0226] In some embodiments relating to any disclosure herein, the immune cell activator(s) (such as NK 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 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). [0227] In some embodiments, 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. See, for example, Zamai L, Del Zotto G, Buccella F, et al. Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy. Cells. 2020;9(3):753. Published 2020 Mar 19. doi: 10.3390/cells9030753.

[0228] In some embodiments, the aAPC is an aAPC as disclosed herein. In some embodiments, 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 :7 or more, about 1 :8 or more, about 1 :9 or more, about 1 : 10 or more, about 1 :20 or more, about 1 : 50 or more, or about 1 : 100 or more. Additionally or alternatively, 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, about 1 :20 or less, about 1 : 50 or less, or about 1 : 100 or less. In some embodiments, 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. In some embodiments, 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.

[0229] In some embodiments, 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.

[0230] In some embodiments, the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-2. In further embodiments, the cell population is cultured with about 1 lU/mL to about 1000 lU/ml IL2. In some embodiments, 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, about 190 lU/mL or more, about 200 lU/mL or more, about 250 lU/mL or more, about 300 lU/mL or more, about 400 lU/mL or more, about 500 lU/mL or more, about 600 lU/mL or more, about 700 lU/mL or more, about 800 lU/mL or more, about 900 lU/mL or more, or about 1000 lU/mL or more IL2. Additionally or alternatively, 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, about 5 lU/mL or less IL2. In some embodiments, the cytokines comprises an IL-2. In further embodiments, 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. In some embodiments, 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/mL, about 900 lU/mL, or about 1000 lU/mL IL2.

[0231] In some embodiments, the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-15. In further embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0232] In some embodiments, the cytokines comprises, or alternatively consists essentially of, or yet further consists of an IL-21. In further embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0233] In some embodiments, the cell population is cultured with more than one cytokines, such as a combination of the cytokines as disclosed herein. In some embodiments, 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 further embodiments, the cell population is cultured with either or both of 50 lU/ml IL-2 and 0.5 ng/ml IL-15. [0234] In some embodiments, 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. See, for example, Peled et al., Blood (2017) 130 (Supplement 1): 657 and Childs RW, Carlsten M. Nat Rev Drug Discov. 2015;14(7):487-498. Additional examples are mTOR inhibitors, PI3K inhibitors, and/or STING-activating cyclic dinucleotides (CDNs).

[0235] In some embodiments, 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. In some embodiments, the CAR specifically recognizes and binds to a tumor associated antigen (TAA) and/or a viral antigen. In further embodiments, the tumor associated antigen (TAA) and/or the viral antigen recognized and bound by the CAR is the one expressed by aAPC. In some embodiments, therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor.

[0236] In some embodiments, 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. In further embodiments, 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.

[0237] In some embodiments, 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.

[0238] 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. In some embodiments, the pseudotyped gammaretroviral particle is disclosed herein. In some embodiments, 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. In further embodiments, the coding polynucleotide and/or a reverse-complement thereof is flanked by two long terminal repeats (LTRs). Additionally or alternatively, the pseudotyped gammaretroviral particle comprises an RD114TR and/or a BaEVTR as disclosed herein. In some embodiments, 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. In yet further embodiments, the components as disclosed herein are located in the vector genome from 5’ to 3’. In some embodiments, the pseudotyped gammaretroviral particle further comprises either of both of a reverse transcriptase and an integrase. In some embodiments, 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.

[0239] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0240] In some embodiments, 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.

[0241] In some embodiments, 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. In some embodiments, the RetroNectin is coated on the inner surface of a container in which the cell population is transduced. In further embodiments, the container is a bag suitable for culturing a cell. In yet further embodiments, the container is a plate suitable for culturing a cell, In another embodiments, the container is a flask suitable for culturing a cell. In some embodiments, the cell population expresses either or both of Integrin a4pi (VLA-4) and Integrin a5pi (VLA-5).

[0242] In some embodiments, the cell population is isolated from a biological sample (such as a blood sample) of a subject. In some embodiments, 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. In some embodiments, 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.

[0243] In some embodiments, 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. In some embodiments, a method as disclosed herein further comprises enriching, deriving and/or generating CD56^dim cells. In further embodiments, CD56^dim cells express CD56 surface antigen in low density and specializes in cytotoxic function. Additionally or alternatively, a method as disclosed herein further comprises enriching, deriving and/or generating CD56^bnght cells. In further embodiments, CD56^bnght cells express CD56 surface antigen in high density and specializes in cytokine secretion. See, for example, Jacobs et al. Eur J Immunol. 2001 Oct;31(10):3121-7. In some embodiments, 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. In further embodiments, 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. In yet further embodiments, the expression levels of the peripheral blood NK cell and/or the cord blood NK cell are different. In some embodiments, 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. In some embodiments, a method as disclosed herein further comprises enriching a peripheral blood NK cell and/or a cord blood NK cell in the cell population.

[0244] In some embodiments, 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.

[0245] In some embodiments, a cell population of any step and/or embodiment and/or aspect (such as a depleted cell population) 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.

[0246] In some embodiments, 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.

[0247] In some embodiments, one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving NK cells.

[0248] In some embodiments, 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.

[0249] Some embodiments of a method as disclosed herein may further comprises isolating, enriching and/or purifying a cell population. In some embodiments, a cell population of any step and/or embodiment and/or aspect is isolated, enriched or purified.

[0250] In some embodiments, 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 ImmunoCult™-XF Medium from Stemcell technologies in the culturing step.

[0251] 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.

[0252] 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 comprising the cell population; (ix) cell phenotype of the aAPCs; (x) IL- 15 level in a composition comprising the cell population; (xi) Expression of the CAR in the cell population; (xii) endotoxin in the cell population or a composition comprising the cell population; (xiii) residual tumor burden and/or contamination with aAPCs of a composition comprising the cell population; (xiv) potency of the cell population; (xv) IFN-y, IL- 15 and TNF-alpha released by the cell population; (xvi) cytotoxic activity of the cell population; (xvii) degranulation of the cell population; and (xviii) monitoring or determining one or more of the following of the culture: cell aggregation, glucose, or lactate. In further embodiments, 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

[0253] In yet a further aspect, provided is a method for preparing a population of y6 T cells. The method 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). In some embodiments, 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.

[0254] In some embodiments, 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).

[0255] 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. In some embodiments, the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor. In some embodiments, the CAR specifically recognizes and binds to a tumor associated antigen (TAA) and/or a viral antigen. In further embodiments, the tumor associated antigen (TAA) and/or the viral antigen recognized and bound by the CAR is the one expressed on the aAPC.

[0256] 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. In some embodiments, the culturing step is repeated for once, twice, three times, or more times with the same or different activator(s).

[0257] In some embodiments, 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. In some embodiments, 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.

[0258] In some embodiments, one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving y6 T cells. In some embodiments, 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. In some embodiments, the cell population is substantially free of cells expressing TCR a chain or TCR P chain.

Antigen and Chimeric Antigen Receptor (CAR)

[0259] In some embodiments of any aspect as disclosed herein, 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 kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPC AM), B7H3, KIT, IL- 13Ra2, IL-l lRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1

[0260] In some embodiments of any aspect as disclosed herein, 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. In some embodiments, the CAR further comprises a signal peptide.

[0261] In some embodiments of any aspect as disclosed herein, 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 kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPC AM), B7H3, KIT, IL- 13Ra2, IL-l lRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1

[0262] In some embodiments of any aspect as disclosed herein, 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 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPC AM), B7H3, KIT, IL- 13Ra2, IL-l lRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1.

[0263] In some embodiments of any aspect as disclosed herein, 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.

[0264] In some embodiments, the hinge domain comprises a CD8 a hinge domain.

[0265] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain.

[0266] In some embodiments, 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). In further embodiments, the costimulatory signaling region comprises a CD28 costimulatory signaling region or a 4- IBB costimulatory signaling region or both.

[0267] In some embodiments, the signaling domain comprises, or alternatively consists essentially of, or yet further consists of a CD3 zeta signaling domain.

[0268] In some embodiments, 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.

[0269] In further embodiments, 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 MICA (soluble or membrane bound).

[0270] Additionally or alternatively, 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.

[0271] In some embodiments, 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.

Coding Polynucleotide and Vectors

[0272] In some embodiments, 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. In further embodiments, 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”). In some embodiments, the coding polynucleotide further comprises a regulatory sequence directing expression of the suicide gene. In yet further embodiments, the regulatory sequence is inducible.

[0273] In some embodiments, 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. [0274] In some embodiments, the coding polynucleotide is introduced to the cell population via a vector. In further embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the viral vector is selected form a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno- associated viral vector or Herpes viral vector. In a further embodiment, the viral backbone contains essential nucleic acids or sequences for integration of the coding polynucleotide into a target cell’s genome. In some embodiments, 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.

[0275] 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. In some embodiments, the vector is selected for expression in prokaryotic or eukaryotic cells. In some embodiments, the vector comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide as described herein, encoding the modified protein. In some embodiments, the vector comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide as described herein, permitting replication of the polynucleotide. In further embodiments, the vector further comprises a regulatory sequence operatively linked to the polynucleotide and directing the replication of the polynucleotide. In yet a further embodiment, 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).

[0276] In some embodiments, a coding sequence is introduced into a target cell via a pseudotyped gammaretroviral particle as disclosed herein.

Packaging Systems

[0277] 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.

[0278] 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. In some embodiments, 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. Examples of suitable envelope proteins include, but are not limited to those containing the VSVG or RD114 domains. In some embodiments, envelope proteins used herein include, but are not limited to RD114TR and/or BaEVTR.

[0279] This disclosure also provides the suitable packaging cell line. In one aspect, the packaging cell line is the HEK-293 cell line. Other 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. In some embodiments, 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. 2007 Dec;14(24): 1705-l l; Dakiw Piaceski A et al, Eur Cell Mater. 2018 Feb 14;35:73-86; Ghani et al., Hum Gene Ther. 2009 Sep;20(9):966-74; U.S. Patent Publication No. US20060270042; and U.S. Patent No. 8034335.

[0280] 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. [0281] 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.

[0282] 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. In some embodiments, the viral particle is a pseudotyped viral particle. In further embodiments, the particles are separated from the cellular supernatant and conjugated to an antibody for cell-specific targeting.

[0283] Also provided is a method for producing a retroviral particle (such as a gammaretroviral particle). The method 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.

[0284] In some embodiments, the vector is a non-viral vector. In further embodiments, the vector is a plasmid.

[0285] In some embodiments, 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.

[0286] In some embodiments, the method further comprises culturing the transduced second packaging cell line.

[0287] In some embodiments, 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. Such produced retroviral vector is enveloped with a lipid membrane comprising GALV. Also, since the 293 Vec-GALV cell is transiently transfected in this step, 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. Also, since 293 Vec-BaEV cell is stably transduced in this step, the replicated retroviral vector here is also referred to as a stable vector and was tested in FIG. 10.

[0288] Surprisingly, 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. Additionally, these stable virus producer cells (also referred to as producers) generated are stable and integrated with a transgene of interest. In some embodiments, 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.

[0289] In some embodiments, 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.

[0290] In some embodiments, the first packaging cell line and the second packaging cell line expresses components required for packaging a retroviral particle. In further embodiments, 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.

[0291] In some embodiments, 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. [0292] Additionally provided is the transduced second packaging cell line, a cell thereof, or a cell population thereof. In some embodiments, the cell stably produce the retroviral particle.

[0293] In some embodiments, 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.

[0294] In some embodiments, 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.

[0295] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0296] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0297] Accordingly provided is a retroviral particle produced by a method as disclosed herein.

[0298] In a further aspect, provided is 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. [0299] In some embodiments, 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. In some embodiments, 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. In some embodiments, the 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.

[0300] In some embodiments, 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.

[0301] In some embodiments, 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. In some embodiments, the reverse transcriptase polymerase which is generated from the packaging plasmid is also located within the capsid with the RNA transcript. In some embodiments, the capsid encases and protects the full- length RNA transcript.

[0302] In some embodiments, 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. After this final incubation period, 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.

[0304] Thus, in one aspect, provided herein is 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. In some embodiments, the packaging system further comprises a packaging cell line. In further embodiments, the packaging cell line is a 293T cell line.

[0305] In some embodiments, 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. In some embodiments, the therapeutic protein or polypeptide is selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor. In some embodiments, 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. In further embodiments, the coding polypeptide is located between the 5’ cap and the3’ poly-A tail.

[0306] In some embodiments, 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.

[0307] In a further aspect, provided is a method for producing a pseudotyped gammaretroviral particle. The method 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. In further embodiments, the packaging cell line is a 293T cell line.

[0308] In some embodiments, 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. In further embodiments, the packaging system comprises at least two envelope plasmids, one expressing RD114TR and the other expressing BaEVTR. In yet further embodiments, 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. In some embodiments, 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, 1:2:1:1; 1.5:15:1:1, 15:1:1:1, 1:15:1:1; or 1:1:1:1; 2.5 :2.5 : 1 : 1 : 1, 2.5:2:1:1, 2.5:1.5:1:1, 2.5:1:1:1, 2.5:0.5:l:l, 2:2.5:1:1, 1.5:2.5:1:1, 1:2.5:1:1, 0.5:2.5:l:l; 2:2:1:1, 2:1.5:1:1, 2:1:1:1, 2:0.5:1:1, 1.5:2:1:1, 1:2:1:1, 0.5:2:l:l; 1.5:1.5:1:1, 15:1:1:1, 1.5:0.5:l:l, 1:1.5:1:1, 0.5:1.5:1:1; 1 : 1 : 1 : 1, 1:05:1:1, 05:1:1:1; 0.75:0.75:1:1, 0.75:0.5:1:1, 0.5:0.75:1:1; or 0.5:0.5:1:1.

Cells and Cell Populations

[0309] In one aspect, provided is a cell population as disclosed herein and/or a progeny thereof. In some embodiments, the cell population is clonal. In some embodiments, the cell population is isolated and/or enriched and/or engineered.

[0310] In a one aspect, provided is an immune cell (such as an NK cell and/or a y6 T cell) or a population thereof produced or prepared by a method as disclosed herein. Further provided is a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier. In some embodiments, the immune cell or population thereof is isolated and/or enriched and/or engineered.

[0311] Additionally or alternatively, the cell as disclosed herein are derived or differentiated from a stem cell. In some embodiments, 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. In some instances, 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. In some embodiments, the T cell does not express CD3, i.e. is a CD3- T cell. In some embodiments, the T cell does not express CD4, i.e. is a CD4- T cell. In some embodiments, 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.

[0312] In some embodiments, 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). In some embodiments, the cell population comprises substantially a stem cell, such as HSCs, and/or an iPSC, optionally deriving to an immune cell.

[0313] In some embodiments, 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.

[0314] In a further aspect, provided is an NK cell or a population thereof produced or prepared by a method as disclosed herein. Further provided is a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier.

[0315] In a further aspect, provided is a y6 T cell or a population thereof produced or prepared by a method as disclosed herein. Further provided is a composition comprising a cell or a population thereof and a carrier, optionally a pharmaceutical acceptable carrier.

[0316] In some embodiments, 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.

[0317] Additionally or alternatively, 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;

[0318] In some embodiments, the cell and/or a population thereof, and/or the composition as disclosed herein are sterile.

[0319] In some embodiments, 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). Such TPD may be determined based on administration routes. In one embodiment, the TPD is 5 EUKg^-1 h^-1 endotoxin, such as for intravenous or intramuscular administration. In one embodiment, the TPD is 0.2 EUKg^-1 h^-1 endotoxin, such as for intrathecal administration.

[0320] In some embodiments, 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).

[0321] In some embodiments, 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. In further embodiments, 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.

[0322] In some embodiments, 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. In further embodiments, 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.

[0323] In some embodiments, 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

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% cells expressing CD3 among the total cells;

[0324] In some embodiments, 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.

[0325] In some embodiments, 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.

[0326] In some embodiments, 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. Such 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.

[0327] In some embodiments, 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.

[0328] In further embodiments, 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.

[0329] In some embodiments, 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 10⁶ cells per kilogram of body weight of the subject to be treated, including but not limited to more than 2 x 10⁶ cells per kilogram, more than 3 x 10⁶ cells per kilogram, more than 4 x 10⁶ cells per kilogram, more than 5 x 10⁶ cells per kilogram, more than 6 x 10⁶ cells per kilogram, more than 7 x 10⁶ cells per kilogram, more than 8 x 10⁶ cells per kilogram, more than 9 x 10⁶ cells per kilogram, more than 1 x 10⁷ cells per kilogram, more than 2 x 10⁷ cells per kilogram, more than 3 x 10⁷ cells per kilogram, more than 4 x 10⁷ cells per kilogram, more than 5 x 10⁷ cells per kilogram, more than 6 x 10⁷ cells per kilogram, more than 7 x 10⁷ cells per kilogram, more than 8 x 10⁷ cells per kilogram, more than 9 x 10⁷ cells per kilogram, or more than 1 x 10⁸ cells per kilogram.

Method of Treatment

[0330] In one aspect, provided is a method for inhibiting the growth of a cancer cell. The method 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. In some embodiments, the antigen recognized by the CAR is a tumor associated antigen (TAA) expressed by the cancer cell. In some embodiments, the contacting is in vivo or in vitro.

[0331] In a further embodiment, provided is a method for treating a cancer in a subject. The method 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. In some embodiments, the antigen recognized by the CAR is a TAA expressed by a cell of the cancer. In some embodiment, the administration is a first line therapy, a second line therapy, a third line therapy, or a fourth line therapy.

[0332] In some embodiments of a method as disclosed herein, 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.

[0333] In some embodiments, the subject is administered with less than 1 x 10², 2 x 10², 3 x

10², 4 x 10², 5 x 10², 6 x 10², 7 x 10², 8 x 10², 9 x 10², 1 x 10³, 2 x 10³, 3 x 10³, 4 x 10³, 5 x

10³, 6 x 10³, 7 x 10³, 8 x 10³, 9 x 10³, 1 x 10⁴ T cells per kilogram of body weight. [0334] In some embodiments, 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, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising at any site; genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre- tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs;, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or/and neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.

[0335] In some embodiments, 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.

[0336] 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. In some embodiments, the separate therapy comprises, or alternatively consists essentially of, or yet further consists of surgical resection, chemotherapy, radiation therapy, immunotherapy and targeted therapy. In some embodiments, the separate therapy is a first line therapy, a second line therapy, a third line therapy, or a fourth line therapy.

[0337] In some embodiments, 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. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the gene or protein 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. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to provide therapeutic benefit.

[0338] The term 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. The disclosure is not limited by the route of administration, the formulation or dosing schedule. In some embodiments, the administration is performed locally, such as to the bone marrow or in the brain. In some embodiments, the administration is performed systemically. In some embodiments, 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.

[0339] Further provided is a kit 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. In some embodiments, 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.

[0340] The following examples are intended to illustrate, and not limit the embodiments disclosed herein.

Experimental Methods

Experiment No. 1: A distinct use of Gamma Retroviral vector (PCIR) mediated gene transfer to deliver and test function of chimeric antigen receptors in human primary NK cells

[0341] Lack of an efficient method for gene delivery to human primary NK cells, instability of the transgenes, and low viability of the transduced CAR-NK cells are most obstacles for successful CAR-NK developing and manufacturing. Vein-to vein time is critical, since undesirable wait time in patients correlates with poor prognosis and not all eligible patients may receive therapy because of poor T cell function, preparation time and/or time to relapse. Currently available therapies have variable potency and/or toxicity. Briefly, compromised T cells in patients may affect product potency. Such variability may cause unpredictable treatment outcome. Further, not infrequent grade 3-4 cytokine release causes syndrome and neurotoxicity. Production cost is high in view of the complicated logistics, inefficiency of scale and limited availability. Manufacturing failures thus include, but are not limited to, there is no ability to create inventory in individualized therapy and retreatment can be difficult due to limited patient starting material.

[0342] To address these issues, provided herein is a distinct use of Gamma Retroviral vector (PCIR) mediated gene transfer to deliver and test function of chimeric antigen receptors (CARs) in human primary NK cells. [0343] Lentivirus and Retrovirus vector mediated gene delivery have been proven a safety way for genetic engineering. However, many reports and even in Applicant’s firsthand experiments, 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.

[0344] Firstly, Moloney murine leukemia virus derived SFG gamma retroviral vector was tested for gene delivery to Peripheral blood (PB)-derived human primary NK cells.

[0345] As show in FIG. 1, 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. 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 (Invitrogen™ 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. Using 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. Clearly, after isolation and culture 5 days, 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).

[0346] The transduction efficiency was then assessed at post infection day 3. Enrichment, expansion, functional and other therapeutics applications were performed.

[0347] Experimental results showed distinct high viable NK cells ranging from 73.51% - 82.81% (a rough viable cell gate can be drawn by excluding low-forward scatter (FSC) and high-side scatter (SSC) events) after infection (FIG. 3B), EGFR CAR gene (Fab-AF647 positive) transduction efficiency ranging from 65.11% - 72.75% among three donors (FIG. 3C) and yields with great proliferation state of fold 25, 34 and 45 in three donors compared with initial infection NK numbers at post infection day 8 (FIG. 3D). This extensive optimization enables consistent transduction of CAR constructs in primary human NK cells with 50% - 80% efficiency; 25 ~ 45 fold ex vivo expansion of CARNK cells from 10 to 20 million to hundreds of millions in 8 days; and fll retention of CAR with NK cell expansion ex vivo.

[0348] Longer cultures were tested. See, for example FIG. 7. This extensive optimization as disclosed herein enabled expansion from tens of millions to tens of billions of NK cells in 17- 24 days and provided truly off-the-shelf products frozen with excellent viability upon thawing. Cost of commercial manufacturing at least is about $2000 per dose and more than 500 does per batch.

[0349] Retrovirus titration based on direct infection of activated NK cells at same proliferate state with same deliver strategy is provided in FIG. 4.

[0350] Further, at post sorting day 10, and the GFP positive cells were significantly reduced from around 100% to 39% for both PCIL-EGFR-CAR-NK and PCIL-GFP empty vector control (FIGS. 5A and 5C). However, flow analysis demonstrated that there were highly stable transduced gene expression at post sorting day 10, day 14 and day 25 for PCIR-EGFR- CAR-NK (FIGS. 5B and 5C).

[0351] Additionally observed were average 85% (CD19-PE positive) and 79.6% (Goat anti Mouse Fab-AF647 positive) transduction rates for empty vector truncated CD19 (EV-Tcdl9) and EGFR transgene separately (FIG. 6A). Also, there were dynamic real time killing for engineered EGFR-CAR-NK compared to Empty vector mock transduction and nontransduced NK groups with lowest live cells index (FIG. 6B).

[0352] Secondly, 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.

[0353] Thirdly, several studies have indeed shown that 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 ASCT-1 and 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, therefore both BaEVTR and RD114TR were tested. BaEVTR plasmids are practically used to the larger inserts delivery, such as tandem CAR, Dual CAR and BiTE CAR.

[0354] Next, 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.

[0355] Further, 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.

[0356] 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.

[0357] In some embodiments, 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.

[0358] In some embodiments, 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. Experiment No. 2: Cell therapies: process development studies

[0359] Media and/or additives are tested for supporting 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).

[0360] 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.

[0361] 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).

[0362] Various CD3 depletion 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.

[0363] 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.

[0364] Further, 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. Additionally, 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.

[0365] 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.

[0366] 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.

[0368] More than 1 x 10¹¹ CD56+ cells were generated starting from 1 x 10⁸ 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.

[0369] Testing 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, eomesodermin-efluor-660, KLRG-l-PE (eBiosciences San Diego CA), DAP12-PE, CD158-FITC (R&D Minneapolis MN), DAP10-FITC (Antibodies-Online Atlanta GA) , NKG2A-PE-Cy7 (Beckman Coulter Irving, TX).

Table 1 Exemplified Tests

[0370] As stated above, 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

[0371] 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.

[0372] Cord blood contains approximately 5 x 10⁸ total nucleated cells (TNCs), of which 30% are NK cells. Thus, each cord blood unit (60 mL) contains about 1~2 x 10⁸ NK cells.

Table 3 Exemplified characterization

Table 4 Exemplified in process testing

Table 5 Exemplified release testing: NK-FLT3-CAR

[0373] 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 10⁵ T cells/kg body weight and NK cell doses are well tolerated when T-cell contamination is 0.03 x 10⁵ 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.

[0374] Potency Assays were performed. 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).

Experiment No. 3: Stable retrovirus virus producer-293 VEC-BaEV for generating a vector with large size transgenes

[0375] 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.

[0376] Among the retroviruses, 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. In addition to ASCT2, BaEV also uses ASCT1 as a cell entry receptor. Gibbon ape leukemia virus (GALV) uses Sodium-Dependent Phosphate Transporters (Pitl) as their entry receptors. See, FIG. 8A. Immunostaining for ASCT1, ASCT2, and Pitl was performed on two packaging cell lines, 293 Vec-GalV and 293 Vec- BaEV, and two titration cell lines, Jurkat T and HT1080. See the result shown in FIG. 8B. Both cell lines, Jurkat T and HT1080, can be used for titration of the three retrovirus envelop proteins, BaEV, RD114 and GALV, due to the ubiquitous expression of their entry receptors. For the 293 based packaging cell lines, 293 Vec GALV and 293 Vec BaEV, only the GALV- pseudotyped retrovirus can infect themselves using entry receptor Pitl.

[0377] A workflow of transduction strategy for generation of stable retrovirus virus producer-293 VEC-BaEV is provided herein.

[0378] The moloney murine leukemia virus (Mo-MuLV)-based retroviral vector (PCJR) is 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.

[0379] 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.

[0380] Firstly, 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-MEM™ Reduced-Serum Medium. Meanwhile, a total of 10 pg/lOpl of PEI MAX 40K was also diluted in 150 pl of Opti-MEM™ 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.

[0381] This supernatant was then used to transduce a BaEV pseudotyping packaging cell line 293Vec-BaEV to generate a bulk producer. For high-titer clone generation, 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. [0382] Then the supernatant can be continually produced from the high titer stable retrovirus virus producer-293 VEC-BaEV.

[0383] Improved titer of raw supernatant was observed from the stable retrovirus virus producer-293 VEC-BaEV for a vector with large size transgenes. See, FIG. 10.

[0384] “ 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. A representative retrovirus for a plasmid 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 plate should reach 90-100 % confluence. The raw supernatant was then collected and spun at 1500 g for 5 min. Titration of 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 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. The plate was incubated at 37 degrees for 48h, then flow cytometry analysis was performed for determination of the titer (transducing Units/ml) according to the formula of Transducing Units (TU)/mL = [(target cell number) X (%Positive cells)]. For this example, a reporter gene RQR8 was detected by flow antibodies against human CD34 Antibody (QBEnd/10) (Allophycocyanin) (Novus Biologicals, LLC, #FAB7227A). B). There is an improved titer of raw supernatant from the stable retrovirus virus producer-293 VEC -BaEV (TU=7.2E5) for a vector with large size transgenes compared to the virus produced transiently (TU=2.2E5).

Equivalents

[0385] It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0386] It should be understood that although the present disclosure has been specifically disclosed by specific embodiments and optional features, modification, improvement and variation of the embodiments therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of particular embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0387] The scoped of the disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0388] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0389] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

PARTIAL SEQUENCE LISTING

[0390] IgGl hinge domain: LEPKSCDKTHTCPPCPDPKGT (SEQ ID NO: 1)

[0391] CD28 transmembrane and cytoplasmic domain:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP

YAPPRDFAAYRS (SEQ ID NO: 2)

[0392] CD3 zeta signaling domain:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ

EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 3)

[0393] IL 2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 4),

[0394] IgGl signal peptide: MGWSSIILFLVATATGVH (SEQ ID NO: 5)

[0395] CDRs of anti-NKG2D antigen binding domain:

CDRL1 :SGSSSNIGNNAVN (SEQ ID NO: 6)

CDRL2: YDDLLPS (SEQ ID NO: 7)

CDRL3: AAWDDSLNGPV (SEQ ID NO: 8)

CDRH1: GFTFSSY (SEQ ID NO: 9)

CDRH2: RYDGSN (SEQ ID NO: 10)

CDRH3: DRGLGDGTYFDY (SEQ ID NO: 11)

[0396] Anti-NKG2D light chain variable region:

QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSG

VSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL (SEQ ID NO: 12)

[0397] Anti-NKG2D heavy chain variable region:

QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYW GQGTTVTVSS (SEQ ID NO: 13)

[0398] Peptide linker: GGGGSGGGGSGGGGS (SEQ ID NO: 14) [0399] human IgG4 Fc region:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 81)

[0400] 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)

ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 82)

[0401] Human muscle aldolase (HMA) peptide linker: PSGQAGAAASESLFVSNHAY (SEQ ID NO: 83)

[0402] Detectable marker: YPYDVPDYA (SEQ ID NO: 84)

[0403] T2A peptide: HVGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 85)

[0404] A nucleotide sequence encoding a signal peptide:

ATGGGGTGGTCAAGCATTATTCTGTTTCTGGTCGCTACCGCTACAGGCGTCCAT (SEQ ID NO: 86)

[0405] A nucleotide sequence encoding a signal peptide:

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAA ACAGT (SEQ ID NO: 87)

[0406] A nucleotide sequence encoding a linker peptide:

GGTGGGGGCGGCTCTGGTGGCGGTGGCAGCGGCGGAGGTGGCAGT (SEQ ID NO: 88)

[0407] A nucleotide sequence encoding a transmembrane and cytoplasmic domain:

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA

CAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAG TGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCA

GCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 89)

[0408] A nucleotide sequence encoding a signaling domain:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA

CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA

CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC

CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA

GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT

TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 90) or an equivalent thereof;

[0409] A nucleotide sequence encoding a cleavable peptide:

CACGTGGGTTCTGGAGAAGGACGCGGTTCCTTGTTGACGTGTGGCGATGTAGAG

GAAAATCCGGGTCCA (SEQ ID NO: 91)

[0410] A nucleotide sequence encoding a linker:

CCGAGCGGCCAGGCGGGCGCGGCGGCATCGGAGTCCCTGTTTGTGTCAAATCAC

GCCTAC (SEQ ID NO: 92)

[0411] A nucleotide sequence encoding a hinge domain:

CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCA

AAGGTACC (SEQ ID NO: 109)

[0412] peptide linker: (glycine-serine)n wherein n is an integer from 1 to 6 (SEQ ID NO: 110)

[0413] 6x His tag: His His His His His His (SEQ ID NO: 111)

[0414] Hinge domain: IgGl heavy chain hinge coding sequence:

CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO: 112)

[0415] CD28 transmembrane region coding sequence:

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA

CAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 113)

[0416] 4-1BB co-stimulatory signaling region coding sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA

GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA

GAAGGAGGATGTGAACTG (SEQ ID NO: 114)

[0417] CD28 co-stimulatory signaling region coding sequence:

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC (SEQ ID NO: 115)

[0418] CD3 zeta signaling region coding sequence:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA (SEQ ID NO: 116)

[0419] Human CD8 alpha hinge domain: PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 117).

[0420] Mouse CD8 alpha hinge domain: KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 118).

[0421] Cat CD8 alpha hinge domain: PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY (SEQ ID NO: 119).

[0422] Human CD8 alpha transmembrane domain: IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 120).

[0423] Mouse CD8 alpha transmembrane domain: IWAPLAGICVALLLSLIITLI (SEQ ID NO: 121).

[0424] Rat CD8 alpha transmembrane domain: IWAPLAGICAVLLLSLVITLI (SEQ ID NO: 122). [0425] 4- IBB costimulatory signaling region: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 123)

[0426] CD28 Sequence: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLDSAVEVCVVYGNYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO: 124)

[0427] ICOS costimulatory signaling region coding sequence: acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta (SEQ ID NO: 125)

[0428] 0X40 costimulatory signaling region coding sequence:

AGGGACCAG AGGCTGCCCC CCGATGCCCA CAAGCCCCCT GGGGGAGGCA GTTTCCGGAC CCCCATCCAA GAGGAGCAGG CCGACGCCCA CTCCACCCTG GCCAAGATC (SEQ ID NO: 126)

[0429] Linker: GGGGS (SEQ ID NO: 134).

[0430] Linker: (GGGGS)n, wherein 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.

[0431] EFl alpha promoter sequence:

AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCAC AGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAA GGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCC CGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTT CGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCT CCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCG TTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAG TTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCT AGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTT TGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC

(SEQ ID NO: 148)

[0432] A reverse sequence of SEQ ID NO: 71: AVGTIVDQSAK (SEQ ID NO: 151)

[0433] IgGl heavy chain signal peptideDNA

ATGGAATTTGGGCTGCGCTGGGTTTTCCTTGTTGCTATTTTAAAAGATGTCCAGTG

T (SEQ ID NO: 168)

[0434] Protein

MEFGLRWVFLVAILKDVQC (SEQ ID NO: 169)

[0435] IgGl FcDNA

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA

CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA

TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG

ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA

AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC

TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT

CCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC

AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCA

AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC

CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC

CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA

GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT

GCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGAGCTGCAACTGGA

GGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCTGTGGACGACCA

TCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACC

TTCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCTGAAGCAGACCATCA

TCCCCGACTACAGGAACATGATCGGACAGGGGGCC (SEQ ID NO: 170)

[0436] Protein encoded by IgGl FcDNA

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<A

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDL KQTIIPDYRNMIGQGA (SEQ ID NO: 171)

EXEMPLIFIED EMBODIMENTS OF THE DISCLOSURE

[0437] 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.

[0438] 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.

[0439] 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):

(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,

(B) a reverse-complement of (A), or

(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.

[0440] 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. [0441] 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.

[0442] 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.

[0443] 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):

(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,

(ii) 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, 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,

(iii) one or more of cytokines thereby activating or proliferating NK cells, or

(iv) 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).

[0444] 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.

[0445] 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):

(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,

(ii) 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,

(iii) one or more of cytokines thereby activating or proliferating NK cells, or

(iv) 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).

Optionally wherein 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.

[0446] 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.

[0447] Embodiment 11. The method of any one of embodiments 7 to 10, wherein the aAPCs further expresses mb IL-21 and 4-1BBL.

[0448] Embodiment 12. The method of any one of embodiments 7 to 11, wherein the aAPCs are engineered K562 cells.

[0449] 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. [0450] Embodiment 14. The method of embodiment 13, wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy.

[0451] 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] Embodiment 19. The method of embodiment 16, wherein the cell population is cultured with 50 lU/ml IL-2.

[0456] 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.

[0457] 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.

[0458] 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. [0459] Embodiment 23. The method of embodiment 22, wherein 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.

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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. [0467] 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.

[0468] 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).

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] 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.

[0475] 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. [0476] Embodiment 40. The method of embodiment 38, wherein one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving NK cells.

[0477] Embodiment 41. The method of any one of embodiments 7 to 40, wherein any cell population is substantially free of T cells.

[0478] Embodiment 42. The method of any one of embodiments 7 to 41, wherein any cell population is substantially free of T regulatory cells.

[0479] Embodiment 43. The method of any one of embodiments 7 to 42, wherein any cell population is isolated, enriched or purified.

[0480] 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 ImmunoCult™-XF Medium from Stemcell technologies in the culturing step.

[0481] 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.

[0482] Embodiment 46. The method of any one of embodiments 7 to 45, generating more than 1 x 10¹¹ cells from 1 x 10⁸ cells .

[0483] 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:

(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 comprising the cell population;

(ix) cell phenotype of the aAPCs;

(x) IL- 15 level in a composition comprising the cell population;

(xi) Expression of the CAR in the cell population;

(xii) endotoxin in the cell population or a composition comprising the cell population;

(xiii) residual tumor burden and/or contamination with aAPCs of a composition comprising the cell population;

(xiv) potency of the cell population;

(xv) IFN-gamma, IL-15 and TNF-alpha released by the cell population;

(xvi) cytotoxic activity of the cell population;

(xvii) degranulation of the cell population; and

(xviii) monitoring or determining one or more of the following of the culture: cell aggregation, glucose, or lactate.

[0484] 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. [0485] Embodiment 49. The method of embodiment 48, wherein the phenotype detection comprises detecting cell expression level of CD56 by the cell population.

[0486] 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 (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), 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 phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPC AM), B7H3, KIT, IL- 13Ra2, IL-l lRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1. [0487] Embodiment 51. The method of any one of embodiments 8 to 50, wherein the 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 (EGFR VIII), 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 phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (R0R1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPC AM), B7H3, KIT, IL- 13Ra2, IL-l lRa, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, mucin 1 (Mucl), NCAM, Prostase, PAP, ELF2M, Ephrin B2, fibroblast activation protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephrin type-A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FC AR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, glypican 3 (GPC3), FCRL5, or IGLL1

[0488] 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.

[0489] 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.

[0490] Embodiment 54. The method of any one of embodiments 8 to 53, wherein the CAR further comprises a signal peptide.

[0491] Embodiment 55. The method of embodiment 53 or 54, wherein the hinge domain comprises a CD8 a hinge domain.

[0492] Embodiment 56. The method of any one of embodiments 53 to 55, wherein the transmembrane domain comprises a CD8a transmembrane domain.

[0493] 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.

[0494] 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.

[0495] Embodiment 59. The method of any one of embodiments 53 to 58, wherein the signaling domain comprises a CD3 zeta signaling domain.

[0496] 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.

[0497] 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, B7RP1, soluble or membrane bound), or MICA (soluble or membrane bound).

[0498] 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.

[0499] Embodiment 63. The method of any one of embodiments 60 to 62, wherein the antibody expressed by the polynucleotides an immune cell activator.

[0500] Embodiment 64. The method of any one of embodiments 8 to 63, wherein the polynucleotide further encodes a signal peptide.

[0501] Embodiment 65. The method of any one of embodiments 8 to 64, wherein the polynucleotide further comprises a suicide gene.

[0502] 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”).

[0503] 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.

[0504] 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.

[0505] 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.

[0506] Embodiment 70. The method of any one of embodiments 8 to 69, wherein the polynucleotide is introduced to the cell population via a vector. [0507] Embodiment 71. The method of embodiment 70, wherein the vector is a viral vector or a non-viral vector.

[0508] Embodiment 72. The method of embodiment 71, wherein the non-viral vector is a plasmid.

[0509] 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.

[0510] 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.

[0511] 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.

[0512] 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.

[0513] Embodiment 77. The method of embodiment 76, wherein the cell population comprises NK cells and less than or equal to 0.3% CD3+ cells.

[0514] Embodiment 78. The method of embodiment 76 or 77, wherein the subject is administered with less than 3 x 10³ T cells per kilogram of body weight.

[0515] 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, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising at any site; genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre- tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs;, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or/and neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.

[0516] Embodiment 80. The method of any one of embodiments 74 to 79, wherein the cancer cell is a solid tumor cell.

[0517] 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.

[0518] 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.

[0519] 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.

[0520] Embodiment 84. The method of any one of embodiments 75 and 79 to 83, wherein the contacting is in vivo or in vitro.

[0521] Embodiment 85. The method of any one of embodiments 74 to 84, further comprising contacting the cell with or administering a separate therapy.

[0522] Embodiment 86. The method of embodiment 85, wherein the separate therapy comprises surgical resection, chemotherapy, radiation therapy, immunotherapy and targeted therapy. [0523] 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.

[0524] Embodiment 88. An NK cell or a population thereof produced or prepared by the method of any one of embodiments 7 to 74.

[0525] Embodiment 89. A composition comprising the cell or a population thereof of embodiment 88 and a carrier, optionally a pharmaceutical acceptable carrier.

[0526] Embodiment 90. The cell or a population thereof of embodiment 88, or the composition of embodiment 89, comprising less than 0.5% aAPCs.

[0527] 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.

[0528] 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:

(A) no less than 70% viable;

(B) sterile;

(C) containing less than 5 EU Kg^-1 h^-1 endotoxin;

(D) negative for one or more of: mycoplasma, Adventitious viruses, or HHV (HHV6 or HHV7);

(E) comprising more than 1 x 10⁶ cells per kilogram of body weight of a subject to be treated;

(F) comprising more than 40% of cells expressing the CAR among the total cells;

(G) comprising more than 95% cells expressing CD56 among the total cells;

(H) comprising less than 0.3% cells expressing CD3 among the total cells;

(I) comprising less than 4 copies of the polynucleotide per cell; (J) no proliferation in absence of IL-2 or IL-21 ;

(K) having potency, for example, having a more than 10-fold increase in

IFNy secretion or CD 107 assay; and

(L) expressing one or more of an antigen such as CD 19, NKp46, or NKG2D.

[0529] 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 10⁷ cells per kilogram of body weight of a subject to be treated; or

(F’) comprising more than 50% of cells expressing the CAR among the total cells.

[0530] 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.

[0531] Embodiment 95. The aAPC of embodiment 94, wherein the aAPCs are engineered K562 cells.

[0532] Embodiment 96. The aAPC of embodiment 94 or 95, wherein the aAPCs are irradiated, thereby lacking cell proliferation or long-term survival or both.

[0533] Embodiment 97. The aAPC of embodiment 96, wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy.

[0534] Embodiment 98. The aAPC of any one of embodiments 94 to 97, wherein the aAPCs do not substantially survive for more than 14 days.

[0535] 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.

[0536] 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.

[0537] 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):

(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,

(ii) 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,

(iii) one or more of cytokines thereby activating or proliferating y6 T cells, or

(iv) 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).

[0538] 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.

[0539] 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,

(ii) 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,

(iii) one or more of cytokines thereby activating or proliferating y6 T cells, or

(iv) 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);

Optionally wherein the culturing step is repeated for once, twice, three times, or more times with the same or different activator(s).

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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.

[0544] 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.

[0545] 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.

[0546] 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,

(B) a reverse-complement of (A), or

(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.

[0547] 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.

[0548] Embodiment 112. The viral packaging system of any one of embodiments 109 to

111, 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.

[0549] Embodiment 113. The viral packaging system of any one of embodiments 109 to

112, further comprising a packaging cell line.

[0550] Embodiment 114. The viral packaging system of embodiment 113, wherein the packaging cell line is a 293T cell line.

[0551] 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.

[0552] Embodiment 116. The method of embodiment 115, wherein the packaging cell line is a 293T cell line. [0553] 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.

[0554] 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.

[0555] 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.

Claims (31)

WHAT IS CLAIMED IS:

1. A pseudotyped gammaretroviral particle comprising a modified RD 114 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, optionally wherein the RD114TR and the BaEVTR are incorporated into the envelope of the gammaretroviral particle as membrane proteins; and further optionally wherein the pseudotyped gammaretroviral particle further comprises a vector genome encapsulated in the envelope, an optional reverse transcriptase, and an optional integrase, 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 or polypeptide optionally selected from an antibody or a fragment thereof, an enzyme, a ligand or a receptor,

(B) a reverse-complement of (A), or

(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.

2. The pseudotyped gammaretroviral particle of claim 1, 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.

3. The pseudotyped gammaretroviral particle of claim 1 or 2, 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.

4. 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):

(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,

(ii) 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, 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,

(iii) one or more of cytokines thereby activating or proliferating NK cells, or

(iv) 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).

5. The method of claim 4, further comprising one or both of the following: 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; or culturing the cell population with an immune cell activator after the introducing step of claim 4, wherein the immune cell 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, (ii) 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,

(iii) one or more of cytokines thereby activating or proliferating NK cells, or

(iv) 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).

Optionally wherein 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.

6. The method of claim 4 or 5, 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 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, optionally wherein the aAPCs further expresses mb IL-21 and 4- 1BBL, optionally wherein the aAPCs are engineered K562 cells, optionally wherein the aAPCs are irradiated, thereby lacking cell proliferation or lacking long-term survival, further optionally wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy, and optionally 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.

7. The method of any one of claims 4 to 6, 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, optionally 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, optionally wherein the cell population is cultured with either or both of 50 lU/ml IL-2 and 0.5 ng/ml IL- 15, optionally wherein the cell population is cultured with 50 lU/ml IL-2.

8. The method of any one of claims 5 to 7, wherein the activator cultured with the cell population before and after the introducing step is the same, or wherein the activator cultured with the cell population before and after the introducing step is different with each other.

141

9. The method of any one of claims 5 to 8, 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, optionally wherein 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, optionally wherein the pseudotyped gammaretroviral particle further comprises either of both of a reverse transcriptase and an integrase, optionally 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, optionally 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.

10. The method of any one of claims 5 to 9, 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, optionally 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, optionally 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.

11. The method of any one of claims 5 to 10, 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, optionally wherein the RetroNectin is coated on the inner surface of a container in which the cell population is transduced, further optionally wherein the cell population expresses either or both of Integrin a4pi (VLA-4) and Integrin a5pi (VLA-5).

12. The method of any one of claims 4 to 11, 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^bright cells.

13. The method of any one of claims 4 to 12, wherein any cell population comprises natural killer (NK) cells, or substantially purified composition thereof, optionally 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, optionally 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, further optionally wherein one or more of the progenitor cells, HSCs, or iPSCs is capable of deriving NK cells, optionally wherein any cell population is substantially free of T cells, optionally wherein any cell population is substantially free of T regulatory cells, optionally wherein any cell population is isolated, enriched or purified.

14. The method of any one of claims 4 to 13, further comprising either or both of formulating the CAR-expressing population in a composition; or cry opreserving the CAR- expressing population.

15. 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 claims 5 to 14 with the cancer cell, wherein the antigen recognized by the CAR is a TAA expressed by the cancer cell.

16. 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 claims 5 to 14 to the subject, wherein the antigen recognized by the CAR is a TAA expressed by a cell of the cancer.

17. An NK cell or a population thereof produced or prepared by the method of any one of claims 4 to 14.

18. A composition comprising the cell or a population thereof of claim 17 and a carrier, optionally a pharmaceutical acceptable carrier.

19. The cell or a population thereof of claim 17, or the composition of claim 18, comprising less than 0.5% aAPCs, optionally less than 0.1%, or less than 0.2%, or less than 0.3%, less than 0.4%, or less than 0.5% aAPCs.

20. 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, optionally wherein the aAPCs are engineered K562 cells, optionally wherein the aAPCs are irradiated, thereby lacking cell proliferation or longterm survival or both, further optionally wherein the aAPCs were irradiated at 50 Gy, 100 Gy, 150 Gy or 200 Gy, optionally wherein the aAPCs do not substantially survive for more than 14 days.

21. A kit comprising one or more of agents suitable for use in a method as disclosed herein and an optional instruction, optionally 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.

22. 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):

(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,

(ii) 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,

(iii) 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).

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23. The method of claim 22, 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.

24. The method of claim 23, further comprising culturing the cell population after the introducing step of claim 23 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,

(ii) 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,

(iii) one or more of cytokines thereby activating or proliferating y6 T cells, or

(iv) 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); optionally wherein the culturing step is repeated for once, twice, three times, or more times with the same or different activator(s).

25. 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, optionally 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,

(B) a reverse-complement of (A), or

(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,

145 optionally 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, optionally 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.

26. The viral packaging system of claim 25, further comprising a packaging cell line, optionally wherein the packaging cell line is a 293T cell line.

27. A method for producing a pseudotyped gammaretroviral particle, comprising transducing a packaging cell line with the system of claim 25 or 26 under conditions suitable to package the pseudotyped gammaretroviral particle, optionally wherein the packaging cell line is a 293T cell line, optionally wherein the cell line is transduced with plasmids of (a), (b) and (c) at a ratio of 1.5: 1.5: 1, optionally wherein the packaging system comprises at least two envelope plasmids, one expressing RD114TR and the other expressing BaEVTR, further optionally 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.

28. A method for producing a retroviral particle, comprising

(i) introducing a vector, optionally a plasmid, 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.

29. The method of claim 28, wherein 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, optionally wherein the retroviral envelope protein is baboon envelope glycoprotein (BaEV) and its entry receptor is Alanine/Serine/Cysteine/Threonine Transporter 1 (ASCT1) or Sodium-Dependent Neutral Amino Acid Transporter Type 2 (ASCT2), optionally wherein the retroviral envelope protein is the envelope glycoprotein of RD114 retrovirus (RD114), and its entry receptor is ASCT2, optionally wherein the retroviral envelope protein is Gibbon ape leukemia virus envelope

146 glycoprotein (GALV) and its entry receptor is sodium-dependent phosphate transporters (Pitl).

30. A retroviral particle produced by the method of claim 28 or 29.

31. A method for producing an engineered immune cell, comprising transducing the retroviral particle of claim 30 into an immune cell or a precursor cell thereof.

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