WO2023034758A1 - Procédés et compositions destinés à l'expansion de cellules - Google Patents

Procédés et compositions destinés à l'expansion de cellules Download PDF

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Publication number
WO2023034758A1
WO2023034758A1 PCT/US2022/075606 US2022075606W WO2023034758A1 WO 2023034758 A1 WO2023034758 A1 WO 2023034758A1 US 2022075606 W US2022075606 W US 2022075606W WO 2023034758 A1 WO2023034758 A1 WO 2023034758A1
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Prior art keywords
cell
aapc
cells
mol
optionally
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PCT/US2022/075606
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English (en)
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Lei Zhang
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Cytoimmune Therapeutics, Inc.
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Publication of WO2023034758A1 publication Critical patent/WO2023034758A1/fr

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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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Definitions

  • NK cells natural killer cells
  • T cells T cells
  • a clinical dose of natural killer (NK) cells of about IxlO 5 NK cells per kilogram of body weight or higher is necessary.
  • NK natural killer
  • an adult patient of about 70 kilogram would require at least about 7xl0 6 NK cells for a single dose, challenging the currently available capacity of expanding NK cells.
  • a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an azurocidin signal peptide, an interleukin 21 (IL- 21) polypeptide and a transmembrane domain, or an equivalent of each thereof.
  • IL- 21 interleukin 21
  • a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an IL-21 polypeptide and a platelet-derived growth factor receptor beta (PDGFRP) transmembrane domain, or an equivalent of each thereof.
  • the polypeptide further comprises a signal peptide, such as an azurocidin signal peptide.
  • the IL-21 polypeptide comprises, or alternatively consists essentially of, or yet further consists of IL-21 isoform 1 or an equivalent thereof.
  • the equivalent of the IL-21 isoform 1 does not comprise, or consist essentially of, or consist of IL-21 isoform 2.
  • the polypeptide further comprises a Tumor Necrosis Factor Superfamily Member 9 (4-1BBL) polypeptide and a selfcleaving peptide located between the IL-21 polypeptide or the equivalent thereof and the 4- 1BBL polypeptide or the equivalent thereof.
  • 4-1BBL Tumor Necrosis Factor Superfamily Member 9
  • the signal peptide does not comprise, or consist essentially of, or yet further consist of a native IL-21 signal peptide. In some embodiments, the signal peptide does not comprise, or consist essentially of, or yet further consist of a signal peptide of Colony Stimulating Factor 2 Receptor Subunit Alpha (CSF2RA).
  • CSF2RA Colony Stimulating Factor 2 Receptor Subunit Alpha
  • a cell comprising and/or expressing the polypeptide as described herein is provided, the signal peptide (such as the azurocidin signal peptide) directs translocation of the IL-21 polypeptide and the transmembrane domain or the equivalent of each thereof to the cell surface, /. ⁇ ., integrated into the cell membrane.
  • the cell expresses the IL-21 polypeptide and the transmembrane domain or the equivalent of each thereof on the cell surface, /. ⁇ ., integrated into the cell membrane.
  • the cell expresses the 4-1BBL polypeptide or the equivalent thereof on the cell surface, /. ⁇ ., integrated into the cell membrane.
  • a polynucleotide encoding a polypeptide as disclosed herein, or a polynucleotide complementary thereto.
  • a vector comprising, or alternatively consisting essentially of, or yet further consisting of a polynucleotide as disclosed herein.
  • the vector further comprises a regulatory element directing the expression of the polynucleotide.
  • an artificial antigen-presenting cell comprising one or more of the following: a polypeptide as disclosed herein, a polynucleotide as disclosed herein, or a vector as disclosed herein.
  • the aAPC expresses the IL-21 polypeptide and 4-1BBL polypeptide, or an equivalent of each thereof, on the cell surface.
  • the aAPC is or is derived from a cell that lacks of expression of an MHC class I molecule (e.g., neuronal cell line OBL-21, a K562 cell, or a 721.221 cell).
  • the aAPC is or is derived from an AML3 cell.
  • a composition comprising, or consisting essentially of, or yet further consisting of (i) an immune cell, such as a natural killer (NK) cell, and (ii) an artificial antigen-presenting cell (“aAPC” such as an aAPC as disclosed herein) comprising an intracellular hydrogel.
  • the immune cell such as the NK cell
  • the hydrogel comprises, or consists essentially of, or yet further consists of polyethylene glycol or a derivative thereof, optionally poly(ethylene glycol) diacrylate (PEG-DA).
  • a method of expanding an immune cell such as a NK cell or a T cell.
  • the method comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell, a precursor cell thereof, or a cell population with an aAPC as disclosed herein.
  • the cell population comprises, or consists essentially of, or yet further consists of the immune cell, or the precursor cell, or both the immune cell and the precursor cell.
  • a method of sustaining an immune cell’s viability comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell with an aAPC as disclosed herein.
  • a method of activating or expanding or both activating or expanding an immune cell such as an NK cell or a T cell.
  • the method comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell, such as the NK cell or a T cell, or a precursor cell thereof with an artificial antigen-presenting cell (aAPC) that comprises an intracellular hydrogel.
  • aAPC artificial antigen-presenting cell
  • the immune cell such as the NK cell or the T cell, activated or expanded or both activated and expanded using the method as disclosed herein.
  • a cell population comprising, or consisting essentially thereof, or yet further consisting of the immune cell, such as the NK cell.
  • a composition comprising, or consisting essentially of, or yet further consisting of the immune cell, such as the NK cell, or a cell population comprising, or consisting essentially of, or yet further consisting of the immune cell, and a carrier.
  • the carrier is a pharmaceutical acceptable carrier.
  • a method of treating a cancer comprising a tumor- associated antigen (TAA) in a subject in need thereof comprises, or consists essentially of, or yet further consists of administering one or more of: an immune cell as disclosed herein (such as the NK cell), a cell population as disclosed herein, or a composition as disclosed herein to the subject.
  • an immune cell such as the NK cell
  • the immune cell was activated or expanded or both activated and expanded by contacting with the aAPC that comprises the TAA on the cell surface.
  • the immune cell, such as the NK cell comprises a CAR specifically recognizing and binding to the TAA.
  • a method of treating an infection caused by a pathogen comprising a pathogen associated antigen in a subject in need thereof comprises, or consists essentially of, or yet further consists of administering one or more of: an immune cell as disclosed herein (such as the NK cell), a cell population as disclosed herein, or a composition as disclosed herein to the subject.
  • an immune cell such as the NK cell
  • the immune cell was activated or expanded or both activated and expanded by contacting with the aAPC that comprises the pathogen associated antigen on the cell surface.
  • the immune cell, such as the NK cell comprises a CAR specifically recognizing and binding to the pathogen associated antigen.
  • the immune cell is selected from one or more of: a T cell, an NK cell, an NKT cell, a gamma-delta T cell, or a macrophage.
  • the immune cell is isolated from primary human peripheral blood or human umbilical cord blood.
  • the immune cell is derived from a precursor cell.
  • the precursor cell is isolated from primary mammalian peripheral blood or mammalian umbilical cord blood.
  • the method as disclosed herein is an ex vivo or in vitro method.
  • the contacting step is repeated for at least once, or at least twice, or at least three times, or more.
  • kits for use in a method as disclosed herein may comprises, or alternatively consists essentially of, or yet further consists of instructions for use and one or more of the following: a polypeptide as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, an aAPC as disclosed herein, a composition as disclosed herein, an immune cell as disclosed herein, or a cell population as disclosed herein.
  • Another aspect of the disclosure is directed to an artificial antigen-presenting cell (aAPC) comprising IL-21 and/or 4-1 BBL on cell surface, wherein the IL-21 and/or 4-1 BBL is introduced to the cell by a viral or a nonviral vector.
  • aAPC artificial antigen-presenting cell
  • the aAPC comprises an intracellular hydrogel.
  • Figure 1 shows the superior ex vivo expansion of primary NK cells with the aAPCs as disclosed herein (also referred to herein as F3 feeder cells) compared to NK expansion systems developed by other research groups.
  • Data present here is the average ex vivo expansion folds of peripheral blood derived primary NK cells after 3-4 weeks’ culture using the NK expansion systems developed by other groups or using a non-limiting example of the methods as disclosed herein.
  • the exemplified K562-F3-Canonical IL-21-4-1BBL demonstrated the highest expansion fold.
  • Figures 2A-2B provide schematic representations of the retroviral vector used to generate the aAPCs as disclosed herein ( Figure 2A) and the recombinant polypeptide expressed by the vector on cell surface of the aAPCs ( Figure 2B).
  • Figure 2A For tethering human canonical IL-21 (UniProtKB - Q9HBE4-1 (IL21 HUMAN)) on the cell surface, a modified human IgG4 Fc and a transmembrane domain from platelet-derived growth factor receptor beta (PDGFRP) were used.
  • PDGFRP platelet-derived growth factor receptor beta
  • a three-site mutated human IgG4 Fc was used to support the flexibility of the membrane bound IL-21.
  • IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region. Substitutions in its Fc region were aimed to avoid the Fc receptors dimerization.
  • a human 4-1BBL UniProtKB - P41273 (TNFL9 HUMAN) was inserted following a P2A self-cleaving peptide. Both inserts above were cloned into retrovirus vectors PCIR between 5’ and 3’ LTR.
  • Figure 3 provides a diagraph of the PCIR-F3 -IL-21-4-1 BBL transgene.
  • the human IL-21 isoform 1 sequence was from database, see, for example, UniProtKB - Q9HBE4-1 (IL21_HUMAN), while the human 4-1BBL sequence was from UniProtKB - P41273 (TNFL9 HUMAN).
  • a signal peptide derived from human azurocidin was chosen to greatly improve the expression of IL-21 tethered on cell surface.
  • a three-site mutated human IgG4 Fc was used to support the flexibility of the membrane bound IL-21.
  • IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region. Substitutions in its Fc region were aimed to avoid the Fc receptors binding. All the above four inserts were cloned into retrovirus vectors PCIR between 5’ and 3’ LTR. The amino acid and nucleotide sequences for each component are provided in Table 1.
  • Figures 4A-4B show assessment of expression of 4-1BBL and tethered IL-21 on the different feeder cells.
  • Figure 4A provides exemplified flow cytometry dot plots. Control K562 feeder cells and the K562 F3 feeder cells are disclosed herein were expanded and irradiated using a gamma irradiator at lOOGy. As illustrated in Figure 2B, the K562 F3 cells express IL-21 isoform 1 and 4-1BBL. The translocation of IL-21 to the cell surface is directed by an azuroci din signal peptide.
  • the control feeder cells express IL-21 isoform 2 as used in the literature and 4-1BBL, while the translocation of IL-21 to the cell surface is directed by a signal peptide of Colony Stimulating Factor 2 Receptor Subunit Alpha (CSF2RA).
  • CSF2RA Colony Stimulating Factor 2 Receptor Subunit Alpha
  • the cells were assessed for the expression of 4-1BBL and tethered IL-21 using the following antibodies: PE- 4-1BBL and AF-647-IL-21, or the corresponding isotypes serving as negative controls.
  • Cell events were acquired using BD LSR Fortessa and double-positive cells expressing both 4-1BBL and IL-21 were gated based on the corresponding isotype control of each sample.
  • Figure 4B shows median fluorescent intensity of IL-21 and 4-1BBL assessed on the feeder cells. Successful co-expression of 4- 1BBL and tethered IL-21 were observed on the K562 F3 feeder.
  • Figures 5A-5C show that the K562 F3 feeder cells support proliferation of human peripheral blood derived NKs ( Figures 5A and 5C) and sustain NK cell’s viability ( Figure 5B).
  • Freshly purified NK cells from frozen peripheral blood mononuclear cell (PBMC) were mixed with mitomycin treated control feeder cells (plotted as circles), or aAPCs expressing isoform 1 of IL-21 and CD137L (F3 feeder cells, plotted as squares), in the presence of IL-2.
  • the NK cells were stimulated with irradiated aAPCs and cytokines three times: day 3, 10 and 17 in order to support NK cell growth.
  • NK cells were stimulated with feeder cells at a ratio of 2: 1 (2 feeder cells for every 1 NK cell), starting on day 3. Then, every 7 days (i.e., on day 10 and day 17), the NK cells were re-stimulated with feeder cells at the same ratio (2: 1), along with cytokines. . For each week, cell counts and viability were monitored via flow cytometry NovoCyte 3005. A small portion of NK cells was used for subsequent expansion and restimulated with the same batch of mitomycin-treated feeder cells after thawing (Figure 5A, expansion fold; and Figure 5B, viability percentage). The cumulative fold expansion after each week is displayed in a graph having a logarithmic scale in the y axis ( Figure 5C).
  • Figures 6A-6D compare expansion fold (Figure 6A), transduction efficiency ( Figures 6B and 6D), and viability (Figure 6C) of NK cells supported by irradiated (IRR) K562 control feeder cells and IRR K562 F3 feeder cells.
  • NK cells from 3 donors identified as UCSC-33, UCSC-40 and UCSC-41) were co-cultured with the IRR K562 control feeder cells and IRR K562 F3 feeder cells at a ratio of 1 :2 using stem cell growth medium (SCGM) with 50 lU/mL of IL-2.
  • SCGM stem cell growth medium
  • NK cells were transduced with retrovirus expressing soluble IL- 15 and truncated EGFR (tEGFR) on day 7 at a multiplicity of infection (MOI) of 0.1.
  • Transduced NK cells were stimulated with another round of feeder cells at a ratio of 1 :2 on day 10 for further expansion, and harvested on day 16.
  • Figure 6A shows fold change of the NK cells on day 16.
  • Figure 6B shows transduction efficiency of the NK cells on day 16 by assessing the expressed truncated EGFR using the flow cytometry antibody of APC-EGFR.
  • Figure 6C shows viability of the transduced NK cells on day 16.
  • Figures 7A-7B provide assessment of the expression of 4-1BBL and membrane bound IL-21 (isoform 2) on the different feeder cells ( Figure 7A, 721.221; Figure 7B, K562).
  • Cells were assessed for the expression of 4-1BB1 (CD137-L) and membrane-bound IL-21 (mIL-21) using the flow antibodies PE-4-1BBL (Biolegend, Cat# 311504) and AF- 647-IL-21 (BD Biosciences, Cat# 560493) with the respective isotype controls Mouse IgGl, K-PE (BD Biosciences, Cat# 555749) and Mouse IgGl, K-AF647 (BD Biosciences, Cat# 557714).
  • the expression can vary based on the cell lines tested.
  • the IL-21 expresses in K562 cells ( Figure 7B), but its expression in 721.221 feeder cell line was shown to be very poor on 721.221 ( Figure 7A).
  • Figure 8 provides a workflow of the generation of a novel single clonal K562 CYZ1 feeder with surface expression of 4-1BBL and IL-21.
  • Figures 9A - 9B provide schematic representations of a nonviral vector used to generate the aAPCs as disclosed herein. ( Figure 9A), and the recombinant polypeptide expressed by the vector on cell surface of the aAPCs ( Figure 9B).
  • Figure 10 provides a schematic representation of a nonviral transposon vectorused in the instant disclosure. The amino acid and exemplary encoding nucleotide sequences for each component are provided in Table 2.
  • Figure 11 provides a schematic representation of a nonviral transpososase vector used in the instant disclosure.
  • the amino acid and encoding nucleotide sequences for each component are provided in Table 3.
  • Figures 12A - 12B show the mean fluorescence intensity (MFI) of 41BBL ( Figure 12A), and IL-21 ( Figure 12B), expression in candidate single clones of K562 cells transfected with the PiggyBac Transposon Non-Viral system of the instant disclosure.
  • MFI mean fluorescence intensity
  • Figures 13A - 13B show the mean fluorescence intensity (MFI) of IL-21 (Figure 13A), and 41BBL ( Figure 13B), expression in different feeder cells pre- and post-irradiation.
  • MFI mean fluorescence intensity
  • Figures 14A - 14C shows that the instant K562 CYZ1 feeder supports human cord blood-derived NK proliferation and sustains NK cell viability and purity.
  • Figure 14A cumulative fold expansion of NK cells at day 12 of expansion with K562 CYZ1 feeder cells.
  • Figure 14B NK cell viability at day 12 of expansion with K562 CYZ1 feeder cells.
  • NK cell purity measured at day 12 of expansion with K562 CYZ1 feeder cells is defined by CD56+CD3- population. More than 90% of the expanded cells of the population were found to be CD56+CD3-.
  • 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.
  • compositions 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.
  • 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%.
  • comparative terms as used herein can refer to certain variation from the reference.
  • such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference.
  • such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.
  • aqueous solution refers to a system, wherein the aqueous solvent comprises, preferably consists of, water.
  • the term “isotonic solution”, refers to a solution in which its effective osmole concentration is the same as the solute concentration of another solution or another cell and/or tissue with which it is compared.
  • the isotonic solution refers to a physiological isotonic solution having an osmotic pressure in the range of about 270 to 300 mOsm and is generally prepared as a solution containing a salt or a sugar as a main solute.
  • lyophilization refers to freezing of a material at low temperature followed by dehydration by sublimation, usually under a high vacuum. Lyophilization is also known as freeze drying.
  • microparticle refers to a microscopic particle having a size measured in micrometers (pm). In some embodiments, the term “microparticle” refers to a particle having a particle size of about 0.01 pm to about 1000 pm. In some embodiments, the microparticle as a size of about 0.05 pm to about 750 nm, about 0.1 pm to about 500 pm, about 0.25 pm to about 250 pm, or about 0.5 pm to about 100 pm. In one embodiment, the microparticle has a particle size of about 75 pm. As used herein, the term “nanoparticle” refers to particle having a particle size of about 0.1 pm to about 1000 pm.
  • a nanoparticle can have a particle size of about 0.5 pm to about 500 pm, about 1 pm to about 250 pm, about 10 pm to about 150 pm, or about 15 pm to about 100 pm. It will be understood by one of ordinary skill in the art that microparticles or nanoparticles usually exhibit a distribution of particle sizes around the indicated “size.” Unless otherwise stated, the term “size” as used herein refers to the mode of a size distribution of microparticles or nanoparticles, i.e., the value that occurs most frequently in the size distribution.
  • Methods for measuring the microparticle or nanoparticle size are known to a skilled artisan, e.g., by dynamic light scattering (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other techniques (such as rheology, and light or electron microscopy).
  • dynamic light scattering such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)
  • light obscuration methods such as Coulter analysis method
  • other techniques such as rheology, and light or electron microscopy.
  • hydrogel refers to a swellable polymeric matrix, consisting of a three-dimensional network of macromolecules held together by covalent or non-covalent crosslinks, which can absorb a substantial amount of liquid, e.g., water, within its structure without dissolution.
  • Non-limiting examples of such include abiocompatible polymer, such as a poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co- glycolide (PLGA), polyesters, poly(ortho ester), poly(phosphazine), poly(phosphate ester), polycaprolactone, gelatin, collagen, fibronectin, keratin, polyaspartic acid, alginate, chitosan, chitin, hyaluronic acid, pectin, polyhydroxyalkanoates, dextrans, polyanhydrides, polyethylene oxide (PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine, any derivatives thereof and any combinations thereof.
  • the hydrogel comprises, or consists essentially of, or yet further consists of polyethylene glycol or a derivative thereof.
  • the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • a “photoinitiator” refers to a chemical that initiates free radical crosslinking/polymerizing reaction by the use of light.
  • the term “engineered” refers to comprising at least one modification not normally found in a naturally occurring counterpart, wild-type or a parent.
  • Such as an engineered NK cell can comprise a chimeric antigen receptor (CAR) that is not naturally occurring.
  • CAR chimeric antigen receptor
  • the term “engineered” is used interchangeably with “recombinant” refers to being synthetized by human.
  • protein refers 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 aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • the N terminus or “at the C terminus” refers to a relative location of two peptide fragments in one polypeptide.
  • the two peptide fragments are immediately adjacent to each other in the polypeptide. In other embodiments, the two peptide fragments are not immediately adjacent to each other in the polypeptide. In further embodiments, the two peptide fragments can be separated by 1, or 2, or 3, or 4, or 5, or 10, or 20, or 50, or 100, or more amino acid residues.
  • a polypeptide as referred to herein also intend to comprise, or alternatively consist essentially of, or yet further consist of an equivalent of the polypeptide, such as a variant, a derivative or a fragment of the polypeptide.
  • an equivalent protein or polypeptide is one having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the reference protein or polypeptide.
  • an equivalent protein or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polypeptide or protein as disclosed herein.
  • a polypeptide or an equivalent thereof have the same or substantially similar biological activity (also referred to as function).
  • the equivalent is a functional protein that optionally can be identified through one or more assays described herein.
  • variant refers to an equivalent having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) or deletions, so long as the modifications do not destroy biological activity and which are substantially identical to the reference polypeptide.
  • Variants generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic: aspartate and glutamate; (2) basic: lysine, arginine, histidine; (3) non-polar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • the polypeptide of interest can include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the polypeptide remains intact.
  • regions of the polypeptide of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art,.
  • derivative is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective variant, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, so long as the desired biological activity of the native polypeptide is substantially retained.
  • Methods for making polypeptide fragments, variants, and derivatives are generally available in the art.
  • fragment is intended a molecule consisting of only a part of the intact full-length sequence and structure.
  • the fragment can include a C-terminal deletion, an N-terminal deletion, an internal deletion of the native polypeptide, or any combination thereof.
  • Active fragments of a particular protein will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question substantially retains biological activity, such as stimulating the proliferation of NK cells or sustaining the viability of immune cells, such as NK cells.
  • 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.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complementary of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • oligonucleotide or “polynucleotide” or “nucleotide” or “portion,” or “segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
  • the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties e.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • a polynucleotide as referred to herein also intend to comprise, or alternatively consist essentially of, or yet further consist of an equivalent of the polynucleotide.
  • an equivalent polynucleotide is one having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the reference polynucleotide.
  • an equivalent polynucleotide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polynucleotide as disclosed herein.
  • a polynucleotide or an equivalent thereof encodes the same polypeptide.
  • a polynucleotide encodes a polypeptide, and an equivalent of the polynucleotide encodes an equivalent of the polypeptide.
  • an equivalent of a reference polynucleotide hybridizes to the complementary sequence of the reference polynucleotide.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg 2+ normally found in a cell.
  • complementary sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other.
  • the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other.
  • sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other.
  • a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide.
  • Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.
  • Under transcriptional control which is also used herein as “directing expression of’, is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell.
  • a regulatory sequence intends a polynucleotide that is operatively linked to a polynucleotide to be transcribed and/or replicated, and facilitates the expression and/or replication of the polynucleotide.
  • a regulatory sequence include a promoter, an enhancer, or a polyadenylation sequence.
  • promoter refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • Non-limiting examples of promoters include a cytomegalovirus CMV promoter or retroviral long terminal repeat (LTR) promoter. See, for example, Weber et ak. Hum Gene Ther. 2007 Sep;18(9):849-60.
  • 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.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) 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.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • the default setting is used.
  • the program is any one of: Clustal Omega accessible at www.ebi.ac.uk/Tools/msa/clustalo/, Needle (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_needle/, Stretcher (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_stretcher/, Water (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_water/, Matcher (EMBOSS) accessible at www.ebi.ac.uk/Tools/psa/emboss_matcher/, LALIGN accessible at www.ebi.ac.uk/Tools/psa/lalign/.
  • the default setting is used.
  • the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and optionally integrate into the target cell’s genome.
  • vectors include a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc.
  • plasmid vectors may be prepared from commercially available vectors.
  • viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
  • the viral vector is a lentiviral vector.
  • the viral vector is a retroviral vector.
  • 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.
  • MCS multiple cloning site
  • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.
  • a “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • the DNA viruses constitute classes I and II.
  • the RNA viruses and retroviruses make up the remaining classes.
  • Class III viruses have a double-stranded RNA genome.
  • Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA
  • Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis.
  • Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
  • Multiplicity of infection refers to the number of viral particles that are added per cell during infection.
  • RNA usually a dimer RNA comprising a cap at the 5’ end and a polyA tail at the 3’ end flanked by LTRs
  • proteins such as a protease.
  • U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes.
  • LTRs long terminal repeats
  • the LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.
  • Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA
  • U5 is derived from the sequence unique to the 5'end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR.
  • U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • RT reverse transcriptase
  • I integrase
  • the components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell.
  • the set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
  • Gammaretrovirus is a genus in the retroviridae family and may be used in the disclosure herein.
  • Example species are the moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), friend murine embryonic stem cell virus (FMEV), xenotropic MuLB-related virus, feline sarcoma virus, xenotropic murine leukemia virus-related virus (XMRV) and the feline leukemia virus.
  • MMLV moloney murine leukemia virus
  • MSCV murine stem cell virus
  • FMEV friend murine embryonic stem cell virus
  • xenotropic MuLB-related virus feline sarcoma virus
  • XMRV xenotropic murine leukemia virus-related virus
  • Gammaretrovirus is a spherical, enveloped virion ranging from 80-100 nm in diameter. It contains a nucleocapsid, reverse-transcriptase, integrase, capsid,
  • the nucleocapsid is a nucleic acid protein assembly within the virus particle, it is a substructure of the virion.
  • Reversetranscriptase is the enzyme responsible for the transformation of RNA to DNA during the virion replication cycle. Integrase works with reverse transcriptase to convert RNA to DNA.
  • the genome of the gammaretrovirus is a single-stranded RNA (+) genome that is approximately 8.3 kb in size. It has a 5’ cap with a 3’ poly-A tail, and it contains two long terminal repeater regions at both the 5’ and 3’ ends. These long terminal repeat regions have the U5, R, and U3 regions as well as a polypurine tract at the 3’ end and a primer binding site at the 5’ end.
  • the typical gammaretrovirus genome contains the gag gene, pol gene, and an env gene, all of which can be omitted in a gene therapy vector.
  • the capsid is a protein shell that surrounds the genome of a virus particle, its main functions are to protect and deliver the genome to the host cell.
  • the viral envelope is the membrane that surround the viral capsid, it is a host cell derived lipid bilayer.
  • gammaretroviruses have some advantages over HIV as a lentiviral vector. Specifically, the gammaretroviral packaging system does not require the incorporation of any sequences overlapping with coding sequences of gag, pol, or accessory genes. See, for example, Tobias Maetzig et al. Viruses. 2011 Jun; 3 (6): 677-713. Epub 2011 June 3.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art.
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant or synthetic serotypes, e.g., AAV-DJ and AAV PHP.B.
  • the AAV particle comprises, alternatively consists essentially of, or yet further consists of three major viral proteins: VP1, VP2 and VP3.
  • the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. These vectors are commercially available or have been described in the patent or technical literature.
  • Interleukin-21 which is also referred to herein as an IL-21 polypeptide, is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells that can destroy virally infected or cancerous cells. This cytokine induces cell division/proliferation in its target cells.
  • the IL-21 is a human IL-21.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04M122612, HGNC: 6005, NCBI Entrez Gene: 59067, Ensembl: ENSG00000138684, OMIM®: 605384, and UniProtKB/Swiss-Prot: Q9HBE4, each of which is incorporated by reference herein in its entirety.
  • the IL-21 polypeptide or the equivalent thereof comprises, or alternatively consists essentially of, or yet further consists of isoform 1 of the IL-21. In some embodiments, the IL-21 polypeptide or the equivalent thereof does not comprise isoform 2 of the IL-21. In some embodiments, the polypeptide as described herein does not comprise, or consist essentially of, or consist of isoform 2 of the IL-21. In some embodiments, the IL-21 isoform 1 comprises, or consists essentially of, or yet further consists of
  • the IL-21 isoform 2 comprises, or consists essentially of, or yet further consists of
  • HKS S SQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEWS AF SCFQK AQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKVSTLSFI (SEQ ID NO: 2).
  • the IL-21 isoform 1 is encoded by CATAAGTCCTCTAGTCAGGGACAGGACCGGCATATGATCAGGATGCGCCAGCTG ATCGACATAGTAGACCAACTTAAGAACTACGTTAACGATCTCGTGCCAGAGTTCC TTCCGGCCCCCGAGGACGTTGAAACCAATTGCGAATGGTCTGCCTTCAGCTGTTT CCAGAAAGCACAGCTGAAATCCGCCAACACTGGTAATAACGAGCGAATCATTAA CGTGTCCATCAAGAAACTCAAACGAAAGCCACCCAGTACGAACGCCGGCCGGCG TCAAAAGCATCGGTTGACCTGTCCCTCTTGCGACAGTTACGAAAAGAAGCCCCCA AAGGAGTTTCTGGAGCGTTTTAAGTCCTTGCTGCAGAAAATGATCCACCAACACT TGTCCAGTCGGACACACGGTTCCGAGGACTCC (SEQ ID NO: 3) or an equivalent thereof that encodes the IL-21 polypeptide.
  • IL-21 as used herein is an IL-21 isoform 1 or an equivalent thereof.
  • the IL-21 equivalent stimulates the proliferation or sustains the viability of immune cells (such as NK cells) or both significantly similar to the IL-21 isoform 1.
  • 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.
  • Assays are available for evaluating such expression, such as two-dimensional gel electrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation (SDS-PAGE), immunohistochemistry (IHC), western blot, flow cytometry, enzyme linked immunoabsorbant assay (ELISA), immunofluorescence, full proteome assays, or PCR.
  • the IL-21 equivalent comprises, or consists essentially of, or further consists of a fragment of the wildtype IL-21. Additionally or alternatively, the IL-21 equivalent comprises, or consists essentially of, or further consists of a variant of the wildtype IL-21 or a fragment thereof, such as adding an extra Methionine at the N-terminus.
  • the IL-21 equivalent comprises, or consists essentially of, or further consists of QGQDXHMXXMXXXXXXVDXLKNXVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNEXXIXXXXXXLXXXXXXTNAGRRQKHRLTCPSCDSYEKKPPKEFLXX FXXLLXXMXXQHXSSRTHGSEDS (SEQ ID NO: 4), and X is any amino acid. See, for example, US Patent Application No. 20190046611. Additionally or alternative, the IL-21 equivalent comprises, or consists essentially of, or further consists of an IL-21 derivative, such as modified by glycosylation, acetylation, or phosphorylation.
  • native IL-21 signal peptide is used interchangeably with “wildtype IL-21 signal peptide”, referring to a signal peptide that is naturally linked with (such as naturally conjugated to) IL-21.
  • a native IL-21 signal peptide is naturally linked with (such as naturally conjugated to) human IL-21.
  • the native IL-21 signal peptide comprises, or alternatively consists essentially of, or further consists of MRSSPGNMERIVICLMVIFLGTLV (SEQ ID NO: 28).
  • the “transmembrane domain” or “TM domain” means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR, or PDGFRp.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the transmembrane domain comprises, or alternatively consists essentially of, or yet consists of a PDGFRP transmembrane domain.
  • a transmembrane domain in a protein mounts the polypeptide to the cell membrane, permitting the fusion protein to be a membrane bound protein.
  • Assays are available for identifying location of a protein expression, such as immunohistochemistry (IHC), immunofluorescence (IF), or cell fraction (isolating the cell membrane from other cell organelles) combined with two-dimensional gel electrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation (SDS-PAGE) or western blot.
  • Platelet Derived Growth Factor Receptor p which is also referred to herein as an PDGFRP polypeptide, is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. These growth factors are mitogens for cells of mesenchymal origin. The identity of the growth factor bound to a receptor monomer determines whether the functional receptor is a homodimer (PDGFB or PDGFD) or a heterodimer (PDGFA and PDGFB). In some embodiments, the PDGFRP is a human PDGFRp.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC05M150113, HGNC: 8804, NCBI Entrez Gene: 5159, Ensembl: ENSG00000113721, OMIM®: 173410, or UniProtKB/Swiss-Prot: P09619, each of which is incorporated by reference herein in its entirety.
  • PDGFRP comprises, or alternatively consists essentially of, or yet further consists of MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSA PVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERK RLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYD HQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENIT LMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTC NVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTV LWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAE VQLS
  • PDGFRP comprises, or alternatively consists essentially of, or yet further consists of MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSA PVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERK RLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYD HQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENIT LMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTC NVTESVNDHQDEKAINITVRAATCGSWERWAHYNLLSCIGAGHCR (SEQ ID NO: 6).
  • a transmembrane domain as used herein is a PDGFRP transmembrane domain or an equivalent thereof.
  • the PDGFRP transmembrane domain or equivalent thereof comprises, or alternatively consists essentially of, or yet further consists of VVISAILALVVLTIISLIILI (SEQ ID NO: 7).
  • the PDGFRP transmembrane domain further comprises additional amino acid residues (1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or more) at the N terminus or the C terminus or both of SEQ ID NO: 7.
  • the additional amino acid residues are those located at the N terminus or the C terminus of SEQ ID NO: 7 in the sequence of SEQ ID NO: 5 or 6.
  • the additional amino acid residues are random amino acid residues.
  • the PDGFRP transmembrane domain or equivalent thereof comprises, or alternatively consists essentially of, or yet further consists of AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 8) or an equivalent thereof.
  • the PDGFRP transmembrane domain is encoded by GCCGTCGGTCAAGATACACAAGAAGTGATAGTCGTTCCGCATTCTCTTCCTTTCA AAGTTGTTGTAATTAGTGCAATTCTCGCGTTGGTTGTCCTGACAATTATAAGCCTC ATAATACTTATAATGCTGTGGCAAAAGAAACCCAGG (SEQ ID NO: 9) or an equivalent thereof that encodes the PDGFRP transmembrane domain.
  • a membrane-bound IL-21 refers to an IL-21 polypeptide or an equivalent thereof that can be expressed on a cell membrane, for example, on a cell surface.
  • an mb IL-21 comprises, or alternatively consists essentially of, or yet further consists of an IL-21 polypeptide or an equivalent thereof and a transmembrane domain.
  • an mb IL-21 comprises, or alternatively consists essentially of, or yet further consists of an IL-21 polypeptide or an equivalent thereof, a transmembrane domain or an equivalent thereof, and an Fc fragment of an antibody or an equivalent thereof optionally located between the IL-21 polypeptide and the transmembrane domain or the equivalent of each thereof.
  • an mb IL- 21 further comprises a signal peptide directing the cell surface expression.
  • a signal peptide used interchangeably with signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined toward the secretory pathway.
  • the signal peptide is a secretary signal.
  • a secretary signal intends a secretory signal peptide that allows the export of a protein from the cytosol into the secretory pathway. Proteins can exhibit differential levels of successful secretion and often certain signal peptides can cause lower or higher levels when partnered with specific proteins.
  • the signal peptide is a hydrophobic string of amino acids that is recognized by the signal recognition particle (SRP) in the cytosol of eukaryotic cells.
  • SRP signal recognition particle
  • the SRP binds the peptide and stops protein translation.
  • the SRP 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.
  • the signal peptide directs a cell surface expression.
  • the signal peptide directs a cell surface expression of an IL-21 polypeptide or the equivalent thereof.
  • a signal peptide as used herein is an azurocidin signal peptide.
  • a signal peptide as used herein does not comprise, or consist essentially of, or consist of an IL-21 native signal peptide, i.e., the wildtype signal peptide of IL-21.
  • a signal peptide as used herein does not comprise, or consist essentially of, or consist of a Colony Stimulating Factor 2 Receptor Subunit Alpha (CSF2RA) signal peptide.
  • a polypeptide as disclosed herein does not comprise, or consist essentially of, or consist of an IL-21 native signal peptide.
  • a polypeptide as disclosed herein does not comprise, or consist essentially of, or consist of a Colony Stimulating Factor 2 Receptor Subunit Alpha (CSF2RA) signal peptide.
  • Azurocidin is a neutrophil granule-derived antibacterial and monocyte- and fibroblastspecific chemotactic glycoprotein. It is a preproprotein that is proteolytically processed to generate a mature azurophil granule antibiotic protein, with monocyte chemotactic and antimicrobial activity.
  • the azurocidin is a human azurocidin.
  • Nonlimiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC19P000825, HGNC: 913, NCBI Entrez Gene: 566, Ensembl:
  • a signal peptide as used herein is an azurocidin signal peptide, which comprises, or alternatively consists essentially, or further consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 10) or an equivalent thereof.
  • the azurocidin signal peptide is encoded by ATGACTAGGTTGACAGTCCTCGCCTTGCTTGCTGGATTGCTTGCCAGTTCTCGAG CC (SEQ ID NO: 11) or an equivalent thereof that encodes the azuroci din signal peptide.
  • Colony Stimulating Factor 2 Receptor Subunit Alpha is also known as CD116, which is a receptor for granulocyte-macrophage colony-stimulating factor, stimulating the production of white blood cells.
  • the CSF2RA is a human CSF2RA.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC0XP001333, HGNC: 2435, NCBI Entrez Gene: 1438, Ensembl: ENSG00000198223, OMIM®: 306250, OMIM®: 425000, and UniProtKB/Swiss- Prot: P15509, each of which is incorporated by reference herein in its entirety.
  • a CSF2RA signal peptide comprises, or alternatively consists essentially, or further consists of MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 29) or an equivalent thereof.
  • a fragment crystallizable (Fc) region also known as an Fc fragment, 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.
  • an Fc region is inserted between the IL-21 isoform 2 and the transmembrane domain.
  • the Fc region stabilizes the polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of the IL-21 isoform 2, the Fc region, and the transmembrane domain.
  • Fc region provides certain structural flexibility of the IL-21 isoform 2 when expressing on a cell surface.
  • Fc region refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site and ending at the C-terminus of the antibody. Accordingly, a complete Fc region comprises, or alternatively consists essentially of, or yet further consists of at least a hinge, a CH2 domain, and a CH3 domain.
  • Fc regions that are dimerized are referred to as “Fc” or “Fc dimer.”
  • An Fc region may be a naturally occurring Fc region, or a naturally occurring Fc region in which one or more amino acids have been substituted, added or deleted, provided that the Fc region has the desired biological properties, for example stabilizing the polypeptide or providing the flexibility of polypeptide.
  • stabilization of the polypeptide can be reflected by the overall expression level of the protein on the cell surface using two-dimensional gel electrophoresis, such as using sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation (SDS-PAGE), immunohistochemistry (IHC), western blot, flow cytometry, enzyme linked immunoabsorbant assay (ELISA), immunofluorescence, or full proteome assays.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation
  • IHC immunohistochemistry
  • western blot western blot
  • flow cytometry flow cytometry
  • ELISA enzyme linked immunoabsorbant assay
  • immunofluorescence or full proteome assays.
  • the term “flexibility” in context with protein structures refers to a structural feature of the polypeptide that favors disordered configuration, i.e., not inclined to form defined secondary structures. This is due to the fact that a linker sequence or an Fc region with high propensity for forming alpha-helical or beta-strand structures would limit the flexibility of the fusion protein and consequently affect its functional activity.
  • Method of modeling or evaluating a protein structural flexibility can be found, for example, Kmiecik et al. Int J Mol Sci. 2018 Nov 6; 19(11):3496; Amaral et al. Nat Commun 8, 2276 (2017); Khan et al. J Biophys.
  • an Fc region comprises, or alternatively consists essentially of, or yet further consists of a hinge domain, a CH2 domain, and a CH3 domain.
  • the Fc fragment comprises a hinge domain, a CH2 domain and a CH3 domain of IgG4 modified at F234A, L235A and N297Q or an equivalent thereof.
  • IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region.
  • substitutions in its Fc region were aimed to avoid the Fc receptors dimerization. Additionally or alternatively, substitutions in its Fc region were aimed to avoid the Fc receptors binding.
  • the Fc fragment comprises, or alternatively consists essentially of, or further consists of
  • F234A refers to the 234 th amino acid residue in a complete antibody, i.e., the 16 th amino acid residue of SEQ ID NO: 12, which was mutated from wildtype Phenylalanine (F) to Alanine (A) as in SEQ ID NO: 12.
  • L235A refers to the 235 th amino acid residue in a complete antibody, i.e., the 17 th amino acid residue of SEQ ID NO: 12, which was mutated from wildtype Leucine (L) to Alanine (A) as in SEQ ID NO: 12.
  • N297Q refers to the 297 th amino acid residue in a complete antibody, i.e., the 79 th amino acid residue of SEQ ID NO: 12, which was mutated from wildtype Asparagine (N) to Glutamine (Q) as in SEQ ID NO: 12.
  • the Fc region is encoded by GAGTCTAAGTACGGTCCCCCATGTCCCCCTTGTCCAGCTCCTGAGGCCGCTGGTG GGCCAAGTGTCTTCCTTTTCCCGCCCAAGCCCAAGGACACATTGATGATCTCG GACGCCAGAGGTCACTTGTGTTGTCGTCGACGTCACAAGAGGACCCAGAGGT TCAATTCAATTGGTACGTCGACGGGGTTGAGGTTCACAATGCCAAGACAAAGCC TAGAGAGGAGCAATTCCAATCAACATACCGGGTTGTCTCCGTTCTCACTGTCCTG CACCAAGACTGGTTGAATGGGAAGGAGTACAAGTGTAAGGTCTCAAATAAGGGG TTGCCCAGCAGTATCGAGAAGACAATATCTAAGGCTAAGGGGCAACCCCGGGAG CCACAAGTCTACACACTGCCCTTCACAAGAGGAGATGACGAAGAATCAAGTC TCTCTCTCACATGTCTCGTCAAGGGGTTCTACCCATCTGACATCGCAGTAGAGAG
  • Hinge or “hinge region” or “hinge domain” refers to the flexible portion of a heavy chain located between the CHI domain and the CH2 domain. It is approximately 25 amino acid long, and is divided into an “upper hinge,” a “middle hinge” or “core hinge,” and a “lower hinge.”
  • a hinge may be a naturally occurring hinge, or a naturally occurring hinge in which one or more amino acids have been substituted, added or deleted, provided that the hinge has the desired biological properties.
  • a desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to the naturally occurring sequence.
  • CHI domain refers to the heavy chain immunoglobulin constant domain located between the VH domain and the hinge. In some embodiments, it spans EU positions 118-215. Information about EU positions can be found, for example, www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.
  • a CHI domain may be a naturally occurring CHI domain, or a naturally occurring CHI domain in which one or more amino acids (“aas”) have been substituted, added or deleted, provided that the CHI domain has the desired biological properties.
  • a desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to the naturally occurring sequence.
  • CH2 domain refers to the heavy chain immunoglobulin constant domain that is located between the hinge and the CH3 domain. In some embodiments, it spans EU positions 237-340.
  • a CH2 domain may be a naturally occurring CH2 domain, or a naturally occurring CH2 domain in which one or more aas have been substituted, added or deleted, provided that the CH2 domain has the desired biological properties.
  • a desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.
  • CH3 domain refers to the heavy chain immunoglobulin constant domain that is located C-terminally of the CH2 domain and spans approximately 110 residues from the N- terminus of the CH2 domain, e.g., about positions 341 -446b (EU numbering system).
  • a CH3 domain may be a naturally occurring CH3 domain, or a naturally occurring CH3 domain in which one or more aas have been substituted, added or deleted, provided that the CH3 domain has the desired biological properties.
  • a desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.
  • a CH3 domain may or may not comprise a C-terminal lysine.
  • CH4 domain refers to the heavy chain immunoglobulin constant domain that is located C-terminally of the CH3 domain in IgM and IgE antibodies.
  • a CH4 domain may be a naturally occurring CH4 domain, or a naturally occurring CH4 domain in which one or more aas have been substituted, added or deleted, provided that the CH4 domain has the desired biological properties.
  • a desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.
  • a wildtype Fc region can be used.
  • a mutant Fc region can be used.
  • the Fc region can be mutated at any one or any two or all of the three sites, F234A, L235A within hinge region and N297Q within CH2 region.
  • substitutions in its Fc region were aimed to avoid the Fc receptors binding. See, for example, Wang et al. Protein Cell. 2018 Jan;9(l):63-73. Epub 2017 Oct 6.
  • the Fc region comprises ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 12) or an Fc equivalent having mutations at a position corresponding to amino acid (aa) 16, aa 17 and aa 79 of SEQ ID NO: 12, i.e., F234, L235 and N297, respectively.
  • the equivalent binds an Fc receptor on an immune cell or on a platelet or that binds a complement protein, optionally in a level substantially similar to that of SEQ ID NO: 12. In some embodiments, the equivalent binds an Fc receptor on an immune cell or on a platelet or that binds a complement protein, optionally in a level substantially less compared to that of SEQ ID NO: 12. In some embodiments, an Fc or an equivalent thereof as used herein reduces or does not induce antibody-dependent cellular cytotoxicity (ADCC). Additionally or alternatively, an Fc or an equivalent thereof as used herein reduces or does not induce complement-dependent cytotoxicity (CDC). Non-limiting examples of such Fc or an equivalent thereof are disclosed herein, or for example, as shown in WQ2018107058A1.
  • 4-1BBL Tumor Necrosis Factor Superfamily Member 9
  • TNFSF9 Tumor Necrosis Factor Superfamily Member 9
  • 4-1BBL polypeptide is a type 2 transmembrane glycoprotein receptor that is found on APCs (antigen presenting cells) and binds to 4-1BB (also known as CD137).
  • the 4-1BB/4- 1BBL complex belongs to the TNFR:TNF superfamily, which is expressed on activated T Lymphocytes.
  • the 4-1BBL is a human 4-1BBL.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC19P006531, HGNC: 11939, NCBI Entrez Gene: 8744, Ensembl: ENSG00000125657, OMIM®: 606182, or UniProtKB/Swiss-Prot: P41273, each of which is incorporated by reference herein in its entirety.
  • a 4-1BBL polypeptide comprises, or alternatively consists essentially, or further consists of MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAV SGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD PGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARH AWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 13) or an equivalent thereof.
  • an equivalent of SEQ ID NO: 13 binds or activates or both binds and activates its ligand, such as 4-1BB (i.e., CD137), in a substantially similar level compared to SEQ ID NO: 13.
  • 4-1BB i.e., CD137
  • Assays of evaluating binding of two proteins and activation of 4-1BB is known in the art, such as Bartko wiak et al. Clin Cancer Res. 2018 Mar 1;24(5): 1138-1151; and Chin et al. Nat Commun. 2018 Nov 8;9(1):4679, along with AlphaLISA® 4-1BB/4- 1BBL binding kit available from PerkinElmer.
  • the 4-1BBL polypeptide is encoded by ATGGAGTATGCTAGCGATGCAAGCTTGGATCCAGAGGCACCCTGGCCACCAGCA CCTAGAGCTAGGGCTTGTCGTGTGCTCCCATGGGCGCTCGTAGCAGGTCTCCTCT TGCTCCTCTTGCTGGCGGCAGCTTGTGCTGTGTTTCTGGCTTGTCCTTGGGCAGTC TCAGGTGCAAGGGCGTCCCCAGGAAGTGCCGCATCCCCCAGGCTGAGAGAAGGC CCAGAACTGAGTCCTGATGACCCGGCAGGATTGCTTGATCTTCGCCAAGGAATGT TCGCTCAACTCGTAGCACAGAACGTATTGCTCATAGACGGCCCTTTGAGTTGGTA TTCCGATCCCGGACTTGCCGGAGTCAGCCTCACTGGCGGTTTGTCTTATAAGGAA GATACAAAAGAATTGGTAGTTGCGAAAGCCGGTGTTTATTACGTTCTTCCAGC TGGAATTGACGTGTTGTCGCTGGAGAAGGTTCT
  • a ribosomal skip sequence which is also referred to as a cleavable peptide, or a cleavable linker, means a peptide that can be cleaved, for example, by an enzyme or a peptide that can induce ribosomal skipping during translation of a protein in a cell, or both.
  • One translated polypeptide comprising such cleavable peptide can produce two final products, therefore, allowing expressing more than one polypeptides from one open reading frame.
  • a ribosomal skip sequence can be used to express a membrane bound IL-21 (i.e., mIL-21 comprising, or alternatively consisting essentially of, or yet further consisting of an IL-21 isoform 1 and a transmembrane domain) and a 4-1 BBL.
  • a membrane bound IL-21 i.e., mIL-21 comprising, or alternatively consisting essentially of, or yet further consisting of an IL-21 isoform 1 and a transmembrane domain
  • a 4-1 BBL Alternatively, other methods can be used to achieve such co-expression, for example, by a nucleic acid molecule comprising a mIL-21 coding sequence, a 4-1BBL coding sequence, an internal ribosome entry site (IRES) located there between.
  • IRS internal ribosome entry site
  • cleavable peptides is a self-cleaving peptide, such as a 2A selfcleaving peptide.
  • 2A self-cleaving peptides is a class of 18-22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell.
  • the 2 A selfcleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. See, for example, Wang Y, et al. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep. 2015;5: 16273. Published 2015 Nov 5.
  • 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: 15), wherein X refers to any amino acid generally thought to be self-cleaving.
  • the self-cleaving peptide is P2A.
  • the P2A comprises, or alternatively consists essentially of, or yet further consists of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 16) or an equivalent thereof.
  • a polypeptide comprising the P2A equivalent produces one peptide that is the fragment of the polypeptide initially located on the N terminal side of the P2A equivalent and the other peptide that is the fragment of the polypeptide initially located on the C terminal side of the P2A equivalent.
  • P2A is encoded by GGTAGTGGGGCTACGAACTTTTCCCTTCTCAAACAGGCCGGAGACGTCGAGGAG AATCCTGGACCA (SEQ ID NO: 17).
  • IRES internal ribosome entry site
  • mRNA messenger RNA
  • IRES also refers to a polynucleotide sequence (such as an RNA sequence, a DNA sequence or a hybrid thereof) complementary, or reverse, or both complementary and reverse to an IRES RNA sequence.
  • Non-limiting examples of IRES can be found in Hellen CU and Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 2001 Jul 1;15(13): 1593-612.
  • MHC class I molecules are one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) and are found on the cell surface of all nucleated cells in the bodies of vertebrates. They also occur on platelets, but not on red blood cells. Their function is to display peptide fragments of proteins from within the cell to cytotoxic T cells or other immune cells; this will trigger an immediate response from the immune system against a particular non-self antigen displayed with the help of an MHC class I protein. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called cytosolic or endogenous pathway.
  • MHC major histocompatibility complex
  • the MHC class I molecule is a human MHC (i.e., human leukocyte antigen, or HLA).
  • the MHC class I molecule comprises, or alternatively consists essentially of, or yet further consists of any one or any two or all three of HLA- A, HLA-B, or HLA-C.
  • a feeder cell should lack expression of an MHC class I molecule so that co-culture with a feeder cell does not render the immune cells targeting on a non-relevant antigen of the feeder cells.
  • the feeder cells naturally lack expression of an MHC class I molecule before the engineering as disclosed herein.
  • expression of an MHC class I molecule is reduced substantially or eliminate from the feeder cells, for example, by RNA interference (RNAi), transcription activator-like effector nucleases (TALENs), or a clustered regularly interspaced short palindromic repeats (CRISPR) system.
  • RNAi RNA interference
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Immuno cells includes, e.g., white blood cells (leukocytes, such as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells)) which may be derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and NKT cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • leukocytes such as granulocytes (neutrophils, eosinophils, and basophils)
  • monocytes and lymphocytes
  • T cells hematopoietic stem cells
  • NK natural killer cells
  • NKT cells myeloid-derived cells
  • the immune cell is derived from a precursor cell, optionally selected 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), haematopoietic stem cells (HSCs), or immortalized cells.
  • the HSCs are derived from umbilical cord blood of a subject, peripheral blood of a subject, or bone marrow of a subject.
  • an “immature cell” refers to a cell which does not possess the desired (adult) phenotype or genotype.
  • a mature cell is a cell that is being replaced.
  • the immature cell can be subjected to techniques including physical, biological, or chemical processes which changes, initiates a change, or alters the phenotype or genotype of the cell into a “mature cell.”
  • a “mature cell” refers to a cell which possess the desired phenotype or genotype.
  • NK cell also known as natural killer cell, refers to a type of lymphocyte that originates in the bone marrow and play a critical role in the innate immune system. NK cells provide rapid immune responses against viral-infected cells, tumor cells or other stressed cell, even in the absence of antibodies and major histocompatibility complex on the cell surfaces. NK cells can either be isolated or obtained from a commercially available source. In some embodiments, NK cells are isolated from cord blood. Non-limiting examples of commercial NK cell lines include lines NK-92 (ATCC® CRL-2407TM), NK- 92MI (ATCC® CRL-2408TM).
  • NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT 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).
  • an NK cell can be identified by the expression or lack of the expression of the following markers.
  • CD56 also termed as Neural Cell Adhesion Molecule 1 (NCAM)
  • NCAM Neural Cell Adhesion Molecule 1
  • 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.
  • CD94 also known as killer cell lectin-like receptor subfamily D, member 1 (KLRD1) is a lectin, cluster of differentiation and a receptor that is involved in cell signaling and is expressed on the surface of natural killer cells in the innate immune system. CD94 pairs with the NKG2 molecule as a heterodimer. The CD94/NKG2 complex, on the surface of natural killer cells interacts with Human Leukocyte Antigen (HLA)-E on target cells.
  • HLA Human Leukocyte Antigen
  • 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: GC12P010226, HGNC: 6378, NCBI Entrez Gene: 3824, Ensembl: ENSG00000134539, OMIM®: 602894, or UniProtKB/Swiss-Prot: QI 3241, each of which is incorporated by reference herein in its entirety.
  • 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.
  • CD16 also known as FcyRIII, is a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages and a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).
  • CD16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD16b), which participate in signal transduction.
  • Non-limiting exemplary sequences of CD 16a or the underlying gene or suitable antibodies for detection of the protein can be found under Gene Cards ID: GC01M161541, HGNC: 3619, NCBI Entrez Gene: 2214, Ensembl: ENSG00000203747, OMIM®: 146740, or UniProtKB/Swiss-Prot: P08637, each of which is incorporated by reference herein in its entirety.
  • Non-limiting exemplary sequences of CD 16b or the underlying gene can be found under Gene Cards ID: GC01M161623, HGNC: 3620, NCBI Entrez Gene: 2215, Ensembl: ENSG00000162747, OMIM®: 610665, or UniProtKB/Swiss-Prot: 075015, each of which is incorporated by reference herein in its entirety.
  • KIR Killer cell immunoglobulin-like receptors
  • NK plasma membrane of natural killer cells and a minority of T cells. They regulate the killing function of these cells by interacting with major histocompatibility (MHC) class I molecules, which are expressed on all nucleated cell types.
  • MHC major histocompatibility
  • KIR gene family has been mapped on chromosome 19ql 3.4 where KIR genes, each spanning -10-16 kb, are tightly arranged in a head-to-tail orientation, including but not limited to KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1.
  • Suitable antibodies for detecting those KIR members are available to one of skill in the art, such as from R&D SYSTEMS® (e.g., MAB1848), BIO-RAD (e.g., NKVFS1), or abeam (e.g., ab205718).
  • NKG2 also known as CD 159, is a receptor for natural killer cells (NK cells).
  • NK cells There are 7 NKG2 types: A, B, C, D, E, F and H.
  • NKG2A dimerizes with CD94 to make an inhibitory receptor (CD94/NKG2).
  • Non-limiting exemplary sequences ofNKG2A or the underlying gene or suitable antibodies for detection of the protein can be found under Gene Cards ID: GC12M015061, HGNC: 6374, NCBI Entrez Gene: 3821, Ensembl:
  • ENSG00000134545 OMIM®: 161555, or UniProtKB/Swiss-Prot: P26715, each of which is incorporated by reference herein in its entirety.
  • NKG2D is an activating receptor on the NK cell surface.
  • 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: GC12M015056, HGNC: 18788, NCBI Entrez Gene: 22914, Ensembl: ENSG00000213809, OMIM®: 611817, or UniProtKB/Swiss-Prot: P26718, each of which is incorporated by reference herein in its entirety.
  • NKp30 is a natural cytotoxicity receptor (NCR) that may aid NK cells in the lysis of tumor cells. It also interacts with CD3-zeta (CD247), a T-cell receptor.
  • NCR natural cytotoxicity receptor
  • CD3-zeta CD247
  • T-cell receptor a T-cell receptor.
  • 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: GC06M031588, HGNC: 19077, NCBI Entrez Gene: 259197, Ensembl: ENSG00000204475, OMIM®: 611550, or UniProtKB/Swiss-Prot: 014931, each of which is incorporated by reference herein in its entirety.
  • NKp44 also known as NCR2 (Natural Cytotoxicity Triggering Receptor 2), is a cytotoxicity-activating receptor that may contribute to the increased efficiency of activated natural killer (NK) cells to mediate tumor cell lysis.
  • NCR2 Natural Cytotoxicity Triggering Receptor 2
  • 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: GC06P052193, HGNC: 6732, NCBI Entrez Gene: 9436, Ensembl: ENSG00000096264, OMIM®: 604531, or UniProtKB/Swiss-Prot: 095944, each of which is incorporated by reference herein in its entirety.
  • NKp46 is also a cytotoxicity-activating receptor that may contribute to the increased efficiency of activated natural killer (NK) cells to mediate tumor cell lysis.
  • NK natural killer
  • 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: GC19P054906, HGNC: 6731, NCBI Entrez Gene: 9437, Ensembl: ENSG00000189430, OMIM®: 604530, or UniProtKB/Swiss-Prot: 076036, each of which is incorporated by reference herein in its entirety.
  • NKp80 is an activating homodimeric C-type lectin-like receptor (CTLR) expressed on nearly all natural killer (NK) cells and stimulates their cytotoxicity and cytokine release. See, for example, Kuttruff et al. Blood. 2009 Jan 8;113(2):358-69.
  • CLR C-type lectin-like receptor
  • 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: GC12P011003, HGNC: 13342, NCBI Entrez Gene: 51348, Ensembl: ENSG00000150045, OMIM®: 605029, or UniProtKB/Swiss-Prot: Q9NZS2, each of which is incorporated by reference herein in its entirety.
  • T-box transcription factor TBX21 also called T-bet, is a marker for NK cells. See, for example, Simonetta et al. Front Immunol. 2016 Jun 20;7:241.
  • 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: GC17P047733, HGNC: 11599, NCBI Entrez Gene: 30009, Ensembl: ENSG00000073861, OMIM®: 604895, or UniProtKB/Swiss-Prot: Q9UL17, each of which is incorporated by reference herein in its entirety.
  • Eomesodermin is a transcription factor which is crucial for embryonic development of mesoderm and the central nervous system in vertebrates.
  • the protein can also be necessary for the differentiation of effector CD8+ T cells which are involved in defense against viral infections. It also has been used as a marker for NK cells. See, for example, Simonetta et al. Front Immunol. 2016 Jun 20;7:241.
  • 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: GC03M027715, HGNC: 3372, NCBI Entrez Gene: 8320, Ensembl: ENSG00000163508, OMIM®: 604615, or UniProtKB/Swiss-Prot: 095936, each of which is incorporated by reference herein in its entirety.
  • CD3 is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells) and composed of four distinct chains. It has been used as a negative marker for NK cells.
  • Suitable antibodies for detecting such protein is available to one of skill in the art, such as from abeam (e.g., abl35372), BIOLEGEND® (e.g., UCHT1), or Thermo Fisher Scientific (e.g., 14-0037-82).
  • CD 127 is a receptor for interleukin-7 and also acts as a receptor for thymic stromal lymphopoietin (TSLP).
  • TSLP thymic stromal lymphopoietin
  • 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: GC05P035852, HGNC: 6024, NCBI Entrez Gene: 3575, Ensembl: ENSG00000168685, OMIM®: 146661, or UniProtKB/Swiss-Prot: P16871, each of which is incorporated by reference herein in its entirety.
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T- cells may either be isolated or obtained from a commercially available source. “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gammadelta T cells.
  • CD4+ cells T-helper cells
  • CD8+ cells cytotoxic T-cells
  • Reg T-regulatory cells
  • gammadelta T cells gammadelta T cells.
  • a “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.
  • T cells can either be isolated or obtained from a commercially available source.
  • commercially available T-cell lines include lines BCL2 (AAA) Jurkat (ATCC® CRL- 2902TM), BCL2 (S70A) Jurkat (ATCC® CRL-2900TM), BCL2 (S87A) Jurkat (ATCC® CRL- 2901TM), BCL2 Jurkat (ATCC® CRL-2899TM), Neo Jurkat (ATCC® CRL-2898TM), T ALL- 104 cytotoxic human T cell line (ATCC # CRL-11386).
  • T-cell lines e.g., such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and immature T- cell lines, e g., ALL-SIL, Bel3, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB- ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-Tl, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT- 16, MT-1, MT- ALL, P12/Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T
  • mature T-cell lines e
  • Null leukemia cell lines including but not limited to REH, NALL-1, KM-3, L92-221, are a another commercially available source of immune cells, as are cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma.
  • 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).
  • NKT cell Natural killer T cell refers to cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non- polymorphic CD Id molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 1% of all peripheral blood T cells. NKT cells can be isolated and expanded from a sample. See, for example, US Patent Application Publication No. US20210106622A1.
  • gamma-delta T cell refers to a T cell having a TCR made up of one gamma chain (y) and one delta chain (6). T-cells may either be isolated or expanded or both isolated and expanded from a sample, See, for example, US Patent Application Publication No. US20160175358A1.
  • macrophage cell refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. Preferably, the macrophages are splenic macrophages. Macrophages can either be isolated or obtained from a commercially available source, such as SC (ATCC® CRL-9855), MD (ATCC® CRL-9850),
  • 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).
  • host cell refers to a cell which can support the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells, such as E. coli or eukaryotic cells (e.g. yeast, insect, amphibian, bird, or mammalian cells). Creation and isolation of host cell lines capable of producing a protein can be accomplished using standard techniques known in the art.
  • 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.
  • TAA tumor associated antigen
  • cancer antigen cancer antigen
  • tumor antigen cancer relevant antigen
  • tumor relevant antigen are used interchangeably herein, referring to antigenic substance of a cancer or tumor cells.
  • a TAA presents on some tumor or cancer cells and also on some normal cells, optionally at a lower level.
  • a TAA only presents on a tumor or cancer cell but not on a normal cell.
  • a TAA refers to a TAA recognized and bound by a CAR as disclosed herein.
  • a TAA refers to a TAA recognized and bound by an antibody as disclosed herein.
  • a TAA is selected from BCMA, GPRC5D, FLT3, CD 19, mesothelin, human epidermal growth factor receptor 2 (HER2), prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CD33, GTPase- activating protein (GAP), ganglioside G2 (GD2), CD5, prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), CD123, CD70, CD38, mucin 1, (Mucl), ephrin type-A receptor 2 precursor (EphA2), epidermal growth factor receptor variant III (EGFRVIII), interleukin 13 receptor alpha 2 (IL13Ra2), CD 133, glypican 3 (GPC3), epithelial cell adhesion molecule precursor (EpCam), fibroblast activation protein alpha (FAP), vascular endothelial growth factor receptor 2 (VEGFR2),
  • a feeder cell As used herein, a feeder cell, an antigen presenting cell (APC), or an artificial APC (aAPC) are used interchangeably, referring to a cell other than an immune cell, such as an NK cell, that co-exists when culturing the immune cell.
  • APC antigen presenting cell
  • aAPC artificial APC
  • feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate.
  • feeder cells support the growth of target cells by releasing growth factors to the culture media, but this is not the only way that feeder cells promote the growth of target cells.
  • Exemplified feeder cells are irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs), non-irradiated autologous PBMCs, RPMI8866, HFWT, K562, K562 cells, 721.221 cells, or AML3 cells.
  • IRR as used herein refers to irradiated or a grammatical variation thereof.
  • a naturally occurring antigen presenting cell refers to an immune cell that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes such as T cells, Classical APCs include dendritic cells, macrophages, Langerhans cells and B cells.
  • an artificial antigen presenting cells synthetic versions of these APCs and are made by attaching the specific immune cell (such as T cell and/or NK cell) stimulating signals to various macro and micro biocompatible surfaces and/or cells. This can potentially reduce the cost while allowing control over generating large numbers of functional pathogen-specific immune cells for therapy.
  • K562 cells were the first human immortalized myelogenous leukemia cell line to be established, which has been used in as feeder cells. Accordingly, one of skill in the art should have no difficulty in obtaining and optionally engineering the cells for the uses as disclosed herein. See, for example, ATCC® CCL-243TM available at ATCC; US 2020-0061115 Al; US 2020-0061115 Al; W02019213610A1; US 10,144,770; US 2017-0333479 Al; US 9,623,082; US 10,300,089; US 2018-0057795 Al; US 2020-0390815 Al; W02020112493; W02020104676; and US 2020-0199532 Al.
  • 721.221 cells are immortalized B lymphoblastoid cells reported to lack endogenous HLA-A, B, and C molecules. Accordingly, one of skill in the art should have no difficulty in obtaining and optionally engineering the cells for the uses as disclosed herein. See, for example, ATCC® CRL-1855TM available at ATCC and 721.221 available at LONZA.
  • AML3 cells were established from the peripheral blood of a 57-year-old man with acute myeloid leukemia (AML FAB M4) at diagnosis in 1987.
  • the cells carry an NPM1 gene mutation (type A) and the DNMT3 A R882C mutation. Accordingly, one of skill in the art should have no difficulty in obtaining and optionally engineering the cells for the uses as disclosed herein. See, for example, ACC 582 available at DSMZ-German Collection of Microorganisms and Cell Culture GmbH, and Quentmeier, et al. Scientific Reports 9 (1): 8218.
  • the phrase “derived from” means isolated from, purified from, or engineered from, or any combination thereof.
  • the F3 feeder cells as disclosed herein is generated by engineering K562 cells to express a mIL-21 and a 4-1BBL, and accordingly considered as derived from K562 cells.
  • the term “derived” also encompasses cells that are cultured from cells isolated directly from a sample, and cells cultured or expanded from primary isolates.
  • the term “culturing” refers to the in vitro or ex vivo 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. In some embodiments, the descendants of NK cells grown in culture remain as NK cells. In further embodiments, the descendants of NK cells grown in culture have an NK cell marker profile as disclosed herein.
  • 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 (also referred to herein as feeder cells), or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.
  • isolated refers to molecules, biologicals, cellular materials, cells or biological samples being substantially free from other materials.
  • the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide (e.g., an antibody or derivative thereof), or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
  • the term “isolated” is used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
  • isolated cell generally refers to a cell that is substantially separated from other cells of a tissue.
  • the term “enriching” or a grammatical variation thereof in the context of cells refers to increasing the percentage by about 10%, by about 20%, by about 30%, by about 40%, by about 50% or greater than 50% of one type of cells (such as NK cells) in a population of cells as compared to the starting population of cells.
  • a contacting step as disclosed herein can refer to culturing the cells under cell culture conditions.
  • cell culture conditions intend one or more of the following: a suitable vessel with a substrate or medium that supplies the essential nutrients (such as amino acids, carbohydrates, vitamins, or minerals), growth factors, hormones, gases (CO2, O2), or a physio-chemical environment (such as pH, osmotic pressure, or temperature), or a period of time of the culture.
  • the suitable conditions as used herein are suitable for maintaining the cell viability.
  • the suitable conditions as used herein are suitable for cell proliferation.
  • commercially available medium provides the essential nutrients, the physiochemical environment, etc.
  • the medium is further supplemented with serum, such as a human serum, providing suitable conditions as disclosed herein.
  • the medium is further supplemented with a cytokine, such as IL-2, providing suitable conditions as disclosed herein. Accordingly, serum, hormones, cytokines, growth factors, etc. are referred to herein as a supplement to the medium.
  • the term “contacting” means direct or indirect binding or interaction between two or more moieties (such as cells, or molecules on two cells).
  • 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.
  • the contacting step or the cell culture conditions or both further comprise culturing the cells with human AB serum.
  • human AB serum refers to serum from type AB human donors lacking antibodies against the A and B blood-type antigens.
  • Human AB Serum is a cell culture reagent for some human cell types providing growth factors, vitamins, nutrients as well as trace elements and transport factors, ensuring faster growth rates than mixed blood group serum.
  • human AB serum are collected from healthy male AB donors lacking antibodies against A and B blood type antigens, tested and found negative for all required viral markers via FDA-approved methods.
  • Suitable human AB serum is available commercially, for example from Access Cell Culture (VWR Catalog Number: 103039-156), Valley Biomedical (HP1022), and Sigma-Aldrich (Product Number: H4522).
  • human serum without limiting the bloodtype of the donor can be used, such as NIST909C sold by Sigma-Aldrich.
  • fetal bovine serum or serum from any other animals can be used to substitute the human AB serum, such as F2442, F4135, F0926, 12103C, 12306C, H1270, Hl 138, 12449C, R4505, S2263, C5405, or P9783 sold by Sigma-Aldrich.
  • the growth factors, vitamins, nutrients, trace elements, or transport factors can be provided to the cell culture from a source other than serum (such as a recombinant protein produced by a host cell or a synthetic compound).
  • the contacting step or the cell culture conditions or both further comprise culturing the cells in a SCGM medium.
  • a stem cell growth medium (SCGM) is widely used for standardized generation of autologous and allogeneic cell therapies using human hematopoietic stem and progenitor cells (HSCs/CD34+ cells), human natural killer cells (NK cells) and human cytokine induced killer cells (CIK cells).
  • the SCGM medium comprises salts, sugars, amino acids, vitamins, buffers, P -Mercaptoethanol, L-glutamine, albumin, and insulin, and has an osmolality of 290-350 (mOsm/kg H2O), a pH of 7.2 - 7.5, and ⁇ 1 EU/mL endotoxin. It is available from CELLGENIX® as 20802-0500, 20902-0500, 20806-0500, or 20906-0500.
  • the SCGM can be substituted with another cell medium suitable for NK cells, such as X-VIVOTM 15 hematopoietic cell medium from Lonza Bioscience, STEMLINE® II Hematopoietic Stem Cell Expansion Medium from Sigma-Aldrich, Blood Cell Basal Medium from Cell Applications, Inc., or Mononuclear Cell Medium from Sigma-Aldrich.
  • X-VIVOTM 15 hematopoietic cell medium from Lonza Bioscience
  • STEMLINE® II Hematopoietic Stem Cell Expansion Medium from Sigma-Aldrich
  • Blood Cell Basal Medium from Cell Applications, Inc.
  • Mononuclear Cell Medium from Sigma-Aldrich.
  • cytokines are a large group of proteins, peptides or glycoproteins that are secreted by specific cells of the immune system and signaling molecules that mediate and regulate immunity, inflammation and hematopoiesis. In some embodiments, they are about 5 kDa to about 20 kDa. Cytokines comprise chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors, but generally not hormones or growth factors. In some embodiments, the cytokine(s) as used herein promote one or more of the development, differentiation, activation, or expansion of immune cells, such as T cells, NK cells, NKT cells, or any combination thereof.
  • interleukin refers to cytokines that was first seen to be expressed by white blood cells (leukocytes).
  • the function of the immune system depends in a large part on interleukins.
  • the majority of interleukins are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, NK cells, NKT cells, and hematopoietic cells.
  • Interleukin-2 is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity.
  • the IL-2 is a human IL-2.
  • the IL-2 is of other species, such as a chimpanzee IL-2 having an NCBI Reference Sequence of XP_517425.1. Non -limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04M122451, HGNC (6001), NCBI Entrez Gene (3558), Ensembl
  • ENSG00000109471 OMIM® (147680), UniProtKB/Swiss-Prot (P60568), and Open Targets atform(ENSG00000109471), each of which is incorporated by reference herein in its entirety.
  • the IL-2 comprises, or consists essentially of, or yet further consists of MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 18).
  • the IL-2 comprises, or consists essentially of, or yet further consists of amino acid (aa) 21 to aa 153 of SEQ ID NO: 18.
  • IL-2 as used herein is a wildtype IL-2 or an equivalent thereof.
  • IL-2 as used herein is a recombinant IL-2 produced by a host cell (such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris) in vitro.
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • a host cell such as an HEK 293 cell, or a CHO cell, or E. coli, or Pichia pastoris
  • the IL-2 equivalent stimulates the proliferation or activates the cytotoxic function of NK cells or both significantly similar to the wildtype IL
  • Assays for evaluating the proliferation and cytotoxic function of NK cells are available for one of skill in the art, such as ex vivo culturing and cell counting (see, for example Choi et al. J Immunother Cancer. 2019 Jul 5;7(1): 168), 51 chromium release assay (see, for example, Dong et al. Cancer Discov. 2019 Oct;9(10): 1422-1437. doi: 10.1158/2159-8290.CD-18-1259. Epub 2019 Jul 24), colorimetric measurement-based cytotoxicity assay (see, for example, Chava et al. J Vis Exp.
  • the IL-2 equivalent comprises, or consists essentially of, or further consists of a fragment of the wildtype IL-2, such as aa 22 to aa 153 of SEQ ID NO: 19.
  • 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: 18 optionally mutated to methionine, the amino acid residue aligned to aa 39 of SEQ ID NO: 18 optionally mutated to serine, the amino acid residue aligned to aa 58 of SEQ ID NO: 18 optionally mutated to alanine or lysine, the amino acid residue aligned to aa 62 of SEQ ID NO: 18 optionally mutated to lysine or isoleucine or alanine or glutamine, the amino acid residue aligned to aa 65 of SEQ ID NO: 18 optionally mutated to asparagine or glutamic acid or alanine or arginine, the amino acid residue aligne
  • the IL-2 equivalent comprises, or consists essentially of, or further consists of an IL-2 derivative, such as modified by glycosylation, acetylation, or phosphorylation.
  • Interleukin- 15 is a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL- 15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by virus(es). This cytokine induces the proliferation of natural killer cells. In some embodiments, the IL- 15 is a human IL-15.
  • Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC04P141636 , HGNC: 5977, NCBI Entrez Gene: 3600, Ensembl: ENSG00000164136, OMIM®: 600554, or UniProtKB/Swiss-Prot: P40933, each of which is incorporated by reference herein in its entirety.
  • the IL- 15 is a human IL-15 isoform 1. Accordingly, in some embodiments, the IL-15 comprises, or consists essentially of, or yet further consists of
  • the IL-15 comprises, or consists essentially of, or yet further consists of amino acid (aa) 30 to aa 162 of SEQ ID NO: 21.
  • the IL-15 is a human IL-15 isoform 2.
  • the IL-15 comprises, or consists essentially of, or yet further consists of MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV TAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNI KEFLQSFVHIVQMFINTS (SEQ ID NO: 22).
  • the IL-15 is of other species, such as a Rhesus macaque IL- 15 having a UniProtKB reference ID of P48092.
  • IL-15 as used herein is a wildtype IL-15 or an equivalent thereof.
  • IL- 15 as used herein is a recombinant IL- 15 produced by a host cell (such as HEK 293 cells, or E. coli) in vitro. See, for example, human IL- 15 sold by Sigma-Aldrich (SRP6293, or SRP3077), and STEMCELLTM Technologies (78031).
  • the IL- 15 equivalent stimulates the proliferation or activates the cytotoxic function of NK cells or both significantly similar to the wildtype IL-15. Assays for evaluating the proliferation and cytotoxic function of NK cells are available for one of skill in the art, and are described herein.
  • Non-limiting examples of the IL-15 equivalents include those disclosed in US20190263877A1, US10450359B2, and US10537615B2. Additionally or alternative, the IL- 15 equivalent comprises, or consists essentially of, or further consists of an IL- 15 derivative, such as modified by glycosylation, acetylation, or phosphorylation.
  • a cytokine may be served as an immune cell activating agent while expanding the immune cells, for example, IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2.
  • Other immune cell activating agent(s) can substitute or supplement such cytokine(s), and thus is included in the disclosure herein.
  • chimeric antigen receptor refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain.
  • the “chimeric antigen receptor (CAR)” is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor (CIR).”
  • extracellular domain capable of binding to an antigen means any oligopeptide or polypeptide that can bind to a certain antigen.
  • intracellular domain or “intracellular signaling domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
  • the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signaling domains in addition to the primary signaling domain.
  • “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.
  • expansion and proliferation are used interchangeably and refer to a process by which a cell grows and divides to produce two daughter cells.
  • the expansion and proliferation is in vitro or ex vivo.
  • cell viability refers to the ability of cells in culture to survive under culture conditions.
  • the term as used herein also refers to ratio of cells which are alive compared the total number of cells, living and dead.
  • sustaining the cell viability after a cell culture or a contacting step refers to a substantially similar ratio of cells which are alive compared to the total number of cells, living and dead, after the cell culture or contacting step compared to prior to the cell culture or contacting step.
  • 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.
  • the term “detectable marker” refers to at least one marker capable of directly or indirectly, producing a detectable signal.
  • the detectable marker is a truncated protein marker (for example, a truncated CD 19, or a truncated EGFR).
  • CD 19 truncated protein markers
  • B-lymphocyte antigen CD 19 are used interchangeably to refer to a protein known to be a transmembrane protein that in humans is encoded by the gene CD 19. In humans, CD 19 is expressed in all B lineage cells, except for plasma cells, and in follicular dendritic cells.
  • Non-limiting exemplary sequences of this protein or the underlying gene may be found under Gene Cards ID: GC16PO28943, HGNC: 1633, Entrez Gene: 930, Ensembl: ENSG00000177455, OMIM: 107265, and UniProtKB: P15391, which are incorporated by reference herein.
  • An exemplified truncated CD 19 is provided herein and comprising, or consisting essentially of, or yet further consisting of MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESP LKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVN VEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGE PPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLS LELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWH WLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 23).
  • CD 19 Another exemplified truncated CD 19 is provided herein and comprising, or consisting essentially of, or yet further consisting of MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESP LKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVN VEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGE PPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTH (SEQ ID NO: 24).
  • an equivalent of SEQ ID NO: 23 or 24, for example those comprising 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or more mutations compared to SEQ ID NO: 23 or 24 or those having at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 23 or 24, may be used.
  • an equivalent of SEQ ID NO: 23 or 24 is still capable of being recognized and bound to by a moiety, such as an antibody or an antigen binding fragment thereof, specifically recognizing and binding CD19.
  • the equivalent of SEQ ID NO: 23 or 24 does not direct a cell expressing the equivalent to perform a function as a wild type CD 19 does.
  • the polynucleotide encoding SEQ ID NO: 23 or 24 comprises or consists essentially of, or yet further consists of
  • EGFR truncated protein marker
  • EGFR Epidermal Growth Factor Receptor
  • HGNC HGNC
  • NCBI Entrez Gene 1956
  • OMIM® 131550
  • UniProtKB/Swiss-Prot P00533, each of which is incorporated by reference herein by its entirety.
  • An exemplified truncated EGFR comprising, or consisting essentially of, or yet further consisting of LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVA FRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHG QFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISN RGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREF VENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVAL GIGLFM (SEQ ID NO: 26).
  • an equivalent of SEQ ID NO: 26, for example those comprising 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or more mutations compared to SEQ ID NO: 26 or those having at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 26, may be used.
  • an equivalent of SEQ ID NO: 26 is still capable of being recognized and bound to by a moiety, such as an antibody or an antigen binding fragment thereof, specifically recognizing and binding EGFR.
  • the equivalent of SEQ ID NO: 26 does not direct a cell expressing the equivalent to perform a function as a wild type EGFR does.
  • a non-exhaustive list of this marker includes enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, P-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and/or chemical properties, such detection may be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation , the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as 32 P, 35 S or 125 1.
  • enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline
  • 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.
  • 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.
  • the term “immunoconjugate” comprises an antibody or an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
  • a second agent such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.
  • suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, and Texas Red.
  • 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.
  • purification marker refers to at least one marker useful for purification or identification.
  • a non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein.
  • Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.
  • stem cell refers to a cell that is in an undifferentiated or partially differentiated state and has the capacity for self-renewal or to generate differentiated progeny or both. 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.).
  • embryonic stem cell is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue.
  • Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle.
  • exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural or neuronal stem cells (NSCs).
  • the stem or progenitor cells can be embryonic stem cells or an induced pluripotent stem cell (iPSC).
  • the stem or progenitor cells are hematopoietic stem cells (HSCs).
  • embryonic stem cells refers to stem cells derived from tissue formed after fertilization but before the end of gestation, including pre- embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Most frequently, embryonic stem cells are pluripotent cells derived from the early embryo or blastocyst. Embryonic stem cells can be obtained directly from suitable tissue, including, but not limited to human tissue, or from established embryonic cell lines. “Embryonic-like stem cells” refer to cells that share one or more, but not all characteristics, of an embryonic stem cell.
  • “Differentiation” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, immune or muscle cell.
  • “Directed differentiation” refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type.
  • “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell.
  • the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell.
  • 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.
  • the "lineage" of a cell defines the heredity of the cell, i.e. its predecessors and progeny.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a “multi-lineage stem cell” or “multipotent stem cell” refers to a stem cell that reproduces itself and at least two further differentiated progeny cells from distinct developmental lineages.
  • the lineages can be from the same germ layer (i.e. mesoderm, ectoderm or endoderm), or from different germ layers.
  • a “precursor” or “precursor cell” 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.
  • a progenitor cell may be in a later stage of cell differentiation.
  • An example of progenitor cell includes, without limitation, a progenitor nerve cell.
  • a precursor cell as used herein also intends a stem cell, a multi-lineage stem cell, a parthenogenetic stem cell, a hematopoietic stem cell, a pluripotent cell, or an induced pluripotent cell.
  • a “pluripotent cell” defines a less differentiated cell that can give rise to at least two distinct (genotypically or phenotypically or both) further differentiated progeny cells.
  • a “pluripotent cell” includes an Induced Pluripotent Stem Cell (iPSC) which is an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell that has historically been produced by inducing expression of one or more stem cell specific genes.
  • iPSC Induced Pluripotent Stem Cell
  • stem cell specific genes include, but are not limited to, the family of octamer transcription factors, i.e.
  • Oct-3/4 the family of Sox genes, i.e., Soxl, Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, i.e. Klfl, Klf2, Klf4 and Klf5; the family of Myc genes, i.e. c-myc and L-myc; the family of Nanog genes, i.e., OCT4, NANOG and REXI; or LIN28.
  • Examples of iPSCs are described in Takahashi et al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.
  • An “induced pluripotent cell” intends embryonic-like cells reprogrammed to the immature phenotype from adult cells.
  • Various methods are known in the art, e.g., "A simple new way to induce pluripotency: Acid.” Nature, 29 January 2014 and available at sciencedaily. com/releases/2014/01/140129184445, last accessed on February 5, 2014 and U.S. Patent Application Publication No. 2010/0041054.
  • Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.
  • a “parthenogenetic stem cell” refers to a stem cell arising from parthenogenetic activation of an egg. Methods of creating a parthenogenetic stem cell are known in the art. See, for example, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(Suppl. 1)11911-6.
  • 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 3/4, 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).
  • HSCs hematopoietic stem cells
  • an immune cell as disclosed herein is derived from an HSC.
  • phenotype refers to a description of an individual’s trait or characteristic that is measurable and that is expressed only in a subset of individuals within a population.
  • an individual’s phenotype includes the phenotype of a single cell, a substantially homogeneous population of cells, a population of differentiated cells, or a tissue comprised of a population of cells.
  • a population of cells as described herein is substantially homogeneous.
  • substantially homogeneous describes a population of cells in which more than about 50%, or alternatively more than about 60 %, or alternatively more than 70 %, or alternatively more than 75 %, or alternatively more than 80%, or alternatively more than 85 %, or alternatively more than 90%, or alternatively more than 95 %, of the cells are of the same or similar phenotype. Phenotype can be determined by a pre-selected cell surface marker or other marker.
  • a population of cells intends a collection of at least 100 million cells 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.
  • a population of cells comprises at least 100 million, at least 200 million, at least 300 million, at least 400 million, at least 500 million, at least 600 million, at least 700 million, at least 800 million, at least 900 million, at least 1 billion, at least 2 billion, at least 3 billion, or more cells.
  • a population of cells comprise at least 100 million substantially homogenous cells (i.e., where more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more cells in the population have the same or similar phenotype).
  • a biological sample is obtained from a subject.
  • exemplary samples include, but are not limited to, cell sample, tissue sample, tumor biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions/flushing, synovial fluid, mucosal
  • the sample is a tumor biopsy.
  • the sample is a tumor tissue in freshly obtained specimens, or a frozen tumor tissue.
  • the sample comprises, or consists essentially of, or yet further consists of NK cells.
  • the sample comprises, or consists essentially of, or yet further consists of cord blood, and therefore is referred to herein as a cord blood sample.
  • the cord blood sample can further comprise a carrier, such as an anticoagulant.
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, or can be therapeutic in terms of a partial or complete cure for a disorder or adverse effect attributable to the disorder.
  • treatment include but are not limited to: preventing a disorder from occurring in a subject that may be predisposed to a disorder, but has not yet been diagnosed as having it; inhibiting a disorder, i.e., arresting its development; or relieving or ameliorating the symptoms of disorder.
  • treatment is the arrestment of the development of symptoms of the disease or disorder, e.g., a cancer.
  • they refer to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • the disease is cancer
  • the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor.
  • the disease is an immune cell cancer, such as multiple myeloma (MM) or an acute myeloid leukemia (AML)
  • an immune cell cancer such as multiple myeloma (MM) or an acute myeloid leukemia (AML)
  • an immunoglobulin (such as IgG) level or residual cancer cells (for example as measured by flow cytometry, RT-PCR, or other conventional clinical methods), or both
  • a biological sample of a subject such as peripheral blood, plasma or serum
  • CTCs circulating tumor cells
  • a biological sample of a subject for example as measured by PCR or other suitable clinical methods
  • peripheral blood, plasma or serum may be used as a clinical end point.
  • treatment excludes prophylaxis. In one aspect, treatment excludes prophylaxis.
  • a mammal is a human.
  • mammals include humans, nonhuman primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
  • a mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a subject is a human.
  • cord blood is used interchangeably with “umbilical cord blood”, referring to blood that remains in the placenta and in the attached umbilical cord after childbirth.
  • umbilical cord blood refers to blood collected from umbilical veins that connect the placenta and fetus in mammals including humans.
  • the mammal is a human.
  • Peripheral blood cells are the cellular components of blood, comprising, or alternatively consisting essentially of, or yet further consisting of red blood cells (erythrocytes), white blood cells (leucocytes), and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.
  • the peripheral blood cells are mammal peripheral blood cells.
  • the peripheral blood cells are human peripheral blood cells.
  • a “composition” is intended to mean a combination of active agent, such as feeder cells or cell populations as disclosed herein, 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.
  • active agent such as feeder cells or cell populations as disclosed herein
  • inert for example, a detectable agent or label
  • active such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • a “population” as used herein, and in particular when referring to enriched or expanded population of cells includes cells of the same or different phenotype.
  • the population can comprise at least 1 X 10 3 cells, or alternatively at least 1 X 10 4 , 1 X 10 5 , 1 X 10 6 , 1 X 10 7 , 1 X 10 8 , 1 X 10 9 , 1 X 10 10 , or more cells.
  • At least 70%, or alternatively at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% or at least 99% of the cells in the population express the same phenotypic maker, e.g., a CD56+CD3- (CD56 positive, CD3 negative) population or alternatively, express the same of one or more exogenous genes.
  • a CD56+CD3- CD56 positive, CD3 negative
  • a population of cells comprises at least 1 X 10 3 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 4 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 5 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 6 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 7 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 8 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 9 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • a population of cells comprises at least 1 X 10 9 NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population are CD56+CD3-.
  • Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • a carrier comprises, or alternatively consists essentially of, or yet further consists of a preservative or a cryoprotective agent or both.
  • a composition as disclosed herein is a pharmaceutical composition.
  • a “pharmaceutical composition” is intended to include the combination of active agent, such as feeder cells or cell populations as disclosed herein, with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).
  • pharmaceutically acceptable carrier refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein).
  • biodegradable materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable).
  • a biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein.
  • Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
  • preservative refers to pharmaceutically acceptable excipients which prevent the growth of microorganisms within the composition and protects the composition against microbial contamination.
  • Exemplified preservatives include a number of organic acids and their salts, such as lactic acid and lactates, propionic acid and propionates, citric acid, acetic acid, sorbic acid, and sorbates, benzoic acid and benzoates, and methyl and propyl parabens (benzoic acid derivatives).
  • Other preservatives are also used, for example, Dandlin et al. Sci Rep. 2017 Jul 18;7(1):5658.
  • cryopreservation refers to storage of cells or tissue in an environment of less than about 8° C, which allows for extended storage of cells and can be at any temperature below 8° C, including temperatures at or below 4° C, 0° C, -20° C, -70° C, -80° C, -135° C, or in liquid nitrogen (-196° C).
  • Methods for cry opreserving cells with a medium containing choline salts and sucrose is disclosed in US Patent No. 5985538. Additional cryopreservation compositions and methods are disclosed in US Patent No.7935478, US Patent No. 7112576 and US Application No. 20170198251A1.
  • Contacting in vivo can be referred to as administering, or administration.
  • administering or “delivery” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application.
  • the administration is an intratumoral administration, or administration to a tumor microenvironment, or both.
  • the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.
  • administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • the disclosure is not limited by the route of administration, the formulation or dosing schedule.
  • administering can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • IxlO 4 to IxlO 15 or ranges in between of cells as disclosed herein are administrated to a subject, such as IxlO 7 to IxlO 10 .
  • administering or a grammatical variation thereof also refers to more than one doses with certain interval.
  • the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.
  • one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more.
  • cells as disclosed herein may be administered to a subject weekly and for up to four weeks.
  • the compositions and therapies can be combined with other therapies, e.g., lymphodepletion chemotherapy followed by infusions (e.g., four weekly infusions) of the therapy, defining one cycle, followed by additional cycles until a partial or complete response is seen or alternatively utilized as a “bridging” therapy to another modality, such as hematopoietic stem cell transplantation or CAR T cell therapy.
  • 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.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro, or ex vivo, or in vivo tests initially can provide useful guidance on the proper doses for patient administration.
  • one will desire to administer an amount of the agent as disclosed herein (such as a cell) that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro or ex vivo. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.
  • “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent (such as a cell as disclosed herein) that is an amount sufficient to obtain a pharmacological response; 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, as needed to induce a partial or complete effect.
  • a therapeutically effective amount may be administered in one or more administrations.
  • a therapeutically effective amount of cells as disclosed herein is IxlO 4 to IxlO 15 or ranges, such as IxlO 7 to IxlO 10 .
  • a treatment such as an immune cell comprising a polypeptide as disclosed herein, is administered to a subject as disclosed herein in an effective amount.
  • a treatment such as an immune cell comprising a polypeptide as disclosed herein, is administered to a subject as disclosed herein in a therapeutically effective amount.
  • an “anti-cancer 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 anti -metabolites.
  • cryopreserved cells refers to the process of restoring cryopreserved cells to a room temperature or a temperature suitable for cell culture.
  • cryoprotectant and “cryoprotective agent” refer to a substance that prevents or reduces damage to cells during cry opreservation.
  • Exemplified cryoprotective agents include a sugar (such as sucrose, dextrose, trehalose, pectin), glycerol, ethylene glycol, propylene glycol, polyethylene glycol (PEG), 1,2-propanediol, trehalose, carbohydrates (such as hydroxy ethyl starch (HES)), dextran, polylysine and dimethyl sulfoxide (DMSO).
  • a sugar such as sucrose, dextrose, trehalose, pectin
  • glycerol ethylene glycol, propylene glycol, polyethylene glycol (PEG), 1,2-propanediol, trehalose
  • carbohydrates such as hydroxy ethyl starch (HES)
  • dextran polylysine and dimethyl sulfoxide (DMSO).
  • the feeder cells as disclosed herein support a superior ex-vivo expansion of primary NK cells.
  • the 4-1BBL and IL-21 play a critical role in NK cell ex vivo expansion.
  • a K562 F3 feeder cell engineered with 4-1BBL an engineered IL-21 isoform and a superior signal peptide for IL-21, the latter two of which converge to contribute to a superior and surprising expansion capacity when compared to feeder cells reported in the literature.
  • the K562 F3 feeder cells as disclosed herein demonstrated distinct advantages for NK expansion and sustained viability after the expanded NK cells being frozen and thawed.
  • the K562 F3 feeder cells provide a great tool for the rapid production of NK cells or T cells, cell populations and other immune cells in therapeutic manufacturing.
  • a CYZ1 feeder cell which is nonvirally engineered with 4- 1BBL, an engineered IL-21 isoform and a superior signal peptide for IL-21, the latter two of which converge to contribute to a superior and surprising expansion capacity when compared to feeder cells reported in the literature.
  • the CYZ1 feeder cells comprise an intracellular hydrogel.
  • the CYZ1 feeder cells as disclosed herein demonstrated distinct advantages for immune cell (e.g., NK expansion) and sustained viability after the expanded immune cells have been frozen and thawed.
  • NK expansion a peripheral immune cell
  • Applicant observed higher efficacy for expanding cord blood-derived immune cells (e.g., NK cells) and transduced CAR-expressing immune cells (e.g., NK cells) using the CYZ1 feeder cells as disclosed herein expressing an azuroci din signal peptide and IL-21 isoform 1, compared to incubation with control feeder cells as reported in the literature.
  • the CYZ1 feeder cells provide a great tool for the rapid production of immune cells such as NK cells and T cells, immune cell populations, and other immune cells in therapeutic manufacturing.
  • methods of the instant disclosure expand NK cells at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 folds or more from the initial seeding populatoin.
  • stem or immune cells such as NK cells are expanded by at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 fold or more by the use of the feeder cells, compositions and/or methods of the instant disclosure.
  • a polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an azurocidin signal peptide, an interleukin 21 (IL- 21) polypeptide and a transmembrane domain, or an equivalent of each thereof.
  • the signal peptide is at the N terminus of the IL-21 polypeptide or an equivalent thereof.
  • the polypeptide comprises, or alternatively consists essentially of, or yet further consists of, from the N terminus to the C terminus, an azuroci din signal peptide or an equivalent thereof, the interleukin 21 (IL-21) polypeptide or an equivalent thereof, and the transmembrane domain or an equivalent thereof.
  • the polypeptide are mammalian polypeptides, e.g., human polypeptides.
  • polypeptide comprising, or alternatively consisting essentially of, or yet further consisting of an IL-21 polypeptide and a platelet-derived growth factor receptor beta (PDGFRP) transmembrane (TM) domain, or an equivalent of each thereof.
  • the polypeptide further comprises a signal peptide.
  • the signal peptide comprises, or consists essentially of, or yet further consists of an azurocidin signal peptide.
  • the signal peptide is at the N terminus of the IL-21 polypeptide or an equivalent thereof.
  • the polypeptide comprises, or alternatively consists essentially of, or yet further consists of, from the N terminus to the C terminus, the signal peptide or an equivalent thereof, the IL-21 polypeptide or an equivalent thereof, and a PDGFRP transmembrane domain or an equivalent thereof.
  • the polypeptide are mammalian polypeptides, e.g., human polypeptides.
  • the IL-21 polypeptide comprises, or alternatively consists essentially of, or yet further consists of IL-21 isoform 1 or an equivalent thereof. In further embodiments, the IL-21 polypeptide does not comprise an IL-21 isoform 2. In yet further embodiments, the IL-21 isoform 1 comprises, or alternatively consists essentially of, or yet further consists of
  • HKS S SQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWS AF SCFQK AQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 1) or an equivalent thereof.
  • the transmembrane domain comprises, or alternatively consists essentially of, or yet further consists of AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 8) or an equivalent thereof.
  • the signal peptide does not comprise, or consist essentially of, or consist of a wildtype IL-21 signal peptide. In some embodiments, the signal peptide does not comprise, or consist essentially of, or consist of a CSF2RA signal peptide. In some embodiments, the signal peptide comprises, or alternatively consists essentially of, or yet further consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 10) or an equivalent thereof.
  • the polypeptide as disclosed herein further comprises an Fc fragment of an antibody or an equivalent thereof located between the IL-21 polypeptide and the transmembrane domain or the equivalent of each thereof.
  • the Fc fragment comprises, or consists essentially of, or yet further consists of a hinge domain, a CH2 domain and a CH3 domain of IgG4 modified at F234A, L235A and N297Q or an equivalent thereof.
  • the Fc fragment comprises, or alternatively consists essentially of, or yet further consists of ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 12) or an equivalent thereof.
  • the polypeptide as disclosed herein further comprises a Tumor Necrosis Factor Superfamily Member 9 (4-1BBL) polypeptide and a self-cleaving peptide located between the IL-21 polypeptide and the 4-1BBL polypeptide or the equivalent of each thereof.
  • the 4-1BBL polypeptide or an equivalent thereof is located at the N terminus of the IL-21 polypeptide or an equivalent thereof.
  • the 4-1BBL polypeptide or an equivalent thereof is located at the C terminus of the IL-21 polypeptide or an equivalent thereof.
  • the 4-1BBL polypeptide or an equivalent thereof comprises the native 4-1BBL signal peptide.
  • the polypeptide as disclosed herein further comprises a signal peptide located at the N terminus of the 4-1BBL polypeptide or an equivalent thereof.
  • the 4-1BBL polypeptide comprises, or alternatively consists essentially of, or yet further consists of MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAV SGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD PGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARH AWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 13) or an equivalent thereof.
  • the self-cleaving peptide comprises, or alternatively consists essentially of, or yet further consists of a 2A self-cleaving peptide.
  • the 2A self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A or T2A.
  • the self-cleaving peptide comprises, or alternatively consists essentially of, or yet further consists of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 16) or an equivalent thereof.
  • polypeptide as disclosed herein comprises, or alternatively consists essentially of, or yet further consists of any one or more or all of the peptides as disclosed in Table 1.
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier, for example, a pharmaceutical carrier, and a polypeptide as disclosed herein (e.g., polypeptides in Tables 1 and 2).
  • polynucleotide encoding a polypeptide as disclosed herein, or a polynucleotide complementary thereto.
  • the polynucleotide can be a mammalian polynucleotide, e.g. a human polynucleotide.
  • the polynucleotide as disclosed herein comprises CATAAGTCCTCTAGTCAGGGACAGGACCGGCATATGATCAGGATGCGCCAGCTG ATCGACATAGTAGACCAACTTAAGAACTACGTTAACGATCTCGTGCCAGAGTTCC TTCCGGCCCCCGAGGACGTTGAAACCAATTGCGAATGGTCTGCCTTCAGCTGTTT CCAGAAAGCACAGCTGAAATCCGCCAACACTGGTAATAACGAGCGAATCATTAA CGTGTCCATCAAGAAACTCAAACGAAAGCCACCCAGTACGAACGCCGGCCGGCG TCAAAAGCATCGGTTGACCTGTCCCTCTTGCGACAGTTACGAAAAGAAGCCCCCA AAGGAGTTTCTGGAGCGTTTTAAGTCCTTGCTGCAGAAAATGATCCACCAACACT TGTCCAGTCGGACACACGGTTCCGAGGACTCC (SEQ ID NO: 3) or an equivalent thereof that encodes an IL-21 polypeptide.
  • the polynucleotide as disclosed herein further comprises ATGACTAGGTTGACAGTCCTCGCCTTGCTTGCTGGATTGCTTGCCAGTTCTCGAG CC (SEQ ID NO: 11) or an equivalent thereof that encodes an azuroci din signal peptide.
  • the polynucleotide as disclosed herein further comprises
  • the polynucleotide as disclosed herein further comprises GCCGTCGGTCAAGATACACAAGAAGTGATAGTCGTTCCGCATTCTCTTCCTTTCA AAGTTGTTGTAATTAGTGCAATTCTCGCGTTGGTTGTCCTGACAATTATAAGCCTC ATAATACTTATAATGCTGTGGCAAAAGAAACCCAGG (SEQ ID NO: 9) or an equivalent thereof that encodes a PDGFRp transmembrane domain.
  • the polynucleotide as disclosed herein further comprises GGTAGTGGGGCTACGAACTTTTCCCTTCTCAAACAGGCCGGAGACGTCGAGGAG AATCCTGGACCA (SEQ ID NO: 17) or an equivalent thereof that encodes a P2A selfcleaving peptide.
  • the polynucleotide as disclosed herein further comprises ATGGAGTATGCTAGCGATGCAAGCTTGGATCCAGAGGCACCCTGGCCACCAGCA CCTAGAGCTAGGGCTTGTCGTGTGCTCCCATGGGCGCTCGTAGCAGGTCTCCTCT TGCTCCTCTTGCTGGCGGCAGCTTGTGCTGTGTTTCTGGCTTGTCCTTGGGCAGTC TCAGGTGCAAGGGCGTCCCCAGGAAGTGCCGCATCCCCCAGGCTGAGAGAAGGC CCAGAACTGAGTCCTGATGACCCGGCAGGATTGCTTGATCTTCGCCAAGGAATGT TCGCTCAACTCGTAGCACAGAACGTATTGCTCATAGACGGCCCTTTGAGTTGGTA TTCCGATCCCGGACTTGCCGGAGTCAGCCTCACTGGCGGTTTGTCTTATAAGGAA GATACAAAAGAATTGGTAGTTGCGAAAGCCGGTGTTTATTACGTTCTTCCAGC TGGAATTGACGTGTTGTCGCT
  • the polynucleotide as disclosed herein comprises, or alternatively consists essentially of, or yet further consists of any one or more or all of the nucleotides as disclosed in Table 1.
  • a vector comprising a polynucleotide as disclosed herein.
  • the vector further comprises a regulatory element directing the expression of the polynucleotide.
  • the regulatory element comprises, or alternatively consists essentially of, or yet further consists of a long terminal repeat (LTR).
  • the vector is selected from a plasmid, a viral vector, or a retrovirus vector.
  • the vector as disclosed herein comprises, or alternatively consists essentially of, or yet further consists of any one or more or all of the nucleotides as disclosed in Table 1 or Table 2 and Table 3.
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier, for example, a pharmaceutical carrier, and a polynucleotide as disclosed herein or a vector as disclosed herein.
  • a carrier for example, a pharmaceutical carrier, and a polynucleotide as disclosed herein or a vector as disclosed herein.
  • Table 1 provides amino acid and nucleotide sequences of components of the PCIR- F3-IL-21-4-1BBL construct.
  • an artificial antigen-presenting cell comprising one or two or all three of a polypeptide as disclosed herein, a polynucleotide as disclosed herein, or a vector as disclosed herein.
  • the aAPC comprises an IL-21 polypeptide and a 4-1BBL polypeptide on the cell surface.
  • the cell is a mammalian cell, e.g., a murine, a canine, a simian, a feline or a human cell. Further provided are populations of such cells.
  • the aAPC is or is derived from a cell that lacks of expression of an MHC class I molecule (e.g., neuronal cell line OBL-21, a K562 cell, or a 721.221 cell). Additionally or alternatively, the aAPC is or is derived from an AML3 cell. In further embodiments, the cell lacking expression of an MHC class molecule is naturally occurring and then engineered to express the IL-21 polypeptide and the 4-1BB1 polypeptide. In other embodiments, the cell lacking expression of an MHC class I molecule is engineered to reduce or eliminate the expression of the MHC class I molecule, such as by RNA interference or CRISPR/Cas9 technology. In one aspect, the cell is a mammalian cell, e.g., a murine, a canine, a simian, a feline or a human cell. Further provided are populations of such cells.
  • an MHC class I molecule e.g., neuronal cell line OBL-21,
  • the aAPC does not proliferate, or proliferate as a significant lower lever compared to the cell line where the aAPC is derived from.
  • the cell number of aAPC at the end of culture increase less than 0.00001%, or less than 0.0001%, or less than 0.001%, or less than 0.01%, or less than 0.1%, or less than 1%, or less than 2%, or less than 3%, or less than 4%, or less than 5%, or less than 6%, or less than 7%, or less than 8%, or less than 9%, or less than 10%, or less than
  • the cell is a mammalian cell, e.g., a murine, a canine, a simian, a feline or a human cell. Further provided are populations of such cells.
  • the aAPC is irradiated. In some embodiments, the irradiated aAPC does not proliferate, or proliferate as a significant lower lever compared to the cell line where the aAPC is derived from.
  • the cell number of irradiated aAPC at the end of culture increase less than 0.00001%, or less than 0.0001%, or less than 0.001%, or less than 0.01%, or less than 0.1%, or less than 1%, or less than 2%, or less than 3%, or less than 4%, or less than 5%, or less than 6%, or less than 7%, or less than 8%, or less than 9%, or less than 10%, or less than 11%, or less than 12%, or less than 13%, or less than 14%, or less than 15%, or less than 16%, or less than 17%, or less than 18%, or less than 19%, or less than 20%, or less than 21%, or less than 22%, or less than 23%, or less than 24%, or less than 25% of the irradiated aAPC cell number seeded in (i.e., originally provided to) the cell culture.
  • the irradiated aAPCs were cultured for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days or longer.
  • the aAPC is irradiated by an ionizing radiation, for example, at a range from about 1 Gy to about 1000 Gy or any subrange or numbers there between.
  • the ionizing radiation is an irradiation by gamma rays, X- rays or ultraviolet (UV).
  • the aAPC is irradiated by an ionizing radiation of about 1 Gy to about 500 Gy, or about 1 Gy to about 250 Gy, or about 1 Gy to about 200 Gy, or about 1 Gy to about 150 Gy, or about 1 Gy to about 100 Gy, or about 1 Gy to about 75 Gy, or about 1 Gy to about 50 Gy, or about 1 Gy to about 25 Gy, or about 1 Gy to about 20 Gy, or about 1 Gy to about 10 Gy, or about 1 Gy to about 5 Gy, or about 5 Gy to about 1000 Gy, or about 5 Gy to about 500 Gy, or about 5 Gy to about 250 Gy, or about 5 Gy to about 200 Gy, or about 5 Gy to about 150 Gy, or about 5 Gy to about 100 Gy, or about 5 Gy to about 75 Gy, or about 5 Gy to about 50 Gy, or about 5 Gy to about 25 Gy, or about 5 Gy to about 20 Gy, or about 5 Gy to about 10 Gy, or about 10 Gy to about 1000 Gy, or about 10 Gy to about 500 Gy, or about 10 Gy to about 250 Gy, or about 10 Gy to about 1000 Gy, or about
  • the aAPC is irradiated by an ultraviolet radiation, for example at a range from about 5 J/m 2 to about 50 J/m 2 or any subrange or number therebetween.
  • the aAPC is irradiated by an ultraviolet radiation of about 5 J/m 2 to about 50 J/m 2 , or about 5 J/m 2 to about 45 J/m 2 , or about 5 J/m 2 to about 40 J/m 2 , or about 5 J/m 2 to about 35 J/m 2 , or about 5 J/m 2 to about 30 J/m 2 , or about 5 J/m 2 to about 25 J/m 2 , or about 5 J/m 2 to about 20 J/m 2 , or about 5 J/m 2 to about 15 J/m 2 , or about 5 J/m 2 to about 10 J/m 2 , or about 10 J/m 2 to about 50 J/m 2 , or about 10 J/m 2 to about 45 J/m 2 , or about 10 J
  • the aAPC is treated with a chemotherapy drug in order to substantially reduce or eliminate its proliferation.
  • Suitable chemotherapy drugs include, but are not limited to, agents that act in S-phase, including topoisomerase inhibitors and antimetabolites.
  • the chemotherapy drug is mitomycin, which is a family of aziridine-containing natural products isolated from Streptomyces caespitosus or Streptomyces lavendulae, including, but not limited to, mitomycin A, mitomycin B, and mitomycin C.
  • the chemotherapy drug is mitomycin c.
  • suitable chemotherapy drugs can be found, for example, from www.cancerresearchuk.org/about-cancer/cancer-in- general/treatment/ cancer-drugs/ drugs .
  • Topoisomerase inhibitors are drugs that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication, and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.
  • Antimetabolites are usually analogs of normal metabolic substrates, often interfering with processes involved in chromosomal replication. They attack cells at very specific phases in the cycle. Antimetabolites include folic acid antagonists, e.g., methotrexate; pyrimidine antagonist, e.g., 5 -fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonist, e.g., 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitor, e.g., cladribine, fludarabine, nelarabine and pentostatin; and the like.
  • folic acid antagonists e.g., methotrexate
  • pyrimidine antagonist e.g., 5 -fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine
  • purine antagonist e.g., 6-mercaptopur
  • Plant alkaloids are derived from certain types of plants.
  • the vinca alkaloids are made from the periwinkle plant (Catharanthus rosea).
  • the taxanes are made from the bark of the Pacific Yew tree (taxus).
  • the vinca alkaloids and taxanes are also known as antimicrotubule agents.
  • the podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian “Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also classified as topoisomerase inhibitors.
  • the plant alkaloids are generally cell-cycle specific.
  • Examples of these agents include vinca alkaloids, e.g., vincristine, vinblastine and vinorelbine; taxanes, e.g., paclitaxel and docetaxel; podophyllotoxins, e.g., etoposide and tenisopide; and camptothecan analogs, e.g., irinotecan and topotecan.
  • a composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier, such as a pharmaceutical carrier, and a cell or a cell population as disclosed herein.
  • compositions comprising, or consisting essentially of, or yet further consisting of (i) an immune cell, such as a natural killer (NK) cell, and (ii) an artificial antigen-presenting cell (aAPC, such as an aAPC as disclosed herein).
  • an immune cell such as a natural killer (NK) cell
  • aAPC artificial antigen-presenting cell
  • the aAPC comprises an intracellular hydrogel.
  • compositions comprising the polypeptides, polynucleotides, vectors, cells, or population of cells as described herein, and a carrier, such as a pharmaceutically acceptable carrier.
  • the immune cell such as the NK cell
  • the immune cell is derived from a precursor cell.
  • the precursor cell is isolated from primary mammalian peripheral blood or mammalian umbilical cord blood.
  • the precursor cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • the immune cell, such as the NK cell is immature.
  • the immune cell, such as the NK cell is mature.
  • the immune cell is an engineered immune cell.
  • the term “engineered immune cell” refers to an immune cell (e.g., T-cell, NK cell, NKT cell, B cell, dendritic cell, etc.) that is genetically modified.
  • the immune cell is an engineered immune cell expressing a chimeric antigen receptor (CAR).
  • the term “engineered immune cell” refers to an immune cell (e.g., T-cell, NK cell, NKT cell, B cell, dendritic cell, etc.) that is genetically modified.
  • the NK cell is an engineered NK cell expressing a chimeric antigen receptor (CAR).
  • the hydrogel comprises, or consists essentially of, or yet further consists of a biocompatible polymer, such as a poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA), polyesters, poly(ortho ester), poly(phosphazine), poly(phosphate ester), polycaprolactone, gelatin, collagen, fibronectin, keratin, polyaspartic acid, alginate, chitosan, chitin, hyaluronic acid, pectin, polyhydroxyalkanoates, dextrans, polyanhydrides, polyethylene oxide (PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine, any derivatives thereof and any combinations thereof.
  • the hydrogel comprises, or consists essentially of, or yet further consists of polyethylene glycol or a derivative thereof.
  • the derivative is poly(ethylene glycol)
  • the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer.
  • the monomer has a molecular weight of about 200 g/mol to about 2000 g/mol, or any subranges or molecular weight there between, such as about 200 g/mol to about 300 g/mol, about 200 g/mol to about 400 g/mol, about 200 g/mol to about 500 g/mol, about 200 g/mol to about 600 g/mol, about 200 g/mol to about 700 g/mol, about 200 g/mol to about 800 g/mol, about 200 g/mol to about 900 g/mol, about 200 g/mol to about 1000 g/mol, about 200 g/mol to about 1100 g/mol, about 200 g/mol to about 1200 g/mol, about 200 g/mol to about 1300 g/mol, about 200 g/mol to about 1400 g/mol, about 200 g/mol to about 1500 g/mol, about 200 g/mol to about 1600 g/mol, about 200 g/mol,
  • the monomer does not show a phase separation in aqueous solution.
  • the monomer is soluble in aqueous solution.
  • the monomer can penetrate cell membrane. Non-limiting example of accessing the monomer’s ability in penetrating cell membrane can be found, for example, Lin et al., Adv Mater. 2021 Jul;33(30):e2101190.
  • the isotonic buffer comprises about 5% to about 20% monomers, including any subranges or concentration thereof, such as about 5% to about 6%, about 5% to about 7%, about 5% to about 8%, about 5% to about 9%, about 5% to about 10%, about 5% to about 11%, about 5% to about 12%, about 5% to about 13%, about 5% to about 14%, about 5% to about 15%, about 5% to about 16%, about 5% to about 17%, about 5% to about 18%, about 5% to about 19%, about 5% to about 20%, about 6% to about 7%, about 6% to about 8%, about 6% to about 9%, about 6% to about 10%, about 6% to about 11%, about 6% to about 12%, about 6% to about 13%, about 6% to about 14%, about 6% to about 15%, about 6% to about 16%, about 6% to about 17%, about 6% to about 18%, about 6% to about 19%,
  • the isotonic buffer comprises about 10% monomer.
  • % represents a weight percentage, such as x% representing x g in a total of 100 g.
  • % represents a volume percentage, such as x% representing x ml in a total of 100 ml.
  • x% represents x g in a total of 100 mL.
  • the incubation is about 1 minute to about 30 minutes, including any subranges and numbers there between, such as about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes.
  • the hydrogel is cross-linked by exposing to a photoinitiator and UV light.
  • the photoinitiator is 12959.
  • the UV light has a wavelength of about 300 nm to about 400 nm, including any subranges and numbers there between, such as about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, or about 400 nm.
  • the aAPC was frozen and thawed. In further embodiments, the aAPC was lyophilized.
  • the aAPC comprises, or consists essentially of, or yet further consists of a dendritic cell (DC).
  • the DC is isolated from a biological sample of a subject.
  • the biological sample is a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • the subject is a human.
  • the aAPC comprises, or consists essentially of, or yet further consists of a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule, such as those as disclosed herein.
  • the aAPC further comprises an antigen on the cell surface. In further embodiments, the aAPC further comprises an antigen on the cell surface in an MHC and antigen complex. In some embodiments, the antigen is a tumor associated antigen. In some embodiments, the antigen is a pathogen associated antigen.
  • the aAPC is irradiated. In some embodiments, the aAPC is irradiated by an ionizing radiation at a range from about 1 Gy to about 1000 Gy, or any subranges or numbers there between. In some embodiments, the aAPC is irradiated by an ultraviolet radiation at a range from about 5 J/m 2 to about 50 J/m 2 , or any subranges or numbers there between.
  • the ratio of the NK cell and the aAPC cell is about 30: 1 to about 1 : 10, or any subranges or ratio there between, optionally 3: 1.
  • the composition further comprises, a cytokine, such as one or more of: IL-2, IL- 15 or IL-21.
  • the composition further comprises a microparticle.
  • the microparticle comprises, or consists essentially of, or yet further consists of poly(lactide-co-glycolide) (PLGA).
  • the microparticle is directly or indirectly linked with the aAPC.
  • the microparticle is conjugated with poly-l-lysine (PLL) that links with the aAPC via electrostatic interaction.
  • the microparticle releases a cytokine optionally selected from one or more of: IL-2, IL- 15 or IL-21.
  • the composition comprises (i) a natural killer (NK) cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the NK cell is selected from an immature NK cell, a mature NK cell or an engineered NK cell expressing a chimeric antigen receptor (CAR).
  • aAPC artificial antigen-presenting cell
  • the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell- membrane-penetrating hydrogel monomer and crosslinking the monomer, optionally wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol, optionally about 700 g/mol, and optionally wherein the isotonic buffer comprises from about 5% to about 20% optionally about 10% monomer.
  • the incubation is from about 1 minute to about 30 minutes, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,2 6, 27 28, 29 or 30 minutes.
  • the hydrogel is cross-linked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • the composition comprises (i) a natural killer (NK) cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the NK cell is derived from a precursor cell, and optionally wherein the precursor cell has been isolated from primary mammalian peripheral blood or mammalian umbilical cord blood, and optionally wherein the precursor cell is a hematopoietic stem cell (HSC).
  • a natural killer cell derived from a precursor cell
  • aAPC artificial antigen-presenting cell
  • HSC hematopoietic stem cell
  • the composition comprises (i) a natural killer (NK) cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the aAPC is selected from a dentritic cell (DC), a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • the aAPC has been irradiated.
  • the composition comprises (i) a T cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the T cell is selected from an immature T cell, a mature T cell or an engineered T cell expressing a chimeric antigen receptor (CAR).
  • aAPC artificial antigen-presenting cell
  • the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer, optionally wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol, optionally about 700 g/mol, and optionally wherein the isotonic buffer comprises from about 5% to about 20% optionally about 10% monomer.
  • the incubation is from about 1 minute to about 30 minutes, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,2 6, 27 28, 29 or 30 minutes.
  • the hydrogel is crosslinked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • the composition comprises (i) a T cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the T cell is derived from a precursor cell, optionally wherein the precursor cell has been isolated from primary mammalian peripheral blood or mammalian umbilical cord blood, and optionally wherein the precursor cell is a hematopoietic stem cell (HSC).
  • aAPC artificial antigen-presenting cell
  • HSC hematopoietic stem cell
  • the composition comprises (i) a T cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the aAPC is selected from a dentritic cell (DC), a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • the aAPC has been irradiated.
  • Another aspect of the disclosure is directed to artificial antigen-presenting cells (aAPC) as described herein.
  • aAPC artificial antigen-presenting cells
  • the aAPCs of the instant disclosure allows superior ex-vivo expansion of primary NK, CAR-NK, primary T cells and CAR-T cells.
  • the aAPC is a clonal feeder cell line that expresses 4-1BBL on its cell surface. In some embodiments, the aAPC expresses a cytokine on its cell surface (“tethered cytokine”). In some embodiments, the cytokine comprises Interleukin-21 (IL-21). In some embodiments, the IL-21 comprises a human IL-21. In some embodiments, the cytokine comprises Interleukin-2 (IL-2). In some embodiments, the IL-2 comprises a human IL-2. In some embodiments, the cytokine comprises Interleukin-7 (IL-7). In some embodiments, the IL-7 comprises a human IL-7.
  • the cytokine comprises Interleukin- 15 (IL-15). In some embodiments, the IL-15 comprises a human IL- 15. In some embodiments, the cytokine comprises Interleukin- 18 (IL- 18). In some embodiments, the IL-18 comprises a human IL-18. In some embodiments, the tethered cytokine comprises an azurocidin signal peptide.
  • the aAPC is selected from a dentritic cell (DC), a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • the aAPC has been irradiated.
  • the aAPC is irradiated by an ionizing radiation at a range of from about 1 Gy to about 1000 Gy (e.g., about 1 Gy, 10 Gy, 20 Gy, 50 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 500 Gy, 600 Gy, 700 Gy, 800 Gy, 900 Gy, or 1000 Gy).
  • the aAPC is irradiated by an ultraviolet radiation at a range of from about 5 J/m 2 to about 50 J/m 2 (e.g., about 5 J/m 2 , 10 J/m 2 , 15 J/m 2 , 20 J/m 2 , 25 J/m 2 , 30 J/m 2 , 35 J/m 2 , 40 J/m 2 , 45 J/m 2 , or 50 J/m 2 ).
  • the aAPC comprises an intracellular hydrogel.
  • the hydrogel is/has been introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer, optionally wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol (e.g., optionally about 200 g/mol, optionally about 250 g/mol, optionally about 300 g/mol, optionally about 350 g/mol, optionally about 400 g/mol, optionally about 450 g/mol, optionally about 500 g/mol, optionally about 550 g/mol, optionally about 600 g/mol, optionally about 650 g/mol, optionally about 700 g/mol, optionally about 750 g/mol, optionally about 800 g/mol, optionally about 850 g/mol, optionally about 900
  • the incubation is from about 1 minute to about 30 minutes, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,2 6, 27 28, 29 or 30 minutes.
  • the hydrogel is cross-linked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • the cells of the population are substantially homogenous for a phenotypic or genetic marker, e.g., CD56+CD3- when the population of cells are expanded NK cells.
  • the cell population comprises at least 1 X 10 3 , 1 X 10 4 , 1 X 10 5 , I X 10 6 , 1 X 10 7 , 1 X 10 8 , 1 X 10 9 , 1 X 10 10 , or more cells, wherein at least 70%, or 80%, or 90%, 95%, 99% or more of the cells express the same phenotypic or genetic marker.
  • a population of cells comprises at least 1 X 10 3 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3- (CD56 positive, CD3 negative).
  • phenotypic or genetic marker e.g. CD56+CD3- (CD56 positive, CD3 negative).
  • a population of cells comprises at least 1 X 10 4 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 5 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 6 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 7 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 8 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 9 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a population of cells comprises at least 1 X 10 9 cells, e.g., NK cells, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the population express the same phenotypic or genetic marker, e.g. CD56+CD3-.
  • a method of expanding an immune cell comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell, or a precursor cell thereof, or a cell population comprising, or alternatively consisting essentially of, or yet further consisting of the immune cell or the precursor cell or both, with an aAPC as disclosed herein.
  • a method of expanding immune cells comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cells, or precursor cells thereof, or a cell population comprising, or alternatively consisting essentially of, or yet further consisting of the immune cells or the precursor cells or both, with aAPCs as disclosed herein.
  • a method of activating or expanding or both activating or expanding an immune cell such as an NK cell.
  • the method comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell or a precursor cell thereof with an artificial antigen-presenting cell (aAPC) that comprises an intracellular hydrogel.
  • aAPC artificial antigen-presenting cell
  • the hydrogel is as disclosed herein, such as in the Composition section.
  • the aAPC is as disclosed herein, such as in the Composition section.
  • the immune cell is selected from a T cell, an NK cell, an NKT cell, a gamma-delta T cell, or a macrophage.
  • the immune cell can be isolated from a biological sample, such as primary peripheral blood, or umbilical cord blood.
  • the biological sample is of a mammal, such as a human.
  • the immune cell is an engineered immune cell.
  • the immune cell is engineered to express a chimeric antigen receptor (CAR).
  • the immune cell is engineered to express a detectable marker or a cytokine or both.
  • the detectable marker comprises, or alternatively consists essentially of, or further consists of a truncated epidermal growth factor receptor (EGFR).
  • the cytokine comprises, or alternatively consists essentially of, or further consists of an IL-15, such as a secretory IL-15.
  • the immune cell is an immature immune cell. In some embodiments, wherein the immune cell is a mature immune cell.
  • the cell population is isolated from a biological sample, such as primary peripheral blood or umbilical cord blood.
  • the biological sample is of a mammal, such as a human.
  • the cell population is a substantially homogeneous population of the immune cell, such as comprising, or alternatively consisting of, or yet further consisting of at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about
  • the precursor cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • the precursor cell is isolated from a biological sample, such as primary peripheral blood or umbilical cord blood.
  • the biological sample is of a mammal, such as a human.
  • the immune cell or the precursor cell or both have been cryopreserved and recovered for at least once.
  • the ratio between the cell number of the aAPC and the cell number of the immune cell, or the precursor cell, or the cell population is about 5: 1 to about 1 :5 or any subrange or ratio therebetween in the aAPC contacting step.
  • the ratio is about 5: 1 to about 1 :5, or about 5: 1 to about 1 :4, or about 5: 1 to about 1 :3, or about 5: 1 to about 1 :2, or about 5: 1 to about 1 : 1, or about 5: 1 to about 2: 1, or about 5: 1 to about 3:1, or about 5: 1 to about 4: 1, or about 4:1 to about 1 :5, or about 4: 1 to about 1 :4, or about 4: 1 to about 1 :3, or about 4: 1 to about 1 :2, or about 4: 1 to about 1 : 1, or about 4: 1 to about 2: 1, or about 4: 1 to about 3 : 1, or about 3 : 1 to about 1 :5, or about 3 : 1 to about 1 :4, or about 3 : 1 to about 1 :3, or about 3 : 1 to about 1 :2, or about 3 : 1 to about 1 : 1, or about 3 : 1 to about 2: 1, or about 2: 1 to about 1 :5, or about 2:1 to about 1 :
  • the ratio is about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 1 : 1, or about 2: 1, or about 3 : 1, or about 4: 1, or about 5: 1.
  • the ratio between the cell number of the aAPC and cell number of the immune cell, or the precursor cell, or the cell population is about 1 : 1 in the aAPC contacting step.
  • the ratio between the cell number of the aAPC and cell number of the immune cell, or the precursor cell, or the cell population is about 2: 1 in the aAPC contacting step.
  • the method is an ex vivo or in vitro method.
  • the method further comprises contacting with an IL-2 polypeptide or an equivalent thereof while contacting with the aAPC.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 2000 lU/mL, or any subrange or concentration therebetween.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1000 lU/mL, or about 20 lU/mL to about 750 lU/mL, or about 20 lU/mL to about 500 lU/mL, or about 20 lU/mL to about 400 lU/mL, or about 20 lU/mL to about 300 lU/mL, or about 20 lU/mL to about 200 lU/mL, or about 20 lU/mL to about 100 lU/mL, or 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1000 lU/mL
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/mL, or about 100 lU/mL, or about 150 lU/mL, or about 200 lU/mL, or about 250 lU/mL, or about 300 lU/mL, or about 400 lU/mL, or about 500 lU/mL. In some embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 200 lU/mL. In some embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 500 lU/mL.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 10 lU/ml to about 100 lU/ml. In yet further embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/ml. In some embodiments, the IL-2 polypeptide or the equivalent thereof is a secretory polypeptide. In further embodiments, the IL-2 polypeptide or the equivalent thereof is expressed and optionally secreted by one or more of the immune cell, the precursor cell, or the feeder cell.
  • the method further comprises contacting with an IL- 15 polypeptide or an equivalent thereof while contacting with the aAPC.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 2000 lU/mL, or any subrange or concentration therebetween.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1000 lU/mL, or about 20 lU/mL to about 750 lU/mL, or about 20 lU/mL to about 500 lU/mL, or about 20 lU/mL to about 400 lU/mL, or about 20 lU/mL to about 300 lU/mL, or about 20 lU/mL to about 200 lU/mL, or about 20 lU/mL to about 100 lU/mL, or 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1000 lU/m
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/mL, or about 60 lU/mL, or about 70 lU/mL, or about 80 lU/mL, or about 90 lU/mL, or about 100 lU/mL, or about 110 lU/mL, or about 120 lU/mL, or about 130 lU/mL, or about 140 lU/mL, or about 150 lU/mL, or about 160 lU/mL, or about 170 lU/mL, or about 180 lU/mL, or about 190 lU/mL, or about 200 lU/mL, or about 250 lU/mL, or about 300 lU/mL, or about 400 lU/mL, or about 500 lU/mL.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 150 lU/mL IL-15. In some embodiments, the IL- 15 polypeptide or the equivalent thereof is a secretory polypeptide. In further embodiments, the IL- 15 polypeptide or the equivalent thereof is expressed and optionally secreted by one or more of the immune cell, the precursor cell, or the feeder cell.
  • the contacting step is repeated for at least once, or at least twice, or at least three times, or more.
  • a method of sustaining an immune cell’s viability comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cell with an aAPC as disclosed herein.
  • a method of sustaining immune cells’ viability comprises, or alternatively consists essentially of, or yet further consists of contacting the immune cells with aAPCs as disclosed herein.
  • the immune cell is selected from a T cell, an NK cell, an NKT cell, a gamma-delta T cell, or a macrophage.
  • the immune cell can be isolated from a biological sample, such as primary peripheral blood, or umbilical cord blood.
  • the biological sample is of a mammal, such as a human.
  • the immune cell is an engineered immune cell.
  • the immune cell is engineered to express a chimeric antigen receptor (CAR).
  • the immune cell is engineered to express a detectable marker or a cytokine or both.
  • the detectable marker comprises, or alternatively consists essentially of, or further consists of a truncated epidermal growth factor receptor (EGFR).
  • the cytokine comprises, or alternatively consists essentially of, or further consists of an IL-15, such as a secretory IL-15.
  • the immune cell is an immature immune cell. In some embodiments, wherein the immune cell is a mature immune cells.
  • the immune cell is derived from a precursor cell.
  • the precursor cell is isolated from a biological sample, such as primary peripheral blood or umbilical cord blood.
  • the biological sample is of a mammal, such as a human.
  • the biological sample is a primary sample collected from a subject.
  • the biological sample is cultured in vitro before precursor cell isolation.
  • the biological sample is frozen and thawed before precursor cell isolation.
  • the immune cell or the precursor cell or both have been cryopreserved and recovered for at least once.
  • the ratio between the cell number of the aAPCs and the cell number of the immune cells is about 5: 1 to about 1 :5 or any subrange or ratio there between in the aAPC contacting step. In further embodiments, the ratio is about 5: 1 to about 1 :5, or about 5 : 1 to about 1 :4, or about 5 : 1 to about 1 :3, or about 5 : 1 to about 1 :2, or about 5 : 1 to about 1 :1, or about 5: 1 to about 2:1, or about 5: 1 to about 3: 1, or about 5: 1 to about 4: 1, or about 4: 1 to about 1 :5, or about 4: 1 to about 1 :4, or about 4: 1 to about 1 :3, or about 4: 1 to about 1 :2, or about 4: 1 to about 1 : 1, or about 4: 1 to about 2: 1, or about 4: 1 to about 3 : 1, or about 3 : 1 to about 1 :5, or about 3 : 1 to about 1 :4, or
  • the ratio is about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 1 : 1 , or about 2 : 1 , or about 3 : 1 , or about 4 : 1 , or about 5 : 1.
  • the ratio between the cell number of the aAPCs and cell number of the immune cells is about 1 : 1 in the aAPC contacting step. In some embodiments, the ratio between the cell number of the aAPCs and cell number of the immune cells is about 2: 1 in the aAPC contacting step.
  • the method is an ex vivo or in vitro method.
  • the method further comprises contacting with an IL-2 polypeptide or an equivalent thereof while contacting with the aAPC.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 2000 lU/mL, or any subrange or concentration there between.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1000 lU/mL, or about 20 lU/mL to about 750 lU/mL, or about 20 lU/mL to about 500 lU/mL, or about 20 lU/mL to about 400 lU/mL, or about 20 lU/mL to about 300 lU/mL, or about 20 lU/mL to about 200 lU/mL, or about 20 lU/mL to about 100 lU/mL, or 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1000 lU/
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/mL, or about 100 lU/mL, or about 150 lU/mL, or about 200 lU/mL, or about 250 lU/mL, or about 300 lU/mL, or about 400 lU/mL, or about 500 lU/mL. In some embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 200 lU/mL. In some embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 500 lU/mL.
  • the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 10 lU/ml to about 100 lU/ml. In yet further embodiments, the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/ml. In some embodiments, the IL-2 polypeptide or the equivalent thereof is a secretory polypeptide. In further embodiments, the IL-2 polypeptide or the equivalent thereof is expressed and optionally secreted by one or more of the immune cell, the precursor cell, or the feeder cell.
  • the method further comprises contacting with an IL- 15 polypeptide or an equivalent thereof while contacting with the aAPC.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 2000 lU/mL, or any subrange or concentration therebetween.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1500 lU/mL, or about 20 lU/mL to about 1000 lU/mL, or about 20 lU/mL to about 750 lU/mL, or about 20 lU/mL to about 500 lU/mL, or about 20 lU/mL to about 400 lU/mL, or about 20 lU/mL to about 300 lU/mL, or about 20 lU/mL to about 200 lU/mL, or about 20 lU/mL to about 100 lU/mL, or 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1500 lU/mL, or about 50 lU/mL to about 1000 lU/m
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/mL, or about 60 lU/mL, or about 70 lU/mL, or about 80 lU/mL, or about 90 lU/mL, or about 100 lU/mL, or about 110 lU/mL, or about 120 lU/mL, or about 130 lU/mL, or about 140 lU/rnL, or about 150 lU/rnL, or about 160 lU/rnL, or about 170 lU/mL, or about 180 lU/mL, or about 190 lU/mL, or about 200 lU/mL, or about 250 lU/mL, or about 300 lU/mL, or about 400 lU/mL, or about 500 lU/mL.
  • the IL- 15 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 150 lU/mL IL-15. In some embodiments, the IL- 15 polypeptide or the equivalent thereof is a secretory polypeptide. In further embodiments, the IL- 15 polypeptide or the equivalent thereof is expressed and optionally secreted by one or more of the immune cell, the precursor cell, or the feeder cell.
  • the contacting step is repeated for at least once, or at least twice, or at least three times, or more.
  • immune cells or a cell population expanded or sustained by a method as disclosed herein are immune cells or a cell population expanded or sustained by a method as disclosed herein.
  • the immune cell such as the NK cell, activated or expanded or both activated and expanded using a method as disclosed herein.
  • a cell population comprising, or consisting essentially thereof, or yet further consisting of an immune cell as disclosed herein.
  • a composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier, such as a pharmaceutical carrier, and immune cells or a cell population expanded or sustained by a method as disclosed herein.
  • a method of treating a cancer comprising a tumor- associated antigen (TAA) in a subject in need thereof comprises, or consists essentially of, or yet further consists of administering one or more of: an immune cell as disclosed herein, a cell population as disclosed herein, or a composition as disclosed herein to the subject.
  • an effective amount of one or more of: the immune cell as disclosed herein, the cell population as disclosed herein, or the composition as disclosed herein was administered.
  • the immune cell was activated or expanded or both activated and expanded by contacting with the aAPC that comprises the TAA on the cell surface. Additionally or alternatively, the immune cell comprises a CAR specifically recognizing and binding to the TAA.
  • a method of treating an infection caused by a pathogen comprising a pathogen associated antigen in a subject in need thereof comprises, or consists essentially of, or yet further consists of administering one or more of: an immune cell as disclosed herein, a cell population as disclosed herein, or a composition as disclosed herein to the subject.
  • an effective amount of one or more of: the immune cell as disclosed herein, the cell population as disclosed herein, or the composition as disclosed herein was administered.
  • the immune cell was activated or expanded or both activated and expanded by contacting with the aAPC that comprises the pathogen associated antigen on the cell surface.
  • the immune cell comprises a CAR specifically recognizing and binding to the pathogen associated antigen.
  • the method further comprises administering a therapeutic agent, or an agent increasing the expression level of TAA or pathogen associated antigen, or both agents.
  • Another aspect of the disclosure is directed to a method for activating or expanding or both activating or expanding an NK cell, comprising contacting the NK cell or a NK precursor cell with an artificial antigen-presenting cell (aAPC) that comprises an intracellular hydrogel, wherein the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • aAPC artificial antigen-presenting cell
  • the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer, optionally wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol, optionally about 700 g/mol, and optionally wherein the isotonic buffer comprises from about 5% to about 20% optionally about 10% monomer.
  • the incubation is from about 1 minute to about 30 minutes, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,2 6, 27 28, 29 or 30 minutes.
  • the hydrogel is cross-linked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • Another aspect of the disclosure is directed to a method for activating or expanding or both activating or expanding a natural killer (NK) cell comprising contacting the NK cell or a NK precursor cell with an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the aAPC is selected from a dentritic cell (DC), a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • the aAPC has been irradiated.
  • Another aspect of the disclosure is directed to a method for activating or expanding or both activating or expanding a T cell, comprising contacting the T cell or a T precursor cell with an artificial antigen-presenting cell (aAPC) that comprises an intracellular hydrogel, wherein the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • aAPC artificial antigen-presenting cell
  • the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer, optionally wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol, optionally about 700 g/mol, and optionally wherein the isotonic buffer comprises from about 5% to about 20% optionally about 10% monomer.
  • the incubation is from about 1 minute to about 30 minutes, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,2 6, 27 28, 29 or 30 minutes.
  • the hydrogel is cross-linked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • Another aspect of the disclosure is directed to a method for activating or expanding or both activating or expanding a T cell comprising contacting the T cell or a T precursor cell with an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel, wherein the aAPC is selected from a dentritic cell (DC), a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • the aAPC has been irradiated.
  • the subject is a mammal, a canine, a feline, an equine, a murine, or a human patient.
  • kits for use in a method as disclosed herein comprises, or consists essentially of, or yet further consists of optional instructions for use and one or more of the following: a composition as disclosed herein, an aAPC as disclosed herein, an immune cell as disclosed herein, or a cell population as disclosed herein.
  • Figure 2 illustrates a strategy to generate higher expression of tethered IL-21 on K562 cell surface with co-expression of 4-1BBL.
  • K562 feeder cells were engineered with high expression of IL-21 and CD137L/4-1BBL.
  • a canonical IL-21 isoform 1 and a signal peptide derived from human azurocidin were chosen to generate the K562 F3 feeder cells, which greatly improved the expression of tethered IL-21 on K562 cell surface.
  • IL-21 There are two forms of IL-21. Dean A Lee and other groups used the membrane form of IL-21 (isoform 2) to generate the artificial antigen-presenting cell (aAPC) lines.
  • aAPC artificial antigen-presenting cell
  • the canonical IL-21 isoform 1 is naturally higher produced than isoform 2, and tested as described below. Further, the IL-21 tested was conjugated with a PDGFRp transmembrane (TM) domain and successfully expressed on the cell surface of K562 cells.
  • TM PDGFRp transmembrane
  • Hee Gu Lee group reported that the azurocidin signal peptides provide an efficient approach for the high-level production in CHO-K1 cells of IL-21 as a biopharmaceutical agent than IL-21 self-signal peptides and some other signal peptides. It is noted that Hee Gu Lee conjugated the signal peptide with a secretory form of IL-21 for production.
  • K562 cells had not been tested for expressing IL-21 with the azurocidin signal peptides. Additionally, the azurocidin signal peptide had not been conjugated with any transmembrane domain. Thus, it was not clear whether the azurozcidin signal peptides can increase the expression of a membrane-bound protein (such as a membrane bound IL-21) or not, for example in a cell line other than CHO-K1.
  • a membrane-bound protein such as a membrane bound IL-21
  • IgG4 Fc a three-site mutated human IgG4 Fc was incorporated to support the flexibility of the membrane bound IL-21.
  • IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region. Substitutions in its Fc region were aimed to avoid the Fc receptors dimerization and binding.
  • 4-1BBL was inserted following P2A self-cleaving peptides. Both inserts were cloned into retrovirus vectors PCIR between 5’ and 3’ LTR.
  • FIG. 3 provides a diagraph of the PCIR-F3 -IL-21-4-1 BBL transgene.
  • the human IL-21 isoform 1 sequence was from UniProtKB - Q9HBE4-1 (IL21 HUMAN), and the human 4-1BBL was from UniProtKB - P41273 (TNFL9 HUMAN).
  • a signal peptide derived from human azuroci din was chosen to greatly improve the expression of tethered IL-21 on cell surface.
  • a three-site mutated human IgG4 Fc was used to support the flexibility of the membrane bound IL-21. IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region.
  • Figures 4A-4B show successful co-expression of 4-1BBL and tethered IL-21 on the K562 F3 feeder cells. Assessment of the expression of 4-1BBL and tethered IL-21 (i.e., membrane bound IL-21 or mIL-21) on the different feeder cells was assessed.
  • 4-1BBL and tethered IL-21 i.e., membrane bound IL-21 or mIL-21
  • the K562 F3 cells express IL-21 isoform 1 and 4-1BBL, and the translocation of IL-21 to the cell surface is directed by an azurocidin signal peptide; while the control feeder cells (which is also referred to herein as a control) express IL-21 isoform 2 as used in the literature and 4-1BBL, and the translocation of IL-21 to the cell surface is directed by a CSF2RA signal peptide.
  • Control K562 feeder cells and the K562 F3 feeder cells were expanded and irradiated using a gamma irradiator at lOOGy.
  • mIL-21 4- 1BBL and membrane-bound IL-21 (mIL-21) using the flow antibodies PE- 4-1BBL and AF- 647-IL-21 or their respective isotype controls.
  • Cell events were acquired using BD LSR Fortessa and the double positive cells expressing both 4-1BBL and mIL-21 were gated based on the respective isotype control of each sample.
  • Figures 5A-5C provide distinct advantages of novel K562 F3 feeder cells for supporting primary human peripheral blood derived NK cells proliferation and sustains their viability.
  • NK cells were purified from frozen PBMC, and mixed with mitomycin treated control feeder cells, or the K562 F3 feeder cells expressing isoform 1 of IL-21 and CD137L, in the presence of IL-2.
  • the NK cells were stimulated with irradiated aAPCs and cytokines three times: day 3, 10 and 17 in order to support NK cell growth.
  • NK cells were stimulated with feeder cells at a ratio of 2: 1 (2 feeder cells for every 1 NK cell), starting on day 3.
  • NK cells were re-stimulated with feeder cells at the same ratio (2:1), along with cytokines. . Every week, cell counts and viability were monitored via flow cytometry NovoCyte 3005.
  • a small portion of NK cells were used for subsequent expansion and re-stimulated with the same batch of mitomycin-treated feeder cells after thawing ( Figures 5A-5B).
  • the cumulative fold expansion after each week is plotted in the graph having a logarithmic scale ( Figure 5C). Data is presented as an average from 4 independent donors as cumulative fold changes ⁇ SEM. Paired T test was used to evaluate the differences of impacts of control and F3 feeder groups. *p ⁇ 0.05, **p ⁇ 0.01, ns: no significance.
  • the F3 feeder cells demonstrated both the higher cumulative fold change and higher viability compared to control since second week.
  • Figures 6A-6D compare the expansion fold and transduction efficiency of the irradiated (IRR) control K562 feeder cells v.s. the IRR K562 F3 feeder cells, and show an enhanced expansion efficacy, a sustained transgene expression, and a high viability of cord blood derived primary NK and CAR-NK using the F3 feeder cells.
  • IRR irradiated
  • NK cells from 3 donors were cocultured with the IRR control K562 feeder cells and the IRR K562 F3 feeder cells at a ratio of 1 :2 using SCGM media supplemented with 50 lU/ml of IL-2.
  • NK cells were transduced with retrovirus expressing soluble IL-15 and truncated EGFR (tEGFR) at day 7 at an MOI of 0.1.
  • Transduced NK cells were stimulated with another round of feeder cells at a ratio of 1 :2 at day 10 and further expanded and harvested on Day 16.
  • isoform 2 (used by Dean Lee and others) expresses in K562 cells, but its expression in 721.221 feeder cell line was shown to be very poor.
  • the expression can vary based on the cell lines tested.
  • the IL-21 expresses in K562 cells ( Figure 7B), but its expression in 721.221 feeder cell line was shown to be very poor on 721.221 ( Figure 7A).
  • engineered K562 cells tested as disclosed herein alternative engineered cells are under investigation.
  • a 721.221 cell, an AML3 cell, a cell lack of expression of an MHC class I molecule are engineered to comprise one or more of: the polypeptide, the polynucleotide, or the vector as disclosed herein.
  • the engineered cells express a membrane-bound IL-21 isoform 1 and a 4-1BBL on the cell surface. The expression of IL-21 and 4-1BBL is evaluated, optionally compared to a control and a K562 F3 cell.
  • the engineered cells are further assessed in supporting primary human peripheral blood derived NK cells proliferation and sustaining the viability as well as in the expansion fold and transduction efficiency of the NK cells, optionally compared to a control and a K562 F3 cell.
  • the experimental setting used in assessing the K562 F3 cells are utilized.
  • JAWSII cells the immature murine dendritic cell line (ATCC, CRL-11904), are grown in alpha minimum essential medium containing ribonucleosides, deoxyribonucleosides, 4 x 10“ 3 M L-glutamine, 1 x 10“ 3 M sodium pyruvate, 5 ng mL -1 murine granulocyte-macrophage colony-stimulating factor (GM- CSF), and 20% fetal bovine serum (FBS).
  • JAWSII cells are seeded onto a 100 mm Petri dish with 10 mL of media at a density of 1 x 10 s per dish.
  • the cells are treated with 1 pg mL -1 of lipopolysaccharide (LPS) for 16 h at 37 °C and then pulsed with 10 pg mL -1 a peptide comprising, or consisting essentially of, or yet further consisting of an antigen, for 4 h in complete media.
  • LPS lipopolysaccharide
  • Human primary DCs are prepared from human PBMCs. Using Ficoll-Paque Plusbased centrifugal separation, PBMCs are isolated from healthy donor blood samples. The PBMCs are further purified by plastic adherence to obtain enriched monocytes.
  • the monocytes are cultured in a Dendritic Cell Medium (CellGenix GmbH, Freiburg, Germany) containing 2% human AB serum, 2 x 10“ 3 M L- glutamine, 50 pg mL -1 gentamicin, 500 IU mL -1 recombinant human GM-CSF (rh GM-CSF, CellGenix GmbH, Freiburg, Germany), and 250 IU mL -1 IL-4 (CellGenix GmbH, Freiburg, Germany).
  • the monocytes are cultured at 37 °C in a CO 2 -humidified incubator for 4 days.
  • the prepared iDCs are further matured with 0.1 KE mL -1 OK432 (Picibanil) and 1000 IU mL -1 interferon gamma (IFN-y) for 8 h to produce mature DCs.
  • DMEM modified Eagle medium
  • the cell debris is spun down via centrifugation at 30 000 x g for 5 min, and the supernatants are collected and mixed with BaCb and iodine solutions in an 8:2: 1 ratio for 15 min of color development.
  • PEG-DA concentrations in the samples are determined by measuring the light absorbance at 535 nm.
  • the standard curve is prepared by serial dilution of PEG-DA.
  • PEG content for theoretical PEG-DA saturation is calculated based on an estimated cell volume of -500 pm 3 per cell.
  • 2 mL of 1 : 1 1-octanol/water mixture is applied to dissolve 10 mg of PEG-DAs.
  • the solutions are settled for phase separation for 2 h.
  • PEG-DA in the two phases is quantified via BaCh and iodine solutions. Partition coefficient is calculated with the formula: log ([PEG-DA in octanol]/[PEG-DA in water]).
  • gelation buffer containing 10 wt% PEG-DA is prepared by mixing 20 pL of 1.5 g mL-1 of 2-hydroxy- 4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure D-2959, Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO) with 180 pL of 700 Da PEG-DA (liquid; Sigma-Aldrich).
  • DMSO dimethyl sulfoxide
  • 18 mg mL -1 of fluorescein O,O'-diacrylate is introduced into the gelation buffer.
  • the solution is then mixed with 1 mL of water to attain a 15 wt% PEG-DA solution with -333 mosmol in osmolarity.
  • 5 x 10 s DCs are collected and suspended in 500 pL of phenol- red-free DMEM (CA21063-029; Thermo Fisher Scientific) containing lx protease inhibitor.
  • 1 mL of the gelation buffer is added to reach a 10 wt% PEG-DA concentration.
  • the cells are pelleted and resuspended in 500 pL phenol-red-free DMEM without gelation buffer and subjected to 365 nm bombardment for 5 min in a UV oven (UVP Crosslinker, Analytik Jena, USA).
  • UV oven UV oven
  • the resulting GCs are washed with PBS prior to stability characterization, cryogenic TEM, and flow cytometric analysis of membrane proteins.
  • GC storage by freezing is achieved by suspending GCs in 500 pL of 10% sucrose (w/v). The samples are placed in a controlled rate freezing box, which enabled gradual temperature decrease by -1 °C min -1 upon placement in a -80 °C freezer.
  • GC lyophilization GC samples frozen in 10% sucrose at -80 °C for a minimum of 24 h are transferred to a lyophilizer. Frozen GCs are thawed in a 37 °C water bath, and lyophilized GCs are reconstituted with water prior to subsequent experiments.
  • Flow Cytometric Analysis of Membrane Proteins A total number of 1 x 10 s live or gelated DCs of mice or of human origin are stained with indicated antibodies as following. JAWSII cells are stained with antibodies anti-H2Kb/SIINFEKL (Thermo Fisher Scientific, #17-5743-82) and human-derived dendritic cells are stained with antibodies antihuman MHC class I (Biolegend, #311409), antihuman MHC class II (Biolegend, #307609), antihuman CD80 (Biolegend, #305219), antihuman CD86 (Biolegend, #305411) for 30 min, respectively. Cells are then washed with PBS twice and subjected to flow cytometric analysis by FACSCanto (BD Biosciences) and analyzed by FlowJo software (Tree Star) to investigate the levels of membrane protein expression.
  • FACSCanto BD Biosciences
  • antigen specific cells CD8+ T cells specific for the antigen in the H2Kb context or NK cells
  • spleens from mice are removed and placed into RPMI1640 complete medium with 10% FBS.
  • the spleens are ground and strained with 5 mL syringe against a sterile 40 pm nylon cell strainer (BD Biosciences Falcon, #352340).
  • Splenocytes are incubated with BD Pharm Lyse lysing buffer (BD Biosciences, #555899) for 3 min for erythrocyte depletion.
  • Antigen specific cells are subsequently isolated from the splenocytes by Mouse CD8a+ T Cell Isolation Kit (BD Biosciences, #19853 A).
  • Antigen specific T/NK cells are stained with CFSE by incubating the cells with PBS containing 5 x 10 -6 M of CFSE (Sigma-Aldrich, #21888) at 37 °C for 5 min. The cells are washed three times with complete medium. CFSE-labeled antigen specific cells are harvested for subsequent studies on T/NK-cell expansion.
  • CFSE-labeled antigen specific cells are co-cultured with stored G-DCs and G-DCs at a fixed number of 8 x 10 4 per well with a T/NK cell/G-DC ratio of 3: 1.
  • Co-cultured cells in 96-well v-bottomed plates re cultured at 37 °C for 3 days. After harvesting, co-cultured cells are stained with allophycocyanin-conjugated rat antimouse CD8a antibodies (eBioscience, #100712, Clone 53-6.7, 1 : 100) and analyzed by flow cytometry. Proliferation analysis performed in FlowJo is used to investigate T/NK-cell division after co-culturing with G-DC.
  • PLGAMPs are synthesized by a double-emulsion method followed by solvent evaporation. Carboxylated PLGA with two different molecular weights is used to modulate the release kinetics of the MPs.
  • a series of polymer blends are first prepared with PLGA(A):PLGA(B) ratios at 100:0, 65:35, 50:50, 35:65, and 20:80.
  • the polymer blends are dissolved in di chloromethane (DCM) at 300 mg mL" 1 , and for optimization of MP release kinetics, 1 mg mL -1 sulfo-cyanine5 fluorophore (sulfo-cyanine5 carboxylic acid; Lumiprobe) dissolved in 100 x 10 -3 M sodium bicarbonate buffer is prepared as the inner aqueous phase.
  • DCM di chloromethane
  • sulfo-cyanine5 fluorophore sulfo-cyanine5 carboxylic acid; Lumiprobe
  • IL-2-loaded microparticles 2 mg mL -1 of recombinant IL-2 protein (Research and Diagnostic Systems, #402-ML) or Alexa-647- conjugated IL-2 dissolved in 100 x 10 -3 M sodium phosphate buffer is prepared.
  • the first emulsion 50 pL of the inner aqueous phase is added to 500 pL of the polymer solution, which is then emulsified by probe sonicator under the pulse mode with 50% amplitude and on-off durations of 1 and 2 s for 20 s on ice.
  • the first emulsion is then added dropwise to 10 mL of ice-cold 10 x 10 -3 M sodium bicarbonate buffer (pH 8) being stirred with a magnetic bar at 1000 rpm to disperse the emulsion into microdroplets. Following 5 min of stirring at 1000 rpm, the stirring speed is reduced to 200 rpm, and DCM is evaporated by nitrogen bombardment for 20 min. Following solvent evaporation, the microparticles are collected by centrifugation at 100 x g followed by resuspension in 2 mL of PBS.
  • GC-MPs are then retrieved and suspended in 200 pL of PBS for subsequent characterizations.
  • the sizes of particles are measured by ImageJ analysis with bright-field microscopy images.
  • the size distributions are analyzed based on three independent figures of GCs, MPs, and GC-MPs.
  • the GC-MPs loaded with Alexa-647-conjugated IL-2 are stained with a Dil dye for visualization.
  • GC-MPs are loaded with antigen specific peptide in a solution containing 2 mg mL -1 of the peptide for 1 h followed by removal of unbound peptides.
  • Encapsulation efficiency of IL-2 is measured using Alexa-647-conjugated IL-2. Following MP preparation, the particles are broken down by 80% acetone. Upon particle dissolution, water is added to dilute the acetone to 20%, and the residual acetone is evaporated under vacuum. Precipitate PLGA is removed by centrifugation at 30,000 x g. The supernatant is collected and measured for fluorescence to assess encapsulation efficiency. The release of Alexa-647 dye and Alexa-647- conjugated IL-2 is studied at 37 and 4 °C.
  • MPs About 3 mg of MPs is suspended in 10 mL of pH7.4 PBS at either 37 °C in a heated water bath or 4 °C in an ice bath at a stirring rate of 150 rpm. To determine the amount of released dye, the MPs are settled for 10 min, and the supernatant is collected at predetermined time points for fluorescence quantification.
  • Tumor Model and Adoptive T/NK-Cell Transfer Therapy C57BL/6 mice are obtained and bred. Mice with 8 weeks of age are used in all experiments.
  • antigen specific cells are co-cultured with MP-IL-2, GC-MP, or GC-MP-IL-2 at a fixed number of MPs at 1 x 10 4 per well with a T/NK cell/MP ratio of 20: 1.
  • Co-cultured cells in 96-well v-bottomed plates are cultured at 37 °C for 3 days.
  • Retrieval of expanded antigen specific T/NK cells is achieved by passing the co-culture medium through a 40 pm cell strainer (Falcon), which effectively trapped the larger MP-IL-2, GC-MP, or GC- MP-IL-2.
  • Mice are subcutaneously inoculated with 100 pL of antigen expressing tumor cell suspension (1 x 10 5 ) subcutaneously in the dorsal flank regions, and then expanded antigenspecific T/NK cells are administered intravenously through the tail vein. Tumor sizes are monitored and calculated as (W 2 x L)/2, where W is the width and L is the length of the tumor.
  • mice are subcutaneously injected with 5 x 10 4 antigen expressing tumor cells for 17 days and then GC-MP expanded, CFSE-labeled wild-type (WT) or antigen specific CD8+ T cells or antigen specific NK cells are administrated intravenously. After 3 days, the tumors are excised and subjected to 1 mL RPMI (Roswell Park Memorial Institute) medium that contained 100 pL of 10 mg mL -1 DNase (D5025, Merck) and 10 mg mL -1 collagenase (C0130, Merck) for 30 min.
  • RPMI Roswell Park Memorial Institute
  • Cells are then centrifuged at 500 x g, and pellets are collected for flow cytometry analysis to investigate the presence of CD8+ T cells or NK cells and CD8+ CFSE + T cells or NK cells in the tumor.
  • the supernatant is subjected to ELISA analysis to investigate the cytokine levels in the tumor microenvironment.
  • a non-virally produced, clonal feeder cell line (K562 CYZ1) expressing 4-1BBL and a high level of tethered human Interleukin-21 and optionally having an azuroci din signal peptide that results in superior ex-vivo expansion of primary NK, CAR-NK, primary T cells and CAR-T cells was produced.
  • Figure 8 shows an exemplary workflow for generation of a single clonal aAPC (K562 CYZ1 feeder) with surface expression of 4-1BBL and IL-2L
  • Figures 9A-9B show a schematic representation of the Nonviral PiggyBac Transposon and Transposase vectors used to generate the aAPCs as disclosed herein ( Figure 9A) and the recombinant polypeptide expressed by the vector on cell surface of the aAPCs ( Figure 9B).
  • Figure 9A For tethering human canonical IL-21 (UniProtKB - Q9HBE4-
  • IgG4 Fc modified human IgG4 Fc and the transmembrane domain from platelet-derived growth factor receptor beta (PDGFRP).
  • PDGFRP platelet-derived growth factor receptor beta
  • a three-site mutated human IgG4 Fc was used to support the flexibility of the membrane bound IL-21.
  • IgG4-Fc was mutated at three sites: F234A, L235A within hinge region, and N297Q within CH2 region. Substitutions in its Fc region were aimed to avoid the Fc receptors dimerization.
  • the human 4-1BBL UniProtKB - P41273 (TNFL9 HUMAN) was inserted following the P2A self-cleaving peptides. Both inserts above were cloned into Cytolmmune developed PiggyBac transposon vector between 5’ and 3’ ITR.
  • FIG. 10 provides a schematic representation of the PiggyBac transposon vector used in the instant disclosure.
  • the synthesized DNA fragments 5’ Invert Terminal Repeats (ITRs), CAG Promoter, human IL-21, and 4-1BBL and 3’ Invert Terminal Repeats (ITRs) (Genscript, Order U791EGK100, U1982HA140, and U8489GL280) were ligated.
  • ITRs Invert Terminal Repeats
  • the amino acid and nucleotide sequences for each component are provided in Table 2.
  • FIG 11 provides a schematic representation of the PiggyBac transpososase vector used in the instant disclosure.
  • the Super PiggyBac Transposase Gene (GenBank: EF587698.1) was synthesized in pcDNA3.1(-) vector (Genscript, Order #U7136GL020 ), then further subcloned into the pCI Mammalian Expression Vector ( Promega,E1731).
  • the amino acid and nucleotide sequences for each component are provided in Table 3.
  • Figures 12A-12B show the mean fluorescence intensity (MFI) of 41BBL ( Figure 12A) and IL-21 ( Figure 12B) expression in candidate single clones of K562 cells transfected with the PiggyBac Transposon Non- Viral system of the instant disclosure.
  • the non-viral PiggyBac transposon (plasmid record # P142) and Transposase (# Pl 13) vectors are delivered into K562 cells (Roswell Park Cancer Institute or RPCI) via Transient transfection reagent (GibcoTM CTSTM LV-MAXTM Transfection Kit, A4132601).
  • the IL-21 and 4-1BBL doublepositive population was sorted via S3e Cell Sorter (Bio-Rad, 12007059). Further, the sorted P142 Bulk was performed with limited dilution with seeding 30 cells per 96 well plate and cultured for about two weeks. Screen the IL-21 and 4-1BBL expressions on these single clonal cells, and some candidate clones with similar or higher Mean Fluorescence Index of IL-21 and 4-1BBL compared to control feeders were further expanded in serum-free media X- VIVO 10 for additional two weeks.
  • FIGS. 13A-13B show the mean fluorescence intensity (MFI) of IL-21 ( Figure 13A) and 41BBL ( Figure 13B) expression in different feeder ells pre and post irradiation.
  • K562 wild type (WT), Control K562 Feeder cells, and novel K562 Feeder CYZ1 were expanded at serum-free conditions with X-VIVO10 for 11 days (Lonza, Catalog #: BEBP02-055Q ) and irradiated using an X-ray irradiator (XRad-320) at lOOGy.
  • Pre-irradiation (Pre IRR) and Postirradiation (Post IRR) were assessed for the expression of 4-1BBL and tethered of IL- 21 using the flow antibodies PE- 4-1BBL and AF-647-IL-21 with their respective isotype controls.
  • FIGs 14A-14B shows that the instant K562 CYZ1 feeder supports human Cord blood-derived NK proliferation and sustains NK cell viability and purity.
  • Cord blood NK cells were isolated using CD56 MicroBeads (Miltenyi Biotec, 130-050-401). NK cells from 2 donors were co-cultured with the IRR K562 wild type (WT), control Feeder cells, and IRR K562 CYZ1 Feeder cells at a ratio of 1 :2 using SCGM media with 200 lU/ml of IL-2. Every week, cell counts, and viability was monitored via the NovoCyte Penteon U7V7B6Y6R4 Flow Cytometer (Agilent).
  • NK cells a small portion of NK cells was used for subsequent expansion and restimulated with the same batch of X-Ray irradiated feeder cells. Data presented as an average from 2 individual counts ⁇ STDEV.
  • Figure 14A The cumulative fold expansion of NK cells at day 12.
  • Figure 14B NK cell viability at day 12.
  • Figure 14C NK cell Purity was measured at day 12.
  • the NK cells phenotype is defined by CD56+CD3- population.
  • Table 2 provides amino acid and nucleotide sequences of components of the PB-IL- 21-4-1 BBL transposon construct (aka. Pl 42).
  • Table 3 provides amino acid and nucleotide sequences of components of the pCI- CMV PiggyBac transposase construct (aka. Pl 13)
  • Embodiment 1 A polypeptide comprising an azurocidin signal peptide, an interleukin 21 (IL-21) polypeptide and a transmembrane domain, or an equivalent of each thereof.
  • IL-21 interleukin 21
  • Embodiment 2 A polypeptide comprising an IL-21 polypeptide and a platelet- derived growth factor receptor beta (PDGFRP) transmembrane domain or an equivalent of each thereof.
  • PDGFRP platelet- derived growth factor receptor beta
  • Embodiment s The polypeptide of embodiment 2, further comprising a signal peptide.
  • Embodiment 4. The polypeptide of any one of embodiments 1 to 3, wherein the IL-21 polypeptide comprises IL-21 isoform 1 or an equivalent thereof.
  • Embodiment 5 The polypeptide of embodiment 4, wherein the IL-21 isoform 1 comprises
  • Embodiment 6 The polypeptide of any one of embodiments 1 to 5, wherein the transmembrane domain comprises
  • AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 8) or an equivalent thereof.
  • Embodiment 7 The polypeptide of any one of embodiments 1 and 3 to 6, wherein the signal peptide comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 10) or an equivalent thereof.
  • Embodiment 8 The polypeptide of any one of embodiments 1 to 7, further comprising an Fc fragment of an antibody or an equivalent thereof located between the IL-21 polypeptide and the transmembrane domain or the equivalent of each thereof.
  • Embodiment 9 The polypeptide of embodiment 8, wherein the Fc fragment comprises a hinge domain, a CH2 domain and a CH3 domain of IgG4 modified at F234A, L235A and N297Q or an equivalent thereof.
  • Embodiment 10 The polypeptide of embodiment 8 or 9, wherein the Fc fragment comprises
  • Embodiment 11 The polypeptide of any one of embodiments 1 to 10, further comprising a Tumor Necrosis Factor Superfamily Member 9 (4-1BBL) polypeptide and a self-cleaving peptide located between the IL-21 polypeptide and the 4-1BBL polypeptide or the equivalent of each thereof.
  • Embodiment 12 The polypeptide of embodiment 11, wherein the 4-1BBL polypeptide comprises MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAV
  • Embodiment 13 The polypeptide of embodiment 11 or 12, wherein the selfcleaving peptide comprises a 2A self-cleaving peptide selected from the group consisting of P2A, E2A, F2A or T2A.
  • Embodiment 14 The polypeptide of any one of embodiments 11 to 13, wherein the self-cleaving peptide comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 16) or an equivalent thereof.
  • Embodiment 15 A polynucleotide encoding the polypeptide of any one of embodiments 1 to 14 or a polynucleotide complementary thereto.
  • Embodiment 16 The polynucleotide of embodiment 15, comprising
  • ATGACTAGGTTGACAGTCCTCGCCTTGCTTGCTGGATTGCTTGCCAGTTCTCGAG CC (SEQ ID NO: 11) or an equivalent thereof that encodes the azuroci din signal peptide.
  • Embodiment 17 The polynucleotide of embodiment 15 or 16, comprising
  • TGTCCAGTCGGACACACGGTTCCGAGGACTCC (SEQ ID NO: 3) or an equivalent thereof that encodes the IL-21 polypeptide.
  • Embodiment 18 The polynucleotide of any one of embodiments 15 to 17, comprising
  • Embodiment 19 The polynucleotide of any one of embodiments 15 to 18, comprising
  • Embodiment 20 The polynucleotide of any one of embodiments 15 to 19, comprising
  • GGTAGTGGGGCTACGAACTTTTCCCTTCTCAAACAGGCCGGAGACGTCGAGGAG AATCCTGGACCA (SEQ ID NO: 17) or an equivalent thereof that encodes the P2A selfcleaving peptide.
  • Embodiment 21 The polynucleotide of any one of embodiments 15 to 20, comprising ATGGAGTATGCTAGCGATGCAAGCTTGGATCCAGAGGCACCCTGGCCACCAGCA CCTAGAGCTAGGGCTTGTCGTGTGCTCCCATGGGCGCTCGTAGCAGGTCTCCTCT
  • Embodiment 22 A vector comprising the polynucleotide of any one of embodiment 15 to 21.
  • Embodiment 23 The vector of embodiment 22, wherein the vector is selected from a plasmid, a viral vector, or a retrovirus vector.
  • Embodiment 24 The vector of embodiment 22 or 23, further comprising a regulatory element directing the expression of the polynucleotide.
  • Embodiment 25 The vector of embodiment 24, wherein the regulatory element comprises a long terminal repeat (LTR).
  • LTR long terminal repeat
  • Embodiment 26 An artificial antigen-presenting cell (aAPC) comprising the polypeptide of any one of embodiments 1 to 14.
  • Embodiment 27 An artificial antigen-presenting cell (aAPC) comprising the polynucleotide of any one of embodiments 15 to 21.
  • aAPC artificial antigen-presenting cell
  • Embodiment 28 An artificial antigen-presenting cell (aAPC) comprising the vector of any one of embodiments 22 to 25.
  • aAPC artificial antigen-presenting cell
  • Embodiment 29 The aAPC of any one of embodiments 26 to 28, expressing the IL-21 polypeptide and 4-1BBL polypeptide on the cell surface.
  • Embodiment 30 The aAPC of any one of embodiments 26 to 29, wherein the aAPC is a K562 cell, a 721.221 cell, an AML3 cell, or a cell lack of expression an MHC class I molecule.
  • Embodiment 31 The aAPC of any one of embodiments 26 to 30, wherein the aAPC is irradiated.
  • Embodiment 32 The aAPC of embodiment 31, wherein the aAPC is irradiated by an ionizing radiation at a range from about 1 Gy to about 1000 Gy.
  • Embodiment 33 The aAPC of embodiment 31, wherein the aAPC is irradiated by an ultraviolet radiation at a range from about 5 J/m 2 to about 50 J/m 2 .
  • Embodiment 34 A method of expanding an immune cell, comprising contacting the immune cell or a precursor cell thereof or a cell population comprising the immune cell or the precursor cell or both with the aAPC of any one of embodiments 26 to 33.
  • Embodiment 35 The method of embodiment 34, wherein the immune cell is selected from a T cell, an NK cell, an NKT cell, a gamma-delta T cell, or a macrophage.
  • Embodiment 36 The method of embodiment 34 or 35, wherein the cell population is isolated from primary human peripheral blood or human umbilical cord blood.
  • Embodiment 37 The method of any one of embodiments 34 to 36, wherein the immune cell is an engineered immune cell, optionally expressing a chimeric antigen receptor.
  • Embodiment 38 The method of any one of embodiments 34 to 37, wherein the precursor cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • Embodiment 39 The method of any one of embodiments 34 to 38, wherein the immune cell or the precursor cell or both have been cryopreserved and recovered for at least once.
  • Embodiment 40 The method of any one of embodiments 34 to 39, wherein the ratio between the cell number of the aAPC and cell number of the precursor cell or the cell population is about 5: 1 to about 1 :5 in the aAPC contacting step.
  • Embodiment 41 The method of any one of embodiments 34 to 40, wherein the ratio between the cell number of the aAPC and cell number of the precursor cell or the cell population is about 1 : 1 in the aAPC contacting step.
  • Embodiment 42 The method of any one of embodiments 34 to 40, wherein the ratio between the cell number of the aAPC and cell number of the precursor cell or the cell population is about 2: 1 in the aAPC contacting step.
  • Embodiment 43 A method of sustaining an immune cell’s viability, comprising contacting the immune cell with the aAPC of any one of embodiments 26 to 33.
  • Embodiment 44 The method of any one of embodiments 34 to 43, wherein the immune cell is isolated from primary mammalian peripheral blood or mammalian umbilical cord blood.
  • Embodiment 45 The method of any one of embodiments 34 to 44, wherein the immune cell comprises a T cell, an NK cell, an NKT cell, a gamma-delta T cell, or a macrophage.
  • Embodiment 46 The method of any one of embodiments 34 to 43, wherein the immune cell is derived from the precursor cell.
  • Embodiment 47 The method of embodiment 46, wherein the precursor cell is isolated from primary mammalian peripheral blood or mammalian umbilical cord blood.
  • Embodiment 48 The method of embodiment 46, wherein the immune cell is an engineered immune cell, optionally expressing a chimeric antigen receptor.
  • Embodiment 49 The method of any one of embodiments 46 to 48, wherein the precursor cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • Embodiment 50 The method of any one of embodiments 34 to 49, wherein the immune cell is an immature immune cell.
  • Embodiment 51 The method of any one of embodiments 34 to 49, wherein the immune cell is a mature immune cell.
  • Embodiment 52 The method of any one of embodiments 34 to 51, wherein the method is ex vivo or in vitro.
  • Embodiment 53 The method of any one of embodiments 34 to 52, further comprising contacting with an IL-2 polypeptide or an equivalent thereof while contacting with the aAPC.
  • Embodiment 54 The method of embodiment 53, wherein the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 10 lU/ml to about 100 lU/ml.
  • Embodiment 55 The method of embodiment 53 or 54, wherein the IL-2 polypeptide or the equivalent thereof is present in the contacting step at a concentration of about 50 lU/ml.
  • Embodiment 56 The method of any one of embodiments 34 to 55, further comprising contacting with an IL-15 polypeptide or an equivalent thereof while contacting with the aAPC.
  • Embodiment 57 The method of embodiment 56, wherein the IL-15 polypeptide or the equivalent thereof is expressed by the immune cell.
  • Embodiment 58 The method of any one of embodiments 34 to 57, wherein the contacting step is repeated for at least once, or at least twice, or at least three times, or more.
  • Embodiment 59 The method of any one of embodiments 34 to 58, wherein the immune cell has been cryopreserved and recovered for at least once.
  • Embodiment 60 The method of any one of embodiments 34 to 59, wherein the ratio between the cell number of the aAPC and cell number of the immune cell is about 5: 1 to about 1 :5 in the aAPC contacting step.
  • Embodiment 61 The method of any one of embodiments 34 to 60, wherein the ratio between the cell number of the aAPC and cell number of the immune cell is about 1 : 1 in the aAPC contacting step.
  • Embodiment 62 The method of any one of embodiments 34 to 62, wherein the ratio between the cell number of the aAPC and cell number of the immune cell is about 2: 1 in the aAPC contacting step.
  • Embodiment 63 A composition comprising (i) a natural killer (NK) cell or a T cell, and (ii) an artificial antigen-presenting cell (aAPC) comprising an intracellular hydrogel.
  • NK natural killer
  • aAPC artificial antigen-presenting cell
  • Embodiment 64 The composition of embodiment 63, wherein the NK cell or the T cell is derived from a precursor cell.
  • Embodiment 65 The composition of embodiment 64, wherein the precursor cell has been isolated from primary mammalian peripheral blood or mammalian umbilical cord blood.
  • Embodiment 66 The composition of embodiment 64 or 65, wherein the precursor cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • Embodiment 67 The composition of any one of embodiments 63 to 66, wherein the NK cell is selected from an immature NK cell, a mature NK cell or an engineered NK cell expressing a chimeric antigen receptor (CAR), or wherein the T cell is selected from an immature T cell, a mature T cell or an engineered T cell expressing a chimeric antigen receptor (CAR).
  • the NK cell is selected from an immature NK cell, a mature NK cell or an engineered NK cell expressing a chimeric antigen receptor (CAR)
  • CAR chimeric antigen receptor
  • Embodiment 68 The composition of any one of embodiments 63 to 67, wherein the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • the hydrogel comprises polyethylene glycol or a derivative thereof, optionally wherein the derivative is poly(ethylene glycol) diacrylate (PEG-DA).
  • Embodiment 69 The composition of any one of embodiments 63 to 68, wherein the hydrogel was introduced into the aAPC by incubating the aAPC in an isotonic buffer comprising a cell-membrane-penetrating hydrogel monomer and crosslinking the monomer.
  • Embodiment 70 The composition of embodiment 69, wherein the monomer has a molecular weight of from about 200 g/mol to about 2000 g/mol, optionally about 700 g/mol.
  • Embodiment 71 The composition of embodiment 69 or 70, wherein the isotonic buffer comprises from about 5% to about 20% optionally about 10% monomer.
  • Embodiment 72 The composition of any one of embodiments 69 to 71, wherein the incubation is from about 1 minute to about 30 minutes, optionally about 5 minutes.
  • Embodiment 73 The composition of any one of embodiments 69 to 72, wherein the hydrogel is cross-linked, optionally by a method comprising exposing the hydrogel to a photoinitiator and UV light, and further optionally wherein the photoinitiator is 12959.
  • Embodiment 74 The composition of embodiment 73, wherein the UV light has a wavelength of from about 300 nm to about 400 nm, optionally about 365 nm.
  • Embodiment 75 The composition of any one of embodiments 63 to 74, wherein the aAPC was previously frozen, optionally lyophilized, and thawed.
  • Embodiment 76 The composition of any one of embodiments 63 to 75, wherein the aAPC comprises a dendritic cell (DC).
  • DC dendritic cell
  • Embodiment 77 The composition of embodiment 76, wherein the DC has been isolated from a biological sample of a subject, optionally a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • Embodiment 78 The composition of embodiment 77, wherein the aAPC or the NK cell or the T cell is a human cell.
  • Embodiment 79 The composition of any one of embodiments 63 to 78, wherein the aAPC comprises a K562 cell, a 721.221 cell, an AML3 cell, a JAWSII cell, or a cell lack of expression an MHC class I molecule.
  • Embodiment 80 The composition of any one of embodiments 63 to 79, wherein the aAPC further comprises an antigen on the cell surface, optionally in an MHC and antigen complex, wherein optionally, the antigen is a tumor associated antigen or a pathogen associated antigen.
  • Embodiment 81 The composition of any one of embodiments 63 to 80, wherein the aAPC has been irradiated.
  • Embodiment 82 The composition of embodiment 81, wherein the aAPC is irradiated by an ionizing radiation at a range of from about 1 Gy to about 1000 Gy.
  • Embodiment 83 The composition of embodiment 82, wherein the aAPC is irradiated by an ultraviolet radiation at a range of from about 5 J/m 2 to about 50 J/m 2 .

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Abstract

L'invention concerne des compositions et des procédés destinés à l'expansion de cellules immunitaires. Par exemple, un polypeptide comprenant un peptide signal d'azurocidine, un polypeptide d'interleukine 21 (IL-21) et un domaine transmembranaire, ou un équivalent de chacun de ceux-ci, est divulgué. L'invention concerne en outre un polypeptide comprenant un polypeptide IL-21 et un domaine transmembranaire du récepteur bêta du facteur de croissance dérivé des plaquettes (PDGFRP) ou un équivalent de ceux-ci. L'invention concerne en outre des polynucléotides codant pour le polypeptide, des vecteurs comprenant les polynucléotides, des cellules comprenant le polypeptide, ainsi que des procédés les utilisant. L'invention concerne en outre des cellules nourricières pouvant supporter la culture, l'activation et l'expansion de cellules immunitaires ex vivo. L'invention concerne également des compositions comprenant des cellules nourricières et des cellules immunitaires et des procédés d'expansion de cellules immunitaires.
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