Methods for targeting glycogen synthase kinase 3 beta in NK cells

Abstract

The present disclosure relates to methods of manipulating natural killer cells to treat, prevent, inhibit, reduce, and / or ameliorate diseases, including, but not limited to, cancer and other proliferative diseases.

Description

[Technical Field]

[0001] Related Applications This PCT application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 439,426, filed January 17, 2023, entitled "TARGETING GLYCOGEN SYNTHASE KINASE 3 BETA IN NK CELLS FOR ENHANCED ANTITUMOR ACTIVITY," which is incorporated by reference herein in its entirety.

[0002] Reference to sequence listing The Sequence Listing, created on January 16, 2024, and submitted on January 17, 2024 as an .XML file entitled "10935-026WO1.XML" having a file size of 2010 bytes, is incorporated herein by reference pursuant to 37 CFR § 1.52(e)(5).

[0003] The present disclosure relates to methods of genetically modifying natural killer cells to treat, prevent, inhibit, reduce, diminish, and / or ameliorate diseases, including, but not limited to, cancer and other proliferative diseases. [Background technology]

[0004] The antitumor activity of NK cells has been widely demonstrated across multiple cancer types. This antitumor role can be limited by several factors in the tumor microenvironment that mediate metabolic suppression. Consequently, chemical inhibitors, genetic, and epigenetic alterations are currently being tested to overcome these inhibitory mechanisms in NK cells. One promising target for enhancing NK cell cytotoxic activity has been the serine / threonine kinase GSK3β. While GSK3β has been shown to regulate multiple functions in other cell types, its role in lymphocytes, including NK cells, has not been fully investigated. Several reports have shown that drug inhibition of GSK3β improves the maturation and antitumor activity of NK cells with elevated GSK3β, such as IL-15-expanded NK cells or NK cells isolated from AML patients. However, specific anticancer GSK3β inhibitors have yet to be developed. Given this limitation, there remains a need to develop cancer therapeutics or methods for inhibiting GSK3β activity to promote antitumor activity. Summary of the Invention

[0005] The present disclosure provides methods for generating glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells, which comprise enhanced cellular metabolism and enhanced anti-tumor activity. The present disclosure also provides methods for treating, preventing, inhibiting, reducing, ameliorating, and / or ameliorating cancer in a subject by administering a composition comprising GSK3β-deficient NK cells, wherein the GSK3β-deficient NK cells comprise enhanced cellular metabolism and enhanced anti-tumor activity.

[0006] In one aspect, disclosed herein is a method of generating glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells, comprising isolating NK cells, deleting GSK3β in the NK cells using a gene editing system, and expanding and stimulating the NK cells with feeder cells comprising membrane-bound (mb) IL21 on their surface, wherein the GSK3β-deficient NK cells comprise decreased GSK3β expression and increased cellular metabolism compared to a control.

[0007] In one aspect, disclosed herein is a method of treating, preventing, inhibiting, diminishing, reducing, and / or ameliorating cancer and / or metastasis (e.g., acute myeloid leukemia, chronic myeloid leukemia, medulloblastoma, osteosarcoma, or glioblastoma, etc.), the method comprising administering to a subject a composition comprising glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells and a pharmaceutically acceptable carrier, wherein the GSK3β-deficient NK cells comprise increased cellular metabolism and increased anti-tumor activity.

[0008] Also disclosed herein are methods of generating GSK3β-deficient NK cells of any preceding aspect, or methods of treating, preventing, inhibiting, reducing, reducing, and / or ameliorating cancer and / or metastasis of any preceding aspect, wherein the method uses a gene editing system, including but not limited to a CRISPR / Cas9 gene editing system, to reduce and / or delete GSK3β in NK cells. In some embodiments, the method of any preceding aspect comprises introducing a Cas9 / ribonucleoprotein (RNP) complex into the NK cells.

[0009] In one aspect, disclosed herein is a method of generating GSK-3β-deficient NK cells of any preceding aspect, or a method of treating, preventing, inhibiting, reducing, reducing, and / or ameliorating cancer and / or metastasis of any preceding aspect, wherein the feeder cells further comprise membrane-bound 4-1BBL. In some embodiments, the GSK-3β-deficient NK cells are stimulated with feeder cells at least once a week. In some embodiments, the GSK-3β-deficient NK cells are stimulated with feeder cells for at least two weeks. In some embodiments, the feeder cells are K562 feeder cells, or the feeder line comprises CTSX-002. In some embodiments, the GSK-3β-deficient NK cells are supplemented with at least 50 IU of IL-2. In some embodiments, the IL-2 is human recombinant IL-2 (rIL-2).

[0010] Also disclosed are methods of generating GSK3β-deficient NK cells of any preceding aspect, or methods of treating, preventing, inhibiting, reducing, diminishing, and / or ameliorating cancer and / or metastasis of any preceding aspect, wherein GSK3β expression or activity is inhibited in NK cells. In some embodiments, the GSK3β-deficient NK cells comprise increased mitochondrial metabolism and glycolytic metabolism. In some embodiments, the GSK3β-deficient NK cells comprise increased cytotoxicity of GSK3β-deficient NK cells.

[0011] In one aspect, disclosed herein is a natural killer (NK) cell modified by any one of the methods of any preceding aspect.

[0012] In one aspect, disclosed herein are GSK3β-deficient NK cells, comprising reduced expression of GSK3β and increased mitochondrial metabolism and increased glycolytic metabolism. [Brief explanation of the drawings]

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0014] [Figure 1] Figures 1A, 1B, and 1C show the generation of GSK3β-KO FC-21-expanded NK cells. Figure 1A shows GSK3β expression after expansion on FC-21, assessed by RNA-seq analysis. Figures 1B and 1C show the efficiency of Cas9 / RNP-mediated deletion of GSK3β, assessed by Western blot and RNA-seq. See also Figure 6. [Figure 2] Figures 2A, 2B, 2C, 2D, and 2E show that deletion of GSK3β did not alter the antitumor activity or maturation of FC-21-expanded NK cells. Figures 2A and 2B show that both WT and GSK3β-KO NK cells were cocultured with AML tumor cell lines for 4 hours (Figure 2A shows the Kasumi1 tumor cell line, and Figure 2B shows the HL60 tumor cell line). NK cell killing is shown as the percentage of cytolysis (n = 3). Figure 2C shows that the release of IL2, IFNγ, and TNFα was assessed 4 hours after PHA stimulation of WT and GSK3β-KO NK cells (n = 3). Figure 2D shows that CD57 expression was assessed by flow cytometry on FC-21-expanded WT and GSK3β-KO NK cells (n = 6). Figure 2E shows B3GAT1 expression in WT, FC-21-expanded WT, and FC-21-expanded GSK3β-KO NK cells as measured by RNA-seq. ***p<0.001. See also Figure 7. [Figure 3] Figure 3 shows the transcriptome changes resulting from GSK3β-KO in NK cells. RNA-seq analysis was performed on FC-21-grown WT and GSK3β-KO derived NK cells. Figure 3 also shows the RNA-seq data showing DEGs (WT / KO), a volcano plat representing GO enrichment (WT), and the top down- and up-regulated genes affected by GSK3β deletion. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. [Figure 4]Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show that deletion of GSK3β enhances the metabolic capacity of NK cells. Figures 4A and 4B show summary data of metabolic analysis of paired WT and GSK3β-KO NK cells, respectively, for mitochondrial and glycolytic stress (n=3; mean±SD). Figure 4C shows the OCR / ECAR ratio. Figures 4D and 4E show the maximum respiratory capacity and reserve derived from Figure 4A. Figures 4F, 4G, and 4H show glycolysis, glycolytic capacity, and glycolytic reserve derived from Figure 4B. N=4, *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 5] Figure 5 shows that the down-regulated genes identified in GSK3β-KO NK cells were mostly up-regulated in GSK3β-overexpressing NK cells from patients with AML. RNA-seq analysis was performed on NK cells at different stages of maturation isolated by sorting from healthy donors and patients with AML. *p≦0.05. [Figure 6] FIG. 6 shows that the efficiency of Cas9 / RNP-mediated deletion of GSK3β was assessed by Western blot. [Figure 7] Figures 7A, 7B, 7C, and 7D show NK cell killing of (A) K452-3 donor, (B) U373 (glioblastoma)-3 donor, (C) DAOY (medulloblastoma), and (D) MG63 (osteosarcoma)-3 donor. [Figure 8] FIG. 8 shows the effect of GSK3β-KO on the expression of RelA, RelB, Rel, NFKB1, NFKB2, and ITGAL. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following description of the present disclosure is provided as an enabling teaching of the present disclosure in its best, currently known embodiment. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein while still obtaining beneficial results of the present disclosure. It will also be apparent that some of the desired advantages of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those skilled in the art will recognize that many modifications and adaptations to the present disclosure are possible and may even be desirable in certain circumstances and are a part of this disclosure. Accordingly, the following description is provided by way of illustration of the principles of the present disclosure, but not of limitation.

[0016] Reference will now be made in detail to the embodiments of the present invention illustrated in the drawings and examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0017] term Unless otherwise defined, all 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. As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide more specific embodiments and are disclosed. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] The following definitions are provided for a full understanding of terms used herein.

[0019] The terms "about" and "approximately" are defined as "close to," as understood by one of ordinary skill in the art. In one non-limiting embodiment, these terms are defined to be within 10%. In another non-limiting embodiment, these terms are defined to be within 5%. In yet another non-limiting embodiment, these terms are defined to be within 1%.

[0020] Ranges may be expressed herein as from "about" a particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of skill in the art, when a value is disclosed as "less than or equal to," it is understood that "greater than or equal to" and possible ranges between those values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15, are considered to be disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0021] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include situations where the condition occurs and situations where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include situations where the formulation includes an excipient and situations where the formulation does not include an excipient.

[0022] "Composition" refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. These terms also encompass pharmacologically acceptable active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, vectors, polynucleotides, cells, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "composition" is used, or when a particular composition is identified, it should be understood that this term includes not only the composition itself, but also pharmaceutically acceptable, pharmacologically active vectors, polynucleotides, salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0023] "Comprising" means that the composition, method, etc. includes the recited elements, but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean including the recited elements, but excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharmaceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc., from isolation and purification methods. "Consisting of" shall mean excluding more than trace elements of other ingredients, as well as substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0024] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a statistically significant amount of a condition, symptom, activity, or composition. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or greater increase, so long as the increase is statistically significant.

[0025] As used herein, the terms "enhance," "enhanced," "enhancing," and any grammatical variations thereof, as used herein, refer to the act of strengthening, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.

[0026] "Reduction" can refer to any change that results in a lower amount of a symptom, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product containing the substance is less compared to the output of the gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average decrease in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease, so long as the reduction is statistically significant.

[0027] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, a reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount below, above, or within a given range, compared to native or control levels.

[0028] "Reduce" or other forms of the word, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically with respect to some standard or expected value, in other words, it is relative, although it is understood that a standard or relative value need not necessarily be referenced. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0029] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction and therefore does not require a comparison to a control. As used herein, something may be reduced but not prevented, although something that is reduced may be prevented. Similarly, something may be prevented but not reduced, although something that is prevented may be reduced. It is understood that where reduction or prevention is used, the use of other words is expressly disclosed unless specifically specified otherwise.

[0030] "Treat," "treating," "treatment," and grammatical variations thereof, as used herein, include administration of a composition intended or intended to partially or completely prevent, delay, cure, palliate, alleviate, relieve, alter, remedy, ameliorate, improve, stabilize, mitigate, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject before onset (e.g., before overt signs of cancer), during early onset (e.g., at the time of early signs and symptoms of cancer), or after the development of established cancer. Prophylactic administration may occur from one day (a few days) to several years before the onset of symptoms of infection.

[0031] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. In one aspect, a subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0032] A "control" is a substitute control or sample used in an experiment for comparison purposes. Controls may be "positive" or "negative."

[0033] As used herein, the term "genetically modified" refers to a living cell, tissue, or organism whose genetic material has been altered using genetic engineering techniques. Genetic modification results in a change that does not occur naturally through breeding and / or natural recombination. Modified genes can be transferred within the same species, between species (creating transgenic organisms), and between kingdoms. Novel exogenous genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.

[0034] As used herein, "mutation" refers to a change in the structure of a gene, resulting in a variant form that can be transmitted to subsequent generations. Mutations can be caused by a single nucleotide change in DNA, or by the deletion, insertion, or rearrangement of larger portions of a gene. Mutations can result in the expression of physically or functionally altered proteins, resulting in lethal, non-lethal dysfunctional effects, or no effect.

[0035] As used herein, the term "deletion," also known as a gene deletion, deficiency, or deletion mutation, refers to a portion of a chromosome or a sequence of DNA that is removed during DNA replication. A deletion or gene deletion can involve the deletion of any number of nucleotides, from a single base to an entire piece of a chromosome.

[0036] "Culture" or "cell culture" is the process of growing cells under controlled conditions, generally outside their natural environment. After isolation of cells of interest from living tissue, they can then be maintained under carefully controlled conditions. These conditions vary for each cell type but generally consist of a suitable container containing a substrate or medium that provides essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2) and regulates the physicochemical environment (pH buffer, osmolality, temperature). Most cells require a surface or artificial substrate to form adherent cultures as monolayers (one single cell thick), while other cells can be grown freely suspended in a medium as suspension cultures. "Cell culture" also refers to the culture of cells derived from multicellular eukaryotes, particularly animal cells, as opposed to other types of cultures for growing cells, such as plant tissue cultures, fungal cultures, and microbial cultures (of microorganisms).

[0037] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" means that compounds are administered at the same time in time or essentially immediately after each other. In the latter case, the two compounds are administered sufficiently close in time that the results observed are indistinguishable from those that would be obtained if the compounds were administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, via entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​or immediately adjacent to the point of administration and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal portions of the subject's body. Administration includes self-administration and administration by another.

[0038] An "effective amount" of a drug refers to an amount of the drug sufficient to provide the desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific drug(s), and so forth. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any given subject may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the subject's age, sex, and weight. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0039] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., a component that can be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to human administration, the term generally means that the component has met the necessary standards of toxicology and manufacturing testing or is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0040] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in the preparation of a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0041] "Pharmacological activity" (or simply "activity") can refer to a derivative or analog (e.g., a salt, ester, amide, complex, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound, and to approximately the same extent, in a "pharmacologically active" derivative or analog.

[0042] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., the prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also encompass pharmacologically acceptable active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0043] A "therapeutically effective amount" or "therapeutically effective amount" of a composition (e.g., a composition comprising a drug) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of type 1 diabetes. In some embodiments, the desired therapeutic result is control of obesity. A therapeutically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), and various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition over a period of days, weeks, or years.

[0044] method The present disclosure provides methods for generating glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells, which comprise enhanced cellular metabolism and enhanced anti-tumor activity. The present disclosure also provides methods for treating, preventing, inhibiting, reducing, ameliorating, and / or ameliorating cancer in a subject by administering a composition comprising GSK3β-deficient NK cells, wherein the GSK3β-deficient NK cells comprise enhanced cellular metabolism and enhanced anti-tumor activity.

[0045] In one aspect, disclosed herein is a method of generating glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells, comprising isolating NK cells, deleting GSK3β in the NK cells using a gene editing system, and expanding and stimulating the NK cells with feeder cells comprising membrane-bound (mb) IL21 on their surface, wherein the GSK3β-deficient NK cells comprise decreased GSK3β expression and increased cellular metabolism compared to a control.

[0046] In one aspect, disclosed herein is a method of treating, preventing, inhibiting, diminishing, reducing, and / or ameliorating cancer and / or metastasis (e.g., acute myeloid leukemia, chronic myeloid leukemia, medulloblastoma, osteosarcoma, or glioblastoma, etc.), the method comprising administering to a subject a composition comprising glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells and a pharmaceutically acceptable carrier, wherein the GSK3β-deficient NK cells comprise increased cellular metabolism and increased anti-tumor activity.

[0047] It is understood and contemplated herein that the disclosed methods can be utilized with any cell type, including natural killer cells (NK cells), T cells, B cells, macrophages, fibroblasts, osteoblasts, hepatocytes, neurons, epithelial cells, and / or muscle cells. Human NK cells are a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). NK cells sense and kill target cells lacking major histocompatibility complex (MHC) class I molecules. NK cell activating receptors include, among others, the natural cytotoxicity receptors (NKp30, NKp44, and NKp46) and the lectin-like receptors NKG2D and DNAM-1. Their ligands are expressed on stressed, transformed, or infected cells, but not on normal cells, rendering the latter resistant to NK cell killing. NK cell activation is negatively regulated through inhibitory receptors such as killer immunoglobin (Ig)-like receptors (KIRs), NKG2A / CD94, TGFα, and leukocyte Ig-like receptor-1 (LIR-1).

[0048] Also disclosed herein are methods of generating GSK3β-deficient NK cells of any of the preceding aspects, or methods of treating, preventing, inhibiting, reducing, mitigating, and / or ameliorating cancer and / or metastasis of any of the preceding aspects, wherein the method uses a gene editing system, including but not limited to a CRISPR / Cas9 gene editing system, to reduce and / or delete GSK3β in NK cells. In some embodiments, the method of any of the preceding aspects comprises introducing a Cas9 / ribonucleoprotein (RNP) complex into the NK cells. Generally, a "CRISPR system," "CRISPR gene editing system," or "CRISPR integration system" collectively refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated "Cas" genes. In some embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. CRISPR systems are known in the art. See, for example, US Pat. No. 8,697,359, which is incorporated herein by reference in its entirety.

[0049] An endonuclease / RNP (e.g., Cas9 / RNP) consists of a three-component recombinase endonuclease protein (e.g., Cas9 endonuclease) complexed with a CRISPR locus. The endonuclease complexed with a CRISPR locus can be referred to as a CRISPR / Cas guide RNA. The CRISPR locus contains a synthetic single guide RNA (gRNA) that consists of an RNA that can hybridize to a complementary repeat RNA (crRNA) and a trans-complementary repeat RNA (tracrRNA) complexed with a target sequence. Thus, the CRISPR / Cas guide RNA hybridizes to a target sequence within the genomic DNA of a cell. In some cases, the Class 2 CRISPR / Cas endonuclease is a Type II CRISPR / Cas endonuclease. In some cases, the Class 2 CRISPR / Cas endonuclease is a Cas9 polypeptide, and the corresponding CRISPR / Cas guide RNA is a Cas9 guide RNA. These Cas9 / RNPs can cleave genomic targets with higher efficiency than approaches relying on exogenous DNA because they are delivered as a functional complex. Additionally, the rapid clearance of Cas9 / RNPs from cells can reduce off-target effects such as the induction of apoptosis.

[0050] To generate the RNP complex, the crRNA and tracrRNA can be mixed at a concentration of about 50 μM to about 500 μM (e.g., 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, 425 μM, 450 μM, 475 μM, or 500 μM), preferably 100 μM to about 300 μM, and most preferably about 200 μM, in a 1:1, 2:1, or 1:2 ratio at 95°C for about 5 minutes to form the crRNA:tracrRNA complex (i.e., guide RNA). The crRNA:tracrRNA complex can then be mixed with a final dilution of about 20 μM to about 50 μM (e.g., 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or 50 μM) of a Cas endonuclease (e.g., Cas9).

[0051] When CRISPR loci are bound to the target sequence in target cells, they can modify the genome by introducing one or more base pair insertions or deletions, heterologous DNA fragments (e.g., donor polynucleotides), endogenous DNA fragment deletions, endogenous DNA fragment inversions or translocations, or combinations thereof, into target DNA, such as GSK3β.Therefore, when combined with DNA for homologous recombination, the method of the present disclosure can be used to generate knockouts or knock-ins.It should be understood that the transduction of CRISPR elements into target DNA can be achieved using viral or non-viral approaches.

[0052] In non-viral approaches, one or more elements of the CRISPR system are electroporated into NK cells. In some embodiments, the method of any preceding aspect comprises introducing Cas9 / ribonucleoprotein (RNP) complex into NK cells. In some embodiments, the method of any preceding aspect comprises electroporating Cas9 / ribonucleoprotein (RNP) complex into NK cells.

[0053] It should also be noted that NK cell activation is negatively regulated through inhibitory receptors, such as killer immunoglobulin (Ig)-like receptors (KIR), NKG2A / CD94, TGFβ, and leukocyte Ig-like receptor-1 (LIR-1). Engagement of a single inhibitory receptor may be sufficient to prevent target lysis. Thus, NK cells efficiently target cells that express many stress-inducing ligands and few MHC class I ligands. TGFβ is a major immunosuppressive cytokine that inhibits NK cell activation and function. Therefore, it is understood and contemplated herein that modification of one of NK cells, e.g., GSK3β-deficient NK cells, is advantageous, as is the suppression of inhibitory receptors, such as killer immunoglobulin (Ig)-like receptors (KIR), NKG2A / CD94, TGFβ, and leukocyte Ig-like receptor-1 (LIR-1), so that negative regulation of NK cells is suppressed. Such modified cells would be highly useful in immunotherapy of any disease or condition that can be treated with the addition of NK cells. Thus, in one aspect, disclosed herein are defective NK cells comprising a knockout of the gene encoding GSK3β.

[0054] The methods disclosed herein also include incubating NK cells in a medium suitable for NK cell expansion. It is understood, and contemplated herein, that the culture conditions may include the addition of cytokines, antibodies, and / or feeder cells. Thus, in one aspect, disclosed herein is a method of genetically modifying NK cells, further comprising incubating the NK cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to transducing the cells in a medium that supports NK cell expansion, wherein the medium further comprises cytokines, antibodies, and / or feeder cells. For example, the medium can include IL-2, IL-12, IL-15, IL-18, and / or IL-21.

[0055] In one embodiment, the feeder cells can be purified from feeder cells that stimulate NK cells. NK cell-stimulating feeder cells for use in the claimed invention disclosed herein can be either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or PBMCs, RPMI8866, HFWT, K562, K562 cells transfected with membrane-bound IL-15 and 41BBL, or IL-21, or any combination thereof, or EBV-LCL. In some embodiments, the NK cell feeder cells are provided in combination with a solution of IL-21, IL-15, and / or 41BBL. The feeder cells can be seeded into the NK cell culture at a ratio of 1:2, 1:1, or 2:1. It is understood and contemplated herein that the culture period can be 1 to 14 days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after electroporation, preferably 3 to 7 days, and most preferably 4 to 6 days.

[0056] In one aspect, disclosed herein is a method of generating GSK-3β-deficient NK cells of any preceding aspect, or a method of treating, preventing, inhibiting, reducing, reducing, and / or ameliorating cancer and / or metastasis of any preceding aspect, wherein the feeder cells further comprise membrane-bound 4-1BBL. In some embodiments, the GSK-3β-deficient NK cells are stimulated with feeder cells at least once a week. In some embodiments, the GSK-3β-deficient NK cells are stimulated with feeder cells for at least two weeks. In some embodiments, the feeder cells are K562 feeder cells. In some embodiments, the feeder line comprises CTSX-002. In some embodiments, the GSK-3β-deficient NK cells are supplemented with at least 50 IU of IL-2. In some embodiments, the IL-2 is human recombinant IL-2 (rIL-2).

[0057] As used herein, "glycogen synthase kinase 3 beta (GSK3β) activity" refers to the addition of a phosphoryl moiety to a key enzyme in various cellular processes, including, but not limited to, glycogen / glucose metabolism, gene regulation, cell proliferation, and cell cycle pathways. It should be noted that the addition / removal of a phosphate group (also referred to as phosphorylation / dephosphorylation) can dramatically affect such cellular processes. A non-limiting example of GSK3β activity is the phosphorylation of glycogen synthase (GS), which inhibits GS from synthesizing glycogen polymers and promotes the release of glucose molecules, leading to hyperglycemia. Because GSK3β is a central hub in cellular metabolism, it has been characterized as a contributor to many diseases, including, but not limited to, cancer, diabetes, obesity, and Alzheimer's disease. Also disclosed are methods of generating GSK3β-deficient NK cells of any of the preceding aspects, or methods of treating, preventing, inhibiting, reducing, diminishing, and / or ameliorating cancer and / or metastasis of any of the preceding aspects, wherein GSK3β expression or activity is inhibited in NK cells. In some embodiments, GSK3β-deficient NK cells comprise increased mitochondrial and glycolytic metabolism.

[0058] As noted throughout this disclosure, the modified NK cells of the present disclosure are ideally suitable for use in immunotherapy, such as adoptively transferring the modified (i.e., engineered) NK cells into a subject in need thereof. Thus, in one aspect, disclosed herein is a method of adoptively transferring engineered NK cells into a subject in need thereof, comprising: a) obtaining target NK cells to be modified; b) deleting, reducing, and / or reducing GSK3β using a gene editing system of any preceding aspect; c) expanding and stimulating the NK cells with feeder cells comprising membrane-bound (mb)IL21 on their surface; and d) transplanting the engineered NK cells into the subject, wherein the modified NK cells comprise reduced GSK3β expression, increased mitochondrial metabolism, and increased glycolytic metabolism. In some embodiments, the GSK3β-deficient NK cells comprise the increased cytotoxicity of GSK3β-deficient NK cells.

[0059] The disclosed methods include GSK3β-deficient NK cells, which can be expanded and stimulated prior to administering the modified (i.e., engineered) NK cells to a subject. For example, disclosed herein is a method of adoptively transferring NK cells into a subject in need thereof, wherein the NK cells are expanded on mbIL-21-expressing feeder cells prior to administration to the subject. It is understood and contemplated herein that in some aspects, stimulation and expansion of modified (e.g., engineered) NK cells can occur in vivo after or simultaneously with administration of the modified NK cells to the subject. Thus, disclosed herein are methods of immunotherapy in which NK cells are expanded in a subject following transfer of the NK cells to the subject via administration of IL-21 or irradiated mbIL-21-expressing feeder cells.

[0060] It is understood, and contemplated herein, that the disclosed modified NK cells and methods of adoptive transfer of modified NK cells can be effective immunotherapies for cancer. The compositions of the present disclosure can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A non-limiting list of different types of cancer is as follows: lymphoma (Hodgkin's and non-Hodgkin's), leukemia, carcinoma, solid tissue cancer, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or cancer in general.

[0061] A representative, but non-limiting list of cancers that can be treated using the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, cancer of the mouth, pharynx, larynx, and squamous cell carcinoma of the lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0062] In one aspect, disclosed herein is a natural killer (NK) cell modified by any one of the methods of any preceding aspect.

[0063] In one aspect, the methods of the disclosure involve generating and / or using GSK3β-deficient NK cells to treat, prevent, inhibit, reduce, reduce, and / or ameliorate cancer and / or metastasis (e.g., acute myeloid leukemia, chronic myeloid leukemia, medulloblastoma, osteosarcoma, or glioblastoma, etc.), wherein the GSK3β-deficient NK cells comprise decreased expression and / or activity of GSK3β, increased mitochondrial metabolism, and increased glycolytic metabolism.

[0064] In some embodiments, the method of any preceding aspect comprises administering a composition comprising GSK-deficient NK cells and a pharmaceutically acceptable carrier.

[0065] The compositions disclosed herein can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier will necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as will be well known to those skilled in the art.

[0066] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically, including by local intranasal administration, or by inhalation. As used herein, "topical intranasal administration" refers to delivery of a composition to the nose and nasal cavity via one or both nostrils and can include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalation can occur through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the subject's species, age, weight, and general condition, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, etc. Therefore, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0067] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution in liquid before injection, or emulsions.Recently revised approaches to parenteral administration involve the use of slow or sustained release to maintain a constant dosage.See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0068] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA through cell-specific ligands, lymphocyte-specific tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in either constitutive or ligand-induced endocytic pathways. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0069] Although a number of embodiments of the present disclosure have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0070] By way of non-limiting illustration, examples of certain specific embodiments of the present disclosure are provided below. [Example]

[0071] The following examples are set forth below to illustrate compositions, devices, methods, and results according to the presently disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which would be apparent to one skilled in the art.

[0072] Example 1: Targeting GSK3β in NK cells for enhanced anti-tumor activity Loss of cytotoxicity and defective metabolism are associated with increased glycogen synthase kinase 3 beta (GSK3β) in natural killer (NK) cells derived from patients with acute myeloid leukemia or from healthy donors after ex vivo expansion with IL-15. Herein, growth of NK cells on feeder cells expressing membrane-bound IL-21 did not alter GSK3β levels. GSK3β was deleted using a Cas9 / RNP approach, and paired donor knockout and wild-type (WT) NK cells were expanded and evaluated for transcriptional and functional changes induced by loss of GSK3β. GSK3β-KO cells demonstrated significant changes in the expression of genes related to rRNA processing, cell proliferation, and metabolic function, indicating metabolic reprogramming. Measured cellular energetics and GSK3β-KO NK cells exhibited a 150% higher spare respiratory capacity (a marker of metabolic fitness). This is particularly important because previous reports of IL15-expanded NK cells indicated upregulation of GSK and the need for the use of drug inhibitors with lower on-target effects. The present disclosure teaches novel advantages of using mblL21 expansion of NK cells and the gene GSK3β in these cells.

[0073] The disclosed technology can be used instead of nonspecific GSK3β inhibitors. This demonstrated the superior advantages of using IL21-expressing feeder cells over IL15-based growth. GSK3β-KO cells can be used to treat several cancers.

[0074] Example 2: Deletion of glycogen synthase kinase 3 beta reprograms NK cell metabolism Drug inhibition of GSK3β in NK cells improves their maturation and cytotoxic activity, but the mechanism of GSK3β-mediated dysfunction has not been fully investigated. Herein, we demonstrate that growth of NK cells on feeder cells expressing membrane-bound IL-21 does not alter GSK3β levels, allowing us to study GSK3β function using CRISPR gene editing. GSK3β was deleted, and paired donor knockout and wild-type (WT) NK cells were expanded and assessed for transcriptional and functional changes induced by the loss of GSK3β. Surprisingly, the data show that deletion of GSK3β did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3β-KO cells showed significant changes in the expression of genes related to rRNA processing, cell proliferation, and metabolic function, demonstrating metabolic reprogramming. Next, key genes were found to be downregulated in GSK3β-KO NK cells and upregulated in GSK3β-overexpressing NK cells from AML patients, confirming their clinical relevance. Finally, we measured cellular energy and observed that GSK3β-KO NK cells exhibited a 150% higher spare respiratory capacity (a marker of metabolic fitness). These findings indicate a role for GSK3β in regulating NK cell metabolism.

[0075] We previously described an approach to study human NK cell biology that combines NK cell expansion with CRISPR gene editing using Cas9 complexed with a guide ribonucleoprotein (Cas9 / RNP). Herein, we applied this approach to study the transcriptional and functional role of GSK3β by generating CRISPR-mediated GSK3β-KO NK cells to avoid confounding off-target effects of small molecule drug inhibition. To confirm the clinical relevance of the differentially expressed genes found in GSK3β-KO NK cells, we compared them with NK cells from patients with AML-NK, previously shown to have elevated GSK3β. Finally, we investigated metabolic changes in NK cells induced by loss of GSK3β.

[0076] Materials and Methods Patient samples: NK cells were isolated from peripheral blood of healthy donors (American Red Cross, Columbus, OH) or untreated AML (Ohio State University Leukemia Tissue Bank). All studies were approved through the Ohio State University Institutional Review Board (protocol number 2009C0019).

[0077] Tumor cell lines: HL60 (AML), Kasumi1 (AML), K562 (CML), DAOY (medulloblastoma), and MG63 (osteosarcoma) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). U373 (glioblastoma) was a generous gift from Kevin Cassady (Nationwide Children's Hospital, Columbus, Ohio, USA). CSTX002 feeder cells (K562 genetically modified to express 4-1BBL and membrane-bound IL-21, hereafter referred to as FC-21) were generated.

[0078] Isolation and expansion of NK cells: Buffy coats from red blood cell donations of healthy volunteers were obtained from the American Red Cross (Columbus, OH, USA), from which NK cells were isolated using RosetteSep™ Human NK Cell Enrichment Cocktail (Stem Cell Technologies, 15065, Vancouver, BC, Canada). Purified NK cells (CD3 陰性 / CD56 陽性 ) were stimulated for 2 weeks with irradiated CSTX002 feeder cells in AIM-V growth medium supplemented with ICSR (CTS™ AIMV™ SFM / CTS™ Immune Cell SR, Thermo Fisher Scientific) and 50 IU of human recombinant IL-2 (rIL-2) (Novartis).

[0079] Generation of CRISPR-edited NK cells: GSK3β-KO NK cells were generated by electroporation of Cas9 / RNP targeting exon 5 of the GSK3β gene (5-CAGTATCAGGATCCAACAAG (SEQ ID NO: 1)) into day 7 expanded NK cells.

[0080] NK functional assay: Calcein-AM was used to assess cytotoxicity. Briefly, tumor cell targets were loaded with 2 μg / mL of Calcein-AM for 30 minutes. Cells were washed and incubated with WT or GSK3β-KO NK cells at multiple effector / target (E:T) ratios as indicated in the figure legends for 4 hours.

[0081] Metabolic assays: Metabolic assays were performed. Briefly, oxygen consumption rate (OCR) was measured using the Seahorse XF Cell Mito Stress Test Kit (catalog number 103015-100, Agilent Technologies, Santa Clara, CA, United States), and extracellular acidification rate (ECAR) was measured using the Seahorse XF Glycolysis Stress Test Kit (catalog number 103020-100, Agilent Technologies, Santa Clara, CA, United States). Cell-Tak coated plates (Corning® Cell-Tak™) (catalog number 354240, Bedford, Massachusetts) were used with an Agilent Extracellular Flux Pak (catalog number 102416-100, Agilent Technologies, Santa Clara, CA, United States) on a Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, United States). Expanded WT and GSK3βKO NK cells were pretreated in XF RPMI at 37°C in a non-CO2 incubator for 1-2 hours before measurement. The medium was supplemented with 10 mM glucose and 1 mM L-glutamine without phenol red, at pH 7.35-7.4. OCR and ECAR were determined under basal conditions after adding 1 mM oligomycin, 1.5 mM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), 0.5 mM rotenone, and 0.5 mM antimycin A to the cell cultures.

[0082] Antibodies: The following antibodies were used for flow cytometry: anti-GSK3β pS9-PE (catalog no. 130-106-964, Miltenyi Biotec), CD57 (catalog no. 130-111-964, Miltenyi Biotec). The following antibodies were used for Western blot: GSK3β rabbit mAb-27C10 (catalog no. 9315S, Cell Signaling, 1:1000), β-actin mouse mAb-8H10D10 (catalog no. 3700S, Cell Signaling, 1:1000), anti-rabbit IgG HRP-conjugated (catalog no. 7074S, Cell Signaling, 1:5000), and anti-mouse IgG HRP-conjugated (catalog no. 7076S, Cell Signaling, 1:5000).

[0083] Cytokine secretion: To induce cytokine secretion, 2 × 10 6 NK cells at 10 μg / mL were stimulated with 10 μg / mL PHA. After a 4-hour incubation, supernatants were collected and stored at -80°C. On the day of the assay, supernatants were thawed and measured in duplicate on a Bio-Rad Bio-plex Pro Human Immunotherapy Panel 20-Plex (catalog no. 12007975) according to the manufacturer's instructions. Data were acquired on a Bio-Rad Bio-Plex 200 system and analyzed with Bio-Plex Manager software using curve fitting by logistic regression (5PL regression).

[0084] RNA-Sequencing of Non-Proliferating Healthy and AML-NK Cells: RNA-sequencing (RNA-seq) analysis was performed. Briefly, freshly sorted NK cells from normal donor peripheral blood (American Red Cross; n = 3 donors) or newly diagnosed AML patients (Ohio State University Leukemia Tissue Bank; n = 5; OSU IRB#2009C0019) were pelleted, and total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen). Directional poly-A RNA sequencing libraries were prepared, ranging from 33.2 to 48.0 × 10 6Sequences were generated as 42-bp paired-end reads on an Illumina NextSeq 500 instrument (Illumina) to a depth of read pairs (Active Motif). Alignment to the human genome (hg19 construct) was performed using TopHat. Transcriptome assembly and analysis were performed using Cufflinks, and expression was reported as FPKM.

[0085] RNA-sequencing of proliferating WT and GSK3β-KO NK cells: For RNA-seq of proliferating WT and GSK3β-KO NK cells, RNA libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc.) and XX × 10 RNA samples per library were analyzed using the Illumina HiSeq 4000 platform (Institute for Genomic Medicine, Nationwide Children's Hospital). 6 Sequence reads (150 bp each) were generated. Reads were aligned and count tables were generated using Kallisto (v0.43.1). Differential expression was then performed using the Bioconductor package DeSeq2 (v1.36.0). Volcano plots were generated using the package Glimma (v2.6.0) in R. Gene Ontology (GO) was performed using GOrillia and then visualized using REVIGO via their online web portal.

[0086] result Deletion of GSK3β in FC-21-expanded NK cells. A gene editing approach for primary NK cells was previously described, in which growth on FC-21 feeder cells enhanced DNA repair mechanisms and expanded edited cells to large numbers. GSK3β was previously shown to be overexpressed when NK cells were expanded in the presence of soluble IL-15, associated with inhibition of their maturation and cytotoxic activity. Therefore, we first assessed the relative gene expression of GSK3β in both naive and FC-21-expanded NK cells to evaluate the stability of GSK3β in this model system. GSK3β expression levels were similar between WT and expanded NK cells (Figure 1A). To study the role of GSK3β and avoid off-target effects of chemical inhibitors, we generated GSK3β-KO NK cells and assessed knockout efficiency at the protein level by Western blot and at the mRNA level by RNA-sequencing. The data demonstrated successful GSK3β gene deletion in FC-21 expanded NK cells (FIGS. 1B, 1C and 6).

[0087] Deletion of GSK3β does not alter the killing capacity, cytokine secretion, or maturation of FC-21-expanded NK cells. Drug inhibition of GSK3β can improve NK cell killing against AML in NK cells with elevated GSK3β, such as those expanded with IL-15 or from patients with AML. Therefore, the effect of knocking out GSK3β in FC-21-expanded NK cells was evaluated based on their anti-AML activity. Unexpectedly, deletion of GSK3β did not alter NK cell killing against the AML cell lines HL60 and Kasumi1 in a standard 4-hour killing assay (Figures 2A and 2B). Additionally, GSK3β-KO NK cells exhibited similar killing capacity to WT NK cells against K562 (CML), DAOY (medulloblastoma), and MG63 (osteosarcoma). Previous reports have also associated improved killing in NK cells treated with GSK3β chemical inhibitors with increased cytokine secretion. Therefore, cytokine secretion was assessed in WT and GSK3β-KO and we found that both WT and GSK3β-KO NK cells secreted high levels of IL-2, IFN, and TNF, with no differences observed between the two (Fig. 2C).

[0088] Additionally, it was previously reported that GSK3β drug inhibition drove the maturation of IL-15-expanded NK cells, as evidenced by CD57 expression (Cichocki, F., et al. GSK3 Inhibition Drives Maturation of NK Cells and Enhances Their Antitumor Activity. Cancer Res, 2017. 77(20):p.5664-5675). Therefore, we analyzed CD57 expression in both FC-21-expanded WT and GSK3β-KO NK cells by flow cytometry and found no difference between them (Figure 2D). FC-21-expanded NK cells were then examined for the expression of B3GAT1, a gene encoding an enzyme involved in catalyzing the CD57 carbohydrate epitope. Interestingly, B3GAT1 was downregulated 30-fold in FC-21-expanded NK cells compared with naive NK cells, and this low expression was not altered by GSK3β deletion (Figure 2E). This explains the low CD57 surface expression in both WT and gene-edited cells and indicates that B3GAT1 is not directly regulated by GSK3β and that other factors may be involved in regulating CD57 expression in NK cells.

[0089] Transcriptional changes in GSK3β-KO NK cells To study the role of GSK3β in NK cells, we performed bulk RNA-seq on WT or GSK3β-KO NK cells after growth in FC-21. Differential gene expression analysis via Deseq2 revealed 55 genes significantly upregulated in WT NK cells (adjusted P value < 0.05, paired Deseq2 test) and 12 genes significantly upregulated in GSK3β-KO cells. Thus, the majority of changes detected were a loss of GSK3β expression concomitant with a loss of GSK3β expression (Figures 3A and 3C), consistent with the lead drop across exon 6 in the GSK3β locus seen in Figure 1C.

[0090] We then used gene ontology (GO) analysis of the 55 WT-specific transcripts to determine which expression programs were regulated by GSK3β. GO results were then visualized via REVIGO, which clustered redundant categories for biological interpretation (Figure 3B). Unique GO clusters included those for ribosomal RNA (rRNA) processing, secretion, metabolic processes, and proliferation (Figure 3B). For example, GSK3β-KO mice lost transcription of genes involved in NK cell homeostasis, such as the high-affinity IL2 trimeric receptor encoded by IL2RA and the survival gene BCL2 (Figure 3C). Downregulation of genes encoding TNFα effectors, namely leukemia inhibitory factor (LIF) and TNFSF10 (encoding TRAIL), was observed in GSK3β-KO mice. Additional expression analysis indicated a global deregulation of ribosomal RNA biogenesis, as several genes involved in ribosomal RNA processing were lost (e.g., WDR3, WDR74, see Figure 3C) and genes involved in mitochondrial function were gained (e.g., MT-ND4 and MT-ND2, see Figure 3C). Together, transcriptome analysis demonstrated a role for GSK3β in regulating NK cell homeostasis, effector function, and metabolism.

[0091] Deletion of GSK3β leads to metabolic reprogramming of NK cells. To explore the effects of upregulating mitochondrial genes identified by RNA-seq, we investigated the cellular metabolism of WT and GSK3β-KO NK cells by assessing both mitochondrial (Figures 4A, 4D, and 4E) and glycolytic stress (Figures 4B, 4F, 4G, and 4H). GSK3β-KO NK cells exhibited an overall shift toward oxidative metabolism with higher OCR and spare respiratory capacity (Figures 4A, 4B, 4C, 4D, and 4E). Spare respiratory capacity is crucial for memory T cell formation and has previously been described as a reliable indicator of metabolic fitness. Additionally, GSK3β-KO NK cells also exhibited moderately higher ECAR, glycolytic capacity, and glycolytic reserve than WT NK cells, indicating that increased oxidative phosphorylation did not compensate for reduced glycolysis. This indicates that GSK3β deletion induces NK cells to enhance their overall metabolic capacity, which is beneficial for maintaining antitumor activity.

[0092] GSK3β is highly expressed in NK cells at stages 5 and 6 of maturation from AML patients. To further explore the role of GSK3β in NK cells, we examined transcriptional changes in NK cells from AML patients, which have been shown to overexpress GSK3β, and validated the key genes identified as differentially expressed in GSK3β-KO NK cells. To correct for confounding effects due to differences in NK cell maturation, we separately studied the expression levels of these genes at NK maturation stages 5 and 6. First, we confirmed that GSK3β was statistically elevated in NK cells from AML patients compared with healthy donor NK cells for both stage 5 and stage 6 cells. The relative expression of key genes identified in GSK3β-KO NK cells (Figure 5), and the strong concordance found for downregulated genes, was then evaluated in GSK3β-KO NK cells that are upregulated in GSK3β-overexpressing NK cells from patients with AML. LIF was overexpressed only at stage 5, but not at stage 6. IL2RA expression was significantly increased in both AML-NK5 and AML-NK6 cells compared with healthy NK cells. No difference was observed in the expression level of WDR74, but WDR3 was significantly increased in AML-NK5. Although we found high levels of MT-ND4 and MT-ND2 transcripts in the GSK3β-KO experiment, MT-ND4 and MT-ND2 mRNA transcripts were not identified in any of these samples.

[0093] Consideration NK cells play an important role in immune surveillance and the prevention of tumor development and progression. NK cells from patients with AML exhibit significant defects in both number and functional activity and are unable to control the onset, progression, and recurrence of AML. Despite well-described phenotypic changes, a specific molecular explanation for these dysfunctions remains necessary. It has previously been reported that high expression of GSK3β on AML NK cells significantly affected NK cell killing capacity (Parameswaran, R., et al., Repression of GSK3 restores NK cell cytotoxicity in AML patients. Nat Commun, 2016.7:p.11154). Herein, we demonstrated that enhanced expression of GSK3β was present in NK cells at both stages 5 and 6 of development in patients with AML. Evidence regarding the biological mechanism of GSK3β-mediated dysfunction in NK cells was previously limited. Using Cas9 / RNP gene editing, we generated GSK3β-KO NK cells, enabling a precise genetic understanding of the effects of GSK3β on primary human NK cells, revealing novel information regarding the influence of GSK3β on homeostasis, effector function, and transcriptional regulation of metabolism. As observed with IL-15-amplified NK cells, we showed that expansion of NK cells on FC-21 did not increase GSK3β expression levels, reducing the need for GSK3β inhibition in adoptive NK cell therapy.

[0094] Deletion of GSK-3β resulted in increased mitochondrial respiratory capacity, which was associated with an increase in the complex 1 genes, MT-ND2 and MT-ND4, that favor OXPHOS metabolism. Complex 1 (also known as NADH dehydrogenase), defined as the "most complex complex," is composed of 46 subunits, seven of which (including MT-ND2 and MT-ND4) are encoded by the mitochondrial genome. Complex 1 catalyzes the transfer of electrons from NADH through the respiratory chain, using ubiquinone as the electron acceptor. Furthermore, expression of mitochondrial respiratory complex 1 has previously been associated with increased mitochondrial oxidative phosphorylation (OXPHOS) activity. Additionally, targeting OXPHOS with complex 1 inhibitors reduced OXPHOS in pancreatic cancer cells, strengthening the correlation between OXPHOS and complex 1. In line with previously published evidence, this data demonstrated that GSK-3β deletion resulted in increased OXPHOS with higher maximal and spare respiratory capacity. The edited cells also shifted their metabolic profile more toward OCR than ECAR, which can improve ATP generation and provide more energy to cells essential for effector function. It has previously been demonstrated that NK cells from both leukemia mice and patients with AML exhibited similar metabolic defects (Bou-Tayeh, B., et al., IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia. Front Immunol, 2021.12:p.730970). Several groups have shown that NK cells from AML patients or expanded with IL-15 have high levels of GSK3β and that chemical inhibition of GSK3β can improve their antitumor activity through an increase in NFKB signaling molecules (RELA, RELB, c-REL, NFKIBA).However, the data herein show that FC21 expansion of NK cells did not increase GSK3β levels, and thus its deletion did not alter their cytotoxicity, at least in the short-term 4-h killing assay used.

[0095] NK cells with high GSK3β have been shown to express low levels of CD57. The benefit of using a GSK inhibitor on these NK cells was increased CD57 expression, indicating maturation to an adaptive memory-like phenotype. This protein is recognized as a maturation / senescence marker, its frequency increases with age, and it has been reported to be absent or weak on fetal and infant NK cells. Several clinical studies have reported that high CD57 expression on NK cells is associated with better clinical outcomes, so CD57 expression, indicating terminal differentiation of NK cells, should be beneficial from an immunotherapy perspective. Drug inhibition of GSK3β has been shown to result in increased CD57 expression. Of note, CD57 is not a DNA-encoded protein but rather a carbohydrate epitope catalyzed by B3GAT1. This example demonstrates a decrease in B3GAT1 levels in FC-21-expanded NK cells, but both CD57 and B3GAT1 remain unchanged after GSK3β deletion. This indicates that GSK3β does not directly regulate B3GAT1, or therefore CD57, suggesting that other regulatory factors and / or pathways control maturation- and memory-associated CD57 expression. Taken together, GSK3β is shown to be a negative metabolic regulator in NK cells.

[0096] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

1. 1. A method for generating glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells, comprising: a. isolating the NK cells; b. deleting GSK3β in said NK cells using a gene editing system; c. Expanding and stimulating the NK cells with feeder cells comprising membrane-bound (mb) IL21 on their surface; The method, wherein the GSK3β-deficient NK cells comprise decreased GSK3β expression and increased cellular metabolism compared to a control.

2. 10. The method of claim 1, wherein the gene editing system comprises a CRISPR / Cas9 gene editing system.

3. 3. The method of claim 1 or 2, wherein the gene editing system comprises introducing a Cas9 / ribonucleoprotein (RNP) complex into the NK cells.

4. The method according to any one of claims 1 to 3, wherein the GSK3β-deficient NK cells are stimulated with feeder cells at least once a week.

5. The method of any one of claims 1 to 4, wherein the feeder cells further comprise membrane-bound 4-1BBL.

6. The method according to any one of claims 1 to 5, wherein the GSK3β-deficient NK cells are stimulated with feeder cells for at least two weeks.

7. The method according to any one of claims 1 to 6, wherein the feeder cells are K562 feeder cells.

8. The method of any one of claims 1 to 7, wherein the feeder line comprises CTSX-002.

9. The method of any one of claims 1 to 8, wherein the GSK3β-deficient NK cells are supplemented with at least 50 IU of IL-2.

10. 10. The method of claim 9, wherein the IL-2 is human recombinant IL-2 (rIL-2).

11. The method of any one of claims 1 to 10, wherein the GSK3β-deficient NK cells comprise increased mitochondrial and glycolytic metabolism.

12. A natural killer (NK) cell modified by any one of the methods according to claims 1 to 11.

13. 1. A method of treating cancer in a subject, the method comprising administering to the subject a composition comprising glycogen synthase kinase 3 beta (GSK3β)-deficient natural killer (NK) cells and a pharmaceutically acceptable carrier, wherein the GSK3β-deficient NK cells have increased cellular metabolism and increased anti-tumor activity.

14. 14. The method of claim 13, wherein GSK3β is deleted from the NK cells using a gene editing system.

15. 15. The method of claim 14, wherein the gene editing system comprises a CRISPR / Cas9 gene editing system.

16. 15. The method of claim 13 or 14, wherein the gene editing system comprises introducing a Cas9 / ribonucleoprotein (RNP) complex into the NK cells.

17. The method of any one of claims 13 to 16, wherein the GSK3β-deficient NK cells are grown on feeder cells comprising membrane (mb)IL21 on the surface thereof.

18. The method according to any one of claims 13 to 17, wherein the GSK3β-deficient NK cells are stimulated with feeder cells at least once a week.

19. The method of any one of claims 13 to 18, wherein the feeder cells further comprise membrane-bound 4-1BBL.

20. The method according to any one of claims 13 to 19, wherein the GSK3β-deficient NK cells are stimulated with feeder cells for at least two weeks.

21. The method according to any one of claims 13 to 20, wherein the feeder cells are K562 feeder cells.

22. The method of any one of claims 13 to 21, wherein the feeder line comprises CTSX-002.

23. The method of any one of claims 13 to 22, wherein the GSK3β-deficient NK cells are supplemented with at least 50 IU of IL-2.

24. 24. The method of claim 23, wherein the IL-2 is human recombinant IL-2 (rIL-2).

25. The method of any one of claims 13 to 24, wherein the method increases mitochondrial metabolism and glycolytic metabolism in the GSK3β-deficient NK cells.

26. The method of any one of claims 13 to 25, wherein the method increases the cytotoxicity of the GSK3β-deficient NK cells.

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