CN117050941B - Method for preparing natural killer cells - Google Patents

Method for preparing natural killer cells Download PDF

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CN117050941B
CN117050941B CN202311309574.2A CN202311309574A CN117050941B CN 117050941 B CN117050941 B CN 117050941B CN 202311309574 A CN202311309574 A CN 202311309574A CN 117050941 B CN117050941 B CN 117050941B
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CN117050941A (en
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刘晓东
贺智勇
周怡辰
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Suzhou Aikailiyuan Biotechnology Co ltd
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Suzhou Aikailiyuan Biotechnology Co ltd
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Abstract

The present invention relates to a method for preparing natural killer cells, comprising inducing differentiation of ipscs into HSPCs, contacting the HSPCs with DLL4, such that the HSPCs differentiate to obtain NK cells. The invention also relates to NK cells prepared by the method, compositions comprising the NK cells and uses of the NK cells or compositions.

Description

Method for preparing natural killer cells
Technical Field
The invention relates to the field of bioengineering, in particular to a method for preparing natural killer cells, and in particular relates to a method for inducing and differentiating natural killer cells from pluripotent stem cells.
Background
Natural Killer (NK) cells are a class of relatively large lymphocytes that have the function of killing infected and tumor cells without being restricted by Human Leukocyte Antigens (HLA) and without prior antigen sensitization. In humans, NK cells are often represented as cd56+cd3 lymphocytes, which can be roughly divided into two subgroups depending on the expression levels of CD56 and CD 16:CD56 bright CD16 cells and CD56 dim Cd16+ cells.
NK cell stimulation and effector function depend on the integration of signals from two different types of receptors (activating and inhibiting). Normal healthy cells express MHC class I molecules on their surface, which act as ligands for inhibitory receptors and contribute to NK cell self-tolerance. However, the surface of virus-infected cells or tumor cells loses MHC class I expression, resulting in a decrease in inhibition signal in NK cells. At the same time, cellular stresses associated with viral infection or tumor growth, such as DNA damage response, senescence programs, or tumor suppressor genes, up-regulate ligands that activate receptors in these cells. Thus, signals from activating receptors in NK cells shift the balance towards NK cell activation and target cell elimination through NK cell mediated cytotoxicity or indirectly through the secretion of pro-inflammatory cytokines.
Once activated, NK cells release preformed cytolytic particles containing granzyme and perforin to lyse the infected or tumor cells. The lysis of NK cells also occurs through CD16 mediated antigen dependent cytotoxicity (ADCC). After activation, NK cells also secrete a variety of cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony stimulating factor (GM-CSF) and chemokines (CCL 1, CCL2, CCL3, CCL4, CCL5 and CXCL 8), which can regulate the function of other innate and adaptive immune cells.
Homogeneous primary NK cells from peripheral blood (PB-NK) and umbilical cord blood NK cells (UCB-NK) have been shown to be safe and effective without significant toxicity, such as Cytokine Release Syndrome (CRS), neurotoxicity or Graft Versus Host Disease (GVHD). However, PB-NK cells and UCB-NK cells used in these experiments are heterogeneous cell products and are different from donor to donor, which limits the potential for developing standardized cellular immunotherapeutic products.
Human embryonic stem cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs) are ideal starting cell types for the development of a variety of cell lineages, including NK cells. Studies have shown that NK cells (iNK) differentiated from hESC-/iPSC exhibit potent anti-tumor and antiviral activity, providing a potential solution for standardized cell therapy of these diseases. iNK cells are powerful and have broad cytotoxic activity against hematological and solid tumors. iNK cells are a uniform, reproducible and unlimited iNK cell source that allows for multiple doses of treatment and eliminates variability from donor to donor. Importantly, genetic engineering of hESCs and iPSCs to differentiate iNK cells with improved antitumor activity or persistence in vivo is also feasible. iNK cells represent a promising new approach to adoptive NK cell immunotherapy, and iNK cells may also provide therapy as an off-the-shelf cell therapy or in combination with antibodies directed against checkpoint inhibitors to enhance anti-tumor responses.
Notch signaling pathways have been shown to play a critical role in the development and function of the innate and adaptive immune systems. Major components of the Notch pathway are expressed in the vasculature and lymphoid organs such as Notch1, notch1/Notch4, jagged1, delta-like ligand 4 (DLL 4), hey1/Hey2 and presenilins. Notch signaling is critical in determining the committed differentiation of hematopoietic progenitor cells, and has been shown to boost the potential of lymphocytes T cells and NK cells at the expense of B cell differentiation. In addition, notch activation at the late stage of NK cell differentiation also enhances expression of CD16 and KIR, as well as functional maturation. Thus, notch signaling has been used to induce NK cell differentiation from hematopoietic stem/progenitor cells in vitro, with Notch ligands typically presented by the mouse bone marrow stromal cell line OP 9. Taken together, these studies suggest that Notch activation may play a key role in NK differentiation.
Disclosure of Invention
Problems to be solved by the invention
How to provide a preparation method of NK cells suitable for mass production and how to prepare NK cells with higher cytotoxicity is a technical problem to be solved by the technicians in the field.
Solution for solving the problem
The present invention provides a method for preparing natural killer cells, comprising the steps of:
a. inducing the differentiation of pluripotent stem cells into embryoid bodies in a culture medium supplemented with CHIR 99021;
b. inducing the EB formed in step a to differentiate into hematopoietic stem and progenitor cells;
c. inducing differentiation of HSPCs formed in step b into NK cells;
wherein, the step a comprises the following steps:
(a1) Induction was performed on day 0 in the presence of CHIR 99021 and Rki;
(a2) Induction was again performed on day 1 in the presence of CHIR 99021.
Preferably, the step b includes:
(b1) Suspension culturing EB in hematopoietic differentiation medium supplemented with BMP4, FGF2, VEGF;
(b2) Suspension culture was continued in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB 431542;
(b3) Further suspension culture in hematopoietic stem cell expansion medium supplemented with SCF, FGF2 and VEGF forms HSPCs.
Preferably, step (a) is performed on days 0-1, step (b 1) is performed on days 2-3, step (b 2) is performed on days 4-5, and step (b 3) is performed on days 6-7.
Preferably, the HSPCs have a cd34+ phenotype.
Preferably, the step c includes:
(c1) Contacting the HSPC formed in step b with a Delta-like ligand 4.
Preferably, the step (c 1) includes:
(c11) Adding the HSPC formed in the step b into a first culture medium for cell culture;
(c12) And (3) adding the cells obtained by the culture in the step (c 11) into a second culture medium for cell culture to obtain NK cells.
Preferably, the first medium contains heat-inactivated human AB serum, a colony stimulating factor, an interleukin, wherein the colony stimulating factor is selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF, EPO, TPO, SCF and FLT3L, and the interleukin is selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27;
preferably, the second medium contains heat-inactivated human AB serum, a colony stimulating factor selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF, EPO, TPO, SCF and FLT3L, and an interleukin selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27.
Preferably, the cell culture in step (c 11) is for 6-8 days;
Preferably, the cell culture in step (c 12) is carried out for 8-10 days, followed by a further 8-10 days in fresh second medium.
The present invention also provides an NK cell prepared according to the method which shows increased expression of one or more of CD45, CD56, CD16, killer immunoglobulin-like receptor, NKG2D, NKp, NKp46, fasL and TRAIL.
The invention also provides a pharmaceutical composition comprising NK cells according to the invention and a pharmaceutically acceptable excipient.
The invention also provides an application of the NK cells prepared by the method, the NK cells or the pharmaceutical composition in preparation of a preparation for treating or preventing immune related diseases.
Preferably, the immune-related disease comprises one or more of a tumor, a viral infection, a graft versus host disease, an autoimmune disease, or leukemia; wherein the viral infection comprises one or more of human immunodeficiency virus, epstein-barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, hepatitis b virus or hepatitis c virus.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention successfully realizes the efficient differentiation to HSPC by carrying out stepwise in vitro culture of iPSC under proper conditions, and the obtained culture product does not contain heterologous substances and has the advantages of shorter differentiation time and higher efficiency.
The present invention also provides a method of inducing differentiation of NK cells from iPSC, which comprises a combination of several reagents and cytokines, is suitable for mass production, and can produce NK cells having higher cytotoxicity.
Drawings
FIG. 1 is a schematic diagram of iNK cell differentiation. Embryoid Body (EB) production hematopoietic EBs derived from human ipscs subsequently differentiated into iNK cells. The differentiation cultures were transferred to DLL 4/recombinant human fibronectin (Retronectin) -coated culture vessels for further differentiation of iNK cells.
Fig. 2 is a schematic representation of a differentiation protocol for producing HSPCs using ipscs, from which HSPCs were produced using a change in cytokines and time of addition.
Fig. 3 shows the cell morphology change during differentiation of ipscs to HSPCs. The timeline of Hematopoietic Stem and Progenitor Cells (HSPCs) differentiated from induced pluripotent stem cells (ipscs) by embryoid bodies is shown, with arrows indicating the presence of cd34+ hematopoietic stem cells in suspension.
Fig. 4 is a flow cytometry analysis of HSPC markers. A is representative of HSPC cell gating for flow cytometry analysis. HSPC cells were identified as (CD 235a-CD 14-cd43+cd34+) on day 8 of condition 6. B is representative of HSPC cell gating for flow cytometry analysis; HSPC cells were identified as (CD 235a-CD 14-cd43+cd34+) on day 10 of condition # 6.
Fig. 5 shows a representative bright field image of the iPSC incubation period.
FIG. 6 shows the results of a comparison of the addition time of CHIR99021 for 1 day vs 2 days.
Fig. 7 shows the results of the cell stability experiment.
FIG. 8 is a graph showing changes in cell morphology during iNK cell differentiation. Representative images from two independent iPSC lines on day 9, day 17 and day 25 of iNK cell differentiation. The DLL 4/recombinant human fibronectin (Retronectin) -immobilized differentiated group significantly increased NK cell numbers on days 17 and 25, as compared to the DLL 4/recombinant human fibronectin (Retronectin) -not immobilized differentiated group. Scale = 200 μm. Enlarged image scale = 50 μm.
FIG. 9 is a graph of kinetics of iNK cell differentiation. Flow cytometry analyzed iNK cell differentiation for CD56, CD45 expression on days 8, 16, 21 and 28. DLL 4/recombinant human fibronectin (Retronectin) exposure was tested for NK cell differentiation for two periods of time: only the first 7 days (7 d) of DLL 4/recombinant human fibronectin (Retronectin) coated or the whole NK cell differentiation period (all times) coated with DLL 4/recombinant human fibronectin (Retronectin). The DLL 4/recombinant human fibronectin (Retronectin) coated significantly enhanced NK cell differentiation, and a minimum of 7 days of transient DLL 4/recombinant human fibronectin (Retronectin) exposure was sufficient to increase CD45+CD56+NK cell production by 4-8 fold. The NK cell fraction peaked at day 21, indicating that DLL 4/recombinant human fibronectin (Retronectin) might accelerate NK cell differentiation.
FIG. 10 is a graph showing the proportion of iNK progenitor cells at an early stage of differentiation. Flow cytometry analyzed iNK cell differentiation for CD7, CD45 expression on day 8, day 16. The DLL 4/recombinant human fibronectin (Retronectin) coated significantly increased the expression of the starter (cd7+) of the lymphoid progenitor cells.
FIG. 11 is a graph showing the expression of NKp44, an NK-activating receptor, in differentiated iNK cells. Flow cytometry analysis was performed on NKp44, CD45 expression on day 28 of iNK differentiation. Coating with DLL 4/recombinant human fibronectin (Retronectin) significantly increased the expression of NKp44, an NK activated receptor, suggesting that DLL 4/recombinant human fibronectin (Retronectin) may agonize iNK cells to a more activated state.
FIG. 12 is an exploded view of CD107a of iNK cells. After incubation with K562 for 4 hours at a target (E: T) ratio of 1:1, 2:1, 5:1 or MOLM13 at E: T ratios of 1:1, 5:1, 10:1, CD107a surface expression was analyzed using a flow cytometer. iNK cells differentiated in the presence of DLL 4/recombinant human fibronectin (Retronectin) expressed higher CD107a in all E: T ratios, while CD107a was a marker of immune cell activation and cytotoxic breakdown, indicating that iNK cells differentiated in the presence of DLL4/Retronectin had higher cancer cytotoxicity.
FIG. 13 is a graph of IFNγ and TNFα produced by iNK cells. After incubation of iNK and K562 for 4 hours at E:T ratios of 1:1, 2:1, 5:1 or MOLM13 at E:T ratios of 1:1, 5:1, 10:1, intracellular IFNγ, TNFα expression levels were analyzed using a flow cytometer. iNK cells differentiated in the presence of DLL 4/recombinant human fibronectin (Retronectin) gave higher IFNγ and TNFα expression levels at lower E:T ratios, indicating a stronger anti-tumor and pro-inflammatory response in cancer cytotoxicity.
Detailed Description
In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or reduced to more clearly show details of the local features; unless defined otherwise, technical and scientific terms used herein have the same meaning as technical and scientific terms in the technical field to which this application belongs.
Unless defined otherwise in the present 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.
The term "a" or "an" means one/one or more/one, for example, "a/a molecule" should be understood to mean one/one or more/a plurality of molecules. Thus, the terms "a" or "an" and "one/or" one/more "and" at least one/at least one "are used interchangeably herein.
In the claims and the description of the invention, unless the context requires otherwise due to the 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 magnitude of a given value. In other embodiments, the term "about" encompasses a range of values in the range of ±20%, ±15%, ±10% or ±5% of the given value. For example, in one embodiment, "about 3 grams" means a value of 2.7-3.3 grams (i.e., 3 grams ± 10%), or the like.
Similarly, while the method of producing NK cells includes ordered, continuous events, the timing of the events may vary by, for example, at least 25%. For example, while a particular step may be disclosed as lasting for one day in one embodiment, the event may last more or less than one day. For example, "one 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 period of time indicated as multiple days may be a multiple of "one day", e.g., two days may span a period of time of about 36 hours to about 60 hours, etc. In another embodiment, the time variation may be reduced, e.g., day 2 is 48±3 hours from day 0; day 4 is 96±3 hours from day 0, and day 5 is 120 hours±3 hours from day 0.
As used herein, "pluripotent stem cells" or "PSC" refer to cells capable of infinitely replicating themselves and differentiating into all cells forming part of a tissue or organ or any of the three germ layers (endodermal, mesodermal or ectodermal). There are two main types of pluripotent stem cells: embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs).
As used herein, "embryonic stem cells" or "ESCs" refer to cells isolated from 5-7 day embryos that have been consented to donation from a patient who has completed in vitro fertilization therapy and has a remaining embryo. Ethical issues in extracting cells from human embryos have prevented the use of ESCs to some extent.
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 problems associated with ESCs, as 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. The reprogramming step typically involves the use of reprogramming factors at specific time intervals and at certain concentrations. Some exemplary methods of reprogramming an adult cell back to a pluripotent state involve the use of RNA, proteins, or other small molecules that are administered to the adult cell culture. Alternatively, the human iPSC line is also commercially available.
Suitable human iPSCs include, but are not limited to, iPSCs 19-9-7T, MIRJT6i-mND1-4 derived from fibroblasts and MIRJT7i-mND2-0 and iPSCs BM119-9 derived from bone marrow mononuclear cells. Other suitable ipscs are available from Cellular Dynamics International of madison, wisconsin, usa.
As used herein, EBs obtained by directional differentiation of ipscs are referred to as "iEB" or induction of EB cells.
As used herein, HSPCs obtained by directional differentiation of ipscs are referred to as "ihpcs" or induced HSPC cells.
As used herein, NK cells obtained from iPSC directed differentiation are referred to as "iNK" or induced NK cells.
As used herein, "differentiation" refers to the process by which cells are transformed from one cell type to another, particularly cells of a less specialized type are transformed into cells of a more specialized type.
As used herein, "medium" or "media" in its plural refers to a liquid or gel designed to support cell growth.
The differentiation of PSCs into NK cells is usually carried out under controlled conditions, especially when the resulting NK cells are intended to be administered to human subjects in accordance with good practice (GMP). The initial step of the process involves culturing the PSC in culture on, for example, a tissue culture plate or dish or within a bioreactor. The use of bioreactors is particularly attractive in view of the ability to expand NK cell production at the clinical level for adoptive transfer. However, protocols utilizing tissue culture plates or dishes can also be scaled up appropriately for adoptive NK cell transfer.
PSC were grown in specific media. Suitable basal media include, but are not limited to, iscove modified Dulbecco's Medium (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, i.e. Essential 8 TM Medium (Life Technologies; also referred to as "E8" medium). The cell culture medium can be supplemented with other growth factors to increase cloning efficiency and single cell survival of the PSC. An exemplary supplement that may be used is CloneR (Stem Cell Technologies). Once PSCs reach the desired confluency, cells can be collected and seeded and plated to form embryoid bodies.
Although the presently disclosed media 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, "embryoid bodies" (EBs) refer to floating three-dimensional aggregates composed of PSCs. EB includes blood-forming EB, which is EB capable of forming endothelial progenitor cells and blood progenitor cells. Various methods of producing EB are known in the art. For example, conventional methods typically involve generating a single cell suspension of PSCs. PSCs can then be cultured in uncoated non-tissue culture treated dishes or microwells to prevent PSCs from adhering, thereby promoting cell aggregation while remaining suspended. Alternatively, PSCs can be cultured in low adhesion dishes or microwells. Newer methods of EB formation involve the use of spin aggregation or bioreactors to improve efficiency and process control. ROCK inhibitors, such as Y-27632, have also been shown to promote PSC aggregation, leading to EB formation. EB has a teratoma-like structure similar to that of a developing embryo, and EB formation is a common platform to establish differentiation into cells (e.g., NK cells) from any of the three germ layers.
As used herein, "hematopoietic stem cells and progenitor cells" (HSPCs) are cells designated as hematopoietic lineages, but capable of further hematopoietic differentiation, and include multipotent hematopoietic stem cells (blood blasts), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells are multipotent stem cells that produce all blood cell types, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells).
In certain embodiments, at about 10,000 cells/cm 2 Up to about 40,000 cells/cm 2 For example 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 PSC was plated to generate EB.
Once the EB is formed, the EB is cultured in a differentiation medium containing 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 EBs 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 certain embodiments, the differentiation medium or expansion medium may be xeno-free and incorporate human proteins isolated from natural sources, such as from placenta or other human tissue, or human proteins produced using recombinant techniques.
In certain embodiments, all proteins described herein are human.
In certain embodiments, all of the proteins used in the differentiation medium or the amplification medium are human proteins.
In certain embodiments, all of the proteins used in the differentiation medium or the amplification medium are human proteins.
In certain embodiments, all of the proteins described herein are human recombinant proteins.
In certain embodiments, all of the proteins used in the differentiation medium or the amplification medium are recombinant human proteins.
All 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.
Those 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 will continue to utilize nutrients, the number of which steadily increases during the expansion phase. Thus, fresh cell culture medium can be used instead of old cell culture medium.
In certain embodiments, the differentiation medium or the expansion medium may be changed about every 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
Once the NK cell population becomes more mature, and/or a sufficient number of PSCs have differentiated into NK cells, more medium exchanges 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 stage, the differentiation medium or the expansion medium may be changed 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 may be stained with markers commonly associated with NK cells, such as CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp, NKp44, NKp46 and NKp80.
In certain embodiments, the supplementation of specific cytokines, chemokines, proteins, signaling factors, and growth factors in the cell culture medium occurs within a defined time interval, after which the supplementation of these factors is stopped.
In certain embodiments, 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, the supplementation with a particular factor may be stopped at 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 certain embodiments, supplementation with 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 culture. Generally, NK cells having spindle-like or elongated morphology are observed around 15-50 days of culture. In certain embodiments, NK cells having 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 commonly associated with NK cells include CD3, CD56, CD45, CD94, CD122, CD127, CD16, KIR, NKG2A, NKG2D, NKp, NKp44, NKp46 and NKp80. Mature NK cells act through granule exocytosis and release of cytotoxic proteins, cytokines and chemokines to induce targeted cell death. An exemplary mechanism of NK cell-mediated cell death is through interactions with caspase enzymatic cascades, apoptosis signaling, and inflammatory signaling. Thus, the maturity and functional characteristics of NK cells, including cytotoxicity and tumor killing capacity, 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 of 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. In addition, 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 cytotoxicity of NK cells produced by the method of the present invention is to co-culture NK cells with 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, etc. 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 fluorescent marker expression, 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 may be further analyzed using flow cytometry, e.g., by Annexin-V (Annexin V) or any other fixable vital dye staining. Co-culture of NK cells and tumor cells is typically performed at a specific effector to target ratio. In certain embodiments, the ratio is about 1:1 to 10:1. In certain embodiments, the ratio is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Methods and assays for determining ADCC are also known in the art, for example, incubating NK cells with target cells and a suitable effector antibody, such as NKp 46. ADCC assays and kits and reagents therefor are also commercially available, for example Promega ADCC Bioassay.
As used herein, the term "pharmaceutical composition" refers to a composition comprising NK cells or NK cell populations described herein that has been formulated for administration to an individual. Preferably, the pharmaceutical composition is sterile. In certain embodiments, the pharmaceutical composition is pyrogen-free.
NK cells or NK cell populations will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the type of 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 these considerations.
NK cells or NK cell populations may be administered to an 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 modifiers intended to enhance innate NK cell characteristics or NK cell activity in vivo. NK cells may also be pretreated or primed with a modifier 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 are also useful in antibody cancer therapies that utilize antibodies, such as monoclonal antibodies, to target tumor cells due to effector properties in inducing ADCC. Indeed, NK cells are able to mediate antibody-dependent tumor killing through cytotoxic granule exocytosis, TNF death receptor signaling, and release of cytokines such as interferon-gamma.
The term "effective amount" refers to an amount of NK cells or NK cell populations effective to treat a disease condition, disease, or disorder in an individual.
The terms "treatment", "treatment" or "treatment" refer to therapeutic treatment and prophylactic or preventative (predictive) measures, wherein the object is to prevent or improve a disease condition, disease or disorder in a subject, or to slow down (lessen) the progression of a disease condition, disease or disorder in a subject. Individuals in need of treatment include individuals already with the disease condition, disease or disorder and individuals who are to be prevented from the disease condition, disease or disorder.
The terms "prevent", "prevention", "prophylactic" or "preventing" refer to preventing the occurrence of a disease condition, disease or disorder, or preventing, defending or resisting the occurrence thereof, including abnormalities or symptoms. A subject in need of prevention may be susceptible to developing the disease condition, disease or disorder.
The term "amelioration" or "amelioration" refers to the reduction, alleviation or elimination of a disease condition, disease or disorder, including abnormalities or symptoms. The individual in need of treatment may already have the disease condition, disease or disorder, or may be susceptible to the disease condition, disease or disorder, or may be in need of prophylaxis of the disease condition, disease or disorder.
The term "individual" as used herein refers to a mammal. The mammal may be a primate, particularly a human, or may be a domestic animal, zoo animal or companion animal. Although the methods disclosed herein and the resulting NK cells or NK cell populations thereof are particularly contemplated as being suitable for medical treatment of humans, they are also suitable for veterinary treatment, including treatment of livestock animals such as horses, cattle and sheep, companion animals such as dogs and cats, or zoo animals such as felines, canines, bovids and ungulates.
The process of the present invention is illustrated by the following specific examples, it being understood that these examples are illustrative of the basic principles, main features and advantages of the present invention, and the present invention is not limited by the scope of the following examples; the implementation conditions employed in the examples may be further adjusted according to specific requirements, and the implementation conditions not specified are generally those in routine experiments. The following table is a list of all or part of the reagents used in this application.
Reagent(s) Suppliers (suppliers) Catalog number
StemScale PSC suspension Medium ThermoFisher A4965001
Y27632 Tocris 1254
CHIR99021 Sigma SML1046
Stemline II hematopoietic stem cell expansion medium Sigma S0192
Penicillin-streptomycin double antibody solution (P/S) ThermoFisher 15140122
Glutamax supplement ThermoFisher 35050061
L-ascorbic acid Sigma-Aldrich A4544
ITS-G ThermoFisher 41400045
BMP4 Miltenyi Biotec 130-111-167
FGF2 Miltenyi Biotec 130-093-564
VEGF Miltenyi Biotec 130-109-396
SB431542 Tocris 1614 / TB1614-GMP
SCF Miltenyi Biotec 130-096-695
DMEM/F-12 GlutaMAX ThermoFisher 10565018
F12+GlutaMAX ThermoFisher 31765-035
Heat-inactivated human AB serum Sigma H3667
L-glutamine ThermoFisher 25030081
Beta-mercaptoethanol ThermoFisher 21985023
Sodium selenite (Se-Na) Sigma 214485
Ethanolamine MP 193845
IL-3 Miltenyi Biotec 130-095-070
IL-7 Miltenyi Biotec 130-095-362
IL-15 Miltenyi Biotec 130-095-764
IL-2 Miltenyi Biotec 130-097-748
IMDM Gibco 12440-053
Cell-Vive T-NK Xeno-free serum substitute, GMP Biolegend 420502
FLT3L Miltenyi Biotec 130-096-477
DPBS ThermoFisher 14190-144
Accutase Stem Cell Technologies 07922
CryoStor® CS10 Stem Cell Technologies 07930
DLL4 Sino Biological 10171-H02H
Recombinant human fibronectin (Retronectin) Takara T1100B
The present invention provides a method of preparing Natural Killer (NK) cells, the method comprising the steps of:
a. inducing pluripotent stem cells (ipscs) to differentiate into Embryoid Bodies (EBs) in a culture medium supplemented with CHIR 99021;
b. inducing the EB formed in step a to differentiate into Hematopoietic Stem and Progenitor Cells (HSPCs);
c. inducing differentiation of HSPCs formed in step b into NK cells;
wherein, the step a comprises the following steps:
(a1) Induction was performed on day 0 in the presence of CHIR 99021 and Rki;
(a2) Induction was again performed on day 1 in the presence of CHIR 99021.
In certain embodiments, the step b comprises:
(b1) Suspension culturing EB in hematopoietic differentiation medium supplemented with BMP4, FGF2, VEGF;
(b2) Suspension culture was continued in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB 431542;
(b3) Further suspension culture in hematopoietic stem cell expansion medium supplemented with SCF, FGF2 and VEGF forms HSPCs.
In certain embodiments, the methods do not require a trophoblast during the preparation.
In certain embodiments, the medium in step (a) is selected from one or more of StemScale PSC, essential 8, KSR/FGF2, mTeSR, AKIT, or B8.
In certain embodiments, the medium in step (a) is a StemScale PSC.
In certain embodiments, the medium of step (b 1) further comprises one or more of P/S, glutaMaX, ascorbic acid, or ITS-G.
In certain embodiments, the medium in step (b 1) contains 1% P/S, 2mM GlutaMaX, 50 ug/mL ascorbic acid, 1% ITS-G, 1-100 ng/mL BMP4, 1-100 ng/mL FGF2, and 1-100 ng/mL VEGF.
In certain embodiments, the concentration of BMP4 in the medium of step (b 1) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of FGF2 in the medium of step (b 1) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of VEGF in the medium in step (b 1) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the medium of step (b 2) further comprises one or more of P/S, glutaMaX, ascorbic acid, and ITS-G.
In certain embodiments, the medium in step (b 2) contains 1% P/S, 2mM GlutaMaX, 50. Mu.g/mL ascorbic acid, 1% ITS-G, 1-100 ng/mL BMP4, 1-100 ng/mL SCF, 1-100 ng/mL FGF2, 1-100 ng/mL VEGF, and 1-20. Mu.M SB431542.
In certain embodiments, the concentration of BMP4 in the medium of step (b 2) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of SCF in the medium in step (b 2) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of FGF2 in the medium of step (b 2) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of VEGF in the medium in step (b 2) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of SB431542 in the medium of step (b 2) is about 1. Mu.M, about 2. Mu.M, about 3. Mu.M, about 4. Mu.M, about 5. Mu.M, about 6. Mu.M, about 7. Mu.M, about 8. Mu.M, about 9. Mu.M, about 10. Mu.M, about 11. Mu.M, about 12. Mu.M, about 13. Mu.M, about 14. Mu.M, about 15. Mu.M, about 16. Mu.M, about 17. Mu.M, about 18. Mu.M, about 19. Mu.M, or about 20. Mu.M.
In certain embodiments, the medium of step (b 3) further comprises one or more of P/S, glutaMaX, ascorbic acid, or ITS-G.
In certain embodiments, the medium in step (b 3) contains 1% P/S, 2mM GlutaMaX, 50 μg/mL ascorbic acid, 1% ITS-G, 1-100 ng/mL SCF, 1-100 ng/mL FGF2, and 1-100 ng/mL VEGF.
In certain embodiments, the concentration of SCF in the medium in step (b 3) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of FGF2 in the medium of step (b 3) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of VEGF in the medium in step (b 3) is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, step (a) is performed on days 0-1, step (b 1) is performed on days 2-3, step (b 2) is performed on days 4-5, and step (b 3) is performed on days 6-7.
In certain embodiments, the EB is iEB.
In certain embodiments, the HSPCs are ihpcs.
In certain embodiments, the NK cells are iNK cells.
In certain embodiments, the HSPCs have a cd34+ phenotype.
In certain embodiments, the step c comprises:
(c1) Contacting the HSPC formed in step b with a Delta-like ligand 4 (DLL 4).
In certain embodiments, the step (c 1) comprises:
(c11) Adding the HSPC formed in the step b into a first culture medium for cell culture;
(c12) And (3) adding the cells obtained by the culture in the step (c 11) into a second culture medium for cell culture to obtain NK cells.
In certain embodiments, the first medium contains heat-inactivated human AB serum, a colony stimulating factor, an interleukin, wherein the colony stimulating factor is selected from one or more of G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, and FLT3L, and the interleukin is selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27;
in certain embodiments, the second medium contains heat-inactivated human AB serum, a colony stimulating factor selected from one or more of G-CSF, M-CSF, GM-CSF, multiCSF (IL-3), EPO, TPO, SCF, and FLT3L, and an interleukin selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27.
In certain embodiments, the colony stimulating factors in the first medium are SCF and FLT3L.
In certain embodiments, the interleukins in the first medium are IL-3, IL-7 and IL-15.
In certain embodiments, the first medium further comprises one or more of DMEM/f12+ GlutaMAX, P/S, L-glutamine, β -mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and recombinant human fibronectin (Retronectin).
In certain embodiments, the first medium contains 56.6% DMEM/F12+GlutataMAX, 28.3% F12+GlutataMAX, 1-20% heat-inactivated human AB serum, 1% P/S, 2 mM L-glutamine, 1. Mu.M beta. -mercaptoethanol, 5-50 ng/mL sodium selenite, 50-200. Mu.M ethanolamine, 20-100 mg/L ascorbic acid, 1-50 ng/mL IL-3, 1-100 ng/mL SCF, 1-100 ng/mL IL-7, 1-100 ng/mL IL-15, 1-100 ng/mL FLT3L, 1-100. Mu.g/mL DLL4, and 1-100. Mu.g/mL recombinant human fibronectin.
In certain embodiments, the concentration of IL-3 in the first medium is about 1/mL, about 2/mL, about 3/mL, about 4/mL, about 5/mL, about 6/mL, about 7/mL, about 8/mL, about 9/mL, about 10/mL, about 11/mL, about 12/mL, about 13/mL, about 14/mL, about 15/mL, about 16/mL, about 17/mL, about 18/mL, about 19/mL, about 20/mL, about 21/mL, about 22/mL, about 23/mL, about 24/mL, about 25/mL, about 26/mL, about 27/mL, about 28/mL, about 29/mL, about 30/mL, about 31/mL, about 32/mL, about 33/mL, about 34/mL, about 35/mL, about 36/mL, about 37/mL, about 38/mL, about 39/mL, about 40/mL, about 41/mL, about 42/mL, about 43/mL, about 44/mL, about 45/mL, about 46/mL, about 48/mL, about 50/mL.
In certain embodiments, the concentration of SCF in the first medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of IL-7 in the first medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of IL-15 in the first medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of FLT3L in the first medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of DLL4 in the first medium is about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL.
In certain embodiments, the concentration of recombinant human fibronectin (Retronectin) in the first medium is about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL.
In certain embodiments, the colony stimulating factors in the second medium are SCF and FLT3L.
In certain embodiments, the interleukins in the second medium are IL-7 and IL-15.
In certain embodiments, the second medium further comprises one or more of DMEM/f12+ GlutaMAX, P/S, L-glutamine, β -mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and recombinant human fibronectin (Retronectin).
In certain embodiments, the second medium contains 56.6% DMEM/F12+GlutataMAX, 28.3% F12+GlutataMAX, 1-20% heat-inactivated human AB serum, 1% P/S, 2 mM L-glutamine, 1. Mu.M beta. -mercaptoethanol, 5-50 ng/mL sodium selenite, 50-200. Mu.M ethanolamine, 20-100 mg/L ascorbic acid, 1-100 ng/mL SCF, 1-100 ng/mL IL-7, 1-100 ng/mL IL-15, 1-100 ng/mL FLT3L, 1-100. Mu.g/mL DLL4, and 1-100. Mu.g/mL recombinant human fibronectin.
In certain embodiments, the concentration of SCF in the second medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of IL-7 in the second medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of IL-15 in the second medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of FLT3L in the second medium is about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
In certain embodiments, the concentration of DLL4 in the second medium is about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL.
In certain embodiments, the concentration of recombinant human fibronectin (Retronectin) in the second medium is about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL.
In certain embodiments, the cell culture in step (c 11) is for 6-8 days.
In certain embodiments, the cell culture in step (c 11) is for about 6 days, about 7 days, or about 8 days.
In certain embodiments, the cell culture in step (c 12) is for 8-10 days, followed by an additional 8-10 days of culture using fresh second medium.
In certain embodiments, the cell culture in step (c 12) is for about 8 days, about 9 days, or about 10 days, followed by a further culture using fresh second medium for about 8 days, about 9 days, or about 10 days.
In certain embodiments, the method further comprises the steps of:
d. amplifying the NK cells prepared in the step c.
In certain embodiments, step d comprises adding the NK cells prepared in step c to a third medium for cell culture.
In certain embodiments, the third medium is selected from IMDM, stemScale PSC, essential 8, KSR/FGF2, mTESR, AKIT, or B8.
In certain embodiments, the third medium is IMDM.
In certain embodiments, the third medium contains an interleukin.
In certain embodiments, the interleukin is selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27.
In certain embodiments, the interleukin is IL-2.
In certain embodiments, the cell culture in step d is for 10-12 days.
In certain embodiments, the cell culture in step d is for about 10 days, about 11 days, or about 12 days.
In certain embodiments, step d further comprises adding IL-2 to the medium on days 4 and 8 of amplification.
In certain embodiments, the method further comprises the steps of:
e. freezing the NK cells amplified in the step d.
In certain embodiments, the NK cells are cryopreserved by a cryopreservation solution.
In certain embodiments, the frozen stock solution is selected from one or more of CryoStor CS10, sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride, human serum albumin, and DMSO.
In certain embodiments, the frozen stock solution is cryoston CS10.
The present invention also provides an NK cell prepared according to the method which shows increased expression of one or more of CD45, CD56, CD16, killer immunoglobulin-like receptor (KIR), NKG2D, NKp44, NKp46, fasL and TRAIL.
In certain embodiments, the NK cells exhibit an increase in NK cell markers or proteins 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.
In certain embodiments, the NK cells exhibit an increase in NK cell marker or protein of 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.
The invention also provides a pharmaceutical composition comprising NK cells according to the invention and a pharmaceutically acceptable excipient.
In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.
In certain embodiments, the additional therapeutic agent is an antibody.
The invention also provides an application of the NK cells prepared by the method, the NK cells or the pharmaceutical composition in preparation of a preparation for treating or preventing immune related diseases.
In certain embodiments, the immune-related disorder includes one or more of a tumor, a viral infection, a graft versus host disease, an autoimmune disease, and leukemia.
In certain embodiments, the viral infection comprises one or more 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).
The invention also provides a kit for preparing NK cells according to the method, which comprises a reagent for culturing somatic cells and instructions for implementing the method.
Example 1: iPSC preparation of HSPC (Condition # 8)
Human ipscs are typically maintained in culture in commercial Essential 8 medium. When iPSC reached 70% cell confluence, cells were examined to have a healthy morphology without spontaneous differentiation. iPSC cells were dissociated into single cells using Accutase. The collected single cells were counted and inoculated in a StemScale PSC suspension complete medium supplemented with 5-50 uM Y27632.
The cells were incubated at 37℃with 5% CO 2 CO resistance in incubator 2 Is incubated with continuous rotation at a speed of 30-100 rpm on an orbital shaker. Half-change cultures were performed within 24 hours (day 0) using StemScale PSC suspension complete medium, and ROCK inhibitor Rki and GSK-3 inhibitor CHIR 99021 were added.
After 24 hours (day 1), CHIR 99021 was added.
After 24 hours (day 2), embryoid Bodies (EBs) formed were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF as hematopoietic differentiation medium 1.
24-48 hours later (day 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF,1-100 ng/ml SCF and 1-20 uM SB431542 as hematopoietic differentiation medium 2.
24-72 hours later (day 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S, 2mM GlutaMaX, 50 ug/ml ascorbic acid, 1% ITS-G, 1-100 ng/ml SCF, 1-100 ng/ml FGF2, 1-100 ng/ml VEGF as hematopoietic differentiation medium 3.
After 24 hours (day 8), EBs and suspension cells were harvested as hematopoietic differentiation medium 4 on Stemline II hematopoietic stem cell expansion medium containing 50 ug/ml ascorbic acid, 1% ITS-G, 1-100 ng/ml SCF, 1-100 ng/ml FGF2, 1-100 ng/ml VEGF, 1-100 ng/ml FLT3 ligand (FLT 3L), 1-100 ng/ml TPO, 1-100 ng/ml IL-11, 1-100 ng/ml IGF1, 1-100 ng/ml IL-3, 1-100 ng/ml IL-6 and supplemented with 1% P/S, 2mM GlutaMaX for downstream differentiation or further expansion. HSPCs were harvested on days 8-10.
Example 2: iPSC preparation of HSPC (Condition # 4)
Human ipscs are typically maintained in culture in commercial Essential 8 medium. When iPSC reached 70% cell confluence, cells were examined to have a healthy morphology without spontaneous differentiation. iPSC cells were dissociated into single cells using Accutase. The collected single cells were counted and inoculated in a StemScale PSC suspension complete medium supplemented with 5-50 uM Y27632.
The cells were incubated at 37℃with 5% CO 2 CO resistance in incubator 2 Is incubated with continuous rotation at a speed of 30-100 rpm on an orbital shaker. Half-change cultures were performed within 24 hours (day 0) using StemScale PSC suspension complete medium and ROCK inhibitor Rki was added.
After 24 hours (day 1), CHIR 99021 (2X) was added.
After 24 hours (day 2), embryoid Bodies (EBs) formed were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF as hematopoietic differentiation medium 1.
24-48 hours later (day 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF,1-100 ng/ml SCF and 1-20 uM SB431542 as hematopoietic differentiation medium 2.
24-72 hours later (day 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml SCF, 1-100 ng/ml FGF2,1-100 ng/ml VEGF as hematopoietic differentiation medium 3.
After 24 hours (day 8), EBs and suspension cells were harvested as hematopoietic differentiation medium 4 on Stemline II hematopoietic stem cell expansion medium containing 50 ug/ml ascorbic acid, 1% ITS-G, 1-100 ng/ml SCF, 1-100 ng/ml FGF2, 1-100 ng/ml VEGF, 1-100 ng/ml FLT3 ligand (FLT 3L), 1-100 ng/ml TPO, 1-100 ng/ml IL-11, 1-100 ng/ml IGF1, 1-100 ng/ml IL-3, 1-100 ng/ml IL-6 and supplemented with 1% P/S, 2mM GlutaMaX for downstream differentiation or further expansion. HSPCs were harvested on days 8-10.
Example 3: iPSC preparation of HSPC (Condition # 6)
Human ipscs are typically maintained in culture in commercial Essential 8 medium. When iPSC reached 70% cell confluence, cells were examined to have a healthy morphology without spontaneous differentiation. iPSC cells were dissociated into single cells using Accutase. The collected single cells were counted and inoculated in a StemScale PSC suspension complete medium supplemented with 5-50 uM Y27632.
The cells were incubated at 37℃with 5% CO 2 CO resistance in incubator 2 Is incubated with continuous rotation at a speed of 30-100 rpm on an orbital shaker. Half-change cultures were performed within 24 hours (day 0) using StemScale PSC suspension complete medium, and ROCK inhibitor Rki and GSK-3 inhibitor CHIR 99021 were added.
After 24 hours (day 1), CHIR 99021 was added.
After 24-48 hours (day 2-3), embryoid Bodies (EBs) formed were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF as hematopoietic differentiation medium 1.
24-48 hours later (day 4-5), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml BMP4,1-100 ng/ml FGF2,1-100 ng/ml VEGF,1-100 ng/ml SCF and 1-20 uM SB431542 as hematopoietic differentiation medium 2.
24-48 hours later (day 6-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium containing 1% P/S,2mM GlutaMaX,50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml SCF, 1-100 ng/ml FGF2,1-100 ng/ml VEGF as hematopoietic differentiation medium 3.
After 24 hours (day 8), EBs and suspension cells were harvested as hematopoietic differentiation medium 4 on Stemline II hematopoietic stem cell expansion medium containing 50 ug/ml ascorbic acid, 1% ITS-G,1-100 ng/ml SCF, 1-100 ng/ml FGF2,1-100 ng/ml VEGF,1-100 ng/ml FLT3 ligand (FLT 3L), 1-100 ng/ml TPO, 1-100 ng/ml IL-11, 1-100 ng/ml IGF1, 1-100 ng/ml IL-3, 1-100 ng/ml IL-6 and supplemented with 1% P/S,2mM GlutaMaX for downstream differentiation or further expansion. HSPCs were harvested on days 8-10 and cell surface markers were assayed and cell morphology and surface marker detection results were shown in figures 4-5.
Examples 4 to 8 (conditions #1 to 3, 5, 7)
The culture methods of examples 4 to 8 (conditions #1 to 3, 5, 7) were substantially the same as those of example 1, and specific conditions are shown in FIG. 2.
Example 9: HSPC stability test
From a morphological view, it was observed that CHIR (condition 6) was added during the iPSC culture stage (i.e., day 0 of fig. 2), and as shown in fig. 5, it was seen that addition of CHIR allowed the iPSC spheres to resume differentiation during the iPSC culture stage and formed, thereby achieving a greater number of spheres with better uniformity for downstream differentiation.
Further attempts to extend the induction time of CHIR99021 to 2d at the stage of induction of formation of mesoderm by iPSC (i.e. days 1-3 of condition 6) resulted in fig. 6, which shows that addition of CHIR99021 for only 1 day would be more conducive to release of HPC cells from HE EB than for 2 days, and also more beneficial for overall cell activity/growth.
As a result of 4 batches of stability experiments performed on condition 6, as shown in fig. 7, most of CD34 cells in hematopoietic EBs were earlier precursor cells (CD 43-), whereas suspension cells that had been released from EB spheres could reach >50% CD34 positive, >90% CD43 positive, indicating that suspension cells released from EB spheres were mostly hematopoietic lineage progenitor cells.
Example 10: preparation of iNK cells
HSPC prepared in examples 1-8 were resuspended in NK differentiation medium 1 containing 56.6% DMEM/F12+GlutataMAX ™ -I, 28.3% F12+GlutataMAX ™ -I, 1-20% heat inactivated human AB serum, 1% P/S, 2 mM L-glutamine, 1. Mu.M beta. -mercaptoethanol, 5 ng/mL sodium selenite, 50. Mu.M ethanolamine, 20ug/mL ascorbic acid, 20 ng/mL IL-3, 50 ng/mL SCF, 50 ng/mL IL-7, 50 ng/mL IL-15, 10 ng/mL FLT 3L. On day 8 of iNK cell differentiation, cultures were resuspended in NK differentiation medium 2 containing 56.6% DMEM/F12+GlutataMAX ™ -I, 28.3% F12+GlutataMAX ™ -I, 1-20% heat inactivated human AB serum, 1% P/S, 2 mM L-glutamine, 1. Mu.M beta. -mercaptoethanol, 5 ng/mL sodium selenite, 50. Mu.M ethanolamine, 20ug/mL ascorbic acid, 50 ng/mL SCF, 50 ng/mL IL-7, 50 ng/mL IL-15, 10 ng/mL FLT 3L. After 10 days (day 18 of iNK cell differentiation), the culture was replaced with fresh NK differentiation medium 2. After 10 days (day 28 of iNK cell differentiation), the cultures were monitored for the presence of spindle iNK-like cells and evaluated for CD45/CD56 expression. The proportion of CD45+/CD56+ cells is expected to be >80%. The entire iNK differentiation process was performed in the presence of 5. Mu.g/mL DLL4 and 5. Mu.g/mL recombinant human fibronectin.
Example 11: expansion of iNK cells
iNK cell expansion was performed on day 28. iNK cells were collected in NK expansion medium (IMDM medium supplemented with 2-20% Cell-Vive ™ T-NK Xeno-Free serum replacement, 1% P/S and 50U/mL IL-2). Culturing 5X 10 in G-Rex 6M culture plates 6 iNK cells and 2.5 x 10 7 K562-mbII21-41BBL/cm 2 (1:5 iNK cells: feeder layer ratio). Cells at 37℃and 5% CO 2 Is cultured. IL-2 (5-500U/ml IL-2) was added to the cultures on days 4 and 8 of iNK cell expansion. iNK cells were collected on day 12 of iNK cell expansion. From this stage, the cultures can be cryopreserved using CryoStor CS 10.
It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims (9)

1. A method of producing natural killer cells, the method comprising the steps of:
a. inducing pluripotent stem cells (ipscs) to differentiate into Embryoid Bodies (EBs) in a culture medium supplemented with CHIR 99021;
b. inducing the EB formed in step a to differentiate into Hematopoietic Stem and Progenitor Cells (HSPCs);
c. inducing differentiation of HSPCs formed in step b into NK cells;
wherein, the step a comprises the following steps:
(a1) Induction was performed on day 0 in the presence of CHIR 99021 and ROCK inhibitor (Rki) Y-27632;
(a2) Induction was again performed on day 1 in the presence of CHIR 99021;
wherein the concentration of the CHIR 99021 is 1-20 mu M, and the concentration of the Y27632 is 5-50 uM;
the step b comprises the following steps:
(b1) Culturing EB in suspension in a culture medium supplemented with BMP4, FGF2 and VEGF;
(b2) Suspension culture was continued in BMP4, SCF, FGF2, VEGF and SB431542 supplemented medium;
(b3) Further suspension culturing in a medium supplemented with SCF, FGF2 and VEGF to form HSPCs;
wherein the concentration of BMP4, FGF2, VEGF and SCF is 1-100 ng/ml, and the concentration of SB431542 is 1-20 uM;
wherein step (a) is performed on days 0-1, step (b 1) is performed on days 2-3, step (b 2) is performed on days 4-5, and step (b 3) is performed on days 6-7;
The step c comprises the following steps: (c1) Contacting the HSPC formed in step b with Delta-like ligand 4 (DLL 4) and recombinant human fibronectin (Retronectin);
wherein the concentration of DLL4 is 1-100 mug/mL, and the concentration of Retronectin is 1-100 mug/mL;
the step (c 1) further comprises:
(c11) Adding the HSPC formed in the step b into a first culture medium for cell culture;
(c12) Adding the cells obtained by the culture in the step (c 11) into a second culture medium for cell culture to obtain NK cells;
wherein the first culture medium contains 1-50ng/mL IL-3, 1-100ng/mL SCF, 1-100ng/mL IL-7, 1-100ng/mL IL-15, 1-100ng/mL FLT3L;
the second culture medium contains 1-100ng/mL SCF, 1-100ng/mL IL-7, 1-100ng/mL IL-15, 1-100ng/mL FLT3L;
the cell culture in step (c 11) is for 6-8 days;
the cell culture in step (c 12) is carried out for 8 to 10 days, and then cultured for 8 to 10 days using a fresh second medium;
the hematopoietic stem cells have a cd34+cd43+ phenotype.
2. The method of claim 1, wherein the medium of step (b 1) is further supplemented with one or more of P/S, glutaMaX, ascorbic acid, or ITS-G.
3. The method of claim 1, wherein the medium of step (b 2) is further supplemented with one or more of P/S, glutaMaX, ascorbic acid, or ITS-G.
4. The method of claim 1, wherein the medium of step (b 3) is further supplemented with one or more of P/S, glutaMaX, ascorbic acid, or ITS-G.
5. The method of any one of claims 2-4, wherein the medium is further supplemented with 1% P/S, 2mM GlutaMaX, 50 ug/ml ascorbic acid, 1% ITS-G.
6. The method of claim 1, wherein the NK cells of step c have a cd45+/cd56+ phenotype.
7. The method according to claim 1, wherein the method further comprises:
d. amplifying the NK cells prepared in the step c.
8. The method of claim 7, wherein the amplifying of step d comprises:
and c, adding the NK cells prepared in the step c into a third culture medium for cell culture, wherein the third culture medium contains IL-2.
9. The method according to claim 1, wherein the method further comprises:
e. freezing the NK cells amplified in the step d.
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