CN115261318A - Method for producing natural killer cells - Google Patents
Method for producing natural killer cells Download PDFInfo
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- CN115261318A CN115261318A CN202211033858.9A CN202211033858A CN115261318A CN 115261318 A CN115261318 A CN 115261318A CN 202211033858 A CN202211033858 A CN 202211033858A CN 115261318 A CN115261318 A CN 115261318A
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Abstract
The present invention relates to a method of producing Natural Killer (NK) cells, the method comprising culturing Pluripotent Stem Cells (PSCs) to produce embryoid bodies, differentiating the embryoid bodies in a differentiation medium comprising an NK differentiation factor to promote differentiation into NK cells; expanding the cells in an expansion medium comprising IL-2 to produce NK cells. The invention also relates to NK cells produced by the method, pharmaceutical compositions comprising NK cells produced by the method, and methods and uses of the NK cells or NK cell populations in the treatment of disease.
Description
Technical Field
The present invention relates to a method of generating natural killer cells from pluripotent stem cells, natural Killer (NK) cells generated by the method, and a pharmaceutical composition comprising the NK cells generated by the method. The invention also relates to methods and uses of NK cells produced by the methods.
Background
Cell therapy or therapy using cell administration such as stem cells and immune cells is increasingly used to treat diseases such as cancer and infectious diseases. An example of an immune cell used in modern cell therapy is a natural killer cell or NK cell. The NK cell is CD3 - CD56 + Innate lymphocytes, classified as type I Innate Lymphocytes (ILC). NK cells account for 5-20% of all circulating lymphocytes in humans and play a crucial role in host defense against infectious agents and malignant tumors. NK cells have been shown to be resistant to viruses or other intracellular pathogens and to inhibit tumor formation. NK cells can kill transformed cells without prior antigen priming, and their cytotoxicity is not limited by the expression of target cell Major Histocompatibility Complex (MHC) molecules.
NK cell activity is regulated by a wide range of stimulatory and inhibitory receptors, and the complex integration of positive and negative signals of these receptors determines the final configuration of NK cell innate killing capacity. NK cells recognize transformed or infected cells through these receptors. Changes in surface marker expression occur in transformed or infected cells, including down-regulation of MHC class I molecules or up-regulation of stress-induced molecules such as MICA/MICB and ULBP. NK cells recognize these molecules by activating receptors such as NKG2D and stimulate NK cells to kill these transformed or infected cells.
NK cells exert a killing effect through a variety of mechanisms. NK cells have been shown to deliver lytic particles and pore-forming proteins directly, including granzymes and perforins. NK cells have also been shown to release cytokines such as IFN-. Gamma.TNF-. Alpha.and chemokines (CCL 3, CCL4 and CCL 5), which form innate and adaptive immune responses. Another pathway utilized by NK cells is the upregulation of FASL and TNF-related apoptosis-inducing ligand (TRAIL) upon NK cell activation. NK cells also express CD16 and recognize the Fc portion of the antibody that binds to tumor cells and triggers antibody-dependent cellular cytotoxicity (ADCC).
Adoptive transfer of allogeneic NK cells has shown clinical benefit in patients with advanced cancer without causing serious adverse side effects, such as graft versus host disease (GvHD) or Cytokine Release Syndrome (CRS). However, there are inherent limitations in the number of NK cells isolated from patients by apheresis, and there are significant differences in the number and quality of NK cells between donors, which makes treatment with multiple doses of cells extremely challenging.
Recently, pluripotent Stem Cells (PSCs) have been used to stably supply multiple cell lineages, including NK cells. NK cells differentiated from PSCs exhibit similar functions to conventional NK cells isolated from patients and have a wide range of cytotoxic activities against hematological and solid tumors. Therefore, the differentiation of NK cells represents a promising new approach to adoptive NK cell immunotherapy. It is expected that NK cells may be provided as off-the-shelf cell therapy, possibly in combination with antibodies directed against inhibitory checkpoint receptors to enhance anti-tumor responses. However, challenges remain with developing NK differentiation protocols that can be scaled up to consistently and reliably produce high yields and turnover rates of NK cells. Another challenge is the inter-culture variability associated with NK cell properties such as the innate killing capacity of NK cells. Thus, there is a need for improved and scalable methods for generating NK cells.
Any publications mentioned in this specification are herein incorporated by reference. However, if any publication is referred to herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in australia or in any other country/region.
Disclosure of Invention
The present inventors have found an improved method for differentiating pluripotent stem cells into natural killer cells in a manner that retains or improves NK cell innate killing ability. This method also allows scaling up NK differentiation to Good Manufacturing Practice (GMP) scale and gains in efficiency compared to methods known in the art.
In a first aspect, the present invention provides a method of generating Natural Killer (NK) cells, the method comprising:
a) Culturing Pluripotent Stem Cells (PSCs) to produce embryoid bodies;
b) Differentiating the embryoid bodies in a differentiation medium comprising an NK differentiation factor to promote differentiation into NK cells; and
c) Expanding the cells of b) in an expansion medium comprising IL-2 to produce NK cells.
The present inventors propose that the inclusion of NK differentiation factors allows for improved efficiency and reliability of NK differentiation. Advantageously, the NK cells produced by this method also exhibit improved properties compared to NK cells produced by differentiation protocols known in the art. In one embodiment, the NK differentiation factor is interleukin-2 (IL-2).
Thus, in one embodiment, the NK cells of the present disclosure express one or more of: CD45, CD56, CD16, killer immunoglobulin-like receptor (KIR), NKG2D, NKp44, NKp46, fas ligand (FasL), and Tumor Necrosis Factor (TNF) -associated apoptosis-inducing ligand (TRAIL).
In another embodiment, the NK cells of the present disclosure exhibit increased expression of one or more of the following relative to a reference NK cell: CD45, CD56, CD16, KIR, NKG2D, NKp44, NKp46, fasL and TRAIL, the reference NK cells differentiated according to the method of the invention except as follows: the differentiation medium in b) comprises NK differentiation factors and/or the expansion medium in c) comprises IL-2.
In one embodiment, the expansion of NK cells of the present disclosure may be performed with a feeder cell line. In a preferred embodiment, the feeder cell line is the K562 myeloid leukemia cell line. In a further preferred embodiment, the K562 myeloid leukemia cell line expresses membrane bound IL-21.
In a second aspect, the invention provides NK cells produced by the method of the first aspect.
In a third aspect, the present invention provides a pharmaceutical composition comprising the NK cell of the second aspect.
In a fourth aspect, the invention provides a method of immunotherapy comprising administering to an individual the NK cells of the second aspect or the pharmaceutical composition of the third aspect.
In a fifth aspect, the invention provides the use of an NK cell of the second aspect in the manufacture of a medicament for immunotherapy of an individual.
In embodiments of the fourth and fifth aspects, the subject has cancer or an infectious disease, such as Human Immunodeficiency Virus (HIV), epstein-barr virus (EBV), herpes Simplex Virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), hepatitis B Virus (HBV), and Hepatitis C Virus (HCV).
Drawings
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings. The NK cells of the present disclosure are identified in the figures as "iCamuno NK cells". The reference NK cells of the present disclosure are identified in the figures as "conventional NK cells".
Fig. 1 includes micrographs of the differentiation of ipscs of example 2 Into NK (iNK) cells. Hematopoietic embryoid bodies (HEEB) were generated from human ipscs and subsequently differentiated into NK cells, comparing NK cells generated by the conventional method with NK cells generated by the method of the present invention. Scale bar =100 μm.
FIG. 2 shows the expression levels of key NK cell markers and contains histograms showing the group assessment of CD45, CD56, CD16, KIR2DL1, KIR2DS2, NKG2D, NKp44, NKp46, fasL and TRAIL. The x-axis illustrates expression levels relative to GAPDH and compares the following groups: ipscs, conventional NK cells, and NK cells produced by the methods of the invention. The expression level of key NK cell markers in NK cells produced by the method of the invention is higher or comparable to that of conventional NK cells.
Figure 3 shows the expression of CD45 and CD56 surface markers, including comparing the flow cytometry plots and histograms of the following groups: ipscs, conventional NK cells, and NK cells produced by the methods of the invention. Flow cytometry plots show that a higher proportion of NK cells produced by the methods of the invention were positive for both CD45 and CD56 (80.3% and 81.1% for NK cells produced by the methods; compared to 46.8% and 49.8% for conventional NK cells).
Figure 4 includes fluorescence micrographs assessing cytotoxicity against K562 myeloid leukemia cell line. These images are time course images comparing 14h cytotoxic co-culture assays of the following groups: ipscs, conventional NK cells, and NK cells produced by the methods of the invention. K562 myeloid leukemia cell line was labeled with mCherry at an effector to target (E: T) ratio of 5. The bottom right panel shows that NK cells produced by the method of the invention show improved cytotoxicity against K562 myeloid leukemia cell line, cell death is evident starting from the 5 hour time point, at the 14 hour time point almost all K562 cells undergo apoptosis. In contrast, the image in the lower left corner shows that in co-culture of conventional NK cells and K562 cells, cell death was only evident near the 9 hour time point, while a higher number of K562 cells were still viable at the 14 hour time point. The uppermost panel shows that co-culture of iPSC and K562 cells did not result in significant apoptosis and illustrates that co-culture of NK cells (whether normal NK cells or NK cells produced by the method of the invention) and K562 cells resulted in cell death.
Figure 5 shows a flow cytometry analysis performed after cytotoxicity assays, including flow cytometry plots comparing mCherry expression for the following groups: ipscs, conventional NK cells, and NK cells produced by the methods of the invention. From left to right, the seeding ratio of NK cells to K562 myeloid leukemia cells increased from 1, 2.5. The top row shows that co-culture of iPSC and K562 cells did not result in significant cell death, and the expression level readings for mCherry were 92.0% (for the ratio of 1. The middle row shows that conventional NK cells are moderately cytotoxic to K562 cells, with mCherry's expression level readings of 49.0% (for a ratio of 1. The bottom row shows that NK cells produced by the method of the invention showed increased cytotoxicity to K562 cells, with mCherry's expression level readings of 67.0% (ratio to 1), 35.8% (ratio to 2.5.
Detailed Description
The present inventors have identified improved methods for generating Natural Killer (NK) cells, in particular methods for improving differentiation and/or expansion of Pluripotent Stem Cells (PSCs). Surprisingly, the improved method provides a highly scalable protocol for the generation of NK cells. Furthermore, the present inventors have also determined a method of producing NK cells with improved functional properties relative to conventional NK cells.
The present inventors utilized the inclusion of cell culture supplements at the initial stage of differentiation to enhance the single cell viability of PSCs differentiating into NK cells. Furthermore, the present inventors have found that the inclusion of NK differentiation factors improves the efficiency of differentiation of PSCs into NK cells.
The present inventors studied the use of NK differentiation factors to improve the efficiency of differentiation of PSCs into NK cells based on principal component analysis performed on gene expression data sets. Differential expression analysis between NK cells and non-immune cells revealed that certain NK differentiation factors are key receptors, which interact with key transcription factors uniquely expressed in NK cells. Based on this information, the present inventors developed methods for producing NK cells using the inclusion of NK differentiation factors during certain time points of the differentiation process to increase the differentiation efficiency. Surprisingly, the inventors found that NK cells produced by the methods described herein exhibit improved expression of key NK cell markers. In addition, NK cells produced by the methods described herein exhibit improved cytotoxicity against leukemia cells during co-culture.
Accordingly, disclosed herein is a method for promoting differentiation into NK cells to produce NK cells using culturing pluripotent stem cells in a differentiation medium containing an NK differentiation factor.
NK cells are associated with several key expression markers, such as CD45 and CD56. Expression of these NK cell markers is thought to correlate with and reflect the maturity and functional properties of these NK cells. NK cells also express a number of inhibitory and activating cell surface receptors that control the state of NK activation. These receptors include killer immunoglobulin-like receptors (KIR) and Killer Activated Receptors (KAR). KIRs recognize "host" molecules, pairing with Human Leukocyte Antigen (HLA) class I molecules to inhibit NK activation, thus allowing tolerance and protection of host cells from NK cell attack. On the other hand, neoplastic or viral-infectious cells express ligands for KAR that are up-regulated in level, triggering NK cell activation and thus triggering cytotoxicity of these diseased cells.
NK cell therapy using NK cells extracted from donors faces many frustrations, including a limited supply of NK cells. To this end, researchers turned to the use of Pluripotent Stem Cells (PSC) as a source of NK cells. PSCs are capable of self-renewal indefinitely and are therefore attractive for use in cell therapy. Some examples of PSCs include human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs). Because of their pluripotency, hescs and ipscs are ideal starting cell types, allowing the development of a variety of cell lineages, including NK cells.
Studies have shown that hESC-/iPSC-derived NK cells exhibit potent antitumor and antiviral activity, suggesting that hescs and ipscs may provide potential solutions for a substantially unlimited source of cell-based standardized therapies for these diseases. Some examples of disease conditions that may benefit from NK cell administration include cancer and infectious diseases such as Human Immunodeficiency Virus (HIV), epstein-barr virus (EBV), herpes Simplex Virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), hepatitis B Virus (HBV) and Hepatitis C Virus (HCV).
NK cell therapy may be administered by a variety of means, such as by the Intravenous (IV) route, or intraperitoneally near the site of disease. In addition, NK cells can be used with immunotherapy, such as antibody cancer therapy, for antibody-dependent cellular cytotoxicity (ADCC).
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs by reference to the disclosure.
It should be noted that the term "a" or "an" refers to one or more than one, e.g., "a molecule" should be understood to mean one or more than one. Thus, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein.
In the claims which follow and in the description of the invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
The term "about" as used herein encompasses a range of values of ± 25% of the amplitude of a given numerical value. In other embodiments, the term "about" encompasses a range of values of ± 20%, ± 15%, ± 10% or ± 5% of the amplitude of the given value. For example, in one embodiment, "about 3 grams" means a value of 2.7 to 3.3 grams (i.e., 3 grams ± 10%), etc.
Similarly, while the method of generating NK cells comprises ordered, sequential events, the timing of the events may vary, for example, by at least 25%. For example, while a particular step may be disclosed as lasting for one day in one embodiment, the event may last for more or less than one day. For example, "a day" may include a period of about 18 to about 30 hours. In other embodiments, the time period may vary by ± 20%, ± 15%, ± 10% or ± 5% of the time period. The time period indicated as multiple days may be a multiple of "one day," e.g., two days may span a time period of about 36 hours to about 60 hours, etc. In another embodiment, the time variation may be reduced, for example, day 2 is 48 ± 3 hours from day 0; day 4 was 96. + -. 3 hours from day 0, and day 5 was 120. + -. 3 hours from day 0.
As used herein, "pluripotent stem cell" or "PSC" refers to a cell that is capable of replicating itself indefinitely and differentiating into all cells that form part of a tissue or organ or any of the three germ layers (endoderm, mesoderm, or ectoderm). There are two main types of pluripotent stem cells: embryonic Stem Cells (ESC) and Induced Pluripotent Stem Cells (iPSC).
As used herein, "embryonic stem cells" or "ESCs" refer to cells isolated from day 5-7 embryos from patients who have completed in vitro fertilization therapy and have had a remaining embryo donation with consent. The use of ESCs has been hampered to some extent by ethical issues in the extraction of cells from human embryos.
Suitable human PSCs include H1 and H9 human embryonic stem cells.
As used herein, "induced pluripotent stem cells" or "ipscs" refer to ESC-like cells derived from adult cells. ipscs have very similar characteristics to ESCs, but avoid the ethical issues associated with ESCs because ipscs are not derived from embryos. In contrast, ipscs are typically derived from fully differentiated adult cells that have been "reprogrammed" back to a pluripotent state. This reprogramming step typically involves the use of reprogramming factors at specific time intervals and at certain concentrations. Some exemplary methods of reprogramming adult cells back to a pluripotent state involve the use of RNA, proteins, or other small molecules administered to an adult cell culture. Alternatively, human iPSC lines are also commercially available.
Suitable human iPSCs include, but are not limited to, iPSC 19-9-7T, MIRJT6i-mND1-4, and MIRJT7i-mND2-0 derived from fibroblasts and iPSC BM119-9 derived from bone marrow mononuclear cells. Other suitable ipscs are available from Cellular Dynamics International, madison, wisconsin.
As used herein, NK cells differentiated from ipscs may be referred to as "iNK" cells.
As used herein, "differentiation" refers to the process of a cell changing from one cell type to another, particularly a less specialized type of cell to a more specialized type of cell.
As used herein, "medium" or plural thereof "medium" refers to a liquid or gel designed to support the growth of cells.
Differentiation of PSCs into NK cells is typically performed under controlled conditions, particularly when the NK cells produced are intended to be administered to human individuals in accordance with Good Manufacturing Practice (GMP). The initial step of the process involves culturing PSCs in culture, for example, on tissue culture plates or dishes, or in bioreactors. The use of bioreactors is particularly attractive in view of the ability to scale up NK cell production at the clinical level for adoptive transfer. However, protocols using tissue culture plates or dishes may also be scaled up appropriately for adoptive NK cell transfer.
The PSC is cultured in a specific medium. Suitable basal media include, but are not limited to, iscove's Modified Dulbecco's Medium/F12 (IMDM/F12), teSR1 basal Medium without FGF2 and TGF-beta (mTeSR 1) TM Basal medium, stem Cell Technologies); DF4S basal Medium, namely Essential 8 TM Media (Life Technologies; also known as "E8" media). The cell culture medium can be supplemented with other growth factors to improve the cloning efficiency and single cell survival of the PSC. An exemplary supplement that may be used is CloneR (Stem Cell Technologies). Once the PSC reaches the desired degree of confluence, the cells can be harvested and seeded and plated to form embryoid bodies.
As used herein, "embryoid body" (EB) refers to a floating three-dimensional aggregate consisting of PSCs. The EBs include hematopoietic EBs, which are EBs capable of forming endothelial progenitor cells and blood progenitor cells. Various methods of generating EBs are known in the art. For example, conventional methods typically involve generating a single cell suspension of PSCs. The PSCs can then be cultured in uncoated, non-tissue culture treated culture dishes or microwells to prevent PSCs from attaching, thereby promoting cell aggregation while maintaining suspension. Alternatively, PSCs can be cultured in low attachment dishes or microwells. Newer methods of EB formation involve the use of spin-focusing or bioreactors, thereby improving efficiency and process control. ROCK inhibitors, such as Y-27632, have also been shown to promote PSC aggregation, leading to the formation of EBs. EBs have teratoma-like structures similar to the developing embryo, and the formation of EBs is a common platform for the establishment of differentiation into cells (e.g., NK cells) from any of the three germ layers.
In some embodiments, at about 10,000 cells/cm 2 To about 40,000 cells/cm 2 Initial density of (2), e.g. 10,000 cells/cm 2 15,000 cells/cm 2 20,000 cells/cm 2 25,000 cells/cm 2 30,000 cells/cm 2 35,000 cells/cm 2 Or 40,000 cells/cm 2 The PSCs were plated to generate EBs.
Once EBs are formed, EBs are cultured in differentiation media including NK differentiation factors to promote differentiation into NK cells. The number of EBs used depends on the type of tissue culture plate, tissue culture dish, or bioreactor used. For example, if a 6-well plate is used, about 10-30 EB plates can be plated. However, one skilled in the art will appreciate that methods of determining the optimal number of EBs to be plated are known in the art, depending on the surface area of the tissue culture plate, tissue culture dish, or bioreactor used.
In some embodiments, the differentiation medium comprises a basal medium, e.g., DMEM, GLUTAMAX TM Human serum, L-glutamine, penicillin-streptomycin, β -mercaptoethanol, sodium selenite (except for any present in the basal medium), ethanolamine, and ascorbic acid.
In some embodiments, the NK differentiation factor is interleukin-2 (IL-2).
In some embodiments, the differentiation medium further comprises one or more of: interleukin-3 (IL-3), SCF, interleukin-7 (IL-7), interleukin-15 (IL-15), FLT3 ligand (FLT 3L), and NK differentiation factor.
As used herein, "differentiation factor" refers to a molecule included in a differentiation medium that promotes differentiation, particularly differentiation of PSCs into NK cells. However, for the purposes of this disclosure, a differentiation factor is not interleukin-3 (IL-3), SCF, interleukin-7 (IL-7), interleukin-15 (IL-15), or FLT3 ligand (FLT 3L).
Although the media disclosed herein may include specific components (e.g., morphogens, small molecules, and hematopoietic cytokines), it is contemplated that other components having the same, equivalent, or similar properties may be used in addition to or in place of those disclosed, as is known in the art.
As used herein, "differentiation medium" refers to a medium designed to support cell differentiation, i.e., the process of supporting the transition of a cell from one cell type to another. According to the method of the present invention, the differentiation medium is used to support the process of converting human PSCs into NK cells.
In some embodiments, the concentration of IL-2 in the differentiation medium is about 1-150U/mL; the concentration of IL-3 in the differentiation medium is about 1-15ng/mL; the concentration of FLT3L in the differentiation medium is about 1-30ng/mL; and/or the concentration of IL-15 in the differentiation medium is about 1-30ng/mL.
In some embodiments, the concentration of IL-2 in the differentiation medium is about 10U/mL, about 15U/mL, about 20U/mL, about 25U/mL, about 30U/mL, about 35U/mL, about 40U/mL, about 45U/mL, about 50U/mL, about 60U/mL, about 70U/mL, about 80U/mL, about 90U/mL, about 100U/mL, about 110U/mL, about 120U/mL, about 130U/mL, about 140U/mL, or about 150U/mL.
In some embodiments, the concentration of IL-2 in the amplification medium is about 10U/mL, about 15U/mL, about 20U/mL, about 25U/mL, about 30U/mL, about 35U/mL, about 40U/mL, about 45U/mL, about 50U/mL, about 60U/mL, about 70U/mL, about 80U/mL, about 90U/mL, about 100U/mL, about 110U/mL, about 120U/mL, about 130U/mL, about 140U/mL, or about 150U/mL.
In some embodiments, the concentration of IL-3 in the differentiation medium is about 1ng/mL, about 2ng/mL, about 3ng/mL, about 4ng/mL, about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, or about 15ng/mL.
In some embodiments, the concentration of FLT3L in the differentiation media is about 1ng/mL, about 2ng/mL, about 3ng/mL, about 4ng/mL, about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, or about 30ng/mL.
In some embodiments, the concentration of IL-15 in the differentiation medium is about 1ng/mL, about 2ng/mL, about 3ng/mL, about 4ng/mL, about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 10ng/mL, about 11ng/mL, about 12ng/mL, about 13ng/mL, about 14ng/mL, about 15ng/mL, about 16ng/mL, about 17ng/mL, about 18ng/mL, about 19ng/mL, about 20ng/mL, about 21ng/mL, about 22ng/mL, about 23ng/mL, about 24ng/mL, about 25ng/mL, about 26ng/mL, about 27ng/mL, about 28ng/mL, about 29ng/mL, or about 30ng/mL.
As referred to herein, the term "defined medium" means that the identity and quantity of each component of the medium is known.
In some embodiments, the differentiation or amplification medium may be xeno-free and incorporate human proteins isolated from natural sources, such as from the placenta or other human tissues, or may be produced using recombinant techniques. In some embodiments, all of the proteins described herein are human. In some embodiments, all of the proteins used in the differentiation or amplification medium are human proteins. In some embodiments, all of the proteins used in the differentiation or amplification medium are human proteins. In some embodiments, all of the proteins described herein are human recombinant proteins. In some embodiments, all of the proteins used in the differentiation or amplification medium are recombinant human proteins.
All of the proteins described herein are known to those of skill in the art, and most, if not all, of the proteins described herein are commercially available.
One skilled in the art will appreciate that the cell culture medium may be replaced at fixed or varying intervals, as the case may be. While the cells remain in culture, toxic metabolites may be produced, and the cultured cells continue to utilize nutrients, the amount of which steadily increases during the expansion phase. Thus, fresh cell culture medium can be used in place of old cell culture medium.
In some embodiments, the differentiation or expansion medium may be replaced about every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
Once the NK cell population becomes more mature, and/or a sufficient number of PSCs have differentiated into NK cells, more medium changes are required to replenish the growing and mature NK cell population. In some embodiments, the period of time is about 10-20 days. After this period, the differentiation or expansion medium may be replaced about every 1 day, about 2 days, or about 3 days.
The number of NK cells in culture can be determined by a variety of methods known in the art. For example, flow cytometry or microscopy may be used. If flow cytometry is used, the cell sample can be stained with markers commonly associated with NK cells, such as CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, and NKp80.
In some embodiments, supplementation of specific cytokines, chemokines, proteins, signaling factors, and growth factors in the cell culture medium occurs within a defined time interval before cessation of such supplementation.
In some embodiments, the supplementation of specific cytokines, chemokines, proteins, signaling factors, and growth factors in the cell culture medium is stopped after about 5-20 days. For example, supplementation with a particular factor may be discontinued on about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days later.
In some embodiments, replenishment of one or more of the following factors is stopped after a defined interval: IL-3, SCF, IL-7, IL-15, FLT3L, and IL-2.
After a period of time, expanded, floating NK cells with a specific spindle-like or elongated morphology begin to appear in the culture. In general, NK cells having spindle-shaped or elongated morphology were observed in about 15 to 50 days of culture. In some embodiments, NK cells having a spindle-like or elongated morphology are observed after about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days of culture.
Verification of NK cell markers and functional properties can be performed using a number of assays known in the art. Cell surface markers typically associated with NK cells include CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, and NKp80. Mature NK cells act through granule exocytosis and the release of cytotoxic proteins, cytokines and chemokines to induce targeted cell death. An exemplary mechanism by which NK cells mediate cell death is through interactions with the caspase enzymatic cascade, apoptosis signaling, and inflammatory signaling. Thus, the maturity and functional properties of NK cells, including cytotoxicity and tumor killing ability, can be measured by measuring the expression levels of proteins such as Fas ligand (FasL), tumor Necrosis Factor (TNF) - α, TNF-related apoptosis-inducing ligand (TRAIL), or other cytokines and chemokines. In addition, cell surface expression of certain markers is also a marker for improved NK cell homing properties. Changes in NK cell functional properties may be indicated by an increase or decrease in the expression level of certain markers or proteins. Furthermore, a change in NK cell functional properties may be indicated by an increase in certain markers or proteins and a concomitant decrease in other markers or proteins.
A suitable method for determining the cytotoxicity of NK cells produced by the method of the present invention is to co-culture NK cells and tumor cell lines as target cells, such as K562, LN-18, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1 or U266 tumor cells, and the like. The tumor cells can then be labeled, for example, with a fluorescent label specific for the tumor cells. Cytotoxicity during co-culture can then be assessed based on the reduction in expression of the fluorescent marker, which would indicate tumor cell apoptosis or cell death. Examples of labels suitable for tagging tumor cells include carboxyfluorescein succinimidyl ester (CFSE), mCherry, or any other suitable fluorescent label known in the art. Alternatively, apoptosis and dead cell populations can be further analyzed using flow cytometry, for example, by Annexin-V (Annexin V) or any other immobilizable viability dye staining. The co-culture of NK cells and tumor cells is usually performed at a specific effector to target ratio. In some embodiments, the ratio is about 1 to 10. In some embodiments, the ratio is about 1, 2, 1, 4. Methods and assays for determining ADCC are also known in the art, e.g., NK cells are incubated with target cells and a suitable effector antibody, e.g., NKp 46. ADCC assays and kits and reagents are also commercially available, for example Promega ADCC Bioassay.
The inventors have surprisingly found that NK cells produced by the present method exhibit increased expression of certain NK cell markers. Without being bound by theory, the present inventors hypothesize that an increase in expression of certain NK cell markers may be associated with an increase in functional properties, such as cytotoxicity against infectious or cancer cells. This may be related to one or more of the following: increased homing capacity to diseased cells, increased efficiency in mediating apoptosis or inflammatory cascades, increased target recognition or increased ADCC. Furthermore, recent studies have shown that NK cells may also play a role in adaptive immunity, including improved activation and response to subsequent secondary challenge to the same antigen. The present inventors hypothesized that an increase in NK cell markers might also play a role in mediating or complementing the ability of NK cells to play a role in adaptive immunity.
In some embodiments, the NK cell expresses one or more of: CD45, CD56, CD16, killer immunoglobulin-like receptor (KIR), NKG2D, NKp44, NKp46, fas ligand (FasL) and Tumor Necrosis Factor (TNF) -associated apoptosis-inducing ligand (TRAIL).
In some embodiments, the NK cell exhibits increased expression of one or more of the following relative to a reference NK cell: CD45, CD56, CD16, KIR, NKG2D, NKp44, NKp46, fasL and TRAIL, the reference NK cells were differentiated according to a method according to the first aspect except: the differentiation medium in b) comprises NK differentiation factors and/or the expansion medium in c) comprises IL-2.
In some embodiments, the NK cell exhibits an increase of about 1%, an increase of about 2%, an increase of about 3%, an increase of about 4%, an increase of about 5%, an increase of about 6%, an increase of about 7%, an increase of about 8%, an increase of about 9%, an increase of about 10%, an increase of about 20%, an increase of about 30%, an increase of about 40%, an increase of about 50%, an increase of about 60%, an increase of about 70%, an increase of about 80%, an increase of about 90%, an increase of about 100% or more. Alternatively, the increase in NK cell marker or protein may be about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or more increase in NK cell marker expression in NK cells or a population of NK cells produced according to the methods of the invention.
NK cell production by the methods of the invention may also be improved or enhanced by co-culturing NK cells with a feeder cell line. Feeder cells are thought to enhance NK cell differentiation and expansion either by interacting directly with NK cells or by releasing beneficial proteins and factors. Some examples of suitable feeder cell lines are K562 cells, epstein-Barr lymphoblast cells (Epstein-Barr lymphoblast cells), RPMI8866 cells, jurkat cells, hep3B cells, raji cells, MCF-7 cells and Ramos cells.
In some embodiments, the feeder cell line is a K562 myeloid leukemia cell line.
Feeder cell lines can be genetically engineered or altered to further improve their ability to increase the efficiency of NK cell differentiation and expansion. For example, feeder cell lines can be genetically engineered to alter the expression levels of specific membrane bound proteins or receptors.
In some embodiments, the K562 cell line expresses membrane bound IL-21.
In some embodiments, the co-culturing of NK cells and feeder cells is performed at a ratio of 5. In some embodiments, the ratio is about 5.
In some embodiments, the methods of the invention comprise culturing in step b) for a period of 5 to 20 days. In some embodiments, the method comprises b) culturing for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days.
In some embodiments, the method comprises culturing EBs in a medium that does not contain NK differentiation factors for a predetermined time interval prior to NK cell expansion. In some embodiments, the predetermined time interval is about 5-10 days. In some embodiments, the predetermined time interval is about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
In some embodiments, the method comprises expanding the NK cells for a period of about 10-50 days. In some embodiments, the method comprises c) amplifying for about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days.
As used herein, the term "pharmaceutical composition" refers to a composition comprising an NK cell or population of NK cells described herein that has been formulated for administration to an individual. Preferably, the pharmaceutical composition is sterile. In one embodiment, the pharmaceutical composition is pyrogen-free.
The NK cells or NK cell population will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the type of the particular disease being treated, the particular individual being treated, the clinical condition of the individual, the site of administration, the method of administration, the timing of administration, possible side effects and other factors known to the physician. Other considerations include maximizing NK cell cytotoxicity and persistence in vivo. The therapeutically effective amount of the NK cell or NK cell population to be administered will be determined by such considerations.
The NK cells or NK cell population can be administered to the individual by any suitable method, including Intravenous (IV) injection and subcutaneous injection. NK cells may also be administered with additional therapeutic agents such as antibodies or modifying agents intended to enhance innate NK cell properties or NK cell activity in vivo. NK cells may also be pretreated or primed with a modifying agent prior to administration to an individual to enhance innate NK cell characteristics or NK cell activity in vivo. Examples of suitable initiators or modifiers are IL-2 or IL-15. Other suitable protein constructs, agonists, antagonists, modulators or inflammatory factors are also contemplated.
NK cells can also be used for antibody cancer therapy, which targets tumor cells with antibodies such as monoclonal antibodies, due to effector properties in inducing ADCC. Indeed, NK cells are able to mediate antibody-dependent tumor killing by cytotoxic granule exocytosis, TNF death receptor signaling, and the release of cytokines such as interferon- γ.
The term "effective amount" refers to an amount of an NK cell or NK cell population effective to treat a disease condition, disease, or disorder in an individual.
The terms "treat," "treating," or "treatment" refer to both therapeutic treatment and prophylactic (preventative) or preventative measures, wherein the object is to prevent or ameliorate a disease state, disease or disorder in an individual, or to slow down (lessen) the progression of a disease state, disease or disorder in an individual. Subjects in need of treatment include subjects already suffering from a disease condition, disease or disorder as well as subjects in whom a disease condition, disease or disorder is to be prevented.
The terms "prevent", "prevention", "preventative" or "preventative" refer to preventing the occurrence of a disease condition, disease or disorder, or arresting, defending or resisting its occurrence, including abnormalities or symptoms. A subject in need of prevention may be susceptible to developing the disease condition, disease or disorder.
The terms "ameliorate" or "amelioration" refer to a reduction, or elimination of a disease condition, disease, or disorder, including an abnormality or symptom. The subject in need of treatment may already have, or may be predisposed to, the disease condition, disease or disorder, or may be the disease condition, disease or disorder to be prevented.
The term "subject" as used herein refers to a mammal. The mammal may be a primate, particularly a human, or may be a domestic, zoo or companion animal. Although it is specifically contemplated that the methods disclosed herein and the resulting NK cells or NK cell populations thereof are suitable for medical treatment of humans, they are also suitable for veterinary treatment, including treatment of domestic animals such as horses, cattle, and sheep, companion animals such as dogs and cats, or zoo animals such as felines, canines, bovines, and ungulates.
Examples
Example 1 reagent
TABLE 1 reagents
| Description of the invention | Suppliers of goods | Directory number |
| Essential 8 medium | ThermoFisher | A1517001 |
| CloneR | Stem Cell Technology | 05889 |
| STEMdiff TM APEL TM 2 culture Medium | Stem Cell Technology | 05275 |
| SCF | Miltenyi Biotech | 130-096-692 |
| BMP4 | Miltenyi Biotech | 130-111-167 |
| VEGF | Peprotech | 100-20 |
| DMEM+GlutaMAX TM -I | ThermoFisher | 10565-018 |
| F12+GlutaMAX TM -I | ThermoFisher | 31765-035 |
| Heat inactivated human AB serum | Sigma | H3667 |
| P/S (penicillin-streptomycin) | ThermoFisher | 15140122 |
| L-Glutamine | ThermoFisher | 25030081 |
| Beta-mercaptoethanol | ThermoFisher | 21985023 |
| Sodium selenite | Sigma | 214485 |
| Ethanolamine | MP Biomedicals | 193845 |
| L-ascorbic acid | Sigma | A4544 |
| IL-3 | Peprotech | 200-03 |
| IL-7 | Miltenyi Biotech | 130-095-361 |
| IL-15 | Miltenyi Biotech | 130-095-762 |
| FLT3L | Miltenyi Biotech | 130-096-477 |
| IL-2 | Peprotech | 200-02 |
| RPMI 1640 medium, glutamine free | ThermoFisher | 21870-084 |
| FBS | ThermoFisher/HyClone | SH30071.03 |
The reagents listed in table 1 are known to those skilled in the art and have acceptable compositions, such as DMEM and Ham's F12.GLUTAMAX comprises L-alanyl-L-glutamine dipeptide and is typically provided in a concentration of 200mM in 0.85% NaCl. GLUTAMAX releases L-glutamine after cleavage of the dipeptide bond by cultured cells. CloneR TM Is a defined serum-free supplement aimed at improving the cloning efficiency and single cell survival of PSCs. STEMdiff TM APEL TM Medium 2 is a fully defined, serum-free and animal component-free medium for differentiation of PSCs, based on the APEL formulation published by Andrew Elefanty, and without undefined components such as protein-free hybridoma medium.
TABLE 2 EB-Forming PSC Medium
TABLE 3 differentiation media
| Description of the preferred embodiment | Final concentration |
| SCF | 20ng/mL |
| DMEM+GlutaMAX TM -I | 56.6％ |
| F12+GlutaMAX TM -I | 28.3％ |
| Heat inactivated human AB serum | 15％ |
| P/S (penicillin-streptomycin) | 1％ |
| L-Glutamine | 2mM |
| Beta-mercaptoethanol | 1μM |
| Sodium selenite | 5ng/mL |
| Ethanolamine | 50μM |
| L-ascorbic acid | 20mg/L |
| IL-3 | 5ng/mL |
| IL-7 | 20ng/mL |
| IL-15 | 10ng/mL |
| FLT3L | 10ng/mL |
| IL-2 | 50U/mL |
TABLE 4 amplification Medium
Example 2 differentiation of PSC into NK cells
Human ipscs were routinely cultured in commercially available Essential 8 medium. Prior to initiating NK cell differentiation, ipscs with a confluency of 40-50% were cultured in Essential 8 medium supplemented with 10% cloner for at least 24 hours until the cell confluency reached 70-80% to increase single cell viability. Once confluency reaches 70-80%, iPSC is ready for differentiation, using trypLE TM Express dissociated cells into single cells and filtered through a 40 μm cell sieve to remove any undissociated cell aggregates. The collected cells were counted and seeded at 8000 cells/well in an ultra-low attachmentRound bottom 96 well plates, STEMdiff supplemented with 40ng/mL SCF, 20ng/mL BMP4, 20ng/mL VEGF, and 10% CloneR TM APEL TM 2 medium, final volume 100. Mu.L. Then the plate was centrifuged at 300 Xg for 5 minutes and 5% CO at 37% 2 And incubated for 6 days to generate hematopoietic embryoid bodies (HEEB). HE EBs formed on day 6 were then pooled into 15ml Falcon tubes and collected by sedimentation. Plating the collected HE EB onto 6-well plates coated or uncoated with 2% gelatin, in NK cell differentiation medium consisting of 56.6% DMEM + GlutaMAX TM -I、28.3％F12+GlutaMAX TM -I, 15% heat-inactivated human AB serum, 1% P/S, 2mM L-glutamine, 1. Mu.M β -mercaptoethanol, 5ng/mL sodium selenite, 50. Mu.M ethanolamine, 20mg/L ascorbic acid, 5ng/mL IL-3, 20ng/mL SCF, 20ng/mL IL-7, 10ng/mL IL-15, 10ng/mL FLT3 ligand (FLT 3L), and 50U/mL IL-2. For each well of a 6-well plate, approximately 16 EBs were plated. The medium was changed every 6 days before 14 days of differentiation and every 3 days after 14 days of differentiation. From day 7 of differentiation, the medium was not supplemented with IL-3. Floating NK cells with spindle-like morphology appeared gradually around 21-35 days of differentiation, collected for subsequent analysis and validation. For expansion, NK cells were compared to the mbIL-21 expressing K562 myeloid leukemia cell line at a ratio of 1 5 Minimum density of individual cells/mL were co-cultured in NK amplification medium (RPMI 1640 medium supplemented with 10% FBS, 2mM L-glutamine, 1% P/S and 50U/mL IL-2).
Example 3 in vitro test protocol
To validate the NK cells generated in example 2, flow cytometry analysis was performed to assess CD45/CD56 expression, or qPCR was performed to assess different NK markers including CD45, CD56, CD16, killer immunoglobulin-like receptor (KIR), NKG2D, NKp44, NKp46, fas ligand (FasL), and Tumor Necrosis Factor (TNF) -associated apoptosis-inducing ligand (TRAIL). For flow cytometry analysis of NK cells, cells were labeled with antibodies (CD 45-APC, CD56-BUV 395) in FACS buffer (DPBS supplemented with 2% (v/v) FBS) for 30 minutes, then used BD FACS X20 analyzer on a Fortessa X20 analyzer TM Software dividesAnd (6) analyzing.
For cytotoxicity assays, NK cells produced in example 2 were co-cultured with mCherry-labeled K562 myeloid leukemia cell line at 1. Time lapse images were obtained throughout the cytotoxicity assay to assess cytotoxicity of live NK cells and the percentage of mCherry positive cells remaining after 14h co-culture was assessed using flow cytometry. Cytotoxicity was assessed as a reduction in mCherry expression, as K562 cells undergoing cell death or apoptosis resulted in a reduction in the observation of mCherry markers in co-culture. For cytotoxicity assays, NK cells were co-cultured with mCherry-labeled K562 cells in NK expansion medium at the respective ratios described above in 96-well plates.
Example 4 differentiation of PSC into NK cells
NK cells were produced according to example 2, and compared with a method of producing NK cells not using IL-2 as an NK differentiation factor at the differentiation and expansion stages.
As a result, as shown in FIG. 1, NK cells produced according to example 2 had improved expansion and differentiation efficiency compared to the scheme without using IL-2 as a differentiation factor.
Example 5 characterization of NK cells
For quantitative reverse transcription polymerase chain reaction (qRT-PCR), total RNA was prepared from NK cells generated in example 2 using a commercially available kit. qRT-PCR was performed using the following steps: 1) 95 ℃ activation step 5 min 2) two cycles (denaturation at 95 ℃ for 10 sec, then annealing/extension at 60 ℃ for 30 sec) were performed for 40 cycles. The primers used for qRT-PCR are described in Table 5 below.
TABLE 5 primers for qRT-PCR
Figure 2 illustrates qRT-PCR analysis, which reveals that NK cells produced according to example 2 exhibit all the key NK cell markers seen in conventional NK cells derived from ipscs (inkcells). Furthermore, the up-regulation of the expression of certain key markers, such as CD56, was significantly higher in the NK cells generated in example 2 compared to conventional inky cells, CD56 being associated with more mature NK cells and being considered to be associated with NK effector properties. Statistical analysis was performed using a two-tailed unpaired student's t-test. * = p <0.05, = p <0.005, = p <0.0005, ns = not significant.
The results shown in figure 3 illustrate flow cytometry analysis of NK cells produced in example 2. Surprisingly, a higher percentage of these NK cells in the presence of IL-2 were CD45+/CD56+, indicating that the addition of IL-2 improved NK cell differentiation. The results also correlate with the results in fig. 4 and 5, where greater cytotoxicity was observed in experiments conducted with NK cells differentiated in the presence of IL-2.
Example 6 cytotoxicity of NK cells
The cytotoxic properties of NK cells produced in example 2 were compared to ipscs (which are not cytotoxic to K562 cells) and to conventional inkcells when co-cultured with the K562 myeloid leukemia cell line.
The results in figure 4 demonstrate that ipscs (in the top panel) are not cytotoxic to K562 cells. Furthermore, there was no significant cell death throughout the 14 hour co-culture experiment, indicating that the culture conditions were sufficient to support the co-cultured cells throughout the process without causing spontaneous cell death. The inky cells (in the bottom left panel) were moderately cytotoxic to K562 cells, with slightly reduced mCherry expression near the 10 hour time point and moderately reduced mCherry expression at the end of the 14 hour time point. In contrast, the co-culture of NK cells and K562 cells produced in example 2 revealed a significant reduction in mCherry expression by the 4 hour time point (compared to 10 hours in conventional inky cells), a moderate reduction in mCherry expression by the 6 hour time point (compared to 14 hours in conventional inky cells), and little mCherry expression by the 13 hour time point, which was surprising.
Next, the cytotoxic properties of NK cells at different seeding densities were evaluated. Ipscs, conventional inkn cells and NK cells generated in example 2 were seeded at a ratio to K562 cells of 1, 2.5.
Figure 5 shows the results of flow cytometry analysis in the form of a flow cytometry plot, revealing the different effects of increasing seeding density on the cytotoxic properties of K562 cells. The top row shows the results of iPSC and K562 co-culture. No significant cytotoxic effect was observed despite increasing the seeding density to 5. At all seeding densities, the percentage of mCherry positive cells was >90%, indicating that almost all K562 cells were still viable.
The middle row shows the results for conventional iNK cells versus K562 cells. At a ratio of 1. At a ratio of 2.5.
The bottom row shows the results of NK cells produced in example 2. At a ratio of 1, 67% of the cells were mCherry positive, indicating moderate cell death or apoptosis of K562 cells. At a ratio of 2.5. This indicates that the NK cells produced in example 2 exhibit high cytotoxic activity against K562 cells, above and beyond the cytotoxic properties of conventional inkcells.
These studies indicate that the NK cells of the present disclosure exhibit increased expression of key NK cell markers, which correlates with a significant increase in cytotoxic activity against tumor cell lines. This indicates that the NK cells of the present disclosure exhibit improved effector properties and may have therapeutic utility for diseases and conditions such as infectious diseases and cancer.
Claims (12)
1. A method of generating Natural Killer (NK) cells, the method comprising:
a) Culturing Pluripotent Stem Cells (PSCs) to produce embryoid bodies;
b) Differentiating said embryoid bodies in a differentiation medium comprising an NK differentiation factor to promote differentiation into said NK cells; and
c) Expanding the cells of b) in an expansion medium comprising IL-2 to produce the NK cells.
2. The method of claim 1, wherein the NK differentiation factor is interleukin 2 (IL-2);
preferably, the differentiation medium further comprises one or more of: fms-like tyrosine kinase 3 ligand (FT 3L), interleukin 3 (IL-3), interleukin 15 (IL-15).
3. The method of claim 2, wherein the differentiation medium comprises IL-2 at a concentration of 1-150U/mL;
preferably, the differentiation medium comprises IL-3 at a concentration of 1-15ng/mL;
preferably, the differentiation medium comprises FT3L at a concentration of 1-30ng/mL;
preferably, the differentiation medium comprises IL-15 at a concentration of 1-30ng/mL.
4. The method of claim 1, wherein the PSCs are human embryonic stem cells (hESCs) or Induced Pluripotent Stem Cells (iPSCs);
preferably, the differentiation medium comprises human serum.
5. The method of claim 1, wherein the NK cells express one or more of: CD45, CD56, CD16, killer immunoglobulin-like receptor (KIR), NKG2D, NKp44, NKp46, fas ligand (FasL), and Tumor Necrosis Factor (TNF) -associated apoptosis-inducing ligand (TRAIL);
preferably, the NK cell exhibits increased expression of one or more of: CD45, CD56, CD16, KIR, NKG2D, NKp44, NKp46, fasL and TRAIL, said reference NK cells being differentiated according to a method according to claim 1 except: said differentiation medium in b) comprises said NK differentiation factor and/or said expansion medium in c) comprises IL-2.
6. The method of claim 1, wherein c) expanding comprises co-culturing the NK cells with a feeder cell line;
preferably, the feeder cell line is a K562 myeloid leukemia cell line;
preferably, wherein the K562 cell line expresses membrane bound IL-21;
preferably, the co-culture is performed at a ratio of NK cells to feeder cell lines of 5.
7. The method according to claim 1, comprising b) culturing for about 5-20 days;
preferably, comprising culturing said embryoid bodies in a medium that does not comprise said NK differentiation factor after b) and before c);
preferably, comprising culturing said embryoid bodies for about 5 to 10 days in a medium not comprising said NK differentiation factor after b) and before c);
preferably, the method comprises c) amplifying for about 10-50 days.
8. A Natural Killer (NK) cell produced according to the method of claim 1;
preferably, said NK cell expresses one or more of: CD45, CD56, CD16, killer immunoglobulin-like receptors (KIRs), NKG2D, NKp44, NKp46, fas ligand (FasL), and Tumor Necrosis Factor (TNF) -associated apoptosis-inducing ligand (TRAIL);
preferably, the NK cell exhibits increased expression of one or more of: CD45, CD56, CD16, KIR, NKG2D, NKp44, NKp46, fasL and TRAIL, said reference NK cells being differentiated according to a method according to claim 1 except: said differentiation medium in b) comprising said NK differentiation factor.
9. A pharmaceutical composition comprising the NK cell of claim 8;
preferably, the pharmaceutical composition comprises an additional therapeutic agent;
preferably, the pharmaceutical composition wherein the additional therapeutic agent is an antibody.
10. A method of immunotherapy comprising administering to an individual an NK cell according to claim 8 or a pharmaceutical composition according to claim 9.
11. Use of an NK cell according to claim 8 in the manufacture of a medicament for immunotherapy of an individual.
12. The method of claim 10 or use of claim 11, wherein the individual has cancer or an infectious disease;
preferably, the infectious disease is selected from the group consisting of: human Immunodeficiency Virus (HIV), epstein-Barr virus (EBV), herpes Simplex Virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), hepatitis B Virus (HBV) and Hepatitis C Virus (HCV).
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| CN117050941A (en) * | 2023-10-11 | 2023-11-14 | 苏州艾凯利元生物科技有限公司 | Method for preparing natural killer cells |
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| US20190359940A1 (en) * | 2017-02-07 | 2019-11-28 | Agency For Science, Technology And Research | Methods and kits for generating mimetic innate immune cells from pluripotent stem cells |
| CN111235105A (en) * | 2020-03-06 | 2020-06-05 | 安徽中盛溯源生物科技有限公司 | Method for differentiating human pluripotent stem cells into natural killer cells and application |
| CN112608895A (en) * | 2020-12-18 | 2021-04-06 | 深圳市安棣生物科技有限责任公司 | Natural killer cell differentiated from human pluripotent stem cell and preparation method and application thereof |
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| US20120148553A1 (en) * | 2010-07-13 | 2012-06-14 | Hariri Robert J | Methods of generating natural killer cells |
| US20130287751A1 (en) * | 2012-04-24 | 2013-10-31 | Dan S. Kaufman | Method for developing natural killer cells from stem cells |
| US20190359940A1 (en) * | 2017-02-07 | 2019-11-28 | Agency For Science, Technology And Research | Methods and kits for generating mimetic innate immune cells from pluripotent stem cells |
| CN111235105A (en) * | 2020-03-06 | 2020-06-05 | 安徽中盛溯源生物科技有限公司 | Method for differentiating human pluripotent stem cells into natural killer cells and application |
| CN112608895A (en) * | 2020-12-18 | 2021-04-06 | 深圳市安棣生物科技有限责任公司 | Natural killer cell differentiated from human pluripotent stem cell and preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117050940A (en) * | 2023-10-11 | 2023-11-14 | 苏州艾凯利元生物科技有限公司 | Method for preparing natural killer cells |
| CN117050941A (en) * | 2023-10-11 | 2023-11-14 | 苏州艾凯利元生物科技有限公司 | Method for preparing natural killer cells |
| CN117050940B (en) * | 2023-10-11 | 2024-01-26 | 苏州艾凯利元生物科技有限公司 | Method for preparing natural killer cells |
| CN117050941B (en) * | 2023-10-11 | 2024-01-26 | 苏州艾凯利元生物科技有限公司 | Method for preparing natural killer cells |
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| CN115261318B (en) | 2024-05-28 |
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