Compositions, systems, and methods for treating cancer using tumor treatment sites and killer cells.

Abstract

The present invention discloses compositions, systems, and methods for reducing the viability of cancer cells, treating cancer, and preventing an increase in the volume of tumors present in the body. The systems and methods include administering at least one composition containing at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell, but not limited to these) and simultaneously applying an alternating electric field.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising at least one natural killer (NK) cell for use in a method of treating cancer in a subject, wherein the method is (1) A step of applying an alternating electric field to a target area of ​​the subject for a certain period of time, (2) A composition comprising the step of administering at least one composition to the subject. Regarding.

[0002] This application claims the benefits under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 493,141, filed on 30 March 2023, and U.S. Provisional Application No. 63 / 599,142, filed on 15 November 2023. The entire contents of the above patent applications are expressly incorporated herein by reference. Statement on federally funded research Not applicable. [Background technology]

[0003] A tumor therapeutic field (TT field) is a low-intensity (e.g., 1–3 V / cm) alternating electric field in the medium-frequency range (e.g., 100–500 kHz) that targets solid tumors by inhibiting mitosis. This non-invasive treatment targets solid tumors and is described, for example, in U.S. Patents 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776. The TT field is typically transmitted through two pairs of transducer arrays that generate a field perpendicular to the tumor being treated, with the electrode arrays constituting each of these pairs of transducer arrays located opposite the part of the body being treated. Specifically, in the OPTUNE® system, one pair of electrodes is positioned on the left and right (LR) sides of the tumor, and the other pair of electrodes is positioned on the front and back (AP) sides of the tumor. The TT field is approved for the treatment of glioblastoma multiforme (GBM) and can be delivered, for example, via the OPTUNE® system (Novocure Limited, St. Helier, Jersey), which includes a transducer array placed on the patient's shaved scalp.

[0004] In the OPTUNE® device, each transducer array used for TT field delivery consists of a series of ceramic disc electrodes bonded to the patient's skin (e.g., the patient's shaved scalp for GBM treatment) via a layer of conductive medical gel. The purpose of the medical gel is to conform to the body's contours and provide good electrical contact between the array and the skin, thereby allowing the gel interface to crosslink the skin and reduce interference. The device is intended to be worn continuously by the patient for 2-4 days, after which it is removed for hygienic care and reshaving (if necessary), and its new array set is reattached. Thus, the medical gel remains in substantially continuous contact with the area of ​​the patient's skin for 2-4 days at a time. In addition, the array can be shifted a few centimeters in either direction, allowing the skin to heal with each treatment period. Therefore, the area of ​​skin that was covered with electrodes / gel for 2-4 days is exposed for 2-4 days with the replaced electrodes slightly shifted, after which the device can be reapplied to the original area of ​​skin for the next 2-4 days. [Overview of the project] [Problems that the invention aims to solve]

[0005] Natural killer (NK) cells are a type of innate lymphocyte population. NK cells can differentiate between healthy (i.e., self) cells and infected or abnormal cells, including transformed cancer cells. Unlike T cells and B cells, NK cells can attack and kill abnormal cells (including cancer cells) in an antigen-independent manner (Shimasaki et al. (2020) Nat Rev Drug Discov, 19:200-218). Healthy cells express low levels of NK cell activating ligands and high levels of major histocompatibility (MHC) class I (in human HLA (human leukocyte antigen)). MHC class I binds to killer immunoglobulin-like (KIR) receptors on NK cells, preventing NK cells from killing healthy cells (Liu et al. (2021) J Hematol Oncol, 14:7). Downregulation of MHC class I in infected or cancer cells can lead to NK cell activation and lysis of transformed cells (Liu et al., 2021). NK cells have other stimulating and inhibitory signals that are facilitated through receptor-ligand interactions with target cells. For example, HLA-E, a non-classical HLA class I molecule, binds to NK cells via the NKG2A receptor and acts as an inhibitory signal to NK cells (Lauterbach et al. (2015) Human Immunology, 76(8):578-586). Therefore, high HLA-E levels can be found in several cancer types, including non-small cell lung cancer (NSCLC), pancreatic, renal, melanoma, and head and neck cancer (Borst et al. (2020) Clinical Cancer Research, 26(21):5549-5556).

[0006] Most clinical trials using NK cell-based immunotherapy have focused on hematological malignancies. However, clinical trials of NK cells have also been conducted in solid tumors such as breast and ovarian cancer (Chu et al. (2022) J Transl Med, 20:240).

[0007] Furthermore, Silginer et al. (Neuro-Oncology (2018) 20(6):vi133) investigated the interaction of the TT field with molecules involved in the immune response, as well as drugs that can modulate immune cell activity. They found increased NKG2D ligand expression and enhanced NK cell-based death of glioma cells exposed to the TT field, while MHC class I and class II expression remained unchanged.

[0008] Cytokine-induced killer (CIK) cell therapy is a type of adoptive immunotherapy that uses a heterogeneous immune cell population generated in vitro from the patient's peripheral blood mononuclear cells (PBMCs) in the presence of IL-2, anti-CD3 antibodies (e.g., OKT3, but not limited to these), and IFN-gamma (Introna (2017) Journal of Autoimmunity, 85:32-44). CIK cells are phenotypic cytotoxic T-terminal differentiated effector memory (EMRA) lymphocytes with antitumor activity (Franceschetti et al. (2009) Experimental Hematology, 37:616-628.e2), CD3 + CD56 + CD8 +This population expresses different NK markers, including NKG2D, and can recognize and kill tumor cells in a TCR-independent NK-like manner (Sangiolo et al. (2008) International Immunology, 20:841-848). Recent reports from the International Registry of CIK Cells (IRCC) indicate that CIK cells have been tested in 106 clinical trial settings in more than 30 different tumors, resulting in significant improvements in progression-free survival (PFS) and overall survival (OS) in 27 autologous settings, with mild adverse effects. CIK cells have also been evaluated in patients with GBM, the most common primary malignant brain tumor (Kong et al. (2017) Oncotarget, 8:7003-7013). In this Phase III trial, CIK cells were associated with improved PFS, but not OS. This trial is one of several immunotherapy trials in GBM with poor clinical outcomes, including immune checkpoint inhibitors, vaccination, and different adoptive T-cell therapies (Weenink et al. (2020) Cancers (Basel), 12:E751). [Brief explanation of the drawing]

[0009] [Figure 1] This paper presents an analysis of MHC class I expression in human and mouse pancreatic cell lines (1A-AsPc1 human pancreatic cancer cell line and 1B-Panc02 mouse pancreatic cancer cell line) after 72 or 96 hours of exposure to a TT field. [Figure 2] The graphs show transcriptomics analyses after TT field treatment in glioblastoma (GBM) cell lines, malignant pleural mesothelioma (MPM) cell lines, and pancreatic cancer cell lines AsPC1 and BxPC3 compared to controls, in order to identify genes showing differences in expression after TT field treatment. [Figure 3]The proliferation of CIK cells is shown in graph. CIK cells were generated in vitro from HD and counted at different time points. (a) Cells were counted at different time points. The data shown are the median and interquartile range of cell counts from five separate experiments. After 21 days (±1 day) of culture, CIK cells were characterized by flow cytometry for (b) CD3, C56, and (c) CD4 and CD8 expression. The data shown are the mean of five separate experiments. [Figure 4] The graph shows the co-culture of CIK / glioblastoma stem cells (GSCs). After 24 or 48 hours of co-culture, GSC viability was assessed by flow cytometry and plotted as the percentage of dead cells (L / D+) in CellTrace-labeled cells. GSCs showed a T / E ratio-dependent increase in the apoptotic fraction when co-cultured with CIK cells (a) rather than PBMCs (b). The results presented are the mean of overlaps from a single experiment. [Modes for carrying out the invention]

[0010] Before describing in detail, by illustrative language and results, at least one embodiment of the concept(s) of the present invention, it should be understood that the application of the concept(s) of the present invention is not limited to the details of configurations and component arrangements described below. The concept(s) of the present invention can be carried out in other embodiments or in various ways. Therefore, the language used herein is intended to give as broad a scope and meaning as possible. Furthermore, the embodiments are intended to be illustrative, not exhaustive. Also, it should be understood that the expressions and terms used herein are for illustrative purposes only and should not be considered restrictive.

[0011] Unless otherwise defined herein, scientific and technical terms used in connection with the concepts of the present invention shall have the meanings as commonly understood by those skilled in the art. Further, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art and are as described in various general and more specific references cited and described throughout this specification. The nomenclature used in connection with analytical chemistry, organic synthetic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0012] All patents, published patent applications, and non-patent publications described herein represent the skill levels of those skilled in the art relevant to the presently disclosed inventive concepts. All patents, published patent applications, and non-patent publications referenced in any part of this application are hereby expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication were specifically and individually indicated to be incorporated by reference.

[0013] All compositions, assemblies, systems, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions, assemblies, systems, kits, and methods of the concepts of the present invention are described with respect to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods, and the steps or the order of the steps of the methods described herein without departing from the spirit, and scope of the concepts of the present invention. These similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the inventive concepts defined by the appended claims.

[0014] The following terms used in accordance with this disclosure shall be understood to have the following meanings unless otherwise specified:

[0015] When used in a claim and / or specification in combination with the term “includes,” the terms “a” or “an” may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” or “one or more.” Therefore, unless the context explicitly indicates otherwise, the terms “a,” “an,” and “the” include multiple references. Thus, for example, “compound” may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more compounds. The term “plural” refers to “two or more.”

[0016] The use of the term "at least one" is understood to include quantities greater than one, including but not limited to one, such as two, three, four, five, ten, fifteen, twenty, thirty, forty, fifty, and one hundred. The term "at least one" may extend to 100 or even more, depending on the term it is attached to, and the quantity 100 / 1000 is not considered limiting, as satisfactory results may be obtained with higher limits. In addition, the use of the term "at least one of X, Y, and Z" is understood to include X only, Y only, Z only, and any combination of X, Y, and Z. The use of ordinal terms (such as "first," "second," "third," and "fourth") is intended solely to distinguish two or more items and does not imply any order or importance of one item being superior to another, or any additional order.

[0017] The use of the term “or” in a claim is used to mean an inclusive “and / or” unless it is explicitly indicated that it refers only to the alternatives, or unless the alternatives are mutually exclusive. For example, the condition “A or B” is satisfied by any of the following: A is not true (or exists) and B is not false (or does not exist); A is not true (or exists) and B is true (or exists); and both A and B are true (or exist).

[0018] In this specification, any reference to “one embodiment,” “embodiment,” “several embodiments,” “example,” “for example,” or “example” means that any particular element, feature, structure, or characteristic described in relation to an embodiment is encompassed in at least one embodiment. For example, even if the phrases “in some embodiments” or “in one example” appear in different parts of the specification, they do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples shall be construed as not limiting the claims.

[0019] Throughout this application, the term “approximately” is used to indicate that a value includes variations in inherent errors of the composition / apparatus / device, the method used to determine the value, or variations that exist between the subjects of study. For example, but not limited to, where the term “approximately” is used, a given value may vary from a given value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, as is appropriate for performing the disclosed method and as understood by those skilled in the art.

[0020] As used herein and in the claims, the terms “comprising” (and any form of “comprise,” such as “comprises,” etc.), “having” (and any form of “have,” such as “has,” etc.), “including” (and any form of “includes,” such as “include,” etc.), and “containing” (and any form of “contains,” such as “contain,” etc.) are inclusive or unrestricted and do not exclude any additional elements or method steps not mentioned herein.

[0021] As used herein, the term “or any combination thereof” refers to all permutations and combinations of the enumerated items preceding the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, combinations that explicitly include repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise clearly indicated by the context, there is generally no limit to the number of items or terms that can be combined.

[0022] As used herein, the term “substantially” means that the event or situation described thereafter occurs completely, or that the event or situation described thereafter occurs to a large extent or degree. For example, in relation to a particular event or situation, the term “substantially” means that the event or situation described thereafter occurs for at least 80% of the time, at least 85% of the time, at least 90% of the time, or at least 95% of the time. For example, the term “substantially adjacent” could mean that two items are 100% adjacent to each other, that two items are very close to each other but not 100% adjacent, or that part of one of two items is not 100% adjacent to the other item but is very close to the other item.

[0023] The term "pharmaceutically acceptable" refers to compounds and compositions suitable for administration to humans and / or animals without excessive side effects such as toxicity, irritation, and / or allergic reactions, in proportion to a reasonable benefit-to-risk ratio.

[0024] As used herein, the terms “patient” or “subject” include human and veterinary subjects. “Mammal” in therapeutic purposes means any animal classified as a mammal, including, but not limited to, humans, livestock, farm animals, non-human primates, and other animals with mammary gland tissue.

[0025] The term "treatment" refers to both therapeutic and preventive measures. Those who require treatment include, but are not limited to, individuals who already have a particular condition / disease / infection, as well as those at risk of developing that condition / disease / infection (such as those requiring preventive measures). The term "treating" refers to administering a drug / element / method to a patient for therapeutic and / or preventive purposes.

[0026] As used herein, the terms “therapeutic composition” or “pharmaceutical composition” refer to a drug that may be administered in vivo to produce a therapeutic and / or preventive effect.

[0027] Administering a therapeutically effective or prophylactically effective dose is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific therapeutically effective dose can be readily determined by a typical healthcare professional and may vary depending on factors known in the art, such as the type of condition / disease / infection, the patient's medical history and age, the stage of the condition / disease / infection, and the concurrent administration of other drugs.

[0028] The term "effective dose," when used in accordance with the concepts of the present invention, refers to the amount of a biologically active molecule or its conjugate or derivative, or a treatment protocol (e.g., alternating electric field), sufficient to produce a detectable therapeutic effect without excessive side effects (such as toxicity, irritation, or allergic reactions, but not limited to these) commensurate with a reasonable benefit / risk ratio. The therapeutic effect may include, but is not limited to, preventing, inhibiting, or mitigating the occurrence of at least one condition, disease, and / or infection. The effective dose for a subject will vary depending on the type of subject, the subject's physique and health status, the nature and severity of the condition / disease / infection being treated, the method of administration, the duration of treatment, the nature of any concomitant therapy, and the specific formulation used. Therefore, it is not possible to specify an exact effective dose in advance. However, the effective dose in a particular situation can be determined by a person skilled in the art through routine experimentation based on the information provided herein.

[0029] As used herein, the term “concurrent therapy” is to be used interchangeably with the terms “combination therapy” and “adjunctive therapy” and is understood to mean that a patient in need of treatment is treated or administered with another drug for a medical condition / disease / infection in conjunction with the treatment disclosed herein. This concurrent therapy may be sequential therapy in which the patient is treated first with one treatment protocol / pharmaceutical composition, then with another treatment protocol / pharmaceutical composition, or two treatment protocols / pharmaceutical compositions are administered simultaneously. Furthermore, it will be understood that one administration step (e.g., administration in a TT field) may occur over a longer period than other administration steps (i.e., oral or injectable administration of a substance). In these examples of various administration periods, the term “concurrently” will be understood to mean that the shorter administration step completely overlaps with the longer administration step. However, the term “concurrently” includes performing a shorter administration step at any point between longer administration steps (e.g., at the beginning, middle, or end of a longer administration step, or any other period between them), as well as performing a shorter administration step completely one or more times within the duration of a longer administration step. Therefore, the term "simultaneously" does not require that the two administration steps be performed over the exact same duration.

[0030] As used herein, the terms “administration” and “administering” are understood to encompass all known routes of administration in the art, including, but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreous, and intravenous routes, and to encompass both topical and systemic applications. In addition, the compositions of this disclosure (and / or methods of administration thereof) may be designed to provide delayed release, controlled release, or sustained release using formulation techniques well known in the art.

[0031] As used herein, the term “target region” refers to a region containing all or part of the cancer, cancer cells, and / or tumor being treated.

[0032] Turning to the concept(s) of the present invention, a combination therapy for cancer is disclosed herein. The combination therapy involves the use of an alternating electric field (e.g., a TT field) in addition to killer cells (e.g., natural killer (NK) cells or cytokine-induced killer (CIK) cells). The combination of an alternating electric field (e.g., a TT field) and killer cells produces a synergistic effect in the treatment of cancer.

[0033] Certain non-limiting embodiments of this disclosure relate to a method for reducing the viability of cancer cells. The method comprises (1) applying an alternating electric field to cancer cells for a certain period of time, and (2) administering to cancer cells at least one composition comprising at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell, but not limited to these). Such a method may be carried out in vitro or in vivo.

[0034] Certain additional, non-limiting embodiments of this disclosure relate to a method for treating cancer in a subject. The method comprises (1) applying an alternating electric field to a target area of ​​the subject for a certain period of time, and (2) administering to the subject at least one composition comprising at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell, but not limited to these).

[0035] Certain additional non-limiting embodiments of the present disclosure relate to a method for reducing the volume of a tumor present in the body of a living organism, wherein the tumor comprises multiple cancer cells. The method includes (1) applying an alternating electric field to a target area of ​​a subject for a certain period of time, and (2) administering to a subject at least one composition, wherein the at least one composition comprises at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell).

[0036] Certain additional non-limiting embodiments of this disclosure relate to a method for preventing an increase in the volume of a tumor, where the tumor is present in the body of a living organism and comprises a plurality of cancer cells. The method comprises (1) applying an alternating electric field to a target area of ​​a subject for a certain period of time, and (2) administering to the subject at least one composition, wherein the at least one composition comprises at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell, but not limited to these).

[0037] Certain additional non-limiting embodiments of the present disclosure relate to a method comprising the steps of (1) applying an alternating electric field to a target region of a subject for a certain period of time, and (2) administering to a subject at least one composition comprising at least one killer cell (e.g., at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell, but not limited to these).

[0038] Certain additional non-limiting embodiments of the present disclosure relate to a composition comprising at least one natural killer (NK) cell for use in a method of treating cancer in a subject, the method comprising (1) applying an alternating electric field to a target region of a subject for a certain period of time, and (2) administering a composition to the subject, wherein the composition comprises at least one natural killer (NK) cell.

[0039] Certain additional non-limiting embodiments of the present disclosure relate to a kit for reducing the viability of cancer cells, the kit comprising at least one composition comprising at least one natural killer (NK) cell, and an electric field generator configured to apply an alternating electric field to the cancer cells for a certain period of time.

[0040] Certain additional non-limiting embodiments of the present disclosure relate to a kit for reducing the viability of cancer cells, the kit comprising a component configured to isolate and culture at least one natural killer cell or its precursor from a subject, and an electric field generator configured to apply an alternating electric field to cancer cells for a certain period of time.

[0041] In certain (but non-limiting) embodiments, the administration of an alternating electric field in any of the methods described herein or separately herein increases the efficacy and / or cytotoxicity of killer cells against cancer cells in a subject, compared to the administration of killer cells to a subject in the absence of an alternating electric field.

[0042] Steps (1) and (2) of any of the methods of this disclosure may be carried out simultaneously, sequentially, and in particular substantially simultaneously, or sequentially in whole or in part. If the steps are carried out sequentially in whole or in part, at least one composition comprising at least one killer cell may be administered before or after the application of the alternating electric field is initiated.

[0043] The methods of this disclosure may be used to treat any type of cancer cell / cancer / tumor that responds to treatment with an alternating electric field (e.g., a TT field) and / or killer cells. Non-limiting examples of cancer cells / cancer / tumors that can be treated according to this disclosure include hepatocellular carcinoma / cancer cells, glioblastoma / glioblastoma cells, pleural mesothelioma / mesothelioma cells, differentiated thyroid cancer / cancer cells, advanced renal cell carcinoma / cancer cells, ovarian cancer / cancer cells, pancreatic cancer / cancer cells, lung cancer / cancer cells, breast cancer / cancer cells, and any combination thereof.

[0044] Any type of conductive or nonconductive electrode(s) and / or transducer array(s) available for generating an alternating electric field, known in the art or otherwise discussed herein, may be used to generate an alternating electric field in accordance with the methods of this disclosure. Non-limiting examples of electrodes and transducer arrays that can be used to generate an alternating electric field in accordance with this disclosure include, but are not limited to, those that function as part of the TT field systems described in U.S. Patents Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776, as well as those that function as part of the TT field systems described in U.S. Patents Nos. 2018 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016.

[0045] An alternating current electric field can be generated at any frequency in accordance with this disclosure. For example (but not limited to), the alternating current electric field may be generated at 50kHz, 75kHz, 100kHz, 125kHz, 150kHz, 175kHz, 200kHz, 225kHz, 250kHz, 275kHz, 300kHz, 325kHz, 350kHz, 375kHz, 400kHz, 425kHz, 450kHz, 475kHz, 500kHz, 550kHz, 600kHz, 650kHz, 700kHz, 750kHz, 800kHz, 850kHz, 900kHz, 950kHz, 1MHz, 2MHz, 3MHz, 4MHz, The frequencies may include 5MHz, 6MHz, 7MHz, 8MHz, 9MHz, 10MHz, etc., as well as ranges formed from any of the above values ​​(e.g., the range of approximately 50kHz to approximately 10MHz, approximately 50kHz to approximately 1MHz, approximately 50kHz to approximately 500kHz, approximately 100kHz to approximately 500kHz, approximately 150kHz to approximately 300kHz, etc.), and ranges formed by combining two integers between the above two values ​​(e.g., the range of approximately 122kHz to approximately 313kHz, approximately 78kHz to approximately 298kHz, etc.).

[0046] In certain (but not limited) embodiments, the alternating electric field may be applied at two or more different frequencies. If two or more frequencies exist, each frequency is selected from any of the above values, a range formed from any of the above values, or a range of two integers that fall between the two above values.

[0047] In certain (but non-limiting) embodiments, the following frequencies may be used for specific cancers: approximately 200 kHz for GBM, approximately 150 kHz for NCLC, approximately 200 kHz for breast cancer, approximately 150 kHz for pancreatic cancer, approximately 150 kHz for brain metastases from NCLC, and approximately 150 kHz for liver cancer.

[0048] The alternating electric field may have any electric field intensity within the target region / subject / cancer cell, as long as it functions in accordance with this disclosure. For example (but not limited to these), an AC electric field has at least approximately 1 V / cm, approximately 1.5 V / cm, approximately 2 V / cm, approximately 2.5 V / cm, approximately 3 V / cm, approximately 3.5 V / cm, approximately 4 V / cm, approximately 4.5 V / cm, approximately 5 V / cm, approximately 5.5 V / cm, approximately 6 V / cm, approximately 6.5 V / cm, approximately 7 V / cm, approximately 7.5 V / cm, approximately 8 V / cm, approximately 9 V / cm, approximately 9.5 V / cm, approximately 10 V / cm, approximately 10.5 V / cm, approximately 11 V / cm, approximately 11.5 V / cm, approximately 12 V / cm, approximately 12.5 V / cm, approximately 13 V / cm, approximately 13.5 V / cm, approximately 14 V / cm, approximately 14.5 V / cm, approximately 15 V / cm, approximately 15.5 V / cm, approximately 16 V / cm, and approximately 16. The electric field strength in the target region / subject / cancer cells may be in the range of 5V / cm, approximately 17V / cm, approximately 17.5V / cm, approximately 18V / cm, approximately 18.5V / cm, approximately 19V / cm, approximately 19.5V / cm, approximately 20V / cm, etc., as well as ranges formed from any of the above values ​​(e.g., range of approximately 1V / cm to approximately 20V / cm, range of approximately 1V / cm to approximately 10V / cm, range of approximately 1V / cm to approximately 4V / cm, etc.), and ranges formed by combining two integers that fall between two of the above reference values ​​(e.g., range of approximately 1.1V / cm to approximately 18.6V / cm, range of approximately 1.2V / cm to approximately 9.8V / cm, range of approximately 1.3V / cm to approximately 4.7V / cm, etc.). Generally, it is desirable to utilize the highest possible electric field strength without causing overheating, given that the electric field strength is usually limited by temperature measurements.

[0049] In some cases, the electric field in at least a portion of the target region / subject / cancer cells is induced by an applied voltage determined by computer simulations of the target region / subject / cancer cells. In some cases, the electric field in at least a portion of the target region / subject / cancer cells is induced by an applied voltage of at least 50 V RMS (root mean square) or at least 50 V p2p (peak to peak), and optionally, the applied voltage is at least 100 V RMS or at least 100 V p2p. In some embodiments, an applied voltage of at least 50 V induces an electric field having an electric field intensity of at least 1 V / cm (e.g., at least 5 V / cm) in at least a portion of the target region / subject / cancer cells.

[0050] An alternating current electric field may be applied in a unidirectional direction between a pair of arrays, or alternately applied in two or more directions / channels (e.g., front-to-back and left-to-right) between two or more pairs of arrays. For example, certain TT field devices (OPTUNE® system) (Novocure Limited, St. Helier, Jersey) operate in two directions to increase the likelihood that dividing cells will be aligned with the electric field, allowing the electric field to exert a desirable anti-mitotic effect. However, it is understood that the scope of the invention also includes the application of an alternating current electric field in a unidirectional direction. The term “alternating current electric field” as used herein is understood to include not only application in a single direction / channel but also application in two or more directions / channels, and furthermore, the term “alternating current electric field” as used herein is understood to include not only the application of a single alternating current electric field but also the application of multiple alternating current electric fields continuously over a period of time.

[0051] An alternating electric field may be applied for any duration or cumulative period sufficient to achieve a reduction in the viability of cancer cells and / or a decrease in tumor volume (and / or prevention of an increase in tumor volume). The period during which the alternating electric field is applied includes both continuous and cumulative periods. That is, the period during which the alternating electric field is applied includes not only a single session (i.e., continuous application) but also multiple sessions with short breaks in between (i.e., continuous application over a cumulative period). For example, a subject may be allowed to take breaks during treatment with an alternating electric field device, and is expected to keep the device in place and operating on their body for at least about 60%, at least about 70%, or at least about 80% of the entire treatment period (e.g., one day, one week, two weeks, one month, two months, three months, four months, five months, etc.).

[0052] For example, although not limited to these, an alternating electric field can last for at least approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 ​​hours, 51 hours, 54 hours, 57 hours, 60 hours, 63 hours, 66 hours, 69 hours, 72 hours, 75 hours, 78 hours, 81 hours, 84 hours, 87 hours, and 90 hours. It can be applied over a continuous or cumulative period of time, such as approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 21 days, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, etc., as well as a range formed from any of the above values ​​(for example, a range from approximately 1 hour to approximately 6 months, a range from approximately 24 hours to approximately 72 hours, etc.), and a range formed by combining two integers between the two above values ​​(for example, a range from approximately 14 hours to approximately 68 hours, etc.).

[0053] In certain (but non-limiting) embodiments, the period during which an alternating electric field is applied is at least about 24 hours, with the device positioned on the body and operating for at least about 80% of that period.

[0054] In certain (but non-limiting) embodiments, an alternating electric field is applied to a subject's cancer cell / target region for a period sufficient to reduce MHC class I expression in the cancer cell compared to control cancer cells not exposed to the alternating electric field.

[0055] Any killer cells known in the art or discussed separately herein may be used in accordance with this disclosure, insofar as they can function as described herein. Non-limiting examples of killer cells that may be used in accordance with this disclosure include natural killer (NK) cells and cytokine-induced killer (CIK) cells. Killer cells may be autologous (and therefore obtained from the subject being treated) or allogeneic (and therefore obtained from another subject). Autologous or allogeneic killer cells may be cultured directly from the subject and then transplanted / injected into cancer patients. Alternatively (and / or in addition), pluripotent stem cells may be obtained from the subject and subsequently differentiated in vitro into killer cells that are later transplanted / injected into cancer patients.

[0056] In certain (but not limited to) embodiments, killer cells are derived from interleukin-2 (IL-2)-dependent cell lines. For example (but not limited to), NK-92® cells (ImmityBio, Inc., Culver City, CA) are commercially available and may be used in accordance with this disclosure.

[0057] In certain (but not limited) embodiments, killer cells are manipulated. For example, but not limited to, killer cells may include chimeric antigen receptor-modified NK (CAR-NK) cells.

[0058] Various other types of killer cells that may be used in accordance with this disclosure are known in the art, commercially available from multiple sources, and therefore do not require further description.

[0059] Compositions containing at least one killer cell may be provided in any formulation known in the art or otherwise discussed herein. In certain (but non-limiting) embodiments, a composition containing at least one killer cell comprises one or more pharmaceutically acceptable carriers (and thus the composition may also be referred to as a “pharmaceutical composition”). Non-limiting examples of suitable pharmaceutically acceptable carriers include water, saline, dextrose solution, fructose or mannitol, calcium carbonate, cellulose, ethanol, oils of animal, plant or synthetic origin, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, detergents, liposome carriers, buffer solutions such as sodium chloride, saline, or phosphate-buffered saline, and / or other substances, diluents, excipients such as polyethylene glycol (PEG), or any combination thereof that are physiologically acceptable and / or can be safely used. For pharmaceutically acceptable carriers suitable for pharmaceutical formulations, see, for example, Remington: The Science and Practice of Pharmacy, 23rd ed (2020).

[0060] In certain (but not limited) embodiments, a composition comprising at least one killer cell may further contain one or more additional activators. A variety of active drugs that can be used in combination with alternating electric fields or killer cells are known in the art, and certain combination therapies are approved by the FDA or are currently in clinical trials. Non-limited examples of therapeutic agents that can be used concurrently with killer cells pursuant to this disclosure include (but not limited to) anti-PD-1 agents such as pembrolizumab (KEYTRUDA®, Merck & Co., Inc., Rahway, NJ), tislerizumab, nivolumab, and semiprimab; anti-PD-L1 agents such as atezolizumab, avelumab, and durvalumab; and lenvatinib, paclitaxel, docetaxel, ifosamide, and etoposide (VEPESID®, Bristol-Myers Squibb Co., New Zealand). This includes (but not limited to) chemotherapeutic agents such as gemcitabine, lomustine, nab-paclitaxel, temozolomide, and carboplatin; (but not limited to) TKI inhibitors such as everolimus; mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; FGF inhibitors; anti-LAB3 agents; anti-CTLA-4 agents; aromatase inhibitors such as letrozole (but not limited to), biological agents such as monoclonal antibodies (e.g., denosumab and pembrolizumab); and any combination thereof.

[0061] In addition, any composition of this disclosure may contain other drugs that enable the administration of the composition via a particular route of administration. For example, but not limited to, compositions may be formulated for administration via oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreous, and / or intravenous routes. Based on the route of administration, a composition may also contain one or more additional components (e.g., killer cells and / or additional therapeutic agents) in addition to the activator. Examples of additional secondary compounds that may be present include, but are not limited to, fillers, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifiers, dispersants, and other substances well known in the art.

[0062] In certain (but not limited) embodiments, at least one composition comprising at least one killer cell is administered to a subject by injection or implantation. For example (but not limited to), in some cases it may be desirable to administer killer cells at a local / topical level to ensure targeting of the killer cells to a specific location within the subject's body and to inhibit nonspecific interactions in other parts of the body, while in other cases a more systemic administration may be desired.

[0063] At least one composition containing at least one killer cell may be administered before or after the application of an alternating electric field. In certain (but not limited) embodiments, at least one composition containing at least one killer cell may be administered after the application of an alternating electric field has begun. In particular (but not limited to), at least one composition containing at least one killer cell may be administered during the application of the alternating electric field (e.g., before the period during which the alternating electric field is applied has elapsed) and / or after the application of the alternating electric field has elapsed.

[0064] For example (but not limited to these), at least one composition containing at least one killer cell is subjected to an alternating electric field for at least about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, and about 63 hours. , approximately 66 hours, approximately 69 hours, approximately 72 hours, approximately 75 hours, approximately 78 hours, approximately 81 hours, approximately 84 hours, approximately 87 hours, approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, etc., as well as a range formed from any of the above values ​​(e.g., a range of approximately 24 hours to approximately 96 hours), and a range formed by combining two integers between the above two values ​​(e.g., a range of approximately 14 hours to approximately 94 hours). In certain (but non-limiting) embodiments, at least one composition containing at least one killer cell is administered at least approximately 24 hours after the start of application of the alternating electric field.

[0065] In other non-limiting examples, at least one composition containing at least one killer cell may be administered after a period of time in which an alternating electric field has been applied, or at least one composition containing at least one killer cell may be introduced within approximately 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 ​​hours, 51 hours, 54 hours, 57 hours, 60 hours, 63 hours, 66 hours, 69 hours, 72 hours, 75 hours, 78 hours, 81 hours, 84 hours, 87 hours, 90 hours, 93 hours, 96 hours, 5 days, 6 days, 7 days, etc., after a period of time in which an alternating electric field has been applied.

[0066] In certain (but non-limiting) embodiments, at least one composition containing at least one killer cell is administered within approximately 96 hours after the period has elapsed.

[0067] A composition comprising at least one killer cell can be administered to a cancer cell / subject at any concentration that provides a therapeutically effective concentration of the killer cell. In certain non-limiting embodiments, the application of an alternating electric field reduces the amount of killer cells required to be therapeutically effective when compared to the normal therapeutically effective amount administered when no alternating electric field is present. For example, but not limited to these, the therapeutically effective concentration of killer cells can be reduced by at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more relative to the dosage of killer cells known to be therapeutically effective when no alternating electric field is applied. In certain (but non-limiting) embodiments, the therapeutically effective concentration of killer cells is reduced by at least about 50% when compared to the dosage of killer cells known to be therapeutically effective when no alternating electric field is present.

[0068] The killer cell-containing composition(s) can be administered to a subject at any concentration that can induce an inflammatory response against a tumor or cancer cell. For example, but not limited to these, the killer cells can be at about 10 cells / kg body weight, about 100 cells / kg body weight, about 1000 cells / kg body weight, about 10 4 cells / kg body weight, about 10 5 cells / kg body weight, about 10 6 cells / kg body weight, about 10 7 cells / kg body weight, about 10 8 cells / kg body weight, about 10 9 cells / kg body weight, about 10 10 cells / kg body weight, about 10 11 cells / kg body weight, about 10 12 cells / kg body weight, about 10 13 cells / kg body weight, about 10 14 cells / kg body weight, about 10 15 cells / kg body weight, or more, and in ranges formed from any of the above values (e.g., a range from about 10 4 to about 10 9 cells / kg body weight, etc.) and can be administered.

[0069] In certain (but not limited) embodiments, the method includes one or more additional steps. For example (but not limited to), the method may further include step (3) discontinuing the application of an alternating electric field (for example, for purposes such as allowing cells / tissues to recover, but not limited to such purposes). Furthermore, either step (1) and / or (2) may be repeated one or more times.

[0070] In certain (but not limited) embodiments, the killer cell-containing composition may be administered by any dosing regimen known in the art. For example, the killer cell-containing composition may be administered as a single dose or multiple doses over a defined therapeutic period, but is not limited thereto. For example (but is not limited thereto), a therapeutically effective concentration of the killer cell-containing composition may be administered approximately every 4 hours, approximately every 8 hours, approximately every 12 hours, approximately every day, approximately every 2 days, approximately every 3 days, approximately once a week, approximately twice a week, approximately three times a week, approximately every 2 weeks, approximately every 3 weeks, approximately once a month, etc., as well as within a range formed from any of the above values ​​(e.g., the range of approximately every 4 to 8 hours, approximately once a week to approximately once a month).

[0071] In certain (but non-limiting) embodiments, the method involves simultaneous treatment with two or more compositions. Thus, the method may include an additional step of (4) administering at least a second composition to cancer cells / subjects. In certain (but non-limiting) embodiments, at least a second composition may contain one or more active substances disclosed herein or separately considered for use with killer cell-containing compositions.

[0072] Various substances and therapies used in combination with killer cells are known in the art, and certain combination therapies are approved by the FDA or are currently in clinical trials. Examples of therapeutic agents that can be used in concurrent (sequential) therapy with killer cells pursuant to this disclosure include, but are not limited to, anti-PD-1 agents such as pembrolizumab (KEYTRUDA®, Merck & Co., Inc., Rahway, NJ), tislerizumab, nivolumab, and semiprimab;, but are not limited to, anti-PD-L1 agents such as atezolizumab, avelumab, and durvalumab; and lenvatinib, paclitaxel, docetaxel, ifosamide, and etoposide (VEPESID®, Bristol-Myers Squibb Co., New Zealand). Examples include chemotherapeutic agents such as gemcitabine, lomustine, nab-paclitaxel, temozolomide, and carboplatin (but not limited to these); TKI inhibitors such as everolimus (but not limited to these); mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; FGF inhibitors; anti-LAB3 agents; anti-CTLA-4 agents; aromatase inhibitors such as letrozole (but not limited to these); biological agents such as monoclonal antibodies (e.g., denosumab and pembrolizumab, but not limited to these); and any combination thereof.

[0073] If present, step (4) may be carried out substantially simultaneously with, or in whole or in part, the administration of the first composition in step (2), thereby administering the two separate compositions simultaneously or in whole or in part sequentially. In addition, the two compositions administered in steps (2) and (4) may be administered via the same route (e.g., both intravenously), or the two compositions may be administered via different routes (e.g., one composition orally and the other intravenously).

[0074] If present, any additional dosing step (4) may be performed before or after the application of the alternating electric field is initiated, during the application of the alternating electric field, and / or after the application of the alternating electric field has elapsed, in the same manner and time frame as described above for the first composition.

[0075] That is, for example (but not limited to these), the second composition is subjected to the AC electric field for at least about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, and about 66 hours from the start of application of the AC electric field. It may be administered after approximately 69 hours, approximately 72 hours, approximately 75 hours, approximately 78 hours, approximately 81 hours, approximately 84 hours, approximately 87 hours, approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, and similarly, a range formed from any of the above values ​​(e.g., a range of approximately 24 hours to approximately 96 hours), and a range formed by combining two integers that fall between two of the above values ​​(e.g., a range of approximately 14 hours to approximately 94 hours). In certain (but not limited) embodiments, the second composition is administered at least approximately 24 hours after the application of the alternating electric field begins.

[0076] In other non-limiting examples, the second composition may be administered after the period of application of the alternating electric field has elapsed, in which case the second composition is administered within approximately 3 hours, approximately 6 hours, approximately 9 hours, approximately 12 hours, approximately 15 hours, approximately 18 hours, approximately 21 hours, approximately 24 hours, approximately 27 hours, approximately 30 hours, approximately 33 hours, approximately 36 hours, approximately 39 hours, approximately 42 hours, approximately 45 hours, approximately 48 hours, approximately 51 hours, approximately 54 hours, approximately 57 hours, approximately 60 hours, approximately 63 hours, approximately 66 hours, approximately 69 hours, approximately 72 hours, approximately 75 hours, approximately 78 hours, approximately 81 hours, approximately 84 hours, approximately 87 hours, approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, etc. In certain (but non-limiting) embodiments, the second composition is administered within approximately 96 hours after the period has elapsed.

[0077] In addition, for example (but not limited to these), the second composition may be administered for at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, and about 48 hours. It may be administered after approximately 51 hours, approximately 54 hours, approximately 57 hours, approximately 60 hours, approximately 63 hours, approximately 66 hours, approximately 69 hours, approximately 72 hours, approximately 75 hours, approximately 78 hours, approximately 81 hours, approximately 84 hours, approximately 87 hours, approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, etc., as well as a range formed from any of the above values ​​(e.g., a range of approximately 24 hours to approximately 96 hours), and a range formed by combining two integers between the two above values ​​(e.g., a range of approximately 14 hours to approximately 94 hours). In certain (but not limited) embodiments, the second composition is administered at least approximately 12 hours after the first substance is administered.

[0078] In certain (but not limited) embodiments, the method may further include the step of (5) administering at least one additional therapy to the cells / subject. Any therapy known in the art or considered herein for use in combination with alternating electric field (e.g., TT field) and / or killer cell therapy may be utilized in accordance with the method of the present disclosure. Non-limited examples of additional therapies that may be utilized include radiotherapy, photodynamic therapy, transarterial chemoembolization (TACE), or combinations thereof.

[0079] Steps (1) and (2), and any of the optional steps (3), (4), and (5), may be repeated one or more times. Each step may be repeated any number of times as needed. When step (1) is repeated, the transducer array may be positioned in a slightly different location from the subject's original position, and by rearranging the array in this way, the treatment of the tumor / cancer may be further enhanced. In addition, step (2) and any of the optional steps (4) and (5) (if any) of administering the composition / additional therapy may be repeated at varying numbers and intervals to follow known and / or generally accepted dosage / treatment plans for the composition / therapy(s) / therapy(s).

[0080] The use of ordinal numbers in any step is illustrative only, and any one or more of steps (3), (4), and (5) may be included individually or in combination with each other. That is, the method of the disclosure includes performing step (3) if step (4) or (5) is not present, performing step (4) if step (3) or (5) is not present, and performing step (5) if steps (3) and (4) are not present. In other words, the scope of the methods disclosed herein is to carry out steps (1) to (2) (repeating each step as many times as required), to carry out steps (1) to (3) (repeating one or more of steps (1) to (3) as many times as required), to carry out steps (1) to (2) and (4) (repeating one or more of steps (1) to (2) and (4) as many times as required), to carry out steps (1) to (2) and (5) (repeating one or more of steps (1) to (2) and (5) as many times as required), step ( This includes performing steps (1) through (4) (repeating one or more of steps (1) through (4) as many times as needed), performing steps (1) through (3) and (5) (repeating one or more of steps (1) through (3) and (5) as many times as needed), performing steps (1) through (2) and (4) through (5) (repeating one or more of steps (1) through (2) and (4) through (5) as many times as needed), and performing all of steps (1) through (5) (and further, repeating one or more of steps (1) through (5) as many times as needed).

[0081] While the use of combination therapy of two substances is explicitly described above, it is understood that the scope of this disclosure also includes combination therapy of three or more compositions. Accordingly, this method may include one or more additional steps (similar to steps (1) and (4)) in which an additional composition is administered to a subject. The additional substance administered in this manner may be selected from any of the substances disclosed herein or (as disclosed above herein with respect to any step (4)) that are considered for use in combination with at least one killer cell. Furthermore, the administration of the additional substance may be carried out substantially simultaneously with, or entirely or partially sequentially with, the administration of the first and second compositions / substances, in the same manner(s) and timeframe(s) as described above for the first and second compositions / substances.

[0082] Certain non-limiting embodiments of this disclosure are any of the components of AC electric field (e.g., TT field) generating systems disclosed herein or otherwise considered (e.g., U.S. Patents No. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776, and U.S. Patent Applications US2018 / 0160933, US201 The present invention relates to a kit comprising one or more transducer arrays and / or one or more hydrogel compositions (such as those disclosed in 9 / 0117956, US2019 / 0307781, and US2019 / 0308016) in combination with at least one of the compositions comprising at least one killer cell disclosed herein or otherwise considered, and / or a component for isolating and / or culturing at least one killer cell or its precursor from a patient. The kit may optionally further comprise one or more of the compositions disclosed herein or otherwise considered (e.g., one or more of the compositions comprising at least one additional activator, and / or one or more of the compositions for culturing / inducing differentiation of at least one killer cell or its precursor). The kit may optionally further comprise one or more devices (or one or more components of a device) used in one or more additional therapeutic steps.

[0083] In certain (non-limiting) embodiments, the kit may further include instructions for performing any of the methods disclosed herein or otherwise considered. For example (but not limited to), the kit may include instructions for applying one or more components of an alternating electric field (e.g., TT field) generator to a patient's skin; instructions for applying an alternating electric field to a patient; instructions for isolating killer cells or their precursors from a subject; instructions for culturing and / or differentiating killer cells or their precursors in vitro; instructions for formulating a composition comprising at least one killer cell; instructions for the timing and method of administering the composition comprising at least one killer cell, and for the method of administering one or more optional additional compositions; and / or instructions for when to start and stop the alternating electric field in connection with the administration of the composition comprising at least one killer cell, and / or the administration of one or more optional compositions, and / or in connection with the treatment steps.

[0084] In addition to the components described in detail above, the kit may further contain other components / reagents for carrying out any of the specific methods described herein or otherwise considered. For example (but not limited to), the kit may further include (i) components for preparing the skin before discarding the hydrogel composition and / or transducer array on the skin (e.g., razor, cleansing composition, wipe / towel, etc.), (ii) components for removing the gel / transducer array(s), (iii) components for cleansing the skin after removal of the gel / transducer array(s), and / or (iv) other components used in the system (e.g., conductive materials, non-conductive materials, soothing gels or creams, bandages, etc.). The properties of these additional components / reagents depend on the specific processing method, and their identification is within the scope of the art for those skilled in the art, so no further explanation is considered necessary. Furthermore, the components / reagents included in the kit may be in separate containers / compartments, or various components / reagents may be combined into one or more containers / compartments depending on their sterility, cross-reactivity, and stability.

[0085] The kit may be placed in any package that enables the components contained therein to function in accordance with this disclosure. In certain non-limiting embodiments, the kit further includes a sealed package in which the components are placed. In certain (but non-limiting) embodiments, the sealed package is substantially impermeable to air and / or substantially impermeable to light.

[0086] Furthermore, the kit may further include a set of written instructions explaining how to use one or more of the kit's components. Kits of this nature can be used in any of the methods described herein or otherwise considered.

[0087] In certain non-limiting embodiments, the shelf life of the kit is at least about 6 months, for example (but not limited to) at least about 9 months, or at least about 12 months.

[0088] Certain non-limiting embodiments of this disclosure are any of the components of AC electric field generating systems disclosed herein or otherwise considered (e.g., U.S. Patent Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776, and U.S. Patent Application No. 20 The present invention relates to a system comprising one or more transducer arrays and / or one or more hydrogel compositions (such as, but not limited to, those disclosed in Nos. 18 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016) in combination with at least one of any of the compositions comprising at least one killer cell disclosed or otherwise considered herein. The system may optionally further comprise one or more of any compositions disclosed or otherwise considered herein. The system may optionally further comprise one or more devices (or one or more components of a device) used in one or more additional therapeutic steps. Examples

[0089] Examples are shown below. However, it should be understood that this disclosure is not limited to the application to the specific experiments, results, and experimental procedures disclosed below herein. Rather, these examples are provided simply as one of various embodiments and are intended to be illustrative rather than exhaustive. Example 1

[0090] In this example, the effect of the TT site on MHC class I expression in cancer cell lines was investigated. In contrast to the prior art by Silginer et al. (2018), referenced in the background art section above, which found that MHC class I and class II expression remained unchanged after exposure to the TT site, this example found that MHC class I expression was actually reduced in human and mouse pancreatic cancer cell lines. As shown in Figure 1, MHC class I expression decreased in the 1A-AsPc1 human pancreatic cancer cell line after 72 hours of exposure to the TT site (left panel), and MHC class I expression decreased in the 1B-Panc02 mouse pancreatic cancer cell line after 72 or 96 hours of exposure to the TT site (center and right panels).

[0091] Furthermore, transcriptomics analysis was performed to identify class I HLA genes showing differences in expression after TT field treatment in various cancer cell lines compared to controls. As shown in Figure 2, various class I HLAs (including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and HLA-H) tested after TT field treatment in glioblastoma (GBM) cell lines, malignant pleural mesothelioma (MPM) cell lines, and pancreatic cancer cell lines AsPC1 and BxPC3 showed a statistically significant decrease. Therefore, this data demonstrates that TT field treatment reduces MHC class I expression in various cancers.

[0092] While we do not wish to be bound by any theory, it has been previously shown that TT fields induce ER stress (Silginer et al. (2017) Cell Death Dis, 8:e2753). Furthermore, it has also been previously shown that ER stress downregulates class I HLA (Ulianich et al. (2011) Biochemica et. Biophysica Acta, 1812:431-438). Therefore, the induction of ER stress by TT field exposure can be a possible mechanism for the downregulation of class I HLA after TT field exposure. Example 2

[0093] This example relates to the use of the TT field as an immunotherapy to activate adoptive transplanted NK cells and enhance the efficacy of NK cell-based cancer therapies. Combination therapy with the TT field using adoptive transplanted NK cells provides improved synergistic effects compared to either therapy alone.

[0094] Subjects were treated in a TT field by applying the OPTUNE® device (Novocure Limited, St. Helier, Jersey) to the subject's skin with a pair of arrays positioned to the left and right (LR) and / or anterior and posterior (AP) of the tumor. Each subject was then chronically treated in a 150–200 kHz TT field. The device was worn for at least approximately 80% of the time, with only minor interruptions between sessions to allow for slight adjustments to the array placement and cell and skin regeneration.

[0095] Compositions containing NK cells were prepared. In one example, autologous NK cells were obtained from subjects prior to TT field exposure. Alternatively, NK cells were obtained from allogeneic sources. In yet another example, pluripotent stem cells were obtained from autologous or allogeneic sources and differentiated into NK cells in vitro. The autologous or allogeneic NK cells were then cultured prior to transplantation / injection for adoptive cell transfer therapy.

[0096] Two weeks after the start of TT field application, approximately 10 to 10 billion NK cells were transplanted or injected into human subjects about 1 to 3 times per week for 6 to 8 weeks.

[0097] Following combination therapy, the effectiveness of the above combination therapies against tumors(s) was evaluated. Example 3

[0098] This example studies the role of the TT field in enhancing the in vitro death activity of CIK cells (113) against patient-derived GBM cancer stem cells (GSCs). This example provides proof of principle that the TT field can enhance "HLA-independent immunity" against cancer cells and strengthens the basis for novel clinical research to reconsider the potential of CIK therapy combined with the TT field for treating GBM.

[0099] Cytokine-induced killer (CIK) cell therapy is a type of adoptive immunotherapy that uses a heterogeneous immune cell population generated in vitro from the patient's peripheral blood mononuclear cells (PBMCs) in the presence of IL-2, anti-CD3 antibodies (multiple) (e.g., OKT3, but not limited to these), and IFN-gamma (Introna (2017) Journal of Autoimmunity, 85:32-44). CIK cells are phenotypic cytotoxic T-terminal differentiated effector memory (EMRA) lymphocytes with antitumor activity (Franceschetti et al. (2009) Experimental Hematology, 37:616-628.e2), CD3 + CD56 + CD8 + This population expresses different NK markers, including NKG2D, and can recognize and kill tumor cells in a TCR-independent NK-like manner (Sangiolo et al. (2008) International Immunology, 20:841-848). Recent reports from the International Registry of CIK Cells (IRCC) indicate that CIK cells have been tested in 106 clinical trial settings in more than 30 different tumors, resulting in significant improvements in progression-free survival (PFS) and overall survival (OS) in 27 autologous settings, with mild adverse effects. CIK cells have also been evaluated in patients with GBM, the most common primary malignant brain tumor (Kong et al. (2017) Oncotarget, 8:7003-7013). In this Phase III trial, CIK cells were associated with improved PFS, but not OS. This trial is one of several immunotherapy trials in GBM with poor clinical outcomes, including immune checkpoint inhibitors, vaccination, and different adoptive T-cell therapies (Weenink et al. (2020) Cancers (Basel), 12:E751).

[0100] The TT field has been shown to extend survival in patients with both newly diagnosed and recurrent GBM, leading to FDA clinical approval of this treatment (Rominiyi et al. (2020) Br J Cancer, 124(4):697-709). While we do not wish to be bound by any theory, the primary biological mechanism attributable to the TT field in cancer treatment is its ability to disrupt spindle formation in the mitotic apparatus (Kirson et al. (2004) Cancer Res, 1; 64(9):3288-95). However, there is also emerging evidence that the TT field can activate the immune system against tumor cells (Voloshin et al. (2020) Cancer Immunol Immunother, 69(7):1191-1204). Chen et al. (J Clin Invest, 132(8):e149258(2022)) mechanistically demonstrated that the TT field can stimulate the production of pro-inflammatory and interferon-1 cytokines in a syngeneic mouse GBM model via genotoxic stress activation of cGAS / STING- and AIM2 inflammasomes in GBM cells. This pro-inflammatory shift can lead to robust activation of adoptive immunity, resulting in increased T cell clonal proliferation due to memory phenotypes and a decrease in depleted T cells. Although single-cell RNA sequencing of peripheral blood mononuclear cells obtained from GBM patients treated with the TT field also provided clues to activation in NK cells, the role of "HLA-independent immunity" was not further investigated by Chen et al.

[0101] In this example, 20 batches of CIK were successfully propagated from PBMCs. PBMCs were obtained from both healthy donors (HDs) and patients with GBMs, registered at San Raffaele Hospital prior to chemoradiotherapy, according to the protocol described in the Methods section below. Figure 3 summarizes the growth curves of 5 batches obtained from 5 HDs, showing a median growth rate of 42.03 (IQR: 31.15–74.13). On day 21, CD3 + CD56 +The cells represent 38.96% of the total cells, and this group is mostly CD8 + CD4 - CD3 represented 48.83% of the cells. + CD56 - Mostly CD8 + CD4 - Although they became cells (average = 61.43%), CD4 + CD8 - The proportion of cells was higher (average = 28.94%).

[0102] Co-culture of CIK and GSCs: To evaluate the cytotoxic activity of CIK cells, their ability to induce apoptosis in patient-derived GSCs was tested. To do this, GSCs were labeled with the Purple CellTrace Cell Proliferation Kit and then co-cultured with CIK cells at different target-to-effector ratios (T / E ratios). As shown in Figure 4, dose-dependent cytotoxic effects induced by CIK were observed compared to fresh PBMCs. Study design and methods CIK cells

[0103] Blood samples were collected from healthy volunteers using EDTA. PBMCs were separated from whole blood by density centrifugation and cultured for 21 days at 37°C and 5% CO2 according to a reference protocol (Franceschetti et al., 2009). On day 0, cells were supplemented with 1,000 U / mL IFN-γ, followed by 50 ng / mL anti-CD3 antibody and 500 U / mL IL-2 on day 1. Fresh medium and 500 U / mL IL-2 were supplemented every 3-4 days. The phenotypes of PBMCs and CIK cells were examined on days 12 and 21 and characterized by flow cytometry-detected cell surface markers CD3, CD56, CD4, CD8, CD14, and CD19, along with cell viability dyes. To investigate the effect of the TT field, CIK cells were cultured for 48 hours in the presence of the TT field. CIK viability was tested by Live / Dead Aqua cell staining, and the activation state of CIK cells was evaluated by testing the IFN-gamma concentration in the cell-free supernatant using a sandwich ELISA assay. GSC

[0104] Patient-derived CSC lines were isolated from fresh tumors using NeuroSphere Assay and established as long-term regenerative cell lines in the Neural Stem Cell Biology Unit led by Dr. Galli (Galli et al., (2004) Cancer Research, 64:7011-7021). As a first experiment, the known effects of the TT field on GBM were tested by assessing the viability of GSCs evaluated 48 hours after exposure to the TT field. Additionally, the ability of the TT field to alter the immunogenicity of GSCs was assessed by examining the expression of relevant surface molecules such as MHC class I and NKG2D-ligand MICA / B. Co-culture platform for CIK and GSC

[0105] To evaluate the anticancer activity of CIK cells against GBM, a co-culture platform with patient-derived GSCs was developed. Specifically, GSCs were labeled with the CellTrace Cell Proliferation Kit and co-cultured with CIK cells at various target-to-effector ratios in 6-well flat-bottom plates. After 48 hours of co-culture, apoptotic cell load among CellTrace-positive cells, identified by Live / Dead Aqua cell staining or 7-amino-actinomycin D (7AAD), was used as the primary measure of CIK cell death activity. CIK degranulation was used as a secondary outcome to assess CIK cell activation. To achieve this, CIK cells labeled with the CellTrace Cell Proliferation Kit were co-incubated with GSCs at a 2:1 effector-to-tumor ratio in the presence of CD107a (Aktas et al. (2009) Cell Immunol, 254(2):149-54). After 1 hour, Brefeldin A and GolgiStop were added to each well. CIK degranulation was evaluated using flow cytometry, and CD107a cells were identified among those labeled with CellTrace. +The results were expressed as a percentage of cells. Furthermore, the effects of GSC pretreatment and the TT field as co-culture conditions were tested to determine their ability to enhance the described results. Example 4

[0106] This embodiment relates to the use of the TT field as an immunotherapy to activate cytokine-induced killer (CIK) cells and enhance the efficacy of CIK cell-based cancer therapies. Combination therapy of the TT field and CIK cells provides a synergistic effect compared to either therapy alone.

[0107] Subjects were treated in a TT field by applying the OPTUNE® device (Novocure Limited, St. Helier, Jersey) to the subject's skin with a pair of arrays positioned to the left and right (LR) and / or anterior and posterior (AP) of the tumor. Each subject was then chronically treated in a 150–200 kHz TT field. The device was worn for at least approximately 80% of the time, with only minor interruptions between sessions to allow for slight adjustments to the array placement and cell and skin regeneration.

[0108] Compositions containing CIK cells were prepared. In one example, CIK cells were prepared using PBMCs obtained from autologous and / or allogeneic sources as described in Example 3. The CIK cells were then cultured before transplantation / injection for adoptive cell transfer therapy.

[0109] Two weeks after the start of TT field application, approximately 10 to 10 billion CIK cells were transplanted or injected into human subjects approximately 1 to 3 times per week for 6 to 8 weeks.

[0110] Following combination therapy, the effectiveness of the above combination therapies against tumors(s) was evaluated. Non-limiting exemplary embodiments of the concept of the present invention

[0111] Exemplary Embodiment 1. A method for reducing the viability of cancer cells, the method comprising: (1) applying an alternating electric field to the cancer cells for a certain period of time; and (2) administering at least one composition to the cancer cells, wherein the at least one composition comprises at least one killer cell. The method may be an in vitro or in vivo method.

[0112] Exemplary Embodiment 2. A method for treating cancer in a subject, the method comprising: (1) applying an alternating electric field to a target region of the subject for a certain period of time; and (2) administering to the subject at least one composition, wherein the at least one composition comprises at least one killer cell.

[0113] Exemplary Embodiment 3. A method for reducing the volume of a tumor and / or preventing an increase in the volume of a tumor, wherein the tumor is present in the body of a living organism and comprises a plurality of cancer cells, and the method comprises (1) applying an alternating electric field to a target area of ​​a subject for a certain period of time, and (2) administering to the subject at least one composition, wherein the at least one composition comprises at least one killer cell.

[0114] An exemplary embodiment 4. A method comprising the steps of (1) applying an alternating electric field to a target region of a subject for a certain period of time, and (2) administering to the subject at least one composition, wherein the at least one composition comprises at least one killer cell, the administration of the alternating electric field increases the efficacy and / or cytotoxicity of the killer cell against cancer cells in the subject compared to the administration of the killer cell to the subject in the absence of the application of the alternating electric field.

[0115] Exemplary Embodiment 4A. The method according to any one of the exemplary embodiments 1 to 4, wherein the at least one killer cell comprises at least one natural killer (NK) cell.

[0116] Exemplary Embodiment 4B. The method according to any one of Embodiments 1 to 4A, wherein the at least one killer cell comprises at least one cytokine-induced killer (CIK) cell.

[0117] Exemplary Embodiment 4C. The method according to any one of Embodiments 1 to 4B, wherein the at least one killer cell comprises at least one NK cell and at least one CIK cell.

[0118] Exemplary Embodiment 5. The method according to any one of Exemplary Embodiments 1 to 4, wherein the alternating electric field is applied at a frequency in the range of about 50 kHz to about 1 MHz; the alternating electric field has an electric field intensity of at least about 1 V / cm in at least a portion of the cancerous / targeted area of ​​the subject; the alternating electric field is induced by an applied voltage of at least 50 V RMS or at least 50 V p2p; and the period during which the alternating electric field is applied is at least about 50% of a continuous period of at least about 24 hours.

[0119] Exemplary Embodiment 6. The method according to any one of Exemplary Embodiments 1 to 5, wherein the alternating electric field is applied to the cancer cells / target region of the subject for a period of time sufficient to reduce MHC class I expression in the cancer cells compared to control cancer cells not exposed to the alternating electric field.

[0120] Exemplary Embodiment 7. The method according to any one of the exemplary embodiments 1 to 6, wherein the at least one killer cell comprises at least one autologous killer cell.

[0121] Exemplary Embodiment 8. The method according to any one of the exemplary embodiments 1 to 7, wherein the at least one killer cell comprises at least one allogeneic killer cell.

[0122] Exemplary Embodiment 9. The method according to any one of Exemplary Embodiments 1 to 8, wherein the at least one of the following is derived: the at least one killer cell is derived from the in vitro differentiation of a pluripotent stem cell; the at least one killer cell is derived from an interleukin-2 (IL-2)-dependent cell line; and / or the at least one killer cell comprises a chimeric antigen receptor-modified killer cell.

[0123] Exemplary Embodiment 9A. The method according to any one of Exemplary Embodiments 1 to 8, wherein the at least one killer cell is at least one NK cell derived from the in vitro differentiation of a pluripotent stem cell, the at least one killer cell is at least one NK derived from an interleukin-2 (IL-2)-dependent cell line, and / or the at least one killer cell comprises a chimeric antigen receptor-modified NK (CAR-NK) cell.

[0124] Exemplary Embodiment 9B. The method according to any one of Exemplary Embodiments 1 to 9A, wherein the at least one killer cell is at least one CIK cell derived from a peripheral blood mononuclear cell (PBMC) cultured in the presence of at least one of interleukin-2 (IL-2), anti-CD3 antibody, and interferon-gamma (IFN-γ).

[0125] Exemplary Embodiment 10. The method according to any one of Exemplary Embodiments 1 to 9, wherein the composition further comprises at least one substance selected from the group consisting of immune checkpoint inhibitors, interleukin-1 (IL-1), IL-1R, IL-2, IL-11, IL-15, IL-17, IL-18, IL-21, lirirumab, monalizumab, and combinations thereof.

[0126] Exemplary Embodiment 11. The method according to any one of Exemplary Embodiments 1 to 10, wherein the composition further comprises at least one substance selected from the group consisting of anti-PD-1 therapeutic agents, anti-PD-L1 therapeutic agents, chemotherapeutic agents, paclitaxel, docetaxel, ifosfamide, etoposide, gemcitabine, lomustine, nab-paclitaxel, temozolomide, carboplatin, TKI inhibitors, mTOR inhibitors, Akt inhibitors, PI3K inhibitors, PARP inhibitors, FGF inhibitors, anti-LAB3 agents, anti-CTLA-4 therapeutic agents, aromatase inhibitors, denosumab, pembrolizumab, and combinations thereof.

[0127] Exemplary Embodiment 12. The method according to any one of the exemplary embodiments 1 to 11, further comprising the step of (3) administering at least one additional composition to the cancer cells / subject, wherein the at least one additional composition comprises a substance selected from the group consisting of immune checkpoint inhibitors, interleukin-1 (IL-1), IL-1R, IL-2, IL-11, IL-15, IL-17, IL-18, IL-21, lirilumab, monoarizumab and combinations thereof.

[0128] Exemplary Embodiment 13. The method according to any one of Exemplary Embodiments 1 to 12, further comprising the step of administering at least one additional composition to the cancer cells / subject, wherein the at least one additional composition comprises a substance selected from the group consisting of anti-PD-1 therapeutic agents, anti-PD-L1 therapeutic agents, chemotherapeutic agents, paclitaxel, docetaxel, ifosfamide, etoposide, gemcitabine, lomustine, nab-paclitaxel, temozolomide, carboplatin, TKI inhibitors, mTOR inhibitors, Akt inhibitors, PI3K inhibitors, PARP inhibitors, FGF inhibitors, anti-LAB3 agents, anti-CTLA-4 therapeutic agents, aromatase inhibitors, denosumab, pembrolizumab, and combinations thereof.

[0129] Exemplary Embodiment 14. The method according to any one of the exemplary embodiments 1 to 13, wherein steps (1) and (2) are carried out in whole or in part sequentially, and the at least one composition is administered after the application of the alternating electric field has started.

[0130] Exemplary Embodiment 15. The method according to Exemplary Embodiment 14, wherein the at least one composition is administered before the period during which the alternating electric field is applied has elapsed.

[0131] Exemplary Embodiment 16. The method according to Exemplary Embodiment 14 or 15, wherein the at least one composition is administered after the period of time has elapsed.

[0132] Exemplary Embodiment 17. The method according to any one of the exemplary embodiments 1 to 16, wherein steps (1) and (2) are repeated one or more times.

[0133] Exemplary Embodiment 18. The method according to any one of Exemplary Embodiments 1 to 17, wherein the cancer cells / cancer / tumor are in the form of at least one solid tumor.

[0134] Exemplary Embodiment 19. The method according to any one of Exemplary Embodiments 1 to 18, wherein the cancer / cancer cells are selected from the group consisting of hepatocellular carcinoma / cancer cells, glioblastoma / glioblastoma cells, pleural mesothelioma / mesothelioma cells, differentiated thyroid cancer / cancer cells, advanced renal cell carcinoma / cancer cells, ovarian cancer / cancer cells, pancreatic cancer / cancer cells, lung cancer / cancer cells, breast cancer / cancer cells, and combinations thereof.

[0135] Exemplary Embodiment 20. A kit for reducing the viability of cancer cells, the kit comprising a component configured to isolate and culture at least one natural killer cell or its precursor from a subject, and optionally an electric field generator configured to apply an alternating electric field to the cancer cells for a certain period of time.

[0136] This invention describes the concept(s) of the present invention in conjunction with the specific experiments, results, and terms set forth below, but it will be apparent to those skilled in the art that many alternatives, modifications, and variations are obvious. Accordingly, this invention is intended to encompass all alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A method for treating cancer in a subject, wherein the method is (1) The step of applying an alternating electric field to the target area of ​​the subject for a certain period of time, (2) A method comprising the step of administering to a subject at least one composition comprising at least one natural killer (NK) cell.

2. The aforementioned alternating electric field is applied at a frequency in the range of approximately 50 kHz to approximately 1 MHz. The alternating electric field has an electric field intensity of at least about 1 V / cm in at least a portion of the target area of ​​the subject. The AC electric field is induced by an applied voltage of at least 50V p2p, and The method according to claim 1, wherein the certain period during which the alternating electric field is applied is at least about 50% of a continuous period of at least about 24 hours.

3. The method according to claim 1, wherein the alternating electric field is applied to the target region of the subject for a period of time sufficient to reduce MHC class I expression in the cancer cells in the target region compared to control cancer cells not exposed to the alternating electric field.

4. The method according to claim 1, wherein the at least one NK cell comprises at least one autologous NK cell.

5. The method according to claim 1, wherein the at least one NK cell comprises at least one allogeneic NK cell.

6. The at least one NK cell is derived from the in vitro differentiation of a pluripotent stem cell. The at least one NK cell is derived from an interleukin-2 (IL-2)-dependent cell line, and / or The method according to claim 1, wherein at least one of the above is that the at least one NK cell includes a chimeric antigen receptor modified NK (CAR-NK) cell.

7. The method according to claim 1, wherein steps (1) and (2) are carried out in whole or in part sequentially, and the at least one composition is administered after the application of the alternating electric field is started.

8. The method according to claim 1, wherein steps (1) and (2) are repeated once or more times.

9. The method according to claim 1, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, pleural mesothelioma, differentiated thyroid cancer, advanced renal cell carcinoma, ovarian cancer, pancreatic cancer, lung cancer cells, breast cancer, and combinations thereof.

10. A method for reducing the volume of a tumor and / or preventing an increase in the volume of a tumor, wherein the tumor is present in a living organism and comprises a plurality of cancer cells, and the method is (1) The step of applying an alternating electric field to the target area of ​​the subject for a certain period of time, (2) A method comprising the step of administering at least one composition to the subject, wherein the at least one composition comprises at least one natural killer (NK) cell.

11. The aforementioned alternating electric field is applied at a frequency in the range of approximately 50 kHz to approximately 1 MHz. The alternating electric field has an electric field intensity of at least about 1 V / cm in at least a portion of the target area of ​​the subject. The AC electric field is induced by an applied voltage of at least 50V p2p, and The method according to claim 10, wherein the certain period during which the alternating electric field is applied is at least 50% of a continuous period of at least about 24 hours.

12. The method according to claim 10, wherein the alternating electric field is applied to the target region of the subject for a period of time sufficient to reduce MHC class I expression in the cancer cells compared to control cancer cells not exposed to the alternating electric field.

13. The method according to claim 10, wherein the at least one NK cell comprises at least one autologous NK cell.

14. The method according to claim 10, wherein the at least one NK cell comprises at least one allogeneic NK cell.

15. The at least one NK cell is derived from the in vitro differentiation of a pluripotent stem cell. The at least one NK cell is derived from an interleukin-2 (IL-2)-dependent cell line, and / or The method according to claim 10, wherein at least one of the above is that the at least one NK cell includes a chimeric antigen receptor modified NK (CAR-NK) cell.

16. The method according to claim 10, wherein steps (1) and (2) are carried out in whole or in part sequentially, and the at least one composition is administered after the application of the alternating electric field is started.

17. The method according to claim 10, wherein steps (1) and (2) are repeated once or more times.

18. The method according to claim 10, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, pleural mesothelioma, differentiated thyroid cancer, advanced renal cell carcinoma, ovarian cancer, pancreatic cancer, lung cancer cells, breast cancer, and combinations thereof.

19. (1) The step of applying an alternating electric field to the target area of ​​the subject for a certain period of time, (2) A step of administering at least one composition to the subject, wherein the at least one composition comprises at least one natural killer (NK) cell, A method for increasing the cytotoxicity of NK cells against cancer cells in a subject, when compared to the administration of NK cells to the subject in the absence of the applied AC electric field.

20. The aforementioned alternating electric field is applied at a frequency in the range of approximately 50 kHz to approximately 1 MHz. The alternating electric field has an electric field intensity of at least about 1 V / cm in at least a portion of the target area of ​​the subject. The AC electric field is induced by an applied voltage of at least 50V p2p, and The method according to claim 19, wherein the certain period during which the alternating electric field is applied is at least 50% of a continuous period of at least about 24 hours.

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