WO2023019213A1 - Three-dimensional culturing of pluripotent stem cells to produce hematopoietic stem cells - Google Patents

Abstract

Cell culture methods and media for expansion of pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), to a three- dimensional culture and differentiation to hematopoietic stem cells are described. The hematopoietic stem cells can be further differentiated to natural killer cells or macrophages. The natural killer cells can be further transformed to express a chimeric antigen receptor (CAR).

Description

Three-Dimensional Culturing of Pluripotent Stem Cells to Produce Hematopoietic Stem Cells

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/232,911, filed on August 13, 2021. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND

[0002] A major roadblock to current cell immunotherapies in cancer treatment is the high concentration of effector cells required per dose. Various strategies in cultivating hiPSC- derived NK cells involve using an excessive number of plates or flasks for expansion of the final product, which can be a highly inefficient, time-consuming, and expensive process that also increases overall susceptibility to cell culture contamination. Other methods of expanding NK cells involve the use of cancer feeder-cells, which results in the hiPSC-NK product being declassified as GMP-grade and quality.

SUMMARY

[0003] The methods described herein demonstrate that the entire differentiation and expansion of hiPSCs to functionally mature NK cells can take place entirely within an enclosed bioreactor system. The methods described herein streamline the differentiation process and ensure consistent reproducibility and reliability of the resultant NK cells.

[0004] In comparison to other known methods, the methods described herein are performed under xeno-free and feeder-free conditions. Consequently, the final derived cells are GMP-compliant and can be used in, e.g., clinical trials. One small scale-bioreactor can potentially yield >10-50 x 10⁷ total NK cells. This refinement of this hiPSC to NK cell differentiation process allows the opportunity to scale-up enough high-grade cells to meet the stringent requirements needed to proceed with human clinical trials. Additionally, the methods described herein are efficient and cost-effective for generating phenotypically mature, functional, clinical-grade NK cells for use in developing cancer immunotherapies.

[0005] NK cells were observed and specified in medium suspension almost 10 days faster when compared to 2D-controls (FIGs. 3 A-H). In addition, 3D-bioreactor-derived NK cells demonstrate advanced maturation and development. Flow cytometry analysis to quantitate expression levels of CD56, a marker for mature NK cells, demonstrated that the 3D bioreactor-derived cells were over 93% positive at day 27 of differentiation compared to 58% at day 27 of differentiation for 2D-generated cultures (FIGs. 4A-F and 5). To further assess the robustness and timing of cellular differentiation between the 3D-bioreactor and 2D- monolayer platforms, expression levels were measured for NK lineage-specific markers CD94, NKp44, NKG2D, Lampl, and CD 16. Expression levels of these NK lineage markers for differentiation day 27 bioreactor-generated cells were comparable or significantly exceed expression levels from two independent 2D-differentiation rounds that were cultured almost twice as long (53 days and 55 days, respectively) (FIGs. 6A-O, and Table 1). Culturing hiPSCs in a 3D bioreactor using the methods and culture media described herein can generate highly pure and phenotypically mature cells that are similar to primary NK cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0007] FIG. 1 is a summary of a 3D-directed in vitro differentiation protocol for NK cells derived from human induced pluripotent stem cells (hiPSC). Briefly, hiPSCs are scaled-up from a 2D-monolayer to a 3D-small scale bioreactor cell culture system with Y27632 supplementation to enhance iPSC viability and improve sphere formation. Hematopoietic induction of iPSCs to progenitor cells of an NK lineage restricted fate takes 12 days. Then, the 3D-cultures are transferred to NK cell differentiation conditions. Two weeks thereafter, mature and functional NK cells can be continuously collected from the medium for a period of about 35 days.

[0008] FIGs. 2A-C show 3D-adaptation of hiPSCs from 2D-monolayer culture utilizing a small-scale bioreactor. FIG. 2A is a low magnification (lOx) photomicrograph of hiPSCs in 2D-monolayer culture. FIG. 2B is a photograph of a small-scale 30ml bioreactor flask. FIG. 2C is a photomicrograph of hiPSC-spheres 20-hours after 2D-to-3D adaptation (lOx magnification).

[0009] FIGs. 3 A-H is a comparison of the differentiation potential between 2D- monolayer versus 3D-bioreactor generated hiPSC-NK cells. FIGs. 3 A-D are images of adherent-2D derived cell-spheres and derivatives at indicated time points after the initiation of NK differentiation (lOx). Spheres slowly flatten over time with premature NK-like cells observed in cell medium at differentiation day 23. FIGs. 3A-B: 4x magnification; FIGs. 3C- D: lOx magnification. FIGs. 3E-H are images of 3D bioreactor generated cell-spheres and derivatives. Immature NK-like cells are observed two days after NK differentiation initiation on day 14. FIGs. 3E-F: 20x magnification; FIGs. 3G-H: lOx magnification.

[0010] FIGs. 4A-F show characterization of hiPSC-NK cells. Flow cytometry quantification indicate a correlated temporal pattern of expression for the hematopoietic stem cell progenitor marker CD34 (FIG. 4A) and mature NK cell markers CD45 and CD56 (FIGs. 4B-F).

[0011] FIG. 5 shows comparative CD56-positive marker expression between 2D- monolayer and 3D-bioreactor generated cells at specific time points during the differentiation timeline.

[0012] FIGs. 6A-0 show NK lineage flow cytometry panel comparison of 3D-bioreactor and 2D-monolayer derived NK cells. Mature NK cell expression markers assessed: CD56, CD94, NKp44, NKG2D, Lampl, and CD 16. Top row: Flow cytometry panel for bioreactor derived NK cells at differentiation day 27. Second and third row: Flow cytometry panel of two independently conducted 2D-monolayer trials in generating NK cells at differentiation day 53 and day 55, respectively.

[0013] FIGs. 7A-J shows phenotypic comparison of hiPSC-NK vs. CBNK.

[0014] FIGs. 8A-D show functional in vitro killing. In vitro killing using unmodified iPSC-NKs against Jurkat cells (FIG. 8A), HeLa cells (FIG. 8B), and K562 cells (FIG. 8C) as determined using Incucyte Base Analysis immune cell-killing software for live cell analysis. FIG. 8D shows in vitro killing comparing unmodified primary CBNK and iPSC-NKs against K562 cells (effectortarget ratio of 1 :2). Different concentration of target cancer cells was incubated with hiPSC-NK cells at effectortarget ratios of 1 : 1 and 4:1. Data represented as mean ± SEM.

[0015] FIGs. 9A-J show genetic modification of mature hiPSC-NK cells using lentivirus. hiPSC-NK cells can be transduced with lentiviral -based vectors to express a CD19-CAR construct with high affinity. Conversely, primary NK cells demonstrate a persistent resistance to genetic engineering after lentiviral transduction.

[0016] FIGs. 10 A- J show genetic modification of mature hiPSC-NK cells using retrovirus. Both mature hiPSC-NK and primary NK cells can be transduced with retroviralbased vectors to express a CD19-CAR construct with high affinity. [0017] FIGs. 11 A-C show comparative functional analysis between unmodified and CAR19 hiPSC-NK against tumor targets. In vitro killing against Raji cells (FIG. 11 A), NALM6 cells (FIG. 1 IB), and CCRF cells (FIG. 11C) as determined using Incucyte’s immune cell-killing software for live cell analysis. Different concentration of target cancer cells was incubated with hiPSC-NK cells or CAR19-hiPSC NK cells at effectortarget ratios of 2: 1. Data represented as mean ± SEM.

[0018] FIGs. 12A-C show genetic modification of iPSCs using lentivirus. FIG. 12A: The selection and colony-picking of a single iPSC-colony from the surface of the cell culture plate. FIG. 12B: After 2 days in differentiation medium, CD19-expression can be observed in the removed colonies under fluorescent imaging. A wide variation in CD19-expressing positive clones derived from selected colonies observed (top and bottom row, yellow arrows and arrowheads). FIG. 12C: After 5 days in differentiation medium, the picked colonies expressing CD 19 are capable of proliferating and maintaining expression in vitro (yellow arrowheads).

[0019] FIGs. 13A-B show NK-differentiation of lenti-CAR19 iPSCs using 3D bioreactor on day 12. FIG. 13A: Flow cytometry quantification for CD34 and CD45 marker expression. The untransduced hiPSC cell line exhibited 15% CD34-positive expression (middle panel) compared to 12% expression in the CAR19 S001 modified hiPSCs (far right panel). Percent positive for CD45 was <1% in both cell lines. FIG. 13B: CD19-CAR expression detected in differentiating spheres in 3D-bioreactor.

[0020] FIG. 14 shows schematics of viral CAR constructs.

[0021] FIGs. 15A-B show that cryopreserved unmodified hiPSC-derived NK cells demonstrate strong killing efficacy in a subcutaneous K562 myelogenous leukemia mouse model. FIG. 15 A: Frozen peripheral blood derived NK (PBNK) or frozen iPSC-derived NK cells were injected intravenously (4 x 10⁶/mouse) post-tumor engraftment. Optical bioluminescence imaging of tumor load between days 3 and 7 post-treatment. FIG. 15B: Quantification of tumor intensity examined across all control and experimental cohorts four days after NK-infusion.

[0022] FIGs. 16A-B show iPSC-derived NK cells can survive and persist in vivo for at least two weeks post-injection. FIG. 16A: Infused NK cells transduced with retroviral constructs expressing luciferase alone or luciferase + soluble IL 15 then were injected intravenously into the tail vein (2 x 10⁶/mouse). FIG. 16B: Persistence of genetically modified iPSC-NK cells were detectable two weeks in vivo. Transduction of IL15 enhanced persistence of injected iPSC-NK cells, and there were no discernible effects on mice from iPSC-NK infusion.

[0023] FIGs. 17A-D show that NK cells can be generated from a human embryonic stem cell (hESC) source using a 3D-bioreactor platform system. FIGs. 17A-C: High magnification (40x) photomicrographs of cell suspensions collected from the 3D-bioreactor demonstrate a phenotypic maturation of NK cells over developmental time. FIGs. 17D: A purified population of NK cells were collected and maintained over time as indicated by flow cytometry quantification of mature NK cell markers CD45 and CD56.

[0024] FIGs. 18A-F: NK cells generated from a human embryonic stem cell (hESC) source using a 3D-bioreactor platform system show potent endogenous killing activity and can be genetically modified to enhance killing activity using a CAR construct in hepatocellular cancer lines. FIG. 18 A: ESC-CAR NK in vitro killing displayed as percent growth against Huh7 cancer cell line over a 24-hour period. FIG. 18B: Average Huh7 percent cancer growth at 24 hours. FIG. 18C: ESC-CAR NK in vitro killing displayed as percent growth against HepG2 cancer cell line over a 24-hour period. FIG. 18D: Average HepG2 percent cancer growth at 24 hours. FIG.18E: ESC-CAR NK in vitro killing displayed as percent growth against Hep3B cancer cell line over a 24-hour period. FIG. 18F: Average Hep3B percent cancer growth at 24 hours.

DETAILED DESCRIPTION

[0025] A description of example embodiments follows.

Overview

[0026] According to recent statistics provided by the United States Center for Disease Control and Prevention (CDC), deaths from cancer reported within the United States reached approximately 600,000 per year despite advancements in oncological patient care. The development of novel immunotherapies utilizing the potent cytotoxic antitumor properties of Natural Killer (NK) cells are a promising approach in cell-based therapeutics. However, the source of NK cells often poses certain limitations. Since NK cells only compose 5-15% of all lymphocytes circulating in the blood, the isolation of NK cells from the peripheral blood (PBNK) is often inefficient, resulting in a heterogenous lymphocyte population yielding only 10-20% NK cells [1] Additionally, a significant decrease in cell cytotoxicity has been reported following cryopreservation ([2, 3]). Conversely, NK cells isolated from cord blood (CBNK) exhibit higher cell viability after cryopreservation, however, due to the lower number of NK cells detected per cord blood unit, numerous expansion phases are required to obtain multiple units per dose [4], In comparison to PBNKs, several reports suggest CBNKs display a more immature phenotype that is directly correlated to a reduction in cytotoxicity [4, 5], Although these issues of low NK cell yield and cytotoxicity loss can be partially resolved through cytokine support and feeder cell co-culture, these considerations demonstrate that PBNKs and CBNKs are not ideal for an “off-the-shelf’ allogeneic immunotherapy whereby the NK cells are harvested from unrelated donors, cryopreserved, and thawed.

[0027] Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the capacity for indefinite self-renewal and the ability to differentiate into all cell types of the body. An infinite starting cell source is a tremendous advantage in scaling-up the production of a homogenous and high-quality NK cell product. This allows for the development and expansion of GMP-grade iPSC-derived NK cells to be used in cancer immunotherapy clinical trials. Several research groups have published recent advances in methodological strategies to successfully differentiate pluripotent stem cells into the NK lineage that display comparable phenotype and function to primary NK cells [6-10], Some of those methods involve differentiation of NK cells by co-culturing with OP9 mouse stromal cells. As human pluripotent stem cells (PSCs) continue to be intensely studied for their potential clinical applications as a cancer treatment strategy, PSC-derived NK cells are a promising alternative approach to cancer immunotherapy.

[0028] As the differentiation of PSCs into the NK specific lineage continues to be extensively investigated, several cell culturing strategies have been utilized to produce high- quality unadulterated NK cells: 1) cytokine induction with feeder-cell co-culture; and 2) the generation of an embryoid body (EB) intermediary with cytokine induction. The application of these different strategies yields varying degrees of success.

[0029] The co-culture of hPSCs with stromal cells, primarily derived from the bone marrow, has been a commonly used method in the induction of the hematopoietic fate by first obtaining a CD34+ precursor population [8, 9, 11], However, the protocols often require a long co-culture period of approximately 21 days in addition to a cell-sorting step for CD34+ positive cell selection before downstream NK differentiation can proceed. The additional cell-sorting component not only results in a less efficient protocol, but it also results in a decreased NK cell yield by negatively selecting against hematopoietic progenitors necessary for robust NK differentiation.

[0030] The formation of EBs is a commonly used platform in differentiating PSCs to specific cell lineages. However, current methods in EB generation create spheres of unequal size that may lead to irreproducible differentiation results. Additionally, the disintegration of EB-spheres has been observed shortly after the initiation of differentiation. Therefore, the low efficacy and reproducibility in both hematopoietic induction and NK differentiation via the EB step would not be ideal for adaptation into a large scale-production system required in a clinical trial setting.

[0031] Therefore, modifications and optimizations in the biomanufacturing of NK cells for immunotherapies is currently needed[12]. The ability to generate large quantities of GMP-grade NK cells derived from a pluripotent source would prove most useful for cellbased immunotherapies.

[0032] As used herein, pluripotent stem cells (PSC) includes human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). The methods described here are suitable for use with pluripotent stem cells.

3D Bioreactors

[0033] A three-dimensional (3D) bioreactor promotes growth of cells in three dimensions. A 3D bioreactor is distinguished from two-dimensional (2D) growth of cells on a flat surface, such as a cell culture dish. By promoting cell growth in three dimensions, the cells can grow into spheroids, or 3D cell colonies.

[0034] Some 3D bioreactors have scaffolds to which adherent cells can attach. Other 3D bioreactors employ scaffold-free techniques. One example of a scaffold free 3D bioreactor is a 30 ml disposable magnetic stir bioreactor from ABLE Corporation (Japan) and Biott Corporation (Japan) (ABLE Cat No. BWV-S03 A), which uses an impeller with a magnet on each blade to provide low-shear agitation by laminar flow, which can encourage the formation and growth of spheroid cell clusters. An impeller or other mechanism for mixing the culture media provides multiple benefits, including enhanced mass transfer and increased cell surface area exposure to media, nutrients, and differentiation factors. Relative to a static culture, low-shear stress and fluid flow provide more physiologically relevant environments. Other suitable bioreactor magnetic stir systems are available from ABLE Biott and other manufacturers in a variety of sizes. [0035] Typically, the 3D bioreactor is an enclosed bioreactor.

Cell culture medium for pluripotent stem cell expansion

[0036] Pluripotent stem cells are cultured in a cell culture medium that maintains them in a pluripotent state as the cells grow from an initial aliquot to a 3D culture. Many commercially-available feeder-free, xenofree culture media are suitable for use as a basal medium for iPSC expansion. One example is Essential 8 medium (available from ThermoFisher Scientific). Others include mTeSR Plus™ (StemCell Technologies);

NutriStem® XF medium (Biological Industries); Cellartis DEF-CS 500 (Takara); and StemFlex™ Medium (Fisher Scientific).

[0037] Another suitable medium is described in Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424-429 (Methods, Human ES Cell Culture, E8 media composition). The media described therein includes DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/1), sodium selenium (14 pg/1), FGF2 (100 pg/1), insulin (19.4 mg/1), NaHCO₃ (543 mg/1) and transferrin (10.7 mg/1), TGFpi(2 pg/1) or NODAL (100 pg/1). Osmolarity of all media was adjusted to 340 mOsm at pH 7.4. One of skill in the art will appreciate that the concentrations need not be precisely as described in Chen, G. et al.

[0038] The pluripotent stem cell expansion medium can also include vitronectin, which is a glycoprotein that provides a surface coating to promote cell attachment for use in feeder- free culture of pluripotent stem cells.

[0039] The pluripotent stem cell expansion medium can also include a rho-kinase (ROCK) inhibitor. Typically, a ROCK inhibitor is only used when hiPSCs or hESCs need to be enzymatically passaged to help mitigate cell death. One example of a ROCK inhibitor is Y-27632, which is a compound having the following structure:

[0040] Y-27632 is sometimes available as a salt e.g., ■ 2 HC1).

[0041] After culturing in the pluripotent stem cell expansion medium, the cell density increases and the cells form 3D-PSC spheres after bioreactor adaptation. Expression of OCT- 4, a marker of pluripotency, of the 3D-PSCs can be quantified by flow cytometry. Typically, greater than 90% of the 3D-PSCs resulting from the first culture step express OCT-4.

[0042] In this PSC expansion step, the PSCs are typically cultured to a cell density of at least about 750,000 cells/mL. In some instances, the PSCs are cultured to a cell density from about 750,000 cells/mL to about 1,250,000 cells/mL. For the 30 mL bioreactor used in the Examples, the iPSCs were cultured to approximately 25 million cells to 35 million cells.

[0043] Optionally, the cells can be mechanically dissociated during cell culture (e.g., with a pipette) in order to separate cell clumps or aggregates into smaller clumps, smaller aggregates, or single suspended cells.

Cell culture medium for differentiation of pluripotent stem cells to hematopoietic stem cells (HSCs)

[0044] The cells are then cultured to differentiate the 3D PSCs to hematopoietic stem cells (HSCs).

[0045] Typically, the hematopoietic stem cell culture medium includes stem cell factor (SCF), bone morphogenetic protein 4 (BMP4) (or an agonist of BMP4), and vascular endothelial growth factor (VEGF). The hematopoietic stem cell culture medium typically also includes a basal culture medium that promotes hematopoietic differentiation. One suitable basal culture medium is albumin polyvinylalcohol essential lipids (APEL). Suitable APEL culture media are described in US Patent No. 10,894,944 B2 and in Table 1 of Ng et al., A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies, Nature Protocols 3, 768-776 (2008).

[0046] At the conclusion of the hematopoietic stem cell culturing step, about 10-50% of the cells express CD34+, a marker of hematopoietic stem cells (HSCs). Typically, the cells are cultured for about 6 days to about 12 days. After about day 12, the cells begin to transition to a more mature (differentiated) cell type (e.g., natural killer cells). Once the cells are differentiated to HSCs, the cells can be differentiated to natural killer cells.

[0047] One example of a BMP4 agonist is SB 4, which is a compound having the following structure:

Cell culture medium for differentiation of HSCs to natural killer cells

[0048] The cells are then cultured to differentiate them from hematopoietic stem cells (HSCs) to a differentiated cell type, such as natural killer cells.

[0049] One example of a cell culture medium includes: i) Dulbecco's Modified Eagle Medium with Glutamax; ii) Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 with Glutamax containing Human AB serum; iii) ethanolamine; iv) 2-mercaptoethanol (2-BME); v) sodium selenite; and vi) ascorbic acid with interleukin 3 (IL3), interleukin 7 (IL7), interleukin (IL 15), FMS-like tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF).

[0050] Human AB serum refers to Type AB serum typically derived from male donors. Type AB donors lack antibodies against A and B-blood type antigens, therefore more commonly used to mitigate immunoreactivity.

[0051] Without wishing to be bound by theory, FMS-like tyrosine kinase 3 ligand (Flt3L) can enhance IL15 signaling, which can be important for the NK lineage. IL15 is a cytokine involved in the specific maintenance and proliferation of NK cells, and thus, Flt3L may enhance proliferation and/or NK specification as a result.

[0052] As cells mature and adapt an NK-lineage identity, the NK cells are released into the cell medium, from which they can be collected for use in downstream applications. Cells can be cultured indefinitely in this cell culture medium as NK cells are produced. Lentiviral transduction

[0053] In some embodiments, the lentivirus is concentrated with Lenti-X Concentrator (Takara Bio, Cat. Nos. 631231 & 631232).

[0054] In some embodiments, lentiviral transduction can be enhanced by culturing with RetroNectin, which is a 63 kD fragment of recombinant human fibronectin fragment (also referred to as rFN-CH-296).

[0055] Lentiviral transduction provides advantages relative to retroviral transduction. Lentiviruses (LVs) can infect non-dividing as well as actively dividing cell types, whereas retroviruses can only infect actively dividing, mitotically active cells. Accordingly, transduction with lentivirus is, in theory, more efficient as it has the potential to incorporate into all NK cells as opposed to only proliferating ones.

[0056] NK cells are resistant to LV transduction, which hampers their development as an immunotherapy. Vesicular Stomatitis Virus type-G (VSV-G) LVs, which are one of the most commonly used viruses for generating chimeric antigen receptor (CAR)-T cells, do not efficiently transduce NK cells.

[0057] While the preferred method of incorporating genetic modifications into iPSC-NKs is via transduction of iPSCs followed by selection of single cell clones to create stable iPSC lines, this is not ideal for high throughput screening purposes as the timeline for stable iPSC generation and subsequent NK differentiation is relatively long.

[0058] Prior studies by others have involved modifying the structure and/or sequence of specific LVs to improve transduction efficiency, but these studies have yielded few promising results. The method described herein of using Lenti-X concentrator in combination with retronectin produces unexpected results in transducing induced natural killer cells (iNKs) with a lentivirus.

[0059] As used herein, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” shall be given their ordinary meanings and shall include deoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, and ribonucleic acids, and polymeric forms thereof, and includes either single- or double-stranded forms. Nucleic acids include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleic acid analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), locked-nucleic acids (LNAs), and the like. [0060] As used herein, the term “operably linked,” for example in the context of a regulatory nucleic acid sequence being “operably linked” to a heterologous nucleic acid sequence, shall be given its ordinary meaning and shall mean that the regulatory nucleic acid sequence is placed into a functional relationship with the heterologous nucleic acid sequence. In the context of an IRES, “operably linked to” refers to a functional linkage between a nucleic acid sequence containing an internal ribosome entry site and a heterologous coding sequence initiation in the middle of an mRNA sequence resulting in translation of the heterologous coding sequence.

[0061] As used herein, the term “vector” shall be given its ordinary meaning and shall refer to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a genetically engineered cell, so as to transform the genetically engineered cell and promote expression (e.g., transcription and/or translation) of the introduced sequence.

Chimeric Antigen Receptors

[0062] Embodiments described herein related to transduction of cells to express a chimeric antigen receptor (CAR). Cells can be transduced by a vector, such as a lentiviral vector, to express a chimeric antigen receptor. For example, a lentiviral vector can include a nucleic acid sequence encoding a chimeric antigen receptor. Chimeric antigen receptors include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular activation domain.

[0063] The extracellular antigen recognition domain can be, for example, a single-chain Fragment variant (scFv) derived from an antibody. In embodiments described herein, the extracellular antigen recognition domain binds CD 19.

[0064] The transmembrane domain can be, for example, a CD3 transmembrane domain, a CD8 transmembrane domain, or a CD28 transmembrane domain.

[0065] The intracellular activation domain can be, for example, a CD3^ domain (sometimes written as CD3-zeta, CD3Z, or CD3z).

[0066] In some embodiments, the chimeric antigen receptor can include a co-stimulatory domain, such as, for example, a 4- IBB domain. EXEMPLIFICATION

Materials

Cell lines

[0067] hiPSC cell lines were obtained from Al stem that were derived from umbilical cord blood. Human embryonic stem cells (hESCs) were acquired from ESI BIO (Alameda, California, USA). hiPSC culture

[0068] hiPSCs and hESCs were cultured and maintained in Essential 8 medium (ThermoFisher Scientific, Catalog# A2858501). The supplementation of the ROCK Inhibitor Y27632 (Sigma, Cat# SCM075) was added to hiPSC and hESC cultures at lOpM during cell passaging only and removed from the medium within 24 hours of plating.

Hematopoietic stem cell induction medium

[0069] Medium composed of STEMdiff APEL 2 (albumin, polyvinyl alcohol, essential lipids; Stemcell Technologies, Cat# 05275) basal cell culture medium supplemented with Stem Cell Factor (R&D Systems, SCF, 40ng/ml, Cat# 255-SC/CF-050), Bone Morphogenetic Protein 4 (R&D Systems, BMP4, 20ng/ml, Cat# 314-BP-050) and Vascular Endothelial Growth Factor (R&D Systems, VEGF, 20ng/ml, Cat# 293-VE-050).

NK cell differentiation medium

[0070] Basal medium is composed of a mixture of 56.6% DMEM + Glutamax (Life Technologies, Cat# 10566-016) and 28.3% F-12 + Glutamax (Life Technologies, Cat# 31765035) supplemented with 15% human heat-inactivated type-AB serum (Valley Biomedical, Cat# HP1022 HI), 50pM Ethanolamine (MP Biomedicals, Cat# 194658), 25pM 2-Mercaptoethanol (Sigma, Cat# M6250), 5ng/ml sodium selenite (Sigma, Cat# S5261), 20mg/ml ascorbic acid (Sigma, Cat# A-5960), and 1% penicillin-streptomycin (Gibco, Cat# 15140-148). Cytokine addition into the medium consisted of 5ng/ml IL-3 (Peprotech, Cat# 200-03), 20ng/ml IL-7 (Peprotech, Cat# 200-07), lOng/ml IL-15 (Peprotech, Cat# 200-15), lOng/ml Flt3 ligand (Peprotech, Flt3L, Cat# 300-19), and 20ng/ml SCF. Nalm-6, Raji, and Jurkat cancer cells

[0071] Maintained in Stem Cell Growth Medium (CellGenix, SGCM, Cat# 20802-0500) with 10% fetal bovine serum (Gibco, FBS, Cat# 10082-147), and 1% penicillin/ streptomycin.

Other materials

[0072] TrypLE Select (Life Technologies, Cat# 12563011), Accutase (Innovative Cell Technologies, Inc., Cat# AT105-500, Vitronectin (ThermoFisher Scientific, Cat# A14700), Retronectin (Takara, Cat# T100A), Dnase 1 (Worthington Biochemical, Cat# LK003172), 70pm (Fisherbrand, Cat#22363548), Incucyte Nuclight Red Lentivirus Reagent (EFN-alpha; Puro; Cat#4625), 96 well assay plate (Corning, Cat#3610), 30 ml disposable bioreactor (Able Biott, Cat# ABBWVS03A-6).

Methods

Feeder-free pluripotent stem cell culture and maintenance

[0073] Undifferentiated feeder-free hiPSCs and hESCs are cultured on vitronectin coated substrates in Essential 8 medium. When hiPSCs and hESCs cultures are >70% confluent, cells can be enzymatically passaged using either TrypLE or Accutase and re-seeded in a split ratio between 1 : 10 and 1 : 15 with Y27632 supplementation at a final concentration of lOpM. hiPSCs and hESCs can be continually maintained, expanded, or cryopreserved under these outlined conditions. Prior to initiating 2D-monolayer to 3D-bioreactor hiPSC adaptation, hiPSCs and hESCs need to be expanded until sufficient cells needed have been obtained.

3D-bioreactor adaptation

[0074] hiPSCs and hESCs are transferred as a starting 2D-monolayer into small-scale bioreactors to initiate 3D-suspension culture. When 2D-hiPSCs and hESCs expansion has yielded a cell concentration of more than about 30 x 10⁶ live cells, the undifferentiated hiPSCs and hESCs can be dissociated with TrypLE or Accutase. Cell contents should be filtered through a 70pM cell strainer to remove doublets.

[0075] Optimal seeding density into bioreactors should be approximately 1 x 10⁶ cells/ml in Essential 8 medium with Y27632 supplementation at a final concentration of 10 pM. The rotational speed of the bioreactor at all phases of the 3D-differentiation process ranges from 60-80 RPM. Eighteen to twenty-four hours after adaptation, spheres should be examined carefully under the microscope and assessed for viability and morphology. Hematopoietic lineage induction

[0076] As illustrated in FIG. 1, the induction of pluripotent stem cells (hiPSCs and hESCs) towards a hematopoietic fate can be initiated after successful 3D- adaptation performed from the previous day. The complete removal of Essential 8 medium from the suspension culture is critical and can be executed by transferring the entire volume from the bioreactor to a 50ml conical tube. Leave cells undisturbed for 10-12 minutes to let the spheres pellet to the bottom. Remove the cell supernatant leaving the pellet undisturbed and replace with Hematopoietic induction medium described previously. Prepare the induction medium by formulating Stemdiff APEL 2 medium supplemented with SCF at a concentration of 40ng/ml in conjunction with BMP4 and VEGF at a concentration of 20ng/ml each. Medium change and preparation can be conducted as described during hematopoietic induction stage between differentiation days 1 to 12.

[0077] The expression of the hematopoietic stem cell progenitor marker CD34 and mature NK marker CD45 can be quantified at Day 6 and Day 12 to analyze the progression of NK cell development over time.

NK cell differentiation

[0078] After twelve days in HSC induction medium, the 3D-sphere aggregates can be further differentiated into the NK cell lineage by replacing the cultures with the above- mentioned NK cell differentiation medium. The NK cell differentiation base medium is composed of 56.6% DMEM + Glutamax and 28.3% F-12 + Glutamax supplemented with 15% human heat-inactivated type- AB serum, 50pM Ethanolamine, 25 pM 2- Mercaptoethanol, 5ng/ml sodium selenite, 20mg/ml ascorbic acid, and 1% penicillinstreptomycin. Cytokine addition into the medium consisted of 5ng/ml IL-3 (Peprotech, Cat# 200-03), 20ng/ml IL-7, lOng/ml IL- 15, lOng/ml Flt3 ligand, and 20ng/ml SCF. From differentiation day 12 and onwards, only NK differentiation medium is required. Medium in the 3D-bioreactor cultures should be replaced daily or when needed.

[0079] Typically, pluripotent stem cell-derived NK cells delaminate from the differentiating 3D-sphere aggregates and are released into the cell culture medium suspension after detachment. Mature appearing NK cells can be observed floating in the cell medium after 1 week of NK cell differentiation. hiPSC derived NK cells can be collected directly from the medium and phenotyped for surface antigens that include CD45, CD56, CD 16, KIR, CD94, NKp44, NKG2D, and Lampl using flow cytometry. Functional analyses of hiPSC- derived NK cells can be assessed by in vitro killing assays against numerous cell cancer lines at various effector to target (E:T) ratios. NK cells developed using this 3D-bioreactor platform demonstrate a mature NK cell lineage profile and potent cell cytotoxicity.

[0080] The methods outlined indicate an efficient and streamlined approach in deriving mature and functional NK cells from a pluripotent stem cells source that can be used as an infinite cell source for potential cancer cell therapies.

In vitro killing assay

[0081] The cancer target cells are labeled with the Incucyte Nuclight Red lentivirus, which enables the counting of viable cancer cells over time. Puromycin is added to the cancer cell cultures on day 6 at a concentration of 0.5ug/ml to initiate the selection process. Monitor transduction efficiency daily and supplement with puromycin into the cell culture medium with every media change. By day 10-14, a high percentage of the cancer target cells should be Nuclight red.

[0082] Both effector and target cells are cultured in RPMI medium supplemented with 10% FBS for cytotoxicity analysis. Real-time NK cell cytotoxicity was monitored and quantified using Incucyte Base Analysis Software.

Generation and purification of retrovirus for transduction of iPSC-NK

[0083] Plate 3 x 10⁶ 293T cells in a 10 cm dish in 10 ml DMEM medium. Prepare two sterile Eppendorf tubes for each dish. In tube 1, add 420ul DMEM to a sterile Eppendorf tube add 30 pl of FugeneHD directly to the DMEM in the Eppendorf tube. Gently tap the tube to mix and incubate for 5 minutes at room temperature (RT). In tube 2, add plasmid(s) to a combined total lOug DNA. Dropwise add the FugeneHD/DMEM mixture to the tube containing the DNA. Gently tap the tube to mix the FugeneHD/DMEM/DNA mixture, and incubate for 15 minutes at RT. Dropwise, add the FugeneHD/DMEM/DNA solution to the cell with the gentle agitation. Incubate cells at 37oC for 48 hours. Harvest the supernatant and centrifuge at 400x g for 5 minutes and store in 4oC. Filter the supernatant with 0.45 um low binding filter. For long term storage, store as 3 or 6 ml aliquots and at -80°C Virus transduction of iPSC-NK expanded under feeder-free conditions

[0084] Harvest the virus-containing supernatants and filter through a 0.45 pm low binding filter. Transfer supernatant to a sterile container and combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant and mix by gentle inversion. Incubate mixture at 4°C overnight. Centrifuge sample at 1,500 x g for 45 minutes at 4°C. Carefully remove supernatant and gently resuspend the pellet in 1/10 to 1/100th of the original volume using complete media. Immediately titrate sample or store at -70°C in single-use aliquots. Coat 24 well non-tissue culture treated plate with Retronectin (7ug/mL in PBS). The following day, aspirate Retronectin and wash wells with complete RPMI media. Add 2ml of concentrated virus to Retronectin-coated wells. Spin plate at 2000 g for at least 1 hr at room temperature. Prepare NK MACs media with 500IU/ml IL-2 and 140IU/ml IL-15. Add 2.5E5 cells/ml (1ml per 24 well). Culture undisturbed for a minimum of 2 days before subsequent assays. Monitor CAR expression using flow cytometry and use transduced iNKs for subsequent killing assays.

In vivo killing assay

[0085] Subcutaneous injections of K562 cancer cells were prepared at 2 x 10⁵ cells/mouse. K562 cancer cells were allowed to engraft for 3 days before systematic infusion of hiPSC-NK cells. The cryopreserved hiPSC-NK cells were thawed and prepared for infusion on the same day. Sample preparation for both hiPSC-NK and PBNK were identical as 4 x 10⁶ NK cells/mouse were intravenously injected. Intraperitoneal (IP) injections of recombinant human IL-15 at 30 ng/mouse was administered daily for days 1-7 after NK cell infusion.

Results and Discussion

3D-bioreactor adaptation and iPSC-NK differentiation protocol

[0086] As illustrated in FIG. 1, which is a summary of a 3D-directed in vitro differentiation protocol for NK cells derived from human induced pluripotent stem cells (hiPSC), the induction of hiPSCs towards a hematopoietic fate can be initiated after successful 3D-hiPSC adaptation, which requires approximately 12 days, after which time 3D- cultures are transferred to NK cell differentiation conditions. Two weeks thereafter, mature and functional NK cells can be continuously collected from the medium for a period of about 35 days. Briefly, hiPSCs were scaled-up from a 2D-monolayer to a 3D-small scale bioreactor cell culture system with Y27632 supplementation to enhance iPSC viability and improve sphere formation.

[0087] hiPSCs were grown as 2D-monolayer cultures to a starting concentration of 30 x 10⁶ live cells. These undifferentiated hiPSCs were dissociated with TrypLE then filtered through a 70pM cell strainer to achieve single cell suspension. The 3D-adaptation of hiPSCs from 2D-monolayer culture was completed utilizing a small-scale bioreactor. FIG. 2A is a low magnification (lOx) photomicrograph of hiPSCs in 2D-monolayer culture. FIG. 2B is a photograph of a small-scale 30 ml bioreactor flask. FIG. 2C is a photomicrograph of hiPSC- spheres 20-hours after 2D-to-3D adaptation.

Phenotypic characterization of 3D-bioreactor-derived hiPSC-NK cells

[0088] Importantly, methods of hiPSC-NK differentiation have been described using adherent 2D-monolayer cultures. The 3D differentiation protocol described herein was compared to a traditional method of adherent 2D hiPSC-NK differentiation.

[0089] In comparing the differentiation potential between 2D-monolayer versus 3D- bioreactor generated hiPSC-NK cells, NK cells were observed and specified in medium suspension almost 10 days faster when compared to 2D-controls (FIGs. 3A-H). FIGs. 3A-D are images of adherent-2D derived cell-spheres and derivatives at indicated time points after the initiation of NK differentiation (lOx). Spheres slowly flatten over time with premature NK-like cells observed in cell medium at differentiation day 23. FIGs. 3E-H are images of 3D bioreactor generated cell-spheres and derivatives. Immature NK-like cells are observed two days after NK differentiation initiation on day 14.

[0090] In addition, 3D-bioreactor-derived NK cells demonstrate advanced maturation and development. Flow cytometry quantification indicates a correlated temporal pattern of expression for the hematopoietic stem cell progenitor marker CD34 (FIG. 4A) and NK cell markers CD45 and CD56 (FIGs. 4B-F). Expression levels of CD56, a marker for mature NK cells, demonstrated that the 3D bioreactor-derived cells were over 93% positive at day 27 of differentiation compared to 58% at day 27 of differentiation for 2D-generated cultures (FIGs. 5A-F and 5). Comparison of expression levels of CD56 over time reveals that bioreactor- derived cells express consistently higher levels of CD56 across all timepoints tested as compared to similarly staged 2D samples (FIG. 5).

[0091] To further assess the robustness and timing of cellular differentiation between the 3D-bioreactor and 2D-monolayer platforms, expression levels were measured for NK lineage-specific markers CD94, NKp44, NKG2D, Lampl, and CD 16. Expression levels of these NK lineage markers for differentiation day 27 bioreactor-generated cells were comparable or significantly exceed expression levels from two independent 2D- differentiation rounds that were cultured almost twice as long (53 days and 55 days, respectively) (FIG 6, and Table 1).

Table 1. Differential expression of lineage-specific surface markers comparing 2D versus 3D differentiation methods.

[0092] As an additional comparison, 3D-bioreactor-derived hiPSC-NK cells were compared to primary cord blood-derived NK (CBNK) cells (FIGs. 7A-J). hiPSC-NK cells expressed similar levels of mature NK markers CD56, CD 16, CD94, NKG2D, NKp44 and LAMP1 as compared to primary CBNK cells. Culturing hiPSCs in a 3D bioreactor using the methods and culture media described herein can generate highly pure and phenotypically mature cells that are similar to primary NK cells.

Functional in vitro killing capacity of 3D-bioreactor-derived hiPSC-NK cells

[0093] While it is important that NK cells express appropriate maturation markers, the gold standard for validating the phenotype of these cells is by assessing the functional killing capacity of the final NK product. Functional analyses of hiP SC -derived NK cells were assessed by in vitro killing assays against Jurkat (FIG. 8A), HeLa (FIG. 8B), and K562 (FIG. 8C) cancer cells as determined using using Incucyte Base Analysis immune killing software for live cell analysis. Different concentrations of target cancer cells were incubated with hiPSC-NK cells at effectortarget (E:T) ratios of 1 : 1 and 4: 1. For additional comparison, similar killing assays were performed to compare the functionality of hiPSC-NK to primary CBNK at an E:T ratio of 1 :2. Importantly, hiPSC-NK performed as well as (if not better than) CBNK in K562 killing (FIG. 8D). Thus, NK cells developed using this 3D-bioreactor platform demonstrate a mature NK cell lineage profile and potent cell cytotoxicity.

Virus transduction of pre-differentiated hiPSC-NK expanded under feeder-free conditions [0094] One method for incorporating genetic modifications into iPSC-NKs is via transduction of iPSCs followed by selection of single cell clones to create stable iPSC lines. However, this method is not ideal for high throughput screening purposes as the timeline for stable iPSC generation and subsequent NK differentiation is relatively long. The transduction protocol described herein was used to incorporate both lentiviral and retroviral constructs into pre-differentiated hiPSC-NK cells.

[0095] Genetic modification of NK cells can be performed to improve their function, target specific antigens, and/or enhance persistence in vivo. One application in the development of NK therapeutics is the incorporation of different chimeric antigen receptor (CAR) constructs. Introducing CAR constructs into NK cells has been hindered by the relative difficulty of viral modification of NK cells in comparison with T cells. Use of retroviral vectors for NK transduction has become more common. As retroviruses specifically infect proliferating cells, substantial efforts have been made to enhance NK proliferation as a means of enhancing transduction efficiency. In comparison with retroviruses, lentiviral vectors can be advantageous because they reduce the potential for genomic insertion and potential off-target effects. However, transduction of NK cells with lentiviral vectors has been significantly more challenging than transduction with retroviral vectors.

[0096] Differentiated, mature hiPSC-NK cells as well as primary adult peripheral blood- derived NK (PBNK) cells were transduced with either lentivirus (FIGs. 9A-J) or retrovirus (FIGs. 10A-J) using the protocol described herein. hiPSC-NK cells can be transduced with both retroviral and lentiviral -based vectors to express a CD19-CAR construct with high affinity. Conversely, primary PBNK cells demonstrate a persistent resistance to lentiviral transduction. Schematics of the constructs are depicted in FIG. 14. Functional in vitro killing capacity of pre-differentiated hiPSC-NK cells transduced with CAR19

[0097] 3D-bioreactor derived hiPSC-NKs were used to transduce a retroviral construct expressing both CAR19 as well as an IL- 15 overexpression cassette using the transduction method described herein. Resulting CAR19 expression was approximately 47%. Nontransduced (NT) or CAR19 hiPSC-NKs were subjected to an in vitro killing assay against CD19+ Raji cells (FIG. 11 A), CD19+ NALM6 cells (FIG. 1 IB), and CD19-CCRF cells (FIG. 11C) as determined using Incucyte’s immune cell-killing software for live cell analysis. Different concentrations of target cancer cells were incubated with hiPSC-NK cells or CAR19-hiPSC NK cells at effectortarget ratios of 2: 1. Importantly, CAR19-transduced hiPSC-NK cells showed enhanced killing efficiency against cancer cell lines expressing the CD 19 target.

Lentivirus transduction of undifferentiated hiPSCs

[0098] A preferred method of incorporating genetic modifications into iPSC-NKs is via transduction of iPSCs followed by selection of single cell clones to create stable iPSC lines. Stably introducing CAR constructs at the iPSC-stage, allows for the persistence of this genetic modification in all differentiated progenitors, resulting in an ultimate CAR transduction efficiency of 100%. Importantly, some genetic modifications induced during the iPSC-stage may result in impaired differentiation into the desired cell type. As proof of principle that genetic modifications performed at the iPSC-stage do not impair 3D bioreactor- mediated NK differentiation, lentiviral transduction was performed on iPSC colonies, which were subsequently expanded as undifferentiated iPSCs for subsequent differentiation within the enclosed bioreactor system (FIGs. 12A-C). Single colonies from iPSC cultures were selected and replated onto new vitronectin substrates in the presence of concentrated lentivirus expressing a CAR19 construct (FIG. 12 A). After 2 days in differentiation medium, CAR19-expression can be observed in the removed colonies under fluorescent imaging. As the selected colonies propagate over time, an expansion of CAR19-expressing cells is observed, as indicated by the binding of CD19-GFP proteins to iPSCs successfully infected with CAR19. This CAR construct is retained after numerous rounds of symmetric cell division (FIGs. 12B-C). Without wishing to be bound by theory, due to the epigenetic silencing of introduced constructs that naturally occurs during pluripotency, the expression of this CAR construct is variable due to methylation of non-pluripotent genes. After 5 days in differentiation medium, the picked colonies expressing CAR19 are capable of proliferating and maintaining expression in vitro. Using this method demonstrates that most progeny of CAR19-transduced iPSCs express the induced CAR19 construct.

3D bioreactor-based differentiation of lenti-CAR19 hiPSCs

[0099] After generating CAR19-hiPSCs via single colony transduction, both nontransduced (NT) and CAR19 hiPSC lines were induced to differentiate into NK cells using the 3D bioreactor protocol. This approach allowed for the continued monitoring of the response of genetically modified hiPSC lines to the previously established 3D bioreactor differentiation protocol. As such, separate bioreactors were used to differentiate both NT and CAR19 hiPSC lines. On day 12, hematopoietic differentiation as determined by CD34 and CD45 expression were unchanged between NT and CAR19 hiPSC lines (FIG. 13 A). NT hiPSC cell line exhibited 15% CD34-positive expression (FIG. 13 A, middle panel) compared to 12% expression in the CAR19 S001 modified hiPSCs (FIG. 13A, right panel). Percent positive for CD45 was <1% in both cell lines. CAR19-transduced hiPSC lines were assayed for CAR19 expression (FIG. 13B), which demonstrated CD19-CAR expression in differentiating spheres in 3D-bioreactor.

Post-differentiation expansion of iPSC-NK

[00100] In comparison to other known methods, the methods described herein are performed under xeno-free and feeder-free conditions. Consequently, the final derived cells are GMP-compliant and can be used in, e.g., clinical trials. One small-scale (30 ml) bioreactor can potentially yield >10 x 10⁷ to 50 x 10⁷ total NK cells. This refinement of this hiPSC to NK cell differentiation process allows the opportunity to scale-up enough highgrade cells to meet the stringent requirements needed to proceed with human clinical trials. Additionally, the methods described herein are efficient and cost-effective for generating phenotypically mature, functional, clinical-grade NK cells for use in developing cancer immunotherapies. Phenotypic characterization and killing efficacy of 3D-bioreactor derived embryonic stem cell (ESC)-NK cells

[00101] Embryonic stem cells (ESCs) also generated NK cells at a similar relative efficiency to iPSCs following the differentiation protocol in the 3D-bioreactor platform system as previously described. A purified population of NK cells that express mature NK lineage markers were collected in suspension from the bioreactor medium (FIGs. 17A-D). These ESC-NK cells demonstrate robust in vitro killing against HUH7, HEPG2, and HEP3B cancer cell lines compared to the PBNK-control (FIGs. 18A-F). ESC-NK cells genetically modified to express a CAR (ESC-NK CAR) demonstrated a greater reduction in HUH7, HEPG2, and HEP3B cancer cells compared to non-transformed ESC-NK cells (ESC-NK NT).

Functional in vivo killing efficacy of 3D-bioreactor-derived hiPSC-NK cells in a myelogenous K562 cancer mouse model

[00102] To assess the killing efficacy of the iPSC-derived NK cells in vivo, cryopreserved hiPSC-NK cells were thawed and prepared the same day for infusion into K562 xenografted mice. The K562 cancer line was injected subcutaneously and allowed to engraft for three days before a single dose of hiPSC-NK cells was injected intravenously. Bioluminescent imaging four days post-NK injection showed significant tumor regression in the hiPSC-NK mice in comparison to the PBNK and PBS control groups (FIG. 15A-B). Additionally, the hiPSC-NK injected mice demonstrated slower tumor growth in comparison to the PBNK and PBS control groups as illustrated by luciferase expression (FIG. 15B). Importantly, this illustrates that the hiPSC-NK cells derived using the 3D-bioreactor system are functionally potent and capable of short-term anti-tumor efficacy in vivo.

In vivo persistence of hiPSC-NK genetically modified to express IL-15

[00103] To assess the persistence of hiPSC-NK in vivo, post-differentiated hiPSC-NK cells were transduced with retroviral constructs expressing luciferase alone or luciferase + secreted IL15, using methods described herein. Transduced hiPSC-NK cells were intravenously infused via tail vein injection (2 x 10⁶/mouse) then tracked over time using bioluminescent imaging. These genetically modified hiPSC-NK cells persisted in vivo for up to two weeks (FIGs. 16A-B). Those hiPSC-NK cells transduced with IL- 15 demonstrated enhanced persistence in vivo. Importantly, there were no discernible effects on mice resulting from the hiPSC-NK infusion.

REFERENCES

[00104] 1. Koepsell, S .A., et al., Successful ,Auin-flight,Au activation of natural killer cells during long-distance shipping. Transfusion, 2013. 53(2): p. 398-403.

[00105] 2. C, M., et al., - Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells. - Nat Commun. 2020 Oct 16; 11(1):5224. doi: 10.1038/s41467-020- 19094-0., (- 2041-1723 (Electronic)): p. T - epublish.

[00106] 3. Szmania, S., et al., Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients.

[00107] 4. Wang, H., et al., The unexpected effect of cyclosporin A on CD56+CD16- and

CD56+CD16+ natural killer cell subpopulations. (0006-4971 (Print)).

[00108] 5. Luevano, M., A. Madrigal, and A. Saudemont, Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy. Cell Mol Immunol, 2012. 9(4): p. 310-20.

[00109] 6. Hermanson DI and Kaufman Ds, Utilizing chimeric antigen receptors to direct natural killer cell activity. (1664-3224 (Print)).

[00110] 7 Hermanson, D.L., et al., Induced Pluripotent Stem Cell-Derived Natural Killer

Cells for Treatment of Ovarian Cancer.

[00111] 8. Knorr, D.A., et al., Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy.

[00112] 9. Woll, P.S., et al., Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity.

[00113] 10. Ni, Z., et al., Expression of Chimeric Receptor CI)4(E by Natural Killer

Cells Derived from Human Pluripotent Stem Cells Improves In Vitro Activity but Does Not Enhance Suppression of HIV Infection In Vivo. Stem Cells, 2014. 32(4): p. 1021-1031.

[00114] 11. Woll, P.S., et al., Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

[00115] 12. Shankar, K., C.M. Capitini, and K. Saha, Genome engineering of induced pluripotent stem cells to manufacture natural killer cell therapies. Stem Cell Research & Therapy, 2020. 11(1): p. 234. [00116] 13. Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424-429 (Methods, Human ES Cell Culture, E8 media composition).

INCORPORATION BY REFERENCE; EQUIVALENTS

[00117] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00118] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of making hematopoietic stem cells, the method comprising: a) culturing pluripotent stem cells in a three-dimensional (3D) bioreactor in a first cell culture medium to produce a 3D culture of pluripotent stem cells having a cell density of at least about 750,000 cells/mL; and b) culturing the 3D culture of pluripotent stem cells in the three-dimensional (3D) bioreactor in a second cell culture medium to produce a 3D culture of hematopoietic stem cells, wherein the second cell culture medium comprises: i) stem cell factor (SCF); ii) bone morphogenetic protein 4 (BMP4) or an agonist of BMP4; and iii) vascular endothelial growth factor (VEGF).

2. The method of claim 1, wherein a) comprises producing a three-dimensional culture of pluripotent stem cells having a cell density from about 750,000 cells/mL to about 1,250,000 cells/mL.

3. The method of claim 1, wherein b) comprises culturing for at least six days.

4. The method of claim 1, wherein b) comprises culturing for six days to fourteen days.

5. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

6. The method of claim 5, wherein the induced pluripotent stem cells are human induced pluripotent stem cells.

7. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells.

8. The method of claim 7, wherein the embryonic stem cells are human embryonic stem cells.

9. The method of claim 1, wherein the first cell culture medium comprises a rho-kinase (ROCK) inhibitor.

- 26 - The method of claim 9, wherein the ROCK inhibitor is Y27632. The method of claim 1, wherein the 3D bioreactor is a scaffold-free bioreactor. The method of claim 11, wherein the scaffold-free bioreactor is a rotational bioreactor. The method of claim 12, wherein the rotational bioreactor rotates at a speed from 60 revolutions per minute to 80 revolutions per minute. The method of claim 1, wherein the hematopoietic stem cells are CD34+. The method of claim 1, wherein the concentration of stem cell factor is from about 5 ng/ml to about 100 ng/ml. The method of claim 1, wherein the concentration of BMP4 is from about 5 ng/ml to about 100 ng/ml. The method of claim 1, wherein the concentration of VEGF is from about 5 ng/ml to about 100 ng/ml. The method of claim 1, wherein the second culture medium further comprises: iv) albumin polyvinylalcohol essential lipids (APEL). The method of any one of claims 1 through 18, further comprising: c) culturing the hematopoietic stem cells in the 3D bioreactor in a third cell culture medium to produce natural killer (NK) cells. The method of claim 19, further comprising transducing the NK cells with a lentiviral vector. The method of claim 20, wherein the lentiviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor. The method of claim 21, wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular activation domain. The method of claim 22, wherein the extracellular antigen recognition domain is a single-chain Fragment variant (scFv) derived from an antibody. The method of claim 22, wherein the intracellular activation domain is a CD3(^ domain. The method of claim 22, wherein the chimeric antigen receptor further comprises a co-stimulatory domain. The method of claim 25, wherein the co-stimulatory domain is a 4-1BB domain. The method of claim 20, wherein transducing the NK cells with a lentiviral vector comprises culturing with retronectin. The method of claim 19, further comprising quantifying cells expressing CD56, and when at least 75% of the cells express CD56, cryopreserving the NK cells. The method of any one of claims 19 through 28, wherein the third cell culture medium comprises: i) 2-mercaptoethanol (2-BME); ii) sodium selenite; iii) ascorbic acid; iv) interleukin 3 (IL-3); v) interleukin 7 (IL-7); vi) interleukin (IL- 15); vii) FMS-like tyrosine kinase 3 ligand (FLT3L); and viii) stem cell factor (SCF). The method of claim 29, wherein the concentration of 2-BME is from about 5 pM to about 100 pM. The method of claim 29, wherein the concentration of sodium selenite is from about 0.5 ng/ml to about 50 ng/ml. The method of claim 29, wherein the concentration of ascorbic acid is from about 2 pg/ml to about 200 pg/ml. The method of claim 29, wherein the concentration of IL-3 is from about 0.5 ng/ml to about 50 ng/ml. The method of claim 29, wherein the concentration of IL-7 is from about 2 ng/ml to about 200 ng/ml. The method of claim 29, wherein the concentration of IL-15 is from about 2 ng/ml to about 200 ng/ml. The method of claim 29, wherein the concentration of FLT3L is from about 2 ng/ml to about 200 ng/ml. The method of claim 29, wherein the concentration of stem cell factor is from about 2 ng/ml to about 200 ng/ml. The method of claim 29, the third cell culture medium further comprises ethanolamine. The method of claim 38, wherein the concentration of ethanolamine is from about 5 pM to about 500 pM. A cell culture medium comprising: a) stem cell factor (SCF); b) bone morphogenetic protein 4 (BMP4) or an agonist of BMP4; and c) vascular endothelial growth factor (VEGF). The cell culture medium of claim 40, wherein the cell culture medium further comprises: d) albumin polyvinylalcohol essential lipids (APEL).

- 29 -