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

Description

1 / 47 “COMPOSITIONS, SYSTEMS AND METHODS FOR CANCER TREATMENT USING TUMOR AND CELL TREATMENT FIELDS "EXTERMINATORS" REFERENCE TO RELATED ORDERS

[001] The application in question claims benefit under 35 USC § 119(e) of Provisional Application No. US 63 / 493,141, filed March 30, 2023; and Provisional Application No. US 63 / 599,142, filed November 15, 2023. The content of the aforementioned patent applications is expressly incorporated herein by reference. STATEMENT REGARDING RESEARCH SPONSORED BY THE FEDERAL GOVERNMENT

[002] Not Applicable. BACKGROUND OF THE TECHNIQUE

[003] Tumor Treatment Fields (TTFields) are low-intensity alternating electrical fields (e.g., 1-3 V / cm) within the intermediate frequency range (100-500 kHz as an example, but without limitation) that attack solid tumors by disrupting mitosis. This non-invasive process attacks solid tumors and is described, for example, in US 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. TTFields are typically carried through two pairs of transducer arrays that generate perpendicular fields in the treated tumor; The electrode array that makes up each pair is positioned on opposite sides of the body part to be treated. More specifically, for the OPTUNE® system, one pair of electrodes is located to the left and right (LR) of the tumor, and the other pair of electrodes is located anterior and posterior (AP) to the tumor.TTFields are approved for the treatment of glioblastoma multiforme (GBM) and can be administered, for example, via the OPTUNE® system (Novocure Limited, St. Helier, Jersey), which includes sets of... Petition 870250088297, dated 09 / 29 / 2025, page 9 / 66 2 / 47 transducers placed on the patient's shaved head.

[004] Each transducer array used for TTField administration in the OPTUNE® device comprises a set of ceramic disc electrodes coupled to the patient's skin (such as, but not limited to, the patient's shaved head for GBM treatment) via a layer of conductive medical gel. The purpose of the medical gel is to deform to conform to body contours and provide good electrical contact between the arrays and the skin; thus, the gel interface bridges the skin and reduces interference. The device should be worn continuously by the patient for 2 to 4 days before removal for hygienic care and (if necessary) reshaving, followed by reapplication with a new array. In this way, the medical gel remains in substantially continuous contact with an area of ​​the patient's skin for a period of 2 to 4 days at a time.Additionally, the arrays can be moved a few centimeters in either direction to allow the skin to heal from one treatment period to the next. Therefore, the portion of skin covered by electrodes or gel for a period of 2 to 4 days can then be uncovered for 2 to 4 days when the replaced electrodes are slightly displaced; then, the device can be reapplied to the original portion of skin for the next 2 to 4 day period.

[005] Natural killer (NK) cells are a type of innate lymphoid cell population. NK cells can differentiate healthy (i.e., self) cells from infected or abnormal cells, including transformed cancerous cells. Unlike T and B cells, NK cells are able to attack and kill abnormal cells (including cancerous cells) in an antigen-independent manner (Shimasaki et al. (2020) Nat Rev Drug Discov, 19:200-218). Healthy cells have low levels of NK cell-activating ligands and high levels of major histocompatibility (MHC) class I (in humans HLA - human leukocyte antigen). MHC class I binds to immunoglobulin-like killer (IBD) receptors. Petition 870250088297, dated 09 / 29 / 2025, page 10 / 66 3 / 47 (KIR) in NK cells and prevents the NK cell from killing the healthy cell (Liu et al. (2021) J Hematol Oncol, 14:7)). Downregulation of MHC Class I in infected or cancerous cells can lead to NK cell activation and lysis of the transformed cell (Liu et al., 2021). NK cells have other stimulatory and inhibitory signals facilitated by receptor-ligand interactions with the target cell. 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 for NK cells (Lauterbach et al. (2015) Human Immunology, 76(8):578-586)). Consequently, elevated HLA-E levels can be found in several types of cancer, including non-small cell lung carcinoma (NSCLC), pancreatic, kidney, melanoma, head and neck carcinoma, and others (Borst et al. (2020) Clinical Cancer Research, 26(21):5549-5556).

[006] Most clinical trials using NK cell-based immunotherapies have been in 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).

[007] Furthermore, Silginer et al. (Neuro-Oncology (2018) 20(6):vi133) investigated the interaction of TTFields with molecules present in immune responses, as well as drugs that can modulate the activity of immune cells, and found increased expression of the NKG2D ligand and increased death of glioma cells exposed to TTFields by NK cells, while the expression of MHC Class I and Class II remained unchanged.

[008] Cytokine-induced killer (CIK) cell therapy is a type of adoptive immunotherapy that uses a heterogeneous population of immune cells generated ex vivo from the patient's own peripheral blood mononuclear cells (PBMCs) in the presence of IL-2, anti-CD3 antibody (such as, but not limited to, OKT3), and IFN-gamma (Introna (2017) Journal of Autoimmunity, 85:32-44). CIK cells include Petition 870250088297, dated 09 / 29 / 2025, page 11 / 66 4 / 47 a CD3+CD56+CD8+ population, which are phenotypically cytotoxic T-terminal differentiated effector memory lymphocytes (EMRA) (Franceschetti et al. (2009) Experimental Hematology, 37:616-628.e2) with antitumor activity. This population expresses different NK markers, including NKG2D, and is capable of recognizing and killing tumor cells in a TCR-independent manner, similar to NK cells (Sangiolo et al. (2008) International Immunology, 20:841-848). According to a recent report from the International CIK Cell Registry (IRCC), CIK cells have been tested in 106 clinical trials in more than 30 distinct tumors, with a significant improvement in progression-free survival (PFS) and overall survival (OS) in 27 trials in the autologous setting, 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 with negative clinical outcomes in GBM, including immune checkpoint inhibitors, vaccinations, and various adoptive T-cell therapies (Weenink et al. (2020) Cancers (Basel), 12:E751). BRIEF DESCRIPTION OF THE DRAWINGS

[009] Figure 1 graphically represents an analysis of MHC Class I expression in human and mouse pancreatic cell lines (human pancreatic cancer cell line 1A-AsPc1 and mouse pancreatic cancer cell line 1B-Panc02) after exposure to TTFields of 72 or 96 hours.

[010] Figure 2 graphically represents the transcriptomic analysis after treatment with TTFields compared with the control in glioblastoma (GBM) cell lines, malignant pleural mesothelioma (MPM) cell line and pancreatic cancer AsPC1 and BxPC3 cell lines, to identify genes that show a difference in expression after treatment with TTFields.

[011] Figure 3 graphically represents the expansion of CIK cells. As Petition 870250088297, dated 09 / 29 / 2025, page 12 / 66 5 / 47 CIK cells were generated in vitro from healthy donors and counted at different time points. (a) Cells were counted at different time points. Data shown are the median and interquartile range of cell counts from 5 separate experiments. After 21 days (± 1 day) of culture, CIK cells were characterized by flow cytometry for the expression of (b) CD3, C56 and (c) CD4 and CD8. Data shown are the average of 5 separate experiments.

[012] Figure 4 graphically represents 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+) among cells labeled with CellTrace. GSCs showed a T / E ratio-dependent increase in the apoptotic fraction when co-cultured with CIK cells (a), but not with PBMCs (b). The results presented are the average of duplicates in a single experiment. DETAILED DESCRIPTION

[013] Before explaining at least one embodiment of the inventive concept(s) in detail by means of exemplary language and results, it must be understood that the inventive concept(s) is / are not limited in its / their application to the construction details and arrangement of components set out in the description below. The inventive concept(s) is / are capable of other forms of realization or of being practiced or executed in various ways. Thus, the language used here is intended to have the broadest possible scope and meaning; and the embodiments should be exemplary, not exhaustive. Furthermore, it should be understood that the phraseology and terminology employed here are for descriptive purposes and should not be considered limiting.

[014] Unless otherwise defined in this disclosure, the scientific and technical terms used in connection with the inventive concept(s) presently disclosed shall have the meanings commonly understood by those of skill. Petition 870250088297, dated 09 / 29 / 2025, p. 13 / 66 6 / 47 common in the art. Furthermore, unless the context requires otherwise, singular terms will include the plural, and plural terms will include the singular. The techniques and procedures mentioned above are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this descriptive report. Nomenclatures used in connection with laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses and analyses.

[015] All patents, published patent applications and non-patent publications mentioned in the descriptive report are indicative of the level of skill of those skilled in the art to which this / these currently disclosed inventive concept(s) belong. All patents, published patent applications and non-patent publications mentioned anywhere in this disclosure are expressly incorporated herein by reference in their entirety, to the same extent as if each individual patent or publication were specifically and individually indicated for incorporation by reference.

[016] All compositions, assemblies, systems, kits and / or methods disclosed herein can be made and performed without excessive experimentation in the light of the present disclosure. Although the compositions, assemblies, systems, kits and methods of the inventive concept(s) have been described in terms of particular embodiments, it will be evident to those skilled in the art that variations can be applied to the compositions and / or methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the inventive concept(s). All similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the inventive concept(s), as defined by the appended claims. Petition 870250088297, dated 09 / 29 / 2025, page 14 / 66 7 / 47

[017] As used in this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[018] The use of the term “a” or “an” in conjunction with the term “comprising” in the claims and / or descriptive report may mean “one”, but is also consistent with the meaning of “one or more”, “at least one” and “one or more of one”. As such, the terms “a”, “an” and “the” include plural referents, unless the context clearly indicates otherwise. Thus, for example, the reference to “a compound” may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a larger number of compounds. The term plurality refers to two or more.

[019] The use of the term “at least one” will be understood as including one, as well as any quantity greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one”, depending on the term to which it is linked, may extend to 100 or 1000 or more; furthermore, quantities of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. Furthermore, the use of the term “at least one of X, Y, and Z” will be understood as including X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (such as “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of differentiating between two or more items and is not intended to imply any sequence, order, or importance, for example, of one item over another or any order of addition.

[020] The use of the term “or” in claims is used to mean an inclusive “and / or,” unless explicitly stated, to refer only to alternatives or unless the alternatives are mutually exclusive. For example, a condition “A or B” is satisfied by either of: A is true (or is Petition 870250088297, dated 09 / 29 / 2025, p. 15 / 66 8 / 47 present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[021] As used herein, any reference to “an embodiment,” “some embodiments,” “an example,” “for example,” or “an example” means that a specific element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase “in some embodiments” or “an example” in several places in the descriptive report does not necessarily refer to the same embodiment, for example. Furthermore, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[022] Throughout this disclosure, the term “about” is used to indicate that a value includes the inherent error variation for a composition / apparatus / device, the method employed to determine the value, or the variation that exists among the individuals in the study. For example, but not limited to, when the term “about” is used, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent of the specified value, as such variations are appropriate for performing the methods disclosed and as understood by persons of ordinary skill in the art.

[023] As used in this descriptive report and claim(s), the words “comprising” (and any form of comprising, such as “comprising” and “comprising”), “having” (and any form of having, such as “having” and “having”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contains”) are inclusive or open-ended and do not exclude additional elements or method steps not mentioned. Petition 870250088297, dated 09 / 29 / 2025, page 16 / 66 9 / 47

[024] The term “or combinations thereof,” as used herein, refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C or combinations thereof” is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and, if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth, are expressly included. One skilled in the art will understand that there is typically no limit to the number of items or terms in any combination unless the context indicates otherwise.

[025] As used herein, the term “substantially” means that the event or circumstance described below occurs completely or that the event or circumstance described below occurs to a great extent or degree. For example, when associated with a specific event or circumstance, the term “substantially” means that the event or circumstance subsequently described occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term “substantially adjacent” may mean that two items are 100% adjacent to each other, or that the two items are close to each other but not 100% adjacent to each other, or that a part of one of the two items is not 100% adjacent to the other item but is close to the other item.

[026] The term “pharmaceutically acceptable” refers to compounds and compositions that are suitable for administration to humans and / or animals without undue adverse side effects, such as (but not limited to) toxicity, irritation and / or allergic response, consistent with a reasonable benefit / risk ratio.

[027] The term “patient” or “individual” as used herein includes human and veterinary individuals. “Mammal,” for treatment purposes, refers to any Petition 870250088297, dated 09 / 29 / 2025, page 17 / 66 10 / 47 animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, non-human primates and any other animal that has mammary tissue.

[028] The term “treatment” refers to both therapeutic treatment and prophylactic or preventive measures. Those who need treatment include, but are not limited to, individuals who already suffer from a specific condition / disease / infection, as well as individuals who are at risk of acquiring a specific condition / disease / infection (e.g., those who need prophylactic / preventive measures). The term “treat” refers to the administration of an agent / element / method to a patient for therapeutic and / or prophylactic / preventive purposes.

[029] The term “therapeutic composition” or “pharmaceutical composition”, as used herein, refers to an agent that can be administered in vivo to produce a therapeutic and / or prophylactic / preventive effect.

[030] The administration of a therapeutically effective or prophylactically effective amount aims to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific amount that is therapeutically effective can be easily determined by the average physician and may vary depending on factors known in the art, such as (but not limited to) the type of condition / disease / infection, the patient's history and age, the stage of the condition / disease / infection, and the co-administration of other agents.

[031] The term “effective amount” refers to an amount of a biologically active molecule or conjugate or derivative thereof, or an amount of a treatment protocol (e.g., an alternating electric field), sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (such as (but not limited to) toxicity, irritation, and allergic response) consistent with a reasonable benefit / risk ratio when used in the manner of the inventive concept(s)^). The therapeutic effect may include, for example, but not limited to, preventing, Petition 870250088297, dated 09 / 29 / 2025, page 18 / 66 11 / 47 to inhibit or reduce the occurrence of at least one condition, disease, and / or infection. The effective amount for an individual will depend on the type of individual, the size and health of the individual, the nature and severity of the condition / disease / infection being treated, the method of administration, the duration of treatment, the nature of concomitant therapy (if any), the specific formulations employed, and the like. Therefore, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by someone with ordinary skill in the technique using routine experimentation based on the information provided herein.

[032] As used herein, the term “concomitant therapy” is used interchangeably with the terms “concomitant therapy” and “adjuvant therapy” and will be understood to mean that the patient requiring treatment is treated or receives another medication for the condition / disease / infection in conjunction with the treatments of the present disclosure. This concurrent therapy may be sequential therapy, in which the patient is treated first with one treatment protocol / pharmaceutical composition and then with the other treatment protocol / pharmaceutical composition, or both treatment protocols / pharmaceutical compositions are administered simultaneously. Furthermore, it will be understood that one administration step (e.g., but not limited to, administration of TTFields) may occur over a longer period of time than the other administration step (i.e., oral administration or injection of a substance).In cases of variable administration periods, the term "simultaneously" will be understood to mean that the shorter administration phase completely overlaps with the longer administration phase. However, the term "simultaneously" will include the execution of the shorter administration phase at any point during the longer administration phase (for example, the beginning, middle, or end of the longer administration phase, or any other time period in between), as well as the execution of the phase of... Petition 870250088297, dated 09 / 29 / 2025, page 19 / 66 12 / 47 shorter administration one or more times entirely within the time period of the longer administration step. Therefore, the term “simultaneously” does not require that the two administration steps be performed in exactly the same time period.

[033] The terms “administration” and “administer,” as used herein, shall be understood to include all routes of administration known in the art, including, but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, and including local and systemic applications. In addition, the compositions of the present disclosure (and / or the methods of administration thereof) may be designed to provide delayed, controlled, or sustained release using formulation techniques well known in the art.

[034] The term “target region”, as used herein, refers to a region that contains, in whole or in part, the cancer, cancer cells and / or tumor to be treated.

[035] With reference now to the inventive concept(s), a concurrent therapy for cancer is disclosed herein. The concurrent therapy includes the use of alternating electric fields (e.g., TTFields) in addition to killer cells (such as, but not limited to, natural killer (NK) cells or cytokine-induced killer (CIK) cells). The combination of alternating electric fields (e.g., TTFields) with killer cells provides a synergistic result in cancer treatment.

[036] Certain non-limiting embodiments of the present disclosure are directed to a method of reducing the viability of cancer cells. The method includes the steps of: (1) applying an alternating electric field to the cancer cells for a period of time; and (2) administering at least one composition to the cancer cells, wherein the at least one composition comprises at least one Petition 870250088297, dated 09 / 29 / 2025, page 20 / 66 13 / 47 killer cell (such as, but not limited to, at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell). These methods can be performed in vitro or in vivo.

[037] Certain non-limiting embodiments of the present disclosure are directed to a method of treating cancer in an individual. The method includes the steps of: (1) applying an alternating electric field to the cancer cells for a 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 (such as, but not limited to, at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell).

[038] Certain non-limiting embodiments of the present disclosure are directed to a method of reducing the volume of a tumor present in the body of a living individual, wherein the tumor includes a plurality of cancerous cells. The method includes the steps of: (1) applying an alternating electric field to the cancerous cells for a period of time; and (2) administering at least one composition to the cancerous cells, wherein the at least one composition comprises at least one killer cell (such as, but not limited to, at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell).

[039] Certain non-limiting embodiments of the present disclosure are directed to a method of reducing the volume of a tumor present in the body of a living individual, wherein the tumor includes a plurality of cancerous cells. The method includes the steps of: (1) applying an alternating electric field to the cancerous cells for a period of time; and (2) administering at least one composition to the cancerous cells, wherein the at least one composition comprises at least one killer cell (such as, but not limited to, at least one natural killer (NK) cell or at least one induced killer cell). Petition 870250088297, dated 09 / 29 / 2025, page 21 / 66 14 / 47 cytokine (CIK)).

[040] Certain additional embodiments, not limiting to the present disclosure, are directed to a method comprising the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one killer cell (such as, but not limited to, at least one natural killer (NK) cell or at least one cytokine-induced killer (CIK) cell).

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

[042] Certain additional non-limiting embodiments of the present disclosure are directed 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 a field generator device configured to apply an alternating electric field to cancer cells for a period of time.

[043] Certain additional non-limiting embodiments of the present disclosure are directed to a kit for reducing the viability of cancer cells, the kit comprising: components configured to isolate and grow at least one natural killer cell, or a precursor thereof, from an individual; and a field generator device configured to apply an alternating electric field to cancer cells for a period of time.

[044] In certain particular (but not limited) modalities, the Petition 870250088297, dated 09 / 29 / 2025, page 22 / 66 15 / 47 Administration of an alternating electric field in any of the methods described above or otherwise herein increases the effectiveness and / or cytotoxicity of killer cells against cancer cells in the individual when compared to administering killer cells to the individual in the absence of alternating electric field application.

[045] Steps (1) and (2) of any of the methods of the present disclosure may be performed concurrently or in series and, in particular, substantially simultaneously or totally or partially sequentially. When the steps are performed totally or partially sequentially, at least one composition comprising at least one killer cell may be administered before or after the start of the application of the alternating electric field.

[046] The methods of the present disclosure can be used to treat any types of cancer cells / cancers / tumors that respond to treatment with alternating electric fields (e.g., TTFields) and / or killer cells. Non-limiting examples of cancer cells / cancers / tumors that can be treated according to the present disclosure include hepatocellular carcinoma cells / carcinoma, glioblastoma cells / glioblastoma, pleural mesothelioma cells / mesothelioma, differentiated thyroid cancer cells / cancer, advanced renal cell carcinoma cells / carcinoma, ovarian cancer cells / cancer, pancreatic cancer cells / cancer, lung cancer cells / cancer, breast cancer cells / cancer and the like, as well as any combination thereof.

[047] Any type of conductive or non-conductive electrode(s) and / or transducer assemblies that can be used to generate an alternating electric field that are known in the art or otherwise contemplated in this disclosure may be used for generating the alternating electric field according to the methods disclosed herein. Non-limiting examples of electrodes and assemblies of Petition 870250088297, dated 09 / 29 / 2025, p. 23 / 66 16 / 47 Transducers that can be used to generate an alternating electric field according to the present disclosure include those that function as part of a TTFields system, as described, for example, but not as a limitation, 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; and in Patent Applications Nos. U.S. 2018 / 0160933; U.S. 2019 / 0117956; U.S. 2019 / 0307781; and U.S. 2019 / 0308016.

[048] The alternating electric field can be generated at any frequency according to the present disclosure. For example (but not by way of limitation), the alternating electric field may have a frequency of about 50 kHz, about 75 kHz, about 100 kHz, about 125 kHz, about 150 kHz, about 175 kHz, about 200 kHz, about 225 kHz, about 250 kHz, about 275 kHz, about 300 kHz, about 325 kHz, about 350 kHz, about 375 kHz, about 400 kHz, about 425 kHz, about 450 kHz, about 475 kHz, about 500 kHz, about 550 kHz, about 600 kHz, about 650 kHz, about 700 kHz, about 750 kHz, about 800 kHz, approximately 850 kHz, approximately 900 kHz, approximately 950 kHz, approximately 1 MHz, approximately 2 MHz, approximately 3 MHz, approximately 4 MHz, approximately 5 MHz, approximately 6 MHz, approximately 7 MHz, approximately 8 MHz, approximately 9 MHz, approximately 10 MHz and the like, as well as a range formed by any of the above values ​​(for example,a range from about 50 kHz to about 10 MHz, a range from about 50 kHz to about 1 MHz, a range from about 50 kHz to about 500 kHz, a range from about 100 kHz to about 500 kHz, a range from about 150 kHz to about 300 kHz, etc.) and a range that combines two integers that fall between two of the values ​​mentioned above (for example, a range from about 122 kHz to about 313 kHz, a range from about 78 kHz to about 298 kHz, etc.).

[049] In certain specific (but not limited) embodiments, the alternating electric field may be imposed at two or more different frequencies. When two or more frequencies are present, each frequency is selected from any Petition 870250088297, dated 09 / 29 / 2025, p. 24 / 66 17 / 47 is one of the values ​​mentioned above, or an interval formed by any of the values ​​mentioned above, or an interval that combines two integers that lie between two of the values ​​mentioned above.

[050] In particular (but not limiting) modalities, the following frequencies may be used for specific cancers: GBM, approximately 200 kHz; NSCLC, approximately 150 kHz; breast cancer, approximately 200 kHz; pancreatic cancer, approximately 150 kHz; brain metastases of NSCLC, approximately 150 kHz; liver cancer, approximately 150 kHz; and similar.

[051] The alternating electric field can have any field intensity in the target region / individual / in cancer cells, provided that the alternating electric field is capable of functioning in accordance with the present disclosure. For example (but not as a limitation), the alternating electric field can have a field strength in the target region / individual / cancer cells of at least about 1 V / cm, about 1.5 V / cm, about 2 V / cm, about 2.5 V / cm, about 3 V / cm, about 3.5 V / cm, about 4 V / cm, about 4.5 V / cm, about 5 V / cm, about 5.5 V / cm, about 6 V / cm, about 6.5 V / cm, about 7 V / cm, about 7.5 V / cm, about 8 V / cm, about 9 V / cm, about 9.5 V / cm, about 10 V / cm, about 10.5 V / cm, about 11 V / cm, about 11.5 V / cm, about 12 V / cm, about 12.5V / cm, about 13V / cm, about 13.5V / cm, about 14V / cm, about 14.5V / cm, about 15V / cm, about 15.5V / cm, about 16V / cm, about 16.5V / cm, about 17V / cm, about 17.5V / cm,approximately 18 V / cm, approximately 18.5 V / cm, approximately 19 V / cm, approximately 19.5 V / cm, approximately 20 V / cm and the like, as well as a range formed by any of the above values ​​(for example, a range from approximately 1 V / cm to approximately 20 V / cm, a range from approximately 1 V / cm to approximately 10 V / cm, a range from approximately 1 V / cm to approximately 4 V / cm, etc.) and a range that combines two integers that fall between two of the values ​​mentioned above (for example, a range from approximately 1.1 V / cm to approximately 18.6 V / cm, a range from approximately 1.2 V / cm to approximately 9.8 V / cm, etc.) Petition 870250088297, dated 09 / 29 / 2025, p. 25 / 66 18 / 47 V / cm, a range of about 1.3 V / cm to about 4.7 V / cm, etc.). Generally, it is desirable to use the highest possible field strength without causing overheating, with the field strength usually being limited by temperature measurements.

[052] In some cases, the electric field in at least part of the target region / individual / cancer cells is induced by an applied voltage that is determined by computer simulation of the target region / individual / cancer cells. In some cases, the electric field in at least part of the target region / individual / 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 with a field strength of at least 1 V / cm (e.g., at least 5 V / cm) in at least part of the target region / individual / cancer cells.

[053] The alternating electric field can be applied in a single direction between a pair of matrices or it can be alternated in two or more directions / channels between two or more pairs of matrices (e.g., front-back and left-right). For example, certain TTFields devices (such as, but not limited to, the OPTUNE® system (Novocure Limited, St. Helier, Jersey)) operate in two directions to increase the chances of a dividing cell being aligned with the electric field, so that the electric field can have the desired antimitotic effect. However, it should be understood that the scope of the present disclosure also includes the application of the alternating electric field in a single direction. The term “alternating electric field,” as used herein, will be understood as including application in a single direction / channel as well as in two or more directions / channels; furthermore, the term “alternating electric field,” as used herein, will be understood as including both Petition 870250088297, dated 09 / 29 / 2025, page 26 / 66 19 / 47 application of a single alternating electric field versus the application of a plurality of alternating electric fields in succession over a period of time.

[054] The alternating electric field can be applied for any continuous or cumulative period of time sufficient to achieve a reduction in the viability of cancer cells and / or a reduction in tumor volume (and / or a prevention of tumor volume increase). The period of time during which the alternating electric field is applied includes both a continuous period of time and a cumulative period of time. That is, the period of time during which the alternating electric field is applied includes a single session (i.e., continuous application) as well as multiple sessions with short intervals between sessions (i.e., consecutive application over a cumulative period).For example, an individual may take breaks during treatment with an alternating electric field device, and the device is expected to remain positioned on the body and operational for at least 60%, at least 70%, or at least 80% of the total treatment period (e.g., over the course of one day, one week, two weeks, one month, two months, three months, four months, five months, etc.).

[055] For example, but not as a limitation, the alternating electric field may be applied for a continuous or cumulative period of time of at least 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, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, about Petition 870250088297, dated 09 / 29 / 2025, page 27 / 66 20 / 47 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 and the like, as well as an interval formed from any of the above values ​​(for example, an interval from approximately 1 hour to approximately 6 months, an interval from approximately 24 hours to approximately 72 hours, etc.) and an interval that combines two integers that fall between two of the values ​​mentioned above (for example, an interval from approximately 14 hours to approximately 68 hours, etc.).

[056] In a specific (but not limiting) embodiment, the time period during which the alternating electric field is applied is at least about 24 hours, with the device positioned on the body and operational for at least about 80% of that period.

[057] In a specific (but not limited) embodiment, the alternating electric field is applied to the cancer cells / target region of the individual for a period of time sufficient to reduce the expression of MHC Class I in the cancer cells compared with control cancer cells that were not exposed to alternating electric fields.

[058] Any killer cells known in the art or contemplated herein may be used in accordance with the present disclosure, provided that the killer cells are capable of functioning as described herein. Non-limiting examples of killer cells that may be used in accordance with the present disclosure include natural killer (NK) cells and cytokine-induced killer (CIK) cells. Killer cells may be autologous (and therefore obtained from the individual to be treated) or may be allogeneic (and therefore obtained from another individual). Autologous or allogeneic killer cells may be cultured directly from an individual and then implanted / injected into Petition 870250088297, dated 09 / 29 / 2025, page 28 / 66 21 / 47 individual with cancer. Alternatively (and / or additionally), pluripotent stem cells can be obtained from an individual and differentiated ex vivo into killer cells that are subsequently implanted / injected into the cancer patient.

[059] In certain particular (but not limited to) embodiments, killer cells are derived from an interleukin-2 (IL-2)-dependent cell line. For example (but not as a limitation), NK-92® cells (ImmunityBio, Inc., Culver City, CA) are commercially available and can be used in accordance with the present disclosure.

[060] In specific (but not limited) modalities, killer cells are engineered. For example, but not as a limitation, killer cells may include chimeric antigen receptor-engineered NK cells (CARNK).

[061] Several other types of killer cells that can be used according to the present disclosure are known in the art and are commercially available from various sources; therefore, no further description of them is necessary.

[062] The composition comprising at least one killer cell may be provided with any formulation known in the art or otherwise contemplated herein. In certain embodiments (not limited to), the composition comprising at least one killer cell contains one or more pharmaceutically acceptable carriers (and, as such, the composition may also be referred to as a “pharmaceutical composition”). Non-limiting examples of suitable pharmaceutically acceptable carriers include water; saline solution; dextrose solutions; fructose or mannitol; calcium carbonate; cellulose; ethanol; oils of animal, vegetable or synthetic origin; carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposomal carriers; solutions Petition 870250088297, dated 09 / 29 / 2025, page 29 / 66 22 / 47 buffered, such as sodium chloride, saline solution, phosphate-buffered saline solution and / or other substances that are physiologically acceptable and / or safe for use; diluents; excipients, such as polyethylene glycol (PEG); or any combination thereof. Pharmaceutically acceptable vehicles suitable for pharmaceutical formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd ed. (2020).

[063] In specific (but not limited to) embodiments, the composition comprising at least one killer cell may further contain one or more additional active agents. Several active agents that can be used simultaneously with alternating electric fields or killer cells are known in the art, and certain combination therapies are FDA approved or are currently in clinical trials. Non-limiting examples of therapeutic agents that can be used according to the present disclosure simultaneously with killer cells include anti-PD-1 therapeutic agents, such as (but not limited to) Pembrolizumab (KEYTRUDA®, Merck & Co., Inc., Rahway, NJ), Tislelizumab, Nivolumab and Cemiplimab; anti-PD-L1 therapeutic agents, such as atezolizumab, avelumab and durvalumab; chemotherapeutic agents, such as (but not limited to) lenvatinib; Paclitaxel, Docetaxel, Ifosamide, Etoposide (VEPESID®, Bristol-Myers Squibb Co, New York, NY), Gemcitabine, Lomustine, nab Paclitaxel, temozolomide and Carboplatin; TKI inhibitors, such as (but not limited to) Everolimus; mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; FGF inhibitors; anti-LAB3 agents; anti-CTLA-4 therapies; aromatase inhibitors, such as (but not limited to) Letrozole; Biological products, such as monoclonal antibodies (such as, but not limited to, denosumab and pembrolizumab); and the like, as well as any combinations thereof.

[064] In addition, any of the compositions of the present disclosure may contain other agents that allow the administration of the compositions by means of Petition 870250088297, dated 09 / 29 / 2025, page 30 / 66 23 / 47 a specific route of administration. For example, but not as a limitation, the compositions may be formulated for oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal and / or intravenous administration. Based on the route of administration, the compositions may also contain one or more additional components besides the active agent (e.g., killer cell(s) and / or additional therapeutic agent(s)). Examples of additional secondary compounds that may be present include, but are not limited to, fillers, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifying agents, dispersing agents and other materials well known in the art.

[065] In a specific (but not limited to) embodiment, at least one composition comprising at least one killer cell is administered by injection or implantation into the individual. For example (but not as a limitation), in some cases it may be desirable that the killer cell(s) be administered at a local / regional level to ensure targeting of the killer cells to a specific location in the individual’s body and to inhibit non-specific interactions in other parts of the body; in other cases, a more systemic administration may be desired.

[066] At least one composition comprising at least one killer cell may be administered before or after the start of the application of the alternating electric field. In certain particular (but not limiting) embodiments, at least one composition comprising at least one killer cell may be administered after the start of the application of the alternating electric field. In particular (but not as a limitation), at least one composition comprising at least one killer cell may be administered during the application of the alternating electric field (e.g., before the time period during which the alternating electric field is applied has elapsed) and / or after the application of the electric field. Petition 870250088297, dated 09 / 29 / 2025, page 31 / 66 24 / 47 alternating periods have elapsed.

[067] For example (but not as a limitation), at least one composition comprising at least one killer cell may be administered after the application of the alternating electric field has begun for a period of 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, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, and similar values, as well as an interval formed by any of the above values ​​(for example,an interval of about 24 hours to about 96 hours, etc.) and an interval combining two integers that fall between two of the values ​​mentioned above (for example, an interval of about 14 hours to about 94 hours, etc.). In a specific (but not limited) embodiment, at least one composition comprising at least one killer cell is administered at least about 24 hours after the start of the application of the alternating electric field.

[068] In other non-limiting examples, at least one composition comprising at least one killer cell may be administered after the time period during which the alternating electric field is applied has elapsed, wherein at least one composition comprising at least one killer cell is administered within 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 Petition 870250088297, dated 09 / 29 / 2025, page 32 / 66 25 / 47 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, and so on, depending on when the time period has elapsed.

[069] In a particular (but not limited) embodiment, at least one composition comprising at least one killer cell is administered approximately 96 hours after the time period has elapsed.

[070] The composition comprising at least one killer cell may be administered to the cancer cells / individual at any concentration that provides a therapeutically effective concentration of killer cells. In certain non-limiting embodiments, the application of an alternating electric field reduces the amount of killer cells needed to be therapeutically effective when compared to a normal therapeutically effective amount administered in the absence of an alternating electric field. For example, but not as a limitation, the therapeutically effective concentration of killer cells may 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 a killer cell dosage known to be therapeutically effective in the absence of application of an alternating electric field.In one particular (but not limited) embodiment, the therapeutically effective concentration of killer cells is reduced by at least about 50% when compared to a killer cell dosage known to be therapeutically effective in the absence of an alternating electric field.

[071] The composition(s) containing killer cells may be administered to the individual in any concentration capable of inducing an inflammatory response to the tumor or cancer cells. For example, but not Petition 870250088297, dated 09 / 29 / 2025, page 33 / 66 26 / 47 as a form of limitation, killer cells can be administered at approximately 10 cells / kg of body weight, approximately 100 cells / kg of body weight, approximately 1000 cells / kg of body weight, approximately 104 cells / kg of body weight, approximately 105 cells / kg of body weight, approximately 106 cells / kg of body weight, approximately 107 cells / kg of body weight, approximately 108 cells / kg of body weight, approximately 109 cells / kg of body weight, approximately 1010 cells / kg of body weight, approximately 1011 cells / kg of body weight, approximately 1012 cells / kg of body weight, approximately 1013 cells / kg of body weight, approximately 1014 cells / kg of body weight, approximately 1015 cells / kg of body weight or upper, as well as a range formed from any of the above values ​​(for example, a range of about 104 to about 109 cells / kg of body weight, etc.).

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

[073] In certain specific (but not limiting) embodiments, the killer cell-containing composition may be administered by any dosage regimen known in the art. For example, but not as a limitation, a killer cell-containing composition may be administered in a single dosage or in multiple dosages over a defined treatment period. For example (but not as a limitation), a therapeutically effective concentration of the killer cell-containing composition may be administered approximately once every 4 hours, approximately once every 8 hours, approximately once every 12 hours, approximately once daily, approximately once every two days, approximately once every three days, approximately once weekly, approximately twice weekly, approximately three times weekly, approximately once every two weeks, approximately Petition 870250088297, dated 09 / 29 / 2025, page 34 / 66 27 / 47 once every three weeks, about once a month and similar, as well as a range formed from any of the above values ​​(a range of about once every 4 to 8 hours, a range of about once a week to about once a month, etc.).

[074] In certain particular (but not limited to) embodiments, the method involves simultaneous therapy with two or more compositions. As such, the method may include an additional step of (4) administering at least one second composition to the cancer cells / individual. In a particular (but not limited to) embodiment, the at least second composition may contain one or more of any of the active substances disclosed or otherwise contemplated herein for use with the killer cell-containing composition.

[075] Several substances and therapies used concomitantly with killer cells are known in the art, and certain combination therapies are FDA approved or are currently in clinical trials. Non-limiting examples of therapeutic agents that may be used pursuant to the present disclosure in simultaneous (sequential) killer cell therapy include anti-PD-1 therapeutic agents such as (but not limited to) Pembrolizumab (KEYTRUDA®, Merck & Co., Inc., Rahway, NJ), Tislelizumab, Nivolumab and Cemiplimab; anti-PD-L1 therapeutic agents, such as atezolizumab, avelumab and durvalumab; chemotherapeutic agents, such as (but not limited to) lenvatinib; Paclitaxel, Docetaxel, Ifosamide, Etoposide (VEPESID®, Bristol-Myers Squibb Co, New York, NY), Gemcitabine, Lomustine, nab Paclitaxel, temozolomide and Carboplatin; TKI inhibitors, such as (but not limited to) Everolimus; mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; FGF inhibitors; anti-LAB3 agents; anti-CTLA-4 therapies; aromatase inhibitors, such as (but not limited to) Letrozole; Biological products, such as monoclonal antibodies (such as, but not limited to, denosumab and pembrolizumab); and the like, as well as any others. Petition 870250088297, dated 09 / 29 / 2025, page 35 / 66 28 / 47 combinations of these.

[076] When present, step (4) may be performed substantially simultaneously, or totally or partially sequentially with the administration of the first composition in step (2), whereby the two separate compositions are administered simultaneously or totally or partially sequentially. Additionally, the two compositions administered in steps (2) and (4) may be administered by the same route (e.g., both administered intravenously) or the two compositions may be administered by different routes (e.g., one composition administered orally and another composition administered intravenously).

[077] When present, the optional additional administration step (4) may be performed before or after the start of the alternating electric field application, during the alternating electric field application and / or after the alternating electric field application has elapsed, in the same manner(s) and time period(s) described above for the first composition.

[078] That is, for example (but not as a form of limitation), the second composition may be administered after the application of the alternating electric field has begun for a period of 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, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, and so on, as well as an interval formed from any of the above values ​​(for example, an interval from approximately 24 hours to approximately 96 hours, Petition 870250088297, dated 09 / 29 / 2025, page 36 / 66 29 / 47 etc.), and an interval that combines two integers that lie between two of the values ​​mentioned above (for example, an interval of about 14 hours to about 94 hours, etc.). In a particular (but not limiting) embodiment, the second composition is administered at least about 24 hours after the start of the application of the alternating electric field.

[079] In other non-limiting examples, the second composition may be administered after the time period during which the alternating electric field is applied has elapsed, wherein 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 approximately 90 hours, approximately 93 hours, approximately 96 hours, approximately 5 days, approximately 6 days, approximately 7 days, and similar periods, depending on when the time period elapsed.In one particular (but not limited) embodiment, the second composition is administered within approximately 96 hours after the time period has elapsed.

[080] In addition, for example (but not as a limitation), the second composition may be administered after the administration of the first substance for a period of 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, Petition 870250088297, dated 09 / 29 / 2025, page 37 / 66 30 / 47 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 and the like, as well as an interval formed from any of the above values ​​(e.g., an interval from approximately 24 hours to approximately 96 hours, etc.) and an interval that combines two integers that fall between two of the values ​​mentioned above (e.g., an interval from approximately 14 hours to approximately 94 hours, etc.). In a particular (but not limited) embodiment, the second composition is administered at least approximately 12 hours after the administration of the first substance.

[081] In certain particular (but not limited to) embodiments, the method may further comprise the step of (5) administering at least one additional therapy to the cells / individual. Any therapies known in the art or contemplated herein for use with alternating electric fields (e.g., TTFields) and / or killer cell therapy may be used in accordance with the methods of the present disclosure. Non-limiting examples of additional therapies that may be used include radiotherapy, photodynamic therapy, transarterial chemoembolization (TACE), or combinations thereof.

[082] Any of the steps (1) and (2) and the optional steps (3), (4) and (5) may be repeated one or more times. Each of the steps may be repeated as many times as necessary. When repeating step (1), the transducer sets may be placed on the individual in slightly different positions than the original placement; relocation of the sets in this way may further assist in the treatment of the tumor / cancer. In addition, step (2) and the optional steps (4) and (5) (when present) of administering additional compositions / therapies may be Petition 870250088297, dated 09 / 29 / 2025, page 38 / 66 31 / 47 repeated several times and at various intervals to follow any known and / or generally accepted dosage / treatment regimen for the composition(s) / therapy(ies).

[083] The use of ordinal references to optional steps is for illustrative purposes only; the methods of the present disclosure may include one or more of the optional steps (3), (4) and (5), either alone or in combination with each other. Thus, the methods of the present disclosure include performing step (3) in the absence of steps (4) or (5), performing step (4) in the absence of steps (3) or (5), and performing step (5) in the absence of steps (3) and (4). In other words, the scope of the methods disclosed herein includes performing steps (1)-(2) (as well as repeating each step as many times as necessary), performing steps (1)-(3) (as well as repeating one or more of steps (1)-(3) as many times as necessary), performing steps (1)-(2) and (4) (as well as repeating one or more of steps (1)-(2) and (4) as many times as necessary),the execution of steps (1)-(2) and (5) (as well as the repetition of one or more of steps (1)-(2) and (5) as many times as necessary), the execution of steps (1)-(4) (as well as the repetition of one or more of steps (1)-(4) as many times as necessary), the execution of steps (1)-(3) and (5) (as well as the repetition of one or more of steps (1)-(3) and (5) as many times as necessary), the execution of steps (1)-(2) and (4)-(5) (as well as repeating one or more of steps (1)-(2) and (4)-(5) as many times as necessary) and executing all steps (1)-(5) (as well as repeating one or more of steps (1)-(5) as many times as necessary).

[084] Although the use of concomitant therapy with two substances is explicitly described above, it should be understood that the scope of this disclosure also includes concomitant therapy with three or more compositions. As such, the method may include one or more additional steps of administering an additional composition to the individual (similar to steps (1) and (4)). Any additional substances Petition 870250088297, dated 09 / 29 / 2025, p. 39 / 66 32 / 47 administered in the method may be selected from any of the substances disclosed or otherwise contemplated herein for use concomitantly with at least one killer cell (as disclosed herein above with respect to optional step (4)); furthermore, the administration of any additional substances may be carried out substantially simultaneously or wholly or partially sequentially with the administration of the first and / or second compositions / substances and in the same manner(s) and time period(s) described above for the first and second compositions / substances.

[085] Some non-limiting embodiments of the present disclosure relate to kits that include any of the components of alternating electric field generation systems (e.g., TTFields) disclosed or otherwise contemplated herein (such as, but not limited to, one or more sets of transducers and / or one or more hydrogel compositions, as disclosed 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; and in Patent Applications Nos. U.S. 2018 / 0160933; U.S. 2019 / 0117956; U.S. 2019 / 0307781; and US 2019 / 0308016) in combination with at least one of any of the compositions comprising at least one killer cell disclosed or otherwise contemplated herein, and / or components for isolating and / or culturing at least one killer cell or a precursor thereof from a patient.Optionally, the kits may also include one or more of any of the optional compositions disclosed or otherwise contemplated herein (such as, but not limited to, one or more optional compositions containing at least one additional active agent and / or one or more optional compositions to cultivate / induce differentiation of at least one killer or precursor cell). The kits may also optionally include one or more devices (or one or more device components) used in one or more additional therapeutic steps.

[086] In a specific (but not limited) modality, the kit may include Petition 870250088297, dated 09 / 29 / 2025, page 40 / 66 33 / 47 instructions for performing any of the methods revealed here or otherwise contemplated.For example (but not as a limitation), the kit may include instructions for applying one or more components of the alternating electric field generating device (e.g., TTFields) to the patient's skin, instructions for applying the alternating electric field to the patient, instructions for isolating the killer cell(s) or precursor(s) thereof from an individual, instructions for culturing and / or differentiating the killer cell(s) or precursor(s) thereof ex vivo, instructions for formulating the composition comprising at least one killer cell, instructions for when and how to administer the composition comprising at least one killer cell and, optionally, how to administer one or more additional optional compositions, and / or instructions for when to activate and deactivate the alternating electric field in relation to the administration of the composition comprising at least one killer cell and / or administration of one or more optional compositions and / or therapy steps.

[087] In addition to the components described in detail above, the kits may also contain other components / reagents to perform any of the specific methods described or otherwise contemplated in this disclosure. For example (but not limited to), the kits may additionally include: (i) components for preparing the skin before disposal of the hydrogel compositions and / or transducer assemblies on it (e.g., a razor blade, a cleansing composition or moistened wipe / towel, etc.); (ii) components for removing the gel / transducer assembly(ies); (iii) components for cleaning the skin after removal of the gel / transducer assembly(ies); and / or (iv) other components used with the system (e.g., conductive material, non-conductive material, a soothing gel or cream, a bandage, etc.).The nature of these additional components / reagents will depend on the specific treatment format, and their identification is within the ability of someone with common skill in the technique; therefore, none. Petition 870250088297, dated 09 / 29 / 2025, page 41 / 66 34 / 47 Additional description is considered necessary. Furthermore, the components / reagents present in the kits may be in separate containers / compartments, or several components / reagents may be combined in one or more containers / compartments, depending on the sterility, cross-reactivity and stability of the components / reagents.

[088] The kit may be disposed of in any packaging that allows the components contained therein to function in accordance with the present disclosure. In certain non-limiting embodiments, the kit further comprises a sealed package in which the components are disposed of. In certain specific (but not limiting) embodiments, the sealed package is substantially impermeable to air and / or light.

[089] In addition, the kit may include a set of written instructions explaining how to use one or more components of the kit. A kit of this model may be used in any of the methods described or contemplated in this disclosure.

[090] In some non-limiting embodiments, the kit has a shelf life of at least about six months, such as (but not limited to) at least about nine months or at least about 12 months.

[091] Some non-limiting embodiments of the present disclosure relate to systems that include any of the components of the alternating electric field generation systems disclosed or contemplated herein (such as, but not limited to, one or more sets of transducers and / or one or more hydrogel compositions, as disclosed in 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; and in Patent Applications Nos. 2019 / 0308016) in combination with at least one of any of the compositions comprising at least one killer cell disclosed or contemplated herein. Optionally, the systems may also include one or more of any of the optional compositions disclosed or contemplated in this disclosure. The systems Petition 870250088297, dated 09 / 29 / 2025, p. 42 / 66 35 / 47 may also optionally include one or more devices (or one or more device components) used in one or more additional therapeutic steps. EXAMPLES

[092] Examples of the disclosure follow. However, the present disclosure should be understood as not limited in its application to the specific experimentation, results, and laboratory procedures disclosed below. Instead, Examples are provided simply as one of several embodiments and are intended to be exemplary, not exhaustive. Example 1

[093] In the present example, the effects of TTFields on MHC class I expression in cancer cell lines were examined. In contrast to the previous technique by Silginer et al. (2018) mentioned in the Technique Background section, which found that MHC Class I and Class II expression remained unchanged after exposure to TTFields, this Example found that MHC Class I expression was actually reduced in human and mouse pancreatic cancer cell lines. As illustrated in Figure 1, MHC class I expression was reduced in the human pancreatic cancer cell line 1A-AsPc1 after a 72-hour exposure to TTFields (left panel), and MHC class I expression was reduced in the mouse pancreatic cancer cell line 1B-Panc02 after 72- or 96-hour exposures to TTFields (middle and right panels).

[094] Furthermore, a transcriptomic analysis was conducted to identify HLA class I genes that exhibit differences in expression after TTFields treatment in several cancer cell lines compared to the control. As illustrated in Figure 2, there is a statistically significant reduction in several tested HLA class I genes (including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLAG, and HLA-H) after TTFields in glioblastoma (GBM) cell lines, malignant pleural mesothelioma (MPM) cell lines, and cancer cell lines. Petition 870250088297, dated 09 / 29 / 2025, page 43 / 66 36 / 47 pancreatic AsPCI and BxPC3. Therefore, these data demonstrate that TTFields decrease MHC class I expression in various types of cancer.

[095] Although there is no intention to limit ourselves to any one theory, it has been previously demonstrated that TTFields induce ER stress (Silginer et al. (2017) Cell Death Dis, 8:e2753). Furthermore, it has also been previously demonstrated that ER stress negatively regulates HLA class I (Ulianich et al. (2011) Biochemica et Biophysica Acta, 1812:431-438). Therefore, the induction of ER stress by exposure to TTFields may be a possible mechanism for the negative regulation of HLA class I after exposure to TTFields. Example 2

[096] This Example is directed to the use of TTFields as immunotherapy to activate adoptively transferred NK cells to enhance the efficacy of NK cell-based cancer therapies. Concomitant therapy with TTFields and adoptively transferred NK cells provides an enhanced and synergistic effect compared to either treatment alone.

[097] Human subjects are treated with TTFields by applying an OPTUNE® device (Novocure Limited, St. Helier, Jersey) to their skin, with the array pair positioned to the left and right (LR) of the tumor and / or anterior and posterior (AP) to the tumor. Each subject is then treated chronically with TTFields at 150-200 kHz; the device is used at least 80% of the time, with short intervals between sessions and minor adjustments to array positioning to allow cells and skin to recover.

[098] A composition containing NK cells is prepared. In one case, autologous NK cells are obtained from the individual prior to exposure to TTFields. Alternatively, NK cells are obtained from an allogeneic source. In another example, pluripotent stem cells are obtained from an autologous or allogeneic source and differentiated into NK cells ex vivo. The autologous or allogeneic NK cells are then cultured. Petition 870250088297, dated 09 / 29 / 2025, page 44 / 66 37 / 47 before implantation / injection for adoptive cell transfer therapy.

[099] Two weeks after the start of TTFields application, approximately 1 to 10 billion NK cells are implanted or injected into individuals about 1 to 3 times per week for 6 to 8 weeks.

[0100] After concomitant therapeutic treatment, the effects of said concomitant therapy on the tumor(s) are evaluated. Example 3

[0101] This example studies the role of TTFields in potentiating the in vitro scavenging activity of CIK cells against patient-derived GBM cancer stem cells (GSCs). This example provides proof of principle that TTFields can potentiate “HLA-independent immunity” against cancer cells and also strengthens the basis for further clinical studies revisiting the potential of CIK therapy in combination with TTFields to treat GBM.

[0102] Cytokine-induced killer (CIK) therapy is a type of adoptive immunotherapy that uses a heterogeneous population of immune cells generated ex vivo from the patient's peripheral blood mononuclear cells (PBMCs) in the presence of IL-2, anti-CD3 antibody(ies) (such as, but not limited to, OKT3), and IFN-gamma (Introna (2017) Journal of Autoimmunity, 85:32-44). CIK cells include a CD3+CD56+CD8+ population, which are phenotypically cytotoxic T-terminal differentiated effector memory lymphocytes (EMRAs) (Franceschetti et al. (2009) Experimental Hematology, 37:616-628.e2) with antitumor activity. This population expresses different NK markers, including NKG2D, and is able to recognize and kill tumor cells in a TCR-independent manner, similar to NK cells (Sangiolo et al. (2008) International Immunology, 20:841-848).According to a recent report from the International CIK Cell Registry (IRCC), CIK cells have been tested in 106 clinical trials across more than 30 different tumors, with a significant improvement in progression-free survival (PFS) and overall survival. Petition 870250088297, dated 09 / 29 / 2025, page 45 / 66 38 / 47 (SG) in 27 trials in the autologous setting, 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 with negative clinical outcomes in GBM, including immune checkpoint inhibitors, vaccinations, and various adoptive T-cell therapies (Weenink et al. (2020) Cancers (Basel), 12:E751).

[0103] TTFields have been shown to prolong the survival of patients with newly diagnosed and recurrent GBM, leading to FDA clinical approval of this therapy (Rominiyi et al. (2020) Br J Cancer, 124(4):697-709). While we do not wish to limit ourselves to any one theory, the main biological mechanism attributed to TTFields in cancer therapy is their ability to interfere with mitotic spindle formation during mitotic apparatus (Kirson et al. (2004) Cancer Res, 1;64(9):328-895). However, emerging evidence indicates that TTFields are also capable of activating 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, in a syngeneic murine GBM model, that TTFields, through activation of cGAS / STING and AIM2 inflammasome genotoxic stress in GBM cells, are capable of stimulating the production of pro-inflammatory cytokines and type 1 interferon. This pro-inflammatory shift is capable of producing a robust activation of adaptive immunity, showing an increase in clonal expansion of memory phenotype T cells and a reduction in exhausted T cells. Although single-cell RNA sequencing of peripheral blood mononuclear cells obtained from GBM patients treated with TTFields showed clues to activation in NK cells as well, the role of HLA-independent immunity was not further explored in Chen et al.

[0104] In the present example, 20 CIK lots were successfully expanded. Petition 870250088297, dated 09 / 29 / 2025, pp. 46 / 66 39 / 47 from PBMCs. PBMCs were obtained from healthy donors (HD) and GBM patients prior to chemoradiotherapy treatment enrolled at San Raffaele Hospital, following the protocol described in the Methods section below. Figure 3 summarizes the growth curve of the 5 batches obtained from 5 HDs, which showed a median expansion fold of 42.03 (IQR: 31.15 - 74.13). On day 21, CD3+CD56+ cells represented 38.96% of the cells, and this population was predominantly CD8+CD4-. CD3+CD56-, which represented 48.83% of the cells, resulted mainly as CD8+CD4- cells (mean = 61.43%), but showed a more significant proportion of CD4+CD8- cells (mean = 28.94%).

[0105] CIK and GSC Co-culture: To evaluate the cytotoxic activity of CIK cells, their ability to induce apoptosis in patient-derived GSCs was tested. For this, GSCs were labeled with the CellTrace violet cell proliferation kit and then co-cultured with CIK cells with different target-effector ratios (T / E ratio). As illustrated in Figure 4, a dose-dependent cytotoxic effect induced by CIK was observed compared to new PBMCs.

[0106] Research Design and Methods

[0107] CIK Cells

[0108] Blood samples were collected in EDTA from healthy volunteers. PBMCs were separated from whole blood by density centrifugation and cultured for 21 days at 37 °C and 5% CO2 according to the reference protocol (Franceschetti et al., 2009). On day 0, cells were supplemented with 1,000 U / ml of IFN-γ, followed by the addition of 50 ng / ml of anti-CD3 antibody and 500 U / ml of IL-2 on day 1. Every 3-4 days, they were supplemented with fresh medium and 500 U / ml of IL-2. The phenotype of PBMCs and CIK cells was tested on days 12 and 21 and characterized by flow cytometry detecting cell surface markers CD3, CD56, CD4, CD8, CD14, and CD19, along with a cell viability dye. To explore the effect of TTFields, CIK cells were cultured for 48 hours in the presence of Petition 870250088297, dated 09 / 29 / 2025, p. 47 / 66 40 / 47 TTFields. The viability of CIK was tested by staining Live / Dead Aqua cells, and the activation state of CIK cells was assessed by testing the IFN-gamma concentration in the cell-free supernatant using a Sandwich ELISA assay.

[0109] GSC

[0110] Patient-derived CSC cell lines were isolated from new tumors using the NeuroSphere assay and established as long-term turnover cell lines (Galli et al., (2004) Cancer Research, 64:7011-7021) at the Neural Stem Cell Biology Unit led by Dr. Galli. As a first experiment, the known effect of TTFields on GBM is tested by evaluating GSC viability after 48 hours of exposure to TTFields. Additionally, the ability of TTFields to alter GSC immunogenicity is evaluated by examining the expression of relevant surface molecules such as MHC class I and NKG2D MICA / B ligand.

[0111] CIK and GSC co-culture platform

[0112] 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 in various target-effector ratios in 6-well flat-bottom plates. After 48 hours of co-culture, the apoptotic cell load among CellTrace-positive cells identified by Live / Dead Aqua or 7-amino-actinomycin D (7AAD) cell staining was used as a primary measure of CIK destruction activity. CIK degranulation is used as a secondary outcome to assess CIK cell activation. To achieve this, CIK cells labeled with the CellTrace Cell Proliferation Kit are co-incubated with GSCs in a 2:1 effector-tumor ratio in the presence of CD107a (Aktas et al. (2009) Cell Immunol, 254(2):14954). After 1 hour, BrefeldinA and GolgiStop are added to each well.CIK degranulation is assessed using flow cytometry and presented as a percentage. Petition 870250088297, dated 09 / 29 / 2025, pp. 48 / 66 41 / 47 of CD107a+ cells among cells labeled with CellTrace. Furthermore, the impact of TTFields as a pretreatment for GSCs and as a co-culture condition is tested to determine their ability to improve the described results. Example 4

[0113] This example is directed at the use of TTFields as immunotherapy to activate cytokine-induced killer (CIK) cells to increase the efficacy of CIK-based cancer therapies. Concomitant TTFields therapy with CIK cells provides a synergistic effect compared to either treatment alone.

[0114] Human subjects are treated with TTFields by applying an OPTUNE® device (Novocure Limited, St. Helier, Jersey) to their skin, with the array pair positioned to the left and right (LR) of the tumor and / or anterior and posterior (AP) to the tumor. Each subject is then chronically treated with TTFields at 150-200 kHz; the device is used at least 80% of the time, with short intervals between sessions and minor adjustments to array positioning to allow cells and skin to recover.

[0115] A composition containing CIK cells is prepared. In one example, CIK cells are prepared as described in Example 3 using PBMCs obtained from autologous and / or allogeneic source(s). The CIK cells are then cultured prior to implantation / injection for adoptive cell transfer therapy.

[0116] Two weeks after the start of TTFields application, approximately 1 to 10 billion CIK cells are implanted or injected into individuals about 1 to 3 times per week for 6 to 8 weeks.

[0117] After concomitant therapeutic treatment, the effects of said concomitant therapy on the tumor(s) are evaluated. Illustrative, but not limiting, examples of the inventive concept(s) Petition 870250088297, dated 09 / 29 / 2025, page 49 / 66 42 / 47

[0118] Illustrative embodiment 1. A method for reducing the viability of cancer cells, the method comprising the steps of: (1) applying an alternating electric field to the cancer cells for a 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 can be performed in vitro or in vivo.

[0119] Illustrative embodiment 2. A method of treating cancer in an individual, the method comprising the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one killer cell.

[0120] Illustrative embodiment 3. A method for reducing the volume of a tumor and / or preventing an increase in tumor volume, wherein the tumor is present in the body of a living individual and includes a plurality of cancerous cells, the method comprising the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one killer cell.

[0121] Illustrative embodiment 4. Method, comprising the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one killer cell; and wherein the administration of the alternating electric field increases the efficacy and / or cytotoxicity of the killer cells against cancer cells in the individual when compared to the administration of killer cells to the individual in the absence of application of an alternating electric field.

[0122] Illustrative modality 4A. Method of any of the modalities Petition 870250088297, dated 09 / 29 / 2025, pp. 50 / 66 43 / 47 illustrative figures 1-4, wherein at least one killer cell comprises at least one natural killer (NK) cell.

[0123] Illustrative embodiment 4B. Method of any of the illustrative embodiments 1-4A, wherein at least one killer cell comprises at least one cytokine-induced killer cell (CIK).

[0124] Illustrative embodiment 4C. Method of any of the illustrative embodiments 1-4B, wherein at least one killer cell comprises at least one NK cell and at least one CIK cell.

[0125] Illustrative embodiment 5. Method of any of the forms of illustrative embodiments 1 to 4, in which at least one of the following occurs: the alternating electric field is applied at a frequency in a range of about 50 kHz to about 1 MHz; the alternating electric field has a field strength of at least about 1 V / cm in at least a portion of the cancerous cells / target region of the individual; the alternating electric field is induced by an applied voltage of at least 50 V RMS or at least 50 V p2p; and the time period during which the alternating electric field is applied is at least about 50% of a period of at least about 24 consecutive hours.

[0126] Illustrative embodiment 6. Method of any of the illustrative embodiments 1-5, in which the alternating electric field is applied to the cancer cells / target region of the individual for a period of time sufficient to reduce the expression of MHC Class I in the cancer cells compared with control cancer cells that were not exposed to alternating electric fields.

[0127] Illustrative embodiment 7. Method of any of the illustrative embodiments 1-6, wherein at least one killer cell comprises at least one autologous killer cell.

[0128] Illustrative embodiment 8. Method of any of the illustrative embodiments 1-7, in which at least one killer cell comprises Petition 870250088297, dated 09 / 29 / 2025, pp. 51 / 66 44 / 47 minus one allogeneic killer cell.

[0129] Illustrative embodiment 9. Method of any of the illustrative embodiments 1-8, in which at least one of the following occurs: a) at least one killer cell is derived from ex vivo differentiation of pluripotent stem cells; b) at least one killer cell is derived from an interleukin-2 (IL-2)-dependent cell line; and / or c) at least one killer cell comprises a chimeric antigen receptor-engineered killer cell.

[0130] Illustrative embodiment 9A. Method of any of the illustrative embodiments 1-8, in which at least one of the following occurs: a) at least one NK cell is derived from ex vivo differentiation of pluripotent stem cells; b) at least one NK cell is derived from an interleukin-2 (IL-2)-dependent cell lineage; and / or c) at least one killer cell comprises a chimeric antigen receptor-designed killer cell (CAR-NK).

[0131] Illustrative embodiment 9B. Method of any of the illustrative embodiments 1-9A, wherein at least one killer cell is at least one CIK cell derived from peripheral blood mononuclear cells (PBMCs) cultured in the presence of at least one of interleukin-2 (IL-2), anti-CD3 antibody and interferon-gamma (IFN-γ).

[0132] Illustrative embodiment 10. Method of any of the illustrative embodiments 1-9, wherein the composition also comprises at least one substance selected from the group consisting of an immune checkpoint inhibitor, interleukin-1 (IL-1), IL-1R, IL-2, IL-11, IL-15, IL-17, IL-18, IL-21, lirilumab, monalizumab and combinations thereof.

[0133] Illustrative embodiment 11. Method of any of the illustrative embodiments 1-10, wherein the composition also comprises at least one substance selected from the group consisting of an anti-PD-1 therapeutic agent, an anti-PD-L1 therapeutic agent, a chemotherapeutic agent, Paclitaxel, Docetaxel, Petition 870250088297, dated 09 / 29 / 2025, pp. 52 / 66 45 / 47 Ifosamide, Etoposide, Gemcitabine, Lomustine, Paclitaxel nab, Temozolomide, Carboplatin, a TKI inhibitor, an mTOR inhibitor, an Akt inhibitor, a PI3K inhibitor, a PARP inhibitor, an FGF inhibitor, an anti-LAB3 agent, an anti-CTLA-4 therapeutic agent, an aromatase inhibitor, Denosumab, Pembrolizumab, and combinations thereof.

[0134] Illustrative embodiment 12. Method of any of the illustrative embodiments 1-11, wherein the method additionally comprises the step of: (3) administering at least one additional composition to the cancer cells / individual, wherein the at least one additional composition comprises a substance selected from the group consisting of an immune checkpoint inhibitor, interleukin-1 (IL-1), IL-1R, IL-2, IL-11, IL-15, IL-17, IL-18, IL-21, lirilumab, monalizumab and combinations thereof.

[0135] Illustrative embodiment 13. Method of any of the illustrative embodiments 1-12, wherein the method further comprises the step of: (3) administering at least one additional composition to the cancer cells / individual, wherein the at least one additional composition comprises a substance selected from the group consisting of an anti-PD-1 therapeutic agent, an anti-PD-L1 therapeutic agent, a chemotherapeutic agent, Paclitaxel, Docetaxel, Ifosamide, Etoposide, Gemcitabine, Lomustine, Paclitaxel nab, temozolomide, Carboplatin, a TKI inhibitor, an mTOR inhibitor, an Akt inhibitor, a PI3K inhibitor, a PARP inhibitor, an FGF inhibitor, an anti-LAB3 agent, an anti-CTLA-4 therapeutic agent, an aromatase inhibitor, Denosumab, pembrolizumab and combinations thereof.

[0136] Illustrative embodiment 14. Method of any of the illustrative embodiments 1-13, in which steps (1) and (2) are performed sequentially in whole or in part, and in which at least one composition is administered after the application of the alternating electric field has begun. Petition 870250088297, dated 09 / 29 / 2025, pp. 53 / 66 46 / 47

[0137] Illustrative embodiment 15. Method of illustrative embodiment 14, in which at least one composition is administered before the entire time period of application of the alternating electric field has elapsed.

[0138] Illustrative modality 16. Method of illustrative modalities 14 or 15, in which at least one composition is administered after the time period has elapsed.

[0139] Illustrative Modality 17. Method of any of the illustrative modalities 1-16, in which steps (1) and (2) are repeated one or more times.

[0140] Illustrative embodiment 18. Method of any of the illustrative embodiments 1-17, in which the cancerous cells / the cancer / the tumor is in the form of at least one solid tumor.

[0141] Illustrative embodiment 19. Method of any of the illustrative embodiments 1-18, wherein the cancer / cancer cells are selected from the group consisting of hepatocellular carcinoma cells / carcinoma, glioblastoma cells / glioblastoma, pleural mesothelioma cells / mesothelioma, differentiated thyroid cancer cells / cancer, advanced renal cell carcinoma cells / carcinoma, ovarian cancer cells / cancer, pancreatic cancer cells / cancer, lung cancer cells / cancer, breast cancer cells / cancer and combinations thereof.

[0142] Illustrative embodiment 20. A kit for reducing the viability of cancer cells, the kit comprising: components configured to isolate and cultivate at least one natural killer cell, or a precursor thereof, from an individual; and, optionally, a field generator device configured to apply an alternating electric field to the cancer cells for a period of time.

[0143] Although the attached disclosures describe the inventive concept(s) in conjunction with the specific experimentation, results, and language presented below, it is evident that many alternatives, modifications, and variations will be Petition 870250088297, dated 09 / 29 / 2025, pp. 54 / 66 47 / 47 evident to those versed in the technique. Consequently, it is intended to encompass all those alternatives, modifications, and variations that fit within the spirit and broad scope of the present revelation. Petition 870250088297, dated 09 / 29 / 2025, pages 55 / 66

Claims

1 / 5 CLAIMS 1. A method for treating cancer in an individual, CHARACTERIZED in that it comprises the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one natural killer (NK) cell.

2. Method according to claim 1, CHARACTERIZED in that at least one of the following occurs: 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 a field strength of at least about 1 V / cm in at least part of the target region of the individual; the alternating electric field is induced by an applied voltage of at least 50 V p2p; and the time period during which the alternating electric field is applied is at least about 50% of a period of at least about 24 consecutive hours.

3. Method, according to claim 1, CHARACTERIZED in that an alternating electric field is applied to the target region of the individual for a period of time sufficient to reduce the expression of MHC Class I in cancer cells in the target region compared with control cancer cells that were not exposed to alternating electric fields.

4. Method according to claim 1, CHARACTERIZED in that at least one NK cell comprises at least one autologous NK cell.

5. Method according to claim 1, CHARACTERIZED in that at least one NK cell comprises at least one allogeneic NK cell.

6. Method, according to claim 1, CHARACTERIZED by the fact that at least one of the following occurs: at least one NK cell is derived from ex vivo differentiation of pluripotent stem cells; at least one NK cell is derived from an interleukin-2 (IL-2)-dependent cell lineage; and / or at least one NK cell comprises a chimeric antigen receptor-designed NK cell (CAR-NK).

7. Method according to claim 1, CHARACTERIZED in that steps (1) and (2) are performed sequentially in whole or in part, and in which at least one composition is administered after the start of the application of the alternating electric field.

8. Method according to claim 1, CHARACTERIZED in that steps (1) and (2) are repeated one or more times.

9. Method, according to claim 1, CHARACTERIZED in that 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, breast cancer and combinations thereof.

10. Method for reducing the volume of a tumor and / or preventing an increase in tumor volume, CHARACTERIZED in that the tumor is present in the body of a living individual and includes a plurality of cancerous cells, wherein the method comprises the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one natural killer (NK) cell.

11. Method according to claim 10, CHARACTERIZED by the fact that at least one of the following occurs: 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 a field strength of at least about 1 V / cm in at least part of the target region of the individual; the alternating electric field is induced by an applied voltage of at least 50 V p2p; and the time period during which the alternating electric field is applied is at least about 50% of a period of at least about 24 consecutive hours.

12. Method, according to claim 10, CHARACTERIZED in that an alternating electric field is applied to the target region of the individual for a period of time sufficient to reduce the expression of MHC Class I in cancer cells when compared to control cancer cells that were not exposed to alternating electric fields.

13. Method according to claim 10, CHARACTERIZED in that at least one NK cell comprises at least one autologous NK cell.

14. Method according to claim 10, CHARACTERIZED in that at least one NK cell comprises at least one allogeneic NK cell.

15. Method according to claim 10, CHARACTERIZED in that at least one of the following occurs: a) at least one NK cell is derived from ex vivo differentiation of pluripotent stem cells; b) at least one NK cell is derived from an interleukin-2 (IL-2)-dependent cell lineage; and / or c) at least one NK cell comprises a chimeric antigen receptor-engineered NK cell (CAR-NK). Petition 870250088297, dated 09 / 29 / 2025, pp. 58 / 66 4 / 5 16. Method according to claim 10, CHARACTERIZED in that steps (1) and (2) are performed sequentially in whole or in part, and in which at least one composition is administered after the start of the application of the alternating electric field.

17. Method according to claim 10, CHARACTERIZED in that steps (1) and (2) are repeated one or more times.

18. Method, according to claim 10, CHARACTERIZED in that 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, breast cancer and combinations thereof.

19. Method CHARACTERIZED by the fact that it comprises the steps of: (1) applying an alternating electric field to a target region of the individual for a period of time; and (2) administering at least one composition to the individual, wherein the at least one composition comprises at least one natural killer (NK) cell; and wherein the administration of the alternating electric field increases the cytotoxicity of the NK cells against cancerous cells in the individual when compared to the administration of NK cells to the individual in the absence of application of an alternating electric field.

20. Method according to claim 19, CHARACTERIZED in that at least one of the following occurs: 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 a field strength of at least about 1 V / cm in at least part of the target region of the individual; the alternating electric field is induced by an applied voltage of at least 50 V p2p; and the time period during which the alternating electric field is applied is at least about 50% of a period of at least about 24 consecutive hours.

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