CN117836405A

CN117836405A - Generation of hematopoietic stem cells from pluripotent stem cells in three-dimensional culture

Info

Publication number: CN117836405A
Application number: CN202280055442.9A
Authority: CN
Inventors: 克里斯托弗·邱·陈; 达纳·梅雷迪斯·凯恩斯; 张帆; 杨小杰
Original assignee: Xiansheng Innovation Co
Current assignee: Xiansheng Innovation Co
Priority date: 2021-08-13
Filing date: 2022-08-11
Publication date: 2024-04-05
Also published as: WO2023019213A1; WO2023019213A8

Abstract

The present application describes a cell culture method and culture medium for expanding pluripotent stem cells (e.g., induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC)) to three-dimensional cultures and differentiating into hematopoietic stem cells. Hematopoietic stem cells can be further differentiated into natural killer cells or macrophages. Natural killer cells can be further transformed to express chimeric antigen receptors (CARs).

Description

Three-dimensional culture of pluripotent stem cells to produce hematopoietic stem cells

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application No.63/232,911 filed on day 2021, 8 and 13. The entire teachings of the above application are incorporated herein by reference.

Background

One major obstacle to cellular immunotherapy in current cancer treatments is the high concentration of effector cells required per dose. Various strategies for culturing hiPSC-derived NK cells involve the use of an excessive number of plates or flasks to amplify the final product, an extremely inefficient, time consuming and expensive process, while also increasing the overall susceptibility to contamination of the cell culture. Other methods of expanding NK cells involve the use of cancer trophoblasts, which results in the hiPSC-NK product being reduced to GMP grade and quality.

Disclosure of Invention

The methods described herein demonstrate that the entire differentiation and expansion of hipscs into functionally mature NK cells can occur entirely within a closed bioreactor system. The methods described herein simplify the differentiation process and ensure consistent reproducibility and reliability of the resulting NK cells.

In contrast to other known methods, the methods described herein are performed under heterologous-free and trophoblast-free conditions. Thus, the final derived cells meet GMP standards and can be used in clinical trials and the like. The production of a small bioreactor is possible>10-50x10 ⁷ NK cell total number. This improvement in hiPSC to NK cell differentiation process opens the opportunity to expand enough higher cells to meet the stringent requirements required for conducting human clinical trials. In addition, the methods described herein are useful for generating a drug for developing cancerPhenotype matured, functional, clinical grade NK cells for symptomatic immunotherapy are effective and cost effective.

The rate of NK cell production was observed and confirmed to be almost 10 days faster in the medium suspension than in the 2D control (FIGS. 3A-H). In addition, 3D bioreactor derived NK cells showed better maturation and developmental capacity. Flow cytometry analysis quantitatively determined the expression level of CD56 (marker of mature NK cells) indicated that 3D bioreactor-derived cells were more positive than 93% on day 27 of differentiation, in contrast to the 2D-generated cultures which were 58% positive on day 27 of differentiation (fig. 4A-F and fig. 5). To further assess the robustness and time of use of cell differentiation between 3D bioreactor and 2D monolayer platform, the expression levels of NK lineage specific markers CD94, NKp44, NKG2D, lampl and CD16 were measured. NK lineage marker expression levels of the cells produced on day 27 of bioreactor differentiation corresponded to or significantly exceeded the expression levels from two independent 2D differentiation at nearly twice the incubation time (53 days and 55 days, respectively) (fig. 6A-O and table 1). Culturing hipscs in a 3D bioreactor using the methods and media described herein can produce high purity and phenotypically mature cells similar to primary NK cells.

Drawings

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.

FIG. 1 summarizes the 3D directed in vitro differentiation scheme of NK cells derived from human induced pluripotent stem cells (hiPSCs). Briefly, hipscs were scaled up from 2D monolayers to 3D small scale bioreactor cell culture systems and Y27632 was added to enhance iPSC viability and improve spheroid formation. The induction of iPSC hematopoiesis for progenitor cells of NK lineage limited fate takes 12 days. The 3D culture was then transferred to NK cell differentiation conditions. After two weeks, mature and functional NK cells can be continuously collected from the medium for a period of about 35 days.

Figures 2A-C show 3D adaptation of hipscs from 2D monolayer cultures using mini-bioreactors. FIG. 2A is a low magnification (10 x) micrograph of hiPSC in 2D monolayer culture. FIG. 2B is a photograph of a small 30ml bioreactor flask. Fig. 2C is a photomicrograph (10 x magnification) of hiPSC spheres 20 hours after 2D to 3D adaptation.

FIGS. 3A-H are comparisons of differentiation potential between 2D monolayers and hiPSC-NK cells generated by 3D bioreactors. FIGS. 3A-D are images of adherent 2D-derived cytoballs and derivatives at a designated time point (10X) after the start of NK differentiation. Premature NK-like cells were observed to flatten the spheres slowly over time in the cell culture medium on day 23 of differentiation. Fig. 3A-B:4 times of magnification; fig. 3C-D:10 times magnification. FIGS. 3E-H are images of cell spheres and derivatives generated by a 3D bioreactor. On day 14, immature NK-like cells were observed two days after the start of NK differentiation. Fig. 3E-F: magnification of 20 times; fig. 3G-H:10 times magnification.

FIGS. 4A-F show characterization of hiPSC-NK cells. Flow cytometry quantification indicated the relevant temporal pattern of expression of hematopoietic stem cell progenitor markers CD34 (fig. 4A) and mature NK cell markers CD45 and CD56 (fig. 4B-F).

FIG. 5 shows a comparison of CD56 positive marker expression between 2D-monolayer and 3D-bioreactor-generated cells at a particular time point during the differentiation timeline.

FIGS. 6A-O show NK lineage flow cytometry group comparisons of 3D bioreactors and 2D monolayer derived NK cells. Assessment of mature NK cell expression markers: CD56, CD94, NKp44, NKG2D, lamp1 and CD 16. Top row: flow cytometry panels to differentiate day 27 bioreactor-derived NK cells. Second and third rows: flow cytometry panels of two independently performed 2D monolayer assays to generate NK cells on day 53 and day 55 of differentiation, respectively.

FIGS. 7A-J show a phenotypic comparison of hiPSC-NK with CBNK.

Figures 8A-D show functional in vitro killing. In vitro killing of Jurkat cells (fig. 8A), heLa cells (fig. 8B) and K562 cells (fig. 8C) was performed using unmodified iPSC-NK, as determined by live cell analysis by Incucyte Base Analysis immune cell killing software. FIG. 8D shows in vitro killing comparison of unmodified primary CBNK and iPSC-NK against K562 cells (effective target ratio 1:2). Target cancer cells were incubated with hiPSC-NK cells at different concentrations at 1:1 and 4:1 effective target ratios. Data are expressed as mean ± SEM.

FIGS. 9A-J show genetic modification of mature hiPSC-NK cells using lentiviruses. hiPSC-NK cells can be transduced with lentiviral-based vectors to express CD19-CAR constructs with high affinity. In contrast, primary NK cells exhibit sustained resistance to genetic engineering following lentiviral transduction.

FIGS. 10A-J show genetic modification of mature hiPSC-NK cells using retroviruses. Both mature hiPSC-NK and primary NK cells can be transduced with retroviral-based vectors to express CD19-CAR constructs with high affinity.

FIGS. 11A-C show comparative functional analysis between unmodified and CAR19 hiPSC-NK against tumor targets. In vitro killing of Raji cells (fig. 11A), NALM6 cells (fig. 11B) and CCRF cells (fig. 11C) was determined using the living cell analysis immune cell killing software of Incucyte. Target cancer cells were incubated with hiPSC-NK cells or CAR19-hiPSC NK cells at different concentrations at an effective target ratio of 2:1. Data are expressed as mean ± SEM.

FIGS. 12A-C show genetic modification of iPSC using lentiviruses. Fig. 12A: individual iPSC colonies were selected and picked from the cell culture plate surface. Fig. 12B: after 2 days in differentiation medium, CD19 expression can be observed in the removed colonies under fluorescent imaging. A large difference was observed in positive clones expressing CD19 from selected colonies (top and bottom rows, yellow arrow and arrow). Fig. 12C: after 5 days in differentiation medium, selected colonies expressing CD19 were able to proliferate and maintain expression in vitro (yellow arrow).

Figures 13A-B show NK differentiation of the lenti-CAR19 iPSC using a 3D bioreactor on day 12. Fig. 13A: flow cytometry quantification of CD34 and CD45 marker expression. The untransduced hiPSC cell line showed 15% CD34 positive expression (middle panel), whereas CD34 positive expression in CAR 19S 001 modified hiPSC was 12% (right-most panel). The percentage of CD45 positivity in both cell lines was <1%. Fig. 13B: CD19-CAR expression detected in differentiated spheres in 3D bioreactors.

Figure 14 shows a schematic representation of a viral CAR construct.

Figures 15A-B show that cryopreserved unmodified hiPSC-derived NK cells showed strong killing efficacy in a subcutaneous injection K562 myelogenous leukemia mouse model. Fig. 15A: intravenous injection of frozen peripheral blood derived NK (PBNK) or frozen iPSC derived NK cells (4X 10) ⁶ Mice). Tumor burden optical bioluminescence imaging from day 3 to day 7 post treatment. Fig. 15B: quantification of tumor intensity examined four days after NK infusion for all control and experimental groups.

Figures 16A-B show that iPSC-derived NK cells can survive in vivo and last for at least two weeks after injection. Fig. 16A: infused NK cells transduced with luciferase-expressing or luciferase-expressing retroviral constructs for soluble IL15 alone were then intravenously injected into the tail vein (2 x10 ⁶ Mice). Fig. 16B: persistence of the genetically modified iPSC-NK cells was detectable in vivo for two weeks. Transduction of IL15 enhanced the persistence of injected iPSC-NK cells, and iPSC-NK infusion had no significant effect on mice.

Figures 17A-D show that NK cells can be generated from a human embryonic stem cell (hESC) source using a 3D bioreactor platform system. Fig. 17A-C: high magnification (40 x) micrographs of cell suspensions collected from 3D bioreactors demonstrate phenotypic maturation of NK cells over developmental time. Fig. 17D: purified NK cell populations were collected and maintained over time as shown by flow cytometry quantification of the mature NK cell markers CD45 and CD 56.

Fig. 18A-F: NK cells generated from human embryonic stem cell (hESC) sources using the 3D bioreactor platform system exhibit potent endogenous killing activity, and can be genetically modified using CAR constructs to enhance killing activity in liver cancer cell lines. Fig. 18A: ESC-CAR NK in vitro killing was shown as percentage of growth against Huh7 cancer cell line within 24 hours. Fig. 18B: average percent growth of Huh7 cancer cells over 24 hours. Fig. 18C: ESC-CAR NK in vitro killing was shown as percentage of growth against HepG2 cancer cell lines within 24 hours. Fig. 18D: average HepG2 cancer cell growth percentage at 24 hours. Fig. 18E: ESC-CAR NK in vitro killing was shown as percentage of growth against Hep3B cancer cell line within 24 hours. Fig. 18F: average Hep3B cancer cell growth percentage at 24 hours.

Detailed Description

The following is a description of example embodiments.

SUMMARY

According to the latest statistics provided by the U.S. center for disease control and prevention (CDC), cancer deaths reported annually in the united states reach about 60 tens of thousands of people despite advances in oncological patient care. The development of novel immunotherapies using the potent cytotoxic anti-tumor properties of Natural Killer (NK) cells is a promising approach in cell-based therapies. However, there are often limitations to the source of NK cells. Since NK cells account for only 5-15% of all lymphocytes circulating in blood, isolation of NK cells from Peripheral Blood (PBNK) is generally inefficient, resulting in heterogeneous lymphocyte populations producing only 10-20% of NK cells [1]. In addition, it has been reported that cytotoxicity is significantly reduced ([ 2,3 ]) after cryopreservation. In contrast, NK Cells (CBNK) isolated from cord blood show higher cell viability after cryopreservation, however, since the number of NK cells detected per cord blood unit is low, multiple expansion stages are required to obtain multiple units per dose [4]. Some reports suggest that CBNK shows a less mature phenotype compared to PBNK, directly associated with reduced cytotoxicity [4,5]. Although the problems of low NK cell yield and loss of cytotoxicity can be partially addressed by cytokine support and trophoblast co-culture, these considerations suggest that PBNK and CBNK are not ideal choices for "off-the-shelf alloimmunotherapy, where NK cells are harvested from unrelated donors, cryopreserved and thawed.

Human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs) have an indefinite ability to self-renew and differentiate into all cell types in the body. Unlimited starting cell sources have great advantages for expanding the production of homogeneous and high quality NK cell products. This enables the development and expansion of GMP-grade iPSC-derived NK cells for use in cancer immunotherapy clinical trials. Several groups have published recent advances in strategy to successfully differentiate pluripotent stem cells into NK lineages that display phenotypes and functions comparable to primary NK cells [6-10], some of which involve differentiating NK cells by co-culture with OP9 mouse stromal cells. As the potential clinical use of human Pluripotent Stem Cells (PSC) as a cancer treatment strategy continues to be intensively studied, PSC-derived NK cells are a promising alternative to cancer immunotherapy.

With continued extensive research on the differentiation of PSCs into NK-specific lineages, a variety of cell culture strategies have been used to produce high quality, pure NK cells: 1) Cytokine induction by trophoblast co-culture; 2) Embryoid Body (EB) mediators are produced by cytokine induction. The application of these different strategies has met with varying degrees of success.

Co-culture of hPCs with stromal cells (predominantly bone marrow derived) is a common method of inducing hematopoietic fate by first obtaining a CD34+ precursor population [8,9,11]. However, this protocol typically requires a longer co-culture period of about 21 days in addition to the cell sorting step of selecting cd34+ positive cells prior to downstream NK differentiation. The additional cell sorting module not only results in less efficient protocols, but also results in reduced NK cell yield by negative selection of hematopoietic progenitor cells required for robust NK differentiation.

EB formation is a common platform for differentiation of PSCs into specific cell lineages. However, current EB generation methods produce spheres of varying sizes, which may lead to non-reproducible differentiation results. Furthermore, disintegration of EB spheres was observed shortly after the onset of differentiation. Therefore, the efficacy and reproducibility of hematopoietic induction and NK differentiation by EB procedure are low, and are not suitable for adapting to mass production systems required in clinical trial environments.

Thus, there is a current need to modify and optimize the biological production of NK cells for immunotherapy [12]. The ability to generate large numbers of GMP grade NK cells from a pluripotent source has proven to be most useful for cell-based immunotherapy.

As used herein, pluripotent Stem Cells (PSC) include human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs). The methods described herein are applicable to pluripotent stem cells.

3D bioreactor

Three-dimensional (3D) bioreactors promote the growth of cells in three-dimensional space. A 3D bioreactor is distinguished from two-dimensional (2D) growth of cells on a flat surface (e.g., a cell culture dish). By promoting the growth of cells in three dimensions, cells can grow into spheroids or 3D cell colonies.

Some 3D bioreactors have scaffolds to which adherent cells can attach. Other 3D bioreactors use a stentless technique. An example of a stentless 3D bioreactor is a 30ml disposable magnetic stirring bioreactor (ABLE catalog number BWV-S03A) from ABLE Corporation (japan) and Biot Corporation (japan) that uses impellers with magnets on each blade to provide low shear stirring by laminar flow, which can promote the formation and growth of globular cell clusters. Impellers or other mechanisms for mixing media provide a number of benefits including enhanced mass transfer and increased surface area of cells in contact with the media, nutrients and differentiation factors. The low shear stress and fluid flow provide a more physiologically relevant environment relative to static culture. Other suitable bioreactor magnetic stirring systems are available from ABLE Biot and other manufacturers, in a variety of sizes.

Typically, the 3D bioreactor is a closed bioreactor.

Cell culture medium for pluripotent stem cell expansion

Pluripotent stem cells are cultured in a cell culture medium that maintains cells in a pluripotent state as they grow from an initial aliquot of the sample to a 3D culture. Many commercially available nutritionally free and xeno-free media are suitable for use as a basal medium for iPSC amplification. An example is Essential 8 medium (available from ThermoFisher Scientific). Others include mTESR Plus ^TM (StemCell Technologies)；XF medium (Biological Industries); celloartis DEF-CS 500 (Takara); and StemFlex ^TM Culture medium (Fisher Scientific).

Chen, g.et al chemical ly defined conditions for human iPSC derivation and culture. Nature methods.2011;8 (5): 424-429 describes another suitable medium (Methods, human ES Cell Culture, E8 media composition). The medium described therein comprises DMEM/F12, L-ascorbic acid-2-magnesium phosphate (64 mg/L), sodium selenate (14 pg/L), FGF2 (100 pg/L), insulin (19.4 mg/L), sodium bicarbonate (543 mg/L) and transferrin (10.7 mg/L), TG beta 1 (2 pg/L) or NODAL (100 pg/L). The osmotic pressure of all media was adjusted to 340mOsm at pH 7.4. Those skilled in the art will appreciate that the concentrations need not be exactly as described in Chen, g.et al.

The pluripotent stem cell expansion medium may further comprise vitronectin, a glycoprotein that provides a surface coating to promote cell attachment for use in the trophoblast-free culture of pluripotent stem cells.

The pluripotent stem cell expansion medium may further comprise a rho kinase (ROCK) inhibitor. Typically, ROCK inhibitors are only used when hipscs or hescs require enzymatic passaging to help reduce cell death. An example of a ROCK inhibitor is Y-27632, which is a compound having the structure:

y-27632 may sometimes be provided in the form of a salt, for example:.2HCl).

After culturing in pluripotent stem cell expansion medium, cell density increases and cells form 3D-PSC spheres after adaptation to the bioreactor. Expression of OCT-4 (pluripotency marker) of 3D-PSC can be quantified by flow cytometry. Typically, over 90% of the 3D-PSCs produced in the first culturing step express OCT-4.

In this PSC expansion step, the PSC is typically cultured to a cell density of at least about 750,000 cells/mL. In some cases, the PSC is cultured to a cell density of about 750,000 cells/mL to about 1,250,000 cells/mL. For the 30mL bioreactor used in the examples, ipscs were cultured to about 2500 to 3500 ten thousand cells.

Optionally, the cells may be mechanically separated (e.g., with a pipette) during cell culture to separate the cell mass or aggregates into smaller masses, smaller aggregates, or individual suspension cells.

Cell culture medium for differentiation of pluripotent stem cells into Hematopoietic Stem Cells (HSCs)

The cells are then cultured to differentiate 3D PSCs into Hematopoietic Stem Cells (HSCs).

Typically, hematopoietic stem cell media comprise Stem Cell Factor (SCF), bone morphogenic protein 4 (BMP 4) (or an agonist of BMP 4), and Vascular Endothelial Growth Factor (VEGF). Hematopoietic stem cell media also typically include basal media that promote hematopoietic differentiation. One suitable basal medium is albumin polyvinyl alcohol essential lipid (APEL). Suitable APEL media are described in U.S. Pat. No. 10,894,944B2 and Ng et al, aprotocol describing the use of a recombinant protein-based, animal product-free media (APEL) for human embryonic stem cell differentiation as spin embryoid bodies, nature Protocols 3,768-776 (2008) Table 1.

At the end of the hematopoietic stem cell culture step, approximately 10-50% of the cells express cd34+, which is a marker for 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 transform into more mature (differentiated) cell types (e.g., natural killer cells). Once the cells differentiate into HSCs, the cells may differentiate into natural killer cells.

An example of a BMP4 agonist is SB 4, which is a compound having the structure:

cell culture medium for differentiation of HSC into natural killer cells

The cells are then cultured and differentiated from Hematopoietic Stem Cells (HSCs) into differentiated cell types, such as natural killer cells.

Examples of cell culture media include i) DMEM Glutamax medium; ii) DMEM F-12Glutamax human AB serum medium; iii) Ethanolamine; iv) 2-mercaptoethanol (2-BME); v) sodium selenite; vi) ascorbic acid and interleukin 3 (IL 3), interleukin 7 (IL 7), interleukin (IL 15), FMS-like tyrosine kinase 3 ligand (Flt 3L), stem Cell Factor (SCF).

Human AB serum refers to AB type serum typically from a male donor. AB type donors lack antibodies to the a and B blood group antigens and are therefore more commonly used to reduce immunoreactivity.

Without wishing to be bound by theory, FMS-like tyrosine kinase 3 ligand (Flt 3L) may enhance IL15 signaling, which may be important for the NK lineage. IL15 is a cytokine involved in NK cell specific maintenance and proliferation, and therefore Flt3L may enhance proliferation and/or NK specificity.

As the cells mature and adapt to NK lineage characteristics, NK cells are released into the cell culture media from which they can be collected for downstream use. With the production of NK cells, cells can be cultured indefinitely in such cell culture media.

Lentiviral transduction

In some embodiments, lentiviruses are concentrated using a Lenti-X concentrator (Takara Bio, catalog Nos. 631231 and 631232).

In some embodiments, lentiviral transduction may be enhanced by a retroNectin culture, which is a 63kD fragment of a recombinant human fibronectin fragment (also known as rFN-CH-296).

Lentiviral transduction has advantages over retroviral transduction. Lentiviruses (LVs) can infect both non-dividing and actively dividing cell types, whereas retroviruses can only infect actively dividing, mitotically active cells. Thus, lentivirus transduction is theoretically more efficient, as it is likely to integrate into all NK cells, rather than into proliferating cells alone.

NK cells are resistant to LV transduction, which hampers their development as immunotherapy. Vesicular stomatitis virus type G (VSV-G) LV is one of the most commonly used viruses for generating Chimeric Antigen Receptor (CAR) -T cells, but is not able to transduce NK cells efficiently.

Although the preferred method of transferring genetic modifications into iPSC-NK is to create a stable iPSC line by transduction of iPSC followed by selection of single cell clones, this is not ideal for high throughput screening purposes, as the timeline for stable iPSC generation and subsequent NK differentiation is relatively long.

Previous studies by others have involved modification of the structure and/or sequence of a particular LV to increase transduction efficiency, but these studies have rarely achieved promising results. The method described herein using a combination of a Lenti-X concentrator and Retronectin produced unexpected effects in transducing induced natural killer cells with lentivirus (iNK).

The terms "nucleic acid," "nucleotide," and "polynucleotide" as used in this disclosure shall be given their ordinary meaning and shall include deoxyribonucleotides, ribonucleotides, and ribonucleic acids and polymeric forms thereof, and include 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 that contain non-naturally occurring bases, nucleotides linked to other nucleotides than naturally occurring phosphodiester linkages, or nucleic acid analogs that contain bases linked by linkages other than phosphodiester linkages. Thus, nucleic acid analogs include, for example, but are not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, borane phosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide Nucleic Acids (PNAs), locked Nucleic Acids (LNAs), and the like.

The term "operably linked" as used in this disclosure, 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 IRES, "operably linked" refers to a functional linkage between a nucleic acid sequence comprising an internal ribosome entry site and the initiation of a heterologous coding sequence intermediate the mRNA sequences, resulting in translation of the heterologous coding sequence.

The term "vector" as used in this disclosure shall be given its ordinary meaning and shall mean that a DNA or RNA sequence (e.g., an exogenous gene) can be introduced into a genetically engineered cell to transform the genetically engineered cell and facilitate expression (e.g., transcription and/or translation) of the introduced sequence.

Chimeric antigen receptor

Embodiments described herein relate to cell transduction to express Chimeric Antigen Receptors (CARs). The cells can be transduced by a vector (e.g., a lentiviral vector) to express a chimeric antigen receptor. For example, a lentiviral vector may comprise a nucleic acid sequence encoding a chimeric antigen receptor. Chimeric antigen receptors include extracellular antigen recognition domains, transmembrane domains, and intracellular activation domains.

The extracellular antigen recognition domain may 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.

The transmembrane domain may be, for example, a CD3 transmembrane domain, a CD8 transmembrane domain or a CD28 transmembrane domain.

The intracellular activation domain may be, for example, a CD3 zeta domain (sometimes written CD3-zeta, CD3Z or CD 3Z).

In some embodiments, the chimeric antigen receptor can include a costimulatory domain, e.g., a 4-1BB domain.

Examples

Material

Cell lines

hiPSC cell lines were obtained from Alstem (source cord blood). Human embryonic stem cells (hescs) were purchased from ESI BIO (Alameda, california, USA).

hiPSC culture

hiPSC and hESC were cultured and maintained in Essential 8 medium (ThermoFisher Scientific, catalog No. a 2858501). Supplements of ROCK inhibitor Y27632 (Sigma, cat# SCM 075) were added to hiPSC and hESC cultures at 10 μm only during cell passage and removed from the medium within 24 hours of plating.

Hematopoietic stem cell induction medium

The medium consisted of a stem APEL 2 (albumin, polyvinyl alcohol, essential lipids; stemcell Technologies, catalog number 05275) basal cell culture medium supplemented with stem cell factor (R & D Systems, SCF,40ng/ml, catalog number 255-SC/CF-050), bone morphogenic protein 4 (R & D Systems, BMP4,20ng/ml, catalog number 314-BP-050) and vascular endothelial growth factor (R & D Systems, VEGF,20ng/ml, catalog number 293-VE-050).

NK cell differentiation medium

Basal medium contained a mixture of 56.6% DMEM+Glutamax (Life Technologies, catalog No. 10566-016) and 28.3% F-12+Glutamax (Life Technologies, catalog No. 31765035) supplemented with 15% human heat-inactivated AB-type serum (Valley Biomedical, catalog No. HP1022 HI), 50. Mu.M ethanolamine (MP Biomedicals, catalog No. 194658), 25. Mu.M 2-mercaptoethanol (Sigma, catalog No. M6250), 5ng/ml sodium selenite (Sigma, catalog No. S5261), 20mg/ml ascorbic acid (Sigma, catalog No. A-5960) and 1% penicillin-streptomycin (Gibco, catalog No. 15140-148). The cytokines added to the medium consisted of 5ng/ml IL-3 (Peprotech, cat. No. 200-03), 20ng/ml IL-7 (Peprotech, cat. No. 200-07), 10ng/ml IL-15 (Peprotech, cat. No. 200-15), 10ng/ml Flt3 ligand (Peprotech, flt3L, cat. No. 300-19) and 20ng/ml SCF.

Nalm-6, raji and Jurkat cancer cells

Maintained in stem cell growth medium (CellGenix, SGCM, catalog No. 20802-0500) containing 10% fetal bovine serum (Gibco, FBS, catalog No. 10082-147) and 1% penicillin/streptomycin.

Other materials

TrypLE Select (Life Technologies, catalog number 12563011), actutase (Innovative Cell Technologies, inc., catalog number AT 105-500), vitronnectin (ThermoFisher Scientific, catalog number A14700), retronnectin (Takara, catalog number T100A), dnase 1 (Worthington Biochemical), catalog number LK 003172), 70 μm (Fisherbrand, catalog number 22363548), incucyte Nuclight Red lentiviral agents (EFN-alpha; puro; catalog number 4625), 96-well assay plate (Corning, catalog number 3610), 30ml disposable bioreactor (Able, biott catalog number abbwss 03A-6).

Method

Trophoblast-free pluripotent stem cell culture and maintenance

Undifferentiated trophoblast-free hipscs and hescs were cultured on vitronectin coated substrates in Essential 8 medium. When hiPSC and hESC cultures were >70% confluent, cells can be enzymatically passaged using TrypLE or Accutase and re-inoculated at split ratios between 1:10 and 1:15 and Y27632 supplemented at a final concentration of 10 μm. hipscs and hescs can be maintained, amplified or cryopreserved continuously under these outlined conditions. Prior to initiating 2D monolayer to 3D bioreactor hiPSC adaptation, hiPSC and hESC need to be amplified until enough of the desired cells are obtained.

3D-bioreactor adaptation

hipscs and hescs were transferred as starting 2D monolayers into mini-bioreactors to initiate 3D suspension culture. When 2D-hiPSC and hESC amplifications produced in excess of about 30x10 ⁶ At the cell concentration of individual living cells, undifferentiated hiPSCs and hESCs can be dispersed with TrypLE or Acceutase. The cell contents should be filtered through a 70 μm cell strainer to remove the doublet.

The optimal seeding density in the bioreactor should be about 1x10 ⁶ Each cell/ml, Y27632 was added to the Essential 8 medium to a final concentration of 10. Mu.M. The rotational speed of the bioreactor at all stages of the 3D differentiation process ranged from 60-80RPM. The spheres should be carefully examined under a microscope and assessed for viability and morphology 18 to 24 hours after adaptation.

Hematopoietic lineage induction

As shown in fig. 1, after successful 3D adaptation on the previous day, induction of differentiation of pluripotent stem cells (hiPSC and hESC) into hematopoietic cells can begin. Complete removal of Essential 8 medium from the suspension culture is crucial and can be performed by transferring the entire volume from the bioreactor to a 50ml conical tube. The cells were left undisturbed for 10-12 minutes and the spheres were allowed to settle to the bottom. The cell supernatant was removed, the pellet was left undisturbed, and replaced with hematopoietic induction medium as previously described. Induction medium was prepared by preparing Stemdiff APEL 2 medium supplemented with SCF at a concentration of 40ng/ml and BMP4 and VEGF at a concentration of 20ng/ml, respectively. Medium exchange and preparation may be performed as described in the hematopoietic induction phase between day 1 and day 12 of differentiation.

The expression of hematopoietic stem cell progenitor marker CD34 and mature NK marker CD45 was quantified on days 6 and 12 to analyze the progression of NK cell development over time.

NK cell differentiation

After 12 days in HSC induction medium, 3D spheroid aggregates can be further differentiated into NK cell lineages by replacing the cultures with NK cell differentiation medium as described above. NK cell differentiation basal medium contained 56.6% DMEM+Glutamax and 28.3% F-12+Glutamax supplemented with 15% human heat-inactivated type AB serum, 50. Mu.M ethanolamine, 25. Mu.M 2-mercaptoethanol, 5ng/ml sodium selenite, 20mg/ml ascorbic acid and 1% penicillin streptomycin. The cytokines added to the medium consisted of 5ng/ml IL-3 (Peprotech, cat. No. 200-03), 20ng/ml IL-7, 10ng/ml IL-15, 10ng/ml Flt3 ligand and 20ng/ml SCF. From day 12 of differentiation, only NK differentiation medium was required. The medium in the 3D bioreactor culture should be changed daily or when needed.

Typically, pluripotent stem cell-derived NK cells delaminate from the differentiated 3D spheroid aggregates and are released into the cell culture medium suspension after detachment. After NK cells differentiated for 1 week, it was observed that NK cells showing maturation were floating in cell culture medium. hiPSC-derived NK cells can be collected directly from the culture medium and phenotyped using flow cytometry on surface antigens including CD45, CD56, CD 16, KIR, CD94, NKp44, NKG2D and Lamp 1. Functional analysis of hiPSC-derived NK cells can be assessed by in vitro killing assays against a variety of cell carcinoma cell lines of different potency target ratios (E: T). NK cells developed using this 3D bioreactor platform exhibit mature NK cell lineage characteristics and potent cytotoxicity.

The outlined method shows an efficient and simplified method of deriving mature and functional NK cells from a pluripotent stem cell source that can be used as an unlimited cell source for potential cancer cell therapies.

In vitro killing assay

The cancer target cells were labeled with Incucyte Nuclight Red lentivirus, which in turn enabled the counting of viable cancer cells over time. Puromycin was added to the cancer cell culture at a concentration of 0.5ug/ml on day 6 to initiate the selection process. Transduction efficiency was monitored daily and cell culture media was supplemented with puromycin each time the media was changed. By day 10-14, a high percentage of cancer target cells should appear nucleic red.

Both effector cells and target cells were cultured in RPMI medium supplemented with 10% fbs for cytotoxicity analysis. Real-time NK cell cytotoxicity was monitored and quantified using the Incucyte base analysis software.

Production and purification of retroviruses for transduction of iPSC-NK

Will be 3x10 ⁶ 293T cells of (A) were placed in 10cm dishes containing 10ml of DMEM medium. Two sterile Eppendorf tubes were prepared per dish. In tube 1, 420ul of DMEM was added to a sterile Eppendorf tube and 30. Mu.L of FugeneHD was added directly to the DMEM in the Eppendorf tube. The tube was tapped to mix and incubated for 5 minutes at Room Temperature (RT). In tube 2, plasmid(s) was added to the total 10ug dna. The FugeneHD/DMEM mixture was added dropwise to the tubes containing the DNA. The tube was tapped to mix the FugeneHD/DMEM/DNA mixture and incubated for 15 minutes at room temperature. FugeneHD/DMEM/DNA solution is added drop-wise to the cells under gentle agitation. Cells were incubated at 37℃for 48 hours. The supernatant was harvested, centrifuged at 400Xg for 5 minutes and stored at 4 ℃. The supernatant was filtered with a 0.45um low-binding filter. For long term storage, storage was performed in 3 or 6mL aliquots at-80 ℃.

Virus transduction of amplified iPSC-NK in the absence of trophoblasts

The virus-containing supernatant was harvested and filtered through a 0.45 μm low-binding filter. The supernatant was transferred to a sterile container and 1 volume of Lenti-X Concentrator was combined with 3 volumes of clear supernatant and mixed by gentle inversion. The mixture was incubated overnight at 4 ℃. The samples were centrifuged at 1500Xg for 45 minutes at 4 ℃. Carefully remove the supernatant and gently re-suspend the pellet in 1/10 to 1/100 of the original volume using complete medium. The samples were titrated immediately or stored in single use aliquots at-70 ℃. 24-well non-tissue culture treated plates were coated with Retronectin (7 ug/mL in PBS). The next day, retronectin was aspirated and the wells were washed with complete RPMI medium. To the Retronectin coated wells 2ml of concentrated virus was added. The plate was spun at 2000g for at least 1 hour at room temperature. NK MACs medium was prepared at 500IU/ml IL-2 and 140IU/ml IL-15. 2.5E5 cells/ml (1 ml per 24 wells) were added. The cells were incubated undisturbed for a minimum of 2 days prior to subsequent assays. CAR expression was monitored using flow cytometry and transduced iNK was used for subsequent killing assays.

In vivo killing assay

At 2x10 ⁵ Cells/mice were subcutaneously injected with K562 cancer cells. The K562 cancer cells were transplanted 3 days before the system was infused with hiPSC-NK cells. Frozen hiPSC-NK cells were thawed on the same day and ready for infusion. Sample preparation of hiPSC-NK and PBNK was identical, and each mouse was intravenously injected 4X10 ⁶ NK cells. Recombinant human IL-15 was injected Intraperitoneally (IP) daily at a dose of 30 ng/mouse on days 1-7 after NK cell infusion.

Results and discussion

3D bioreactor adaptation and iPSC-NK differentiation protocol

As shown in fig. 1, which summarizes the 3D directed in vitro differentiation protocol of NK cells derived from human induced pluripotent stem cells (hipscs), after successful 3D-hiPSC adaptation, the induction of hiPSC differentiation into hematopoietic differentiation can begin, which takes about 12 days, after which the 3D culture is transferred to NK cell differentiation conditions. After two weeks, 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 2D monolayers to 3D small scale bioreactor cell culture systems by adding Y27632 to enhance iPSC viability and improve spheroid formation.

hipscs were grown as 2D monolayer cultures to 30 x 10 ⁶ Initial concentration of individual living cells. These undifferentiated hipscs were isolated with TrypLE and then filtered through a 70 μm cell filter to obtain single cell suspensions. 3D adaptation of hipscs from 2D monolayer cultures was accomplished using a mini-bioreactor. FIG. 2A is a low magnification (10 x) micrograph of hiPSC in 2D monolayer culture. FIG. 2B is a photograph of a 30ml mini-bioreactor flask. Fig. 2C is a micrograph of hiPSC spheres after 2D to 3D adaptation for 20 hours.

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

Importantly, the method of hiPSC-NK differentiation has been described using adherent 2D monolayer cultures. The 3D differentiation protocol of hiPSC-NK differentiation described herein was compared to the traditional approach of adherent 2D.

In comparing the differentiation potential between 2D-monolayers and hiPSC-NK cells produced by 3D-bioreactors, NK cells were observed and identified in the medium suspension almost 10 days faster than in 2D-controls (FIGS. 3A-H). FIGS. 3A-D are images (10X) of adherent 2D-derived cell spheres and derivatives at designated time points after the start of NK differentiation. On day 23 of differentiation, premature NK-like cells were observed to slowly flatten out the spheres over time in the cell culture medium. FIGS. 3E-H are images of cell spheres and derivatives generated by a 3D bioreactor. Immature NK-like cells were observed two days after the start of NK differentiation on day 14.

In addition, 3D bioreactor derived NK cells showed better maturation and development. Flow cytometry quantification indicated the relevant temporal pattern of expression of hematopoietic stem cell progenitor markers CD34 (FIG. 4A) and NK cell markers CD45 and CD56 (FIGS. 4B-F). The expression level of CD56 (a marker of mature NK cells) indicated that the positive rate of 3D bioreactor-derived cells exceeded 93% on day 27 of differentiation, in contrast to 58% on day 27 of differentiation for 2D-generated cultures (fig. 5A-F and 5). Comparison of CD56 expression levels over time indicated that bioreactor-derived cells had higher levels of CD56 expression at all time points tested compared to similar stage 2D samples (fig. 5).

To further evaluate the stability and time of cell differentiation between 3D bioreactor and 2D monolayer platform, the expression levels of NK lineage specific markers CD94, NKp44, NKG2D, lampl and CD16 were measured. The expression levels of these NK lineage markers of the bioreactor-generated cells at day 27 differentiated were comparable or significantly exceeded the expression levels of two independent 2D differentiation rounds of culture time nearly doubled (53 days and 55 days, respectively) (fig. 6 and table 1).

Table 1.2D compares differential expression of lineage specific surface markers with 3D differentiation methods.

As an additional comparison, 3D bioreactor-derived hiPSC-NK cells were compared with primary cord blood-derived NK (CBNK) cells (7A-J). hiPSC-NK cells expressed similar levels of mature NK markers CD56, CD16, CD94, NKG2D, NKp44 and LAMP1 compared to primary CBNK cells. Culturing hipscs in a 3D bioreactor using the methods and media described herein can produce high purity and phenotypically mature cells similar to primary NK cells.

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

While it is important that NK cells express appropriate maturation markers, the gold standard to verify these cell phenotypes is by assessing the functional killing capacity of the final NK product. Functional analysis of hiPSC-derived NK cells was assessed by in vitro killing assays against Jurkat (fig. 8A), heLa (fig. 8B) and K562 (fig. 8C) cancer cells, which were assayed for viable cell analysis using Incucyte Base Analysis immune killing software. Target cancer cells were incubated with hiPSC-NK cells at different concentrations at 1:1 and 4:1 effective target ratios (E: T). For additional comparison, a similar killing assay was performed to compare the functionality of hiPSC-NK with primary CBNK at a E:T ratio of 1:2. Importantly, hiPSC-NK performed comparable to if not better than CBNK in K562 killing (fig. 8D). Thus, NK cells developed using this 3D bioreactor platform exhibit mature NK cell lineage characteristics and potent cytotoxicity.

Virus transduction of pre-differentiated hiPSC-NK amplified under non-trophoblast conditions

One method of introducing genetic modifications into iPSC-NK is by transduction of iPSC followed by selection of single cell clones to create stable iPSC lines. However, this approach 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 introduce lentiviral and retroviral constructs into pre-differentiated hiPSC-NK cells.

Genetic modifications of NK cells may be made to improve their function, target specific antigens, and/or enhance persistence in vivo. One application in NK therapy development is the integration of different Chimeric Antigen Receptor (CAR) constructs. The viral modification of NK cells is relatively difficult compared to T cells, preventing the introduction of CAR constructs into NK cells. NK transduction using retroviral vectors has become more common. Since retrovirus specifically infects proliferating cells, a great deal of effort has been made to enhance NK proliferation as a means of improving transduction efficiency. Lentiviral vectors are advantageous over retroviruses because they reduce the likelihood of genome insertion and potential off-target effects. However, transduction of NK cells with lentiviral vectors is more challenging than transduction with retroviral vectors.

Differentiated, mature hiPSC-NK cells as well as primary peripheral blood derived NK (PBNK) cells were transduced with lentiviruses (FIGS. 9A-J) or retroviruses (FIGS. 10A-J) using the protocols described herein. hiPSC-NK cells can be transduced with retroviral and lentiviral-based vectors to express CD19-CAR constructs with high affinity. In contrast, primary PBNK cells exhibit sustained resistance to lentiviral transduction. A schematic of the construct is shown in FIG. 14.

Functional in vitro killing ability of CAR19 transduced pre-differentiated hiPSC-NK cells

The 3D bioreactor derived hiPSC-NK was used to transduce retroviral constructs expressing CAR19 as well as IL-15 overexpression cassettes using the transduction methods described herein. The final CAR19 expression was approximately 47%. In vitro killing assays against CD19+ Raji cells (FIG. 11A), CD19+ NALM6 cells (FIG. 11B) and CD19-CCRF cells (FIG. 11C) were performed on non-transduced (NT) or CAR19 hiPSC-NK, live cell analysis was performed using immune cell killing software of Incucyte. Target cancer cells were incubated with hiPSC-NK cells or CAR19-hiPSC NK cells at different concentrations at an effective target ratio of 2:1. Importantly, CAR19 transduced hiPSC-NK cells exhibited enhanced killing efficiency against cancer cell lines expressing CD19 targets.

Lentiviral transduction of undifferentiated hipscs

The preferred method of introducing genetic modifications into iPSC-NK is by transduction of iPSC, followed by selection of single cell clones to create stable iPSC lines. The stable introduction of the CAR construct at the iPSC stage allows for the continued presence of this genetic modification in all differentiated progenitor cells, ultimately achieving 100% CAR transduction efficiency. Importantly, some of the genetic modifications induced during the iPSC phase may result in impaired differentiation into the desired cell type. As a proof of principle that genetic modification at the iPSC stage did not impair 3D bioreactor mediated NK differentiation, lentiviral transduction was performed on iPSC colonies, which were subsequently amplified into undifferentiated ipscs for subsequent differentiation within a closed bioreactor system (fig. 12A-C). Single colonies from iPSC cultures were selected and re-plated onto new vitronectin substrates in the presence of concentrated lentiviruses expressing CAR19 constructs (fig. 12A). After 2 days of culture in differentiation medium, CAR19 expression can be observed in the removed colonies under fluorescent imaging. As selected colonies proliferate over time, expansion of CAR19 expressing cells can be observed, as indicated by binding of CD19-GFP protein to ipscs that successfully infected CAR 19. The CAR construct was retained after multiple rounds of symmetric cell division (fig. 12B-C). Without wishing to be bound by theory, expression of the CAR construct varies due to methylation of non-pluripotent genes due to epigenetic silencing of the introduced construct that occurs naturally during pluripotency. After 5 days of culture in differentiation medium, the selected colonies expressing CAR19 were able to proliferate and maintain expression in vitro. Using this approach, it was shown that most CAR19 transduced iPSC offspring expressed the induced CAR19 construct.

3D bioreactor-based lenti-CAR19 hiPSC differentiation

After CAR 19-hipscs were generated by single colony transduction, the untransduced (NT) and CAR19 hiPSC lines were induced to differentiate into NK cells using a 3D bioreactor protocol. This method allows continuous monitoring of the response of the genetically modified hiPSC lines to previously established 3D bioreactor differentiation protocols. Thus, separate bioreactors were used to distinguish between NT and CAR19 hiPSC lines. On day 12, there was no change in hematopoietic differentiation between NT and CAR19 hiPSC lines as determined by CD34 and CD45 expression (fig. 13A). The NT hiPSC cell line showed 15% CD34 positive expression (fig. 13A, middle panel), in contrast to 12% in CAR 19S 001 modified hiPSC (fig. 13A, right panel). The percentage of CD45 positivity in both cell lines was <1%. CAR19 expression of CAR19 transduced hiPSC lines was analyzed (fig. 13B), demonstrating expression of CD 19-CARs in differentiated spheres in a 3D bioreactor.

Post-differentiation amplification of iPSC-NK

In contrast to other known methods, the methods described herein are performed under xeno-free and trophoblast-free conditions. Thus, the final derived cells meet GMP standards and can be used in clinical trials and the like. A small (30 ml) bioreactor may be produced >10x10 ⁷ Up to 50x10 ⁷ NK cells total number of (a). This improvement in hiPSC to NK cell differentiation process opens the opportunity to expand enough higher cells to meet the stringent requirements required for conducting human clinical trials. In addition, the methods described herein are effective and cost-effective for generating phenotypically mature, functional, clinical-grade NK cells for the development of cancer immunotherapy.

Phenotypic characterization and killing efficacy of 3D bioreactor derived Embryonic Stem Cells (ESC) -NK cells

Embryonic Stem Cells (ESCs) also produced NK cells with a relative efficiency similar to iPSC following the differentiation protocol in the 3D bioreactor platform system described previously. Purified NK cell populations expressing mature NK lineage markers in suspension were collected from bioreactor medium (fig. 17A-D). These ESC-NK cells showed strong in vitro killing of HUH7, HEPG2 and HEP3B cancer cell lines compared to PBNK controls (FIGS. 18A-F). ESC-NK cells genetically modified to express CAR (ESC-NK CAR) showed a greater degree of reduction in HUH7, HEPG2 and HEP3B cancer cells compared to untransformed ESC-NK cells (ESC-NK NT).

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

To assess the in vivo killing efficacy of iPSC-derived NK cells, cryopreserved hiPSC-NK cells were thawed and prepared on the same day for infusion into K562 xenograft mice. K562 cancer cells were subcutaneously injected and implanted for three days, followed by intravenous injection of a single dose of hiPSC-NK cells. Bioluminescence imaging four days after NK injection showed significant tumor regression in hiPSC-NK mice compared to PBNK and PBS control groups (fig. 15A-B). In addition, hiPSC-NK-injected mice exhibited slower tumor growth compared to PBNK and PBS control, as shown by luciferase expression (fig. 15B). Importantly, this demonstrates that hiPSC-NK cells derived using the 3D bioreactor system are powerful and capable of exerting short-term anti-tumor efficacy in vivo.

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

To assess the persistence of hiPSC-NK in vivo, differentiated hiPSC-NK cells were transduced with retroviral constructs expressing luciferase alone or expressing luciferase+secreted IL15 using the methods described herein. By tail vein injection (2 x 10) ⁶ Mice) were infused intravenously with transduced hiPSC-NK cells and then tracked over time using bioluminescence imaging. These genetically modified hiPSC-NK cells lasted up to two weeks in vivo (FIGS. 16A-B). Those hiPSC-NK cells transduced with IL-15 showed enhanced in vivo persistence. Importantly, hiPSC-NK infusions had no significant effect on mice.

Reference to the literature

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Incorporation by reference and equivalent schemes

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

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

1. A method for preparing 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;

b) culturing the 3D culture of pluripotent stem cells in a 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 generating a three-dimensional culture of pluripotent stem cells at a cell density of 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 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.

10. The method of claim 9, wherein the ROCK inhibitor is Y27632.

11. The method of claim 1, wherein the 3D bioreactor is a scaffold-free bioreactor.

12. The method of claim 11, wherein the scaffold-free bioreactor is a rotating bioreactor.

13. The method of claim 12, wherein the rotary bioreactor rotates at a speed of 60 to 80 revolutions per minute.

14. The method of claim 1, wherein the hematopoietic stem cells are CD34+.

15. The method of claim 1, wherein the concentration of stem cell factor is about 5 ng/ml to about 100 ng/ml.

16. The method of claim 1, wherein the concentration of BMP4 is about 5 ng/ml to about 100 ng/ml.

17. The method of claim 1, wherein the concentration of VEGF is about 5 ng/ml to about 100 ng/ml.

18. The method of claim 1, wherein the second culture medium further comprises: iv) albumin polyvinyl alcohol essential lipids (APEL).

19. The method according to any one of claims 1 to 18, further comprising:

c) culturing hematopoietic stem cells in a 3D bioreactor in a third cell culture medium to generate natural killer (NK) cells.

20. The method of claim 19, further comprising transducing NK cells with a lentiviral vector.

21. The method of claim 20, wherein the lentiviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor.

22. The method of claim 21, wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular activation domain.

23. The method of claim 22, wherein the extracellular antigen recognition domain is a single chain fragment variant (scFv) derived from an antibody.

24. The method of claim 22, wherein the intracellular activation domain is the CD3 zeta domain.

25. The method of claim 22, wherein the chimeric antigen receptor further comprises a co-stimulatory domain.

26. The method of claim 25, wherein the co-stimulatory domain is a 4-1BB domain.

27. The method of claim 20, wherein transducing NK cells with a lentiviral vector comprises culturing with retronectin.

28. The method of claim 19, further comprising quantifying cells expressing CD56, and cryopreserving the NK cells when at least 75% of the cells express CD56.

29. The method of any one of claims 19 to 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).

30. The method of claim 29, wherein the concentration of 2-BME is about 5 μM to about 100 μM.

31. The method of claim 29, wherein the concentration of sodium selenite is from about 0.5 ng/ml to about 50 ng/ml.

32. The method of claim 29, wherein the concentration of ascorbic acid is from about 2 μg/ml to about 200 μg/ml.

33. The method of claim 29, wherein the concentration of IL-3 is about 0.5 ng/ml to about 50 ng/ml.

34. The method of claim 29, wherein the concentration of IL-7 is about 2 ng/ml to about 200 ng/ml.

35. The method of claim 29, wherein the concentration of IL-15 is about 2 ng/ml to about 200 ng/ml.

36. The method of claim 29, wherein the concentration of FLT3L is about 2 ng/ml to about 200 ng/ml.

37. The method of claim 29, wherein the concentration of stem cell factor is about 2 ng/ml to about 200 ng/ml.

38. The method of claim 29, wherein the third cell culture medium further comprises ethanolamine.

39. The method of claim 38, wherein the concentration of ethanolamine is about 5 μM to about 500 μM.

40. 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).

41. The cell culture medium of claim 40, wherein the cell culture medium further comprises: d) albumin polyvinyl alcohol essential lipid (APEL).