Compositions, systems and methods for treating cancer using alternating electric field and apoptotic cancer cell vaccination

Abstract

Immunogenic compositions and systems, as well as methods for producing the immunogenic compositions, are disclosed. Also disclosed are compositions, systems, and methods of reducing cancer cell activity and treating cancer, as well as methods of reducing tumor volume and / or preventing an increase in tumor volume present in a living subject. The systems and methods involve applying an alternating electric field to cancer cells (before or after separation from a target portion of a subject), and irradiating the separated and treated cancer cells. The method may also include administering the irradiated and treated cancer cells back to the subject from which the cancer cells are separated, and / or applying an alternating electric field to the subject. The immunogenic composition includes a population of isolated and apoptotic cancer cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Incorporation by Reference Statement

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 377,951, filed on September 30, 2022, and U.S. Provisional Application No. 63 / 496,831, filed on April 18, 2023, under 35 U.S.C. § 119(e). The entire contents of the above patent applications are expressly incorporated herein by reference. Background Art

[0004] Tumor Treatment Fields (TTFields) are low intensity (e.g., 1-3 V / cm) alternating electric fields in the mid-frequency range (such as, but not limited to, 100-500 kHz) that target solid tumors by disrupting mitosis. This non-invasive treatment targets solid tumors and is described, for example, in U.S. Pat. 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 (or similar alternating electric fields used to treat other conditions) are typically delivered through two pairs of transducer arrays that generate perpendicular fields within the tumor being treated; the electrode arrays that make up each of these pairs are positioned on opposite sides of the body part being treated. More specifically, for In the TTField system, one pair of electrodes is located at the left and right sides (LR) of the tumor, and the other pair of electrodes is located at the anterior and posterior sides (AP) of the tumor. TTField is approved for the treatment of glioblastoma multiforme (GBM) and can be used, for example, by system (Novocure Limited, St. Helier, Jersey), which The system includes a transducer array placed on a light head over the patient.

[0005] Used in Each transducer array in the device that delivers TTFields includes a set of ceramic disc electrodes that are coupled to the patient's skin (such as, but not limited to, the shaved head of a patient being treated for GBM) via a layer of conductive medical gel. The medical gel is intended to deform to match the contours of the body and provide good electrical contact between the array and the skin; thus, the gel interface bridges the skin and reduces interference. The device is intended to be worn continuously by the patient for 2 to 4 days, then removed, allowing the patient to perform hygiene care and re-shave the head (if necessary), followed by reapplication of a new set of arrays. In this way, the medical gel remains in essentially continuous contact with the subject's skin area for 2-4 days at a time, and the skin area is only uncovered and exposed to the environment for a brief period of time before more medical gel is applied to the skin area.

[0006] Another form of cancer treatment involves cancer immunotherapy. The main goal of cancer immunotherapy is to activate pre-existing endogenous immune responses in cancer patients. Some possible targets for cancer immunotherapy include mutation-derived tumor-specific antigens, or neoantigens, which are not present in normal cells and can be recognized by the immune system, thus providing specific targets for anti-tumor therapy. Although significant progress has been made in this field, the effectiveness of treatments still needs to be improved. DETAILED DESCRIPTION

[0007] Before explaining at least one embodiment of the inventive concept in detail through exemplary language and results, it should be understood that the application of the inventive concept is not limited to the details of the construction and arrangement of the components set forth in the following description. The inventive concept can have other embodiments or be practiced or executed in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning; and these embodiments are intended to be exemplary rather than exhaustive. In addition, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0008] Unless otherwise defined herein, scientific and technical terms used in conjunction with the inventive concepts disclosed herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification. Terms and laboratory procedures and techniques associated with analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry as described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0009] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the technical level of those skilled in the art to which the inventive concepts disclosed in the present invention pertain. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, as if each individual patent or publication was specifically and individually indicated to be incorporated herein by reference.

[0010] All compositions, components, systems, kits and / or methods disclosed herein can be prepared and performed according to the present disclosure without excessive experimentation. Although the compositions, components, systems, kits and methods of the present invention have been described according to specific embodiments, it will be understood by those skilled in the art that the steps or step sequences of the compositions and / or methods and methods described herein can be changed without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0011] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0012] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a" or "an" may mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Thus, unless the context clearly indicates otherwise, the terms "a", "an", and "the" include plural referents. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or an increased number of compounds. The term "plurality" means "two or more".

[0013] The use of the term "at least one" should be understood to include one as well as any amount more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; 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 to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used solely for the purpose of distinguishing two or more items and is not meant to imply, for example, any order or sequence or importance of one item relative to another or any order of addition.

[0014] The term "or" as used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" satisfies any of the following conditions: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0015] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. For example, the appearances of the phrase "in some embodiments" or "an example" in different places in the specification are not necessarily all referring to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0016] In this application, the term "about" is used to indicate that a value includes the inherent error variation of the components / devices / equipment, the method used to determine the value, or the variation that exists in the subject matter. For example, but not as a limitation, when the term "about" is used, the specified value can vary within plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent of the specified value, as such variations are suitable for performing the disclosed methods and as understood by those of ordinary skill in the art.

[0017] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of containing, such as "includes" and "include"), or "containing" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0018] As used herein. The term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will appreciate that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0019] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a large extent. For example, when related to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance 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" can mean that two items are 100% adjacent to each other, or that the two items are very close to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0020] 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), which are commensurate with a reasonable benefit / risk ratio.

[0021] As used herein, the term "patient" or "subject" includes both human and veterinary subjects. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including but not limited to humans, domestic and farm animals, non-human primates, and any other animal with mammary tissue.

[0022] The term "treatment" refers to therapeutic treatment as well as prophylactic or preventive measures. Those in need of treatment include, but are not limited to, individuals who already have a particular condition / disease / infection and individuals at risk of acquiring a particular condition / disease / infection (e.g., individuals in need of prophylactic / preventive measures). The term "treatment" refers to the administration of an agent / element / method to a patient for therapeutic and / or prophylactic / preventive purposes.

[0023] As used herein, the term "therapeutic composition" or "pharmaceutical composition" refers to an agent that can be administered in vivo to produce a therapeutic and / or prophylactic / preventive effect.

[0024] Administration of a therapeutically effective amount or a prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, disorder, and / or infection. The specific amount that is therapeutically effective can be readily determined by an ordinary physician and can vary according to factors known in the art, such as, but not limited to, the type of disorder / disease / infection, the patient's medical history and age, the stage of the disorder / disease / infection, and the co-administration of other agents.

[0025] The term "effective amount" refers to the amount of a biologically active molecule or its conjugate or derivative, or the amount of a therapeutic regimen (e.g., an alternating electric field), which is sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (e.g., but not limited to, toxicity, irritation, and allergic reactions), commensurate with a reasonable benefit / risk ratio when used in the manner contemplated by the present invention. The therapeutic effect may include, for example, but not limited to, preventing, inhibiting, or reducing the occurrence of at least one condition, disease, and / or infection. The effective amount for a subject will depend on the type of subject, the size and health of the subject, the nature and severity of the condition / disease / infection to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulation used, and the like. Therefore, it is impossible to predetermine an exact effective amount. However, the effective amount for a given situation can be determined by a person of ordinary skill in the art using routine experiments based on the information provided herein.

[0026] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjuvant therapy" and will be understood to mean that a patient in need of treatment is treated with or administered another drug for the condition / disease / infection in combination with the treatment of the present disclosure. Such concurrent therapy may be sequential therapy, wherein the patient is first treated with one therapeutic regimen / drug composition and then treated with another therapeutic regimen / drug composition, or both therapeutic regimens / drug compositions are administered simultaneously.

[0027] The terms "administration" and "administering" as used herein are 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 include local and systemic administration. In addition, the compositions of the present disclosure (and / or methods of administration thereof) can be designed to provide delayed, controlled or sustained release using formulation techniques well known in the art.

[0028] Turning now to the inventive concepts, disclosed herein is a combination therapy for cancer. The combination therapy combines (i) generating alternating electric field-treated apoptotic cancer cells (such as but not limited to TTField-treated apoptotic cancer cells) by applying an alternating electric field (such as but not limited to TTField) thereto; (ii) irradiating the TTField-treated apoptotic cells; (iii) administering the alternating electric field-treated apoptotic cancer cells to a subject; and (iv) applying an alternating electric field (such as but not limited to TTField) to a target area of ​​the subject. Alternating electric field therapy combined with apoptotic cancer cell vaccination produces a synergistic effect in treating cancer.

[0029] Certain non-limiting embodiments of the present disclosure relate to methods for treating cancer in a subject and / or methods for reducing the volume of a tumor present in a subject and / or preventing the tumor volume from increasing. The method comprises the following steps: (1) isolating cancer cells from a target area of ​​the subject; (2) applying an alternating electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells to which the alternating electric field has been applied; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying an alternating electric field to the target area of ​​the subject.

[0030] Certain non-limiting embodiments of the present disclosure relate to other similar methods of treating cancer in a subject (and / or methods of reducing the volume of a tumor present in a subject and / or preventing the volume of the tumor from increasing), except that an alternating electric field (such as but not limited to inducing an immunogenic response) is applied to the target area of ​​the subject before the cancer cells are separated from the target area of ​​the subject. In other words, the method of the present disclosure includes applying an alternating electric field to the cancer cells in vivo and in vitro, then irradiating the cancer cells treated with the alternating electric field, and administering the cells to the subject.

[0031] The methods of the present disclosure can be used to treat any type of cancer cell / cancer / tumor that responds to alternating electric field / TTField and / or apoptotic cancer cell therapy. Non-limiting examples of cancer cells / cancers / tumors that can be treated according to the present disclosure include hepatocellular carcinoma, glioblastoma, pleural mesothelioma, differentiated thyroid cancer, advanced renal cell carcinoma, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, lung cancer (such as but not limited to non-small cell lung cancer), etc. and any combination thereof.

[0032] According to the methods of the present disclosure, the alternating electric field may be generated using any type of conductive or non-conductive electrodes and / or transducer arrays known in the art or otherwise contemplated herein that can be used to generate an alternating electric field. In accordance with the present disclosure, non-limiting examples of electrodes and transducer arrays that can be used to generate alternating electric fields include those electrodes and transducer arrays that are part of an alternating electric field generating system (e.g., a TTField system), such as, but not limited to, those described below: U.S. Patent Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776; and U.S. Patent Application Nos. US2018 / 0160933, US2019 / 0117956, US2019 / 0307781, and US2019 / 0308016.

[0033] According to the present disclosure, the alternating electric field can be generated at any frequency. For example (but not by way of limitation), the alternating electric field can have a frequency of about 50kHz, about 75kHz, about 100kHz, about 125kHz, about 150kHz, about 175kHz, about 200kHz, about 225kHz, about 250kHz, about 275kHz, about 300kHz, about 325kHz, about 350kHz, about 375kHz, about 400kHz, about 425kHz, about 450kHz, about 475kHz, about 500kHz, about 550kHz, about 600kHz, about 650kHz, about 700kHz, about 750kHz, about 800kHz, about 850kHz, about 900kHz , frequencies of about 950 kHz, about 1 MHz, about 2 MHz, about 3 MHz, about 4 MHz, about 5 MHz, about 6 MHz, about 7 MHz, about 8 MHz, about 9 MHz, about 10 MHz, etc., and ranges 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 100 kHz to about 500 kHz, a range from about 150 kHz to about 300 kHz, etc.), as well as ranges combining two integers that fall between two of the above values ​​(for example, a range from about 122 kHz to about 313 kHz, a range from about 78 kHz to about 298 kHz, etc.).

[0034] In certain specific (but non-limiting) embodiments, an alternating electric field may be applied at two or more different frequencies. When there are two or more frequencies, each frequency is selected from any of the above values, or a range formed by any of the above values, or a range of two integers falling between two of the above values.

[0035] The alternating electric field can have any field strength in the subject / cancer cell as long as the alternating electric field is able to function according to the present disclosure. For example, but not by way of limitation, the alternating electric field may have a value 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 12.5 V / cm, about 13 V / cm, about 13.5 V / cm, about 14 V / cm, about 14.5 V / cm, about 15 V / cm, about The present invention relates to a field strength of about 15.5 V / cm, about 16 V / cm, about 16.5 V / cm, about 17 V / cm, about 17.5 V / cm, about 18 V / cm, about 18.5 V / cm, about 19 V / cm, about 19.5 V / cm, about 20 V / cm, etc., as well as a range formed by any of the above values ​​(for example, a range of about 1 V / cm to about 20 V / cm, a range of about 1 V / cm to about 10 V / cm, a range of about 1 V / cm to about 4 V / cm, etc.), and a range combining two integers falling between two of the above values ​​(for example, a range of about 1.1 V / cm to about 18.6 V / cm, a range of about 1.2 V / cm to about 9.8 V / cm, a range of about 1.3 V / cm to about 4.7 V / cm, etc.).

[0036] According to the present disclosure, the alternating electric field can be applied for any period of time long enough to achieve a desired result (such as, but not limited to, generating an immune response, changing cancer cells to induce an immunogenic response, reducing the activity of cancer cells, and / or reducing tumor volume (and / or preventing tumor volume from increasing, etc.). For example, but not by way of limitation, the alternating electric field can be applied for at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours. , about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, 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 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, etc., and ranges formed by any of the above values ​​(e.g., a range from about 1 minute to about 12 hours, a range from about 1 minute to about 1 hour, a range from about 1 hour to about 7 days, a range from about 24 hours to about 72 hours, etc.), as well as ranges combining two integers falling between two of the above values ​​(e.g., a range from about 14 hours to about 68 hours, etc.).

[0037] In a specific (but non-limiting) embodiment, the alternating electric field is applied in vivo at a frequency in the range of about 50 kHz to about 1 MHz, a field strength of at least about 1 V / cm (e.g., in the range of about 1 V / cm to about 10 V / cm), and a time period of at least about 24 hours.

[0038] In a specific (but non-limiting) embodiment, the alternating electric field is applied in vitro at a frequency in the range of about 50 kHz to about 1 MHz, a field strength of at least about 1 V / cm (e.g., in the range of about 1 V / cm to about 10 V / cm), and a time period in the range of at least about 1 hour to about 7 days.

[0039] According to the present disclosure, any method of irradiating cancer cells known in the art or otherwise contemplated herein can be used, as long as the cancer cells are irradiated with sufficient irradiation intensity and sufficient time to render the cancer cells inactive / unable to proliferate. For example, but not limited to, the cancer cells can be irradiated with an intensity of at least about 1 Gray (Gy). The ability to select sufficient irradiation intensity and sufficient time period is well within the capabilities of those of ordinary skill in the art, and therefore it is deemed unnecessary to describe it further.

[0040] The cancer cells treated with the alternating electric field are irradiated for any sufficient time so that the cancer cells reach apoptotic state and substantially lose activity. For example, but not limited to, the cells can be irradiated for at least about 30 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 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, etc., and ranges formed by any of the above values ​​(e.g., a range of about 30 minutes to about 24 hours, etc.), and ranges combining two integers falling between two of the above values ​​(e.g., a range of about 45 minutes to about 20.5 hours, etc.).

[0041] In a specific (but non-limiting) embodiment, the time period for irradiating the alternating electric field treated cancer cells is in the range of about 24 hours to about 96 hours.

[0042] In certain specific (but non-limiting) embodiments, non-viable, irradiated cancer cells are separated prior to administration to a subject (i.e., between steps (3) and (4) of the above method). This optional separation step can ensure that the composition administered to the subject does not contain any cancer cells that may not have received sufficient irradiation and are still viable. For example (but not by way of limitation), the cells can be subjected to one or more sorting steps to separate non-viable cells and ensure that there are no viable cells among the sorted cells.

[0043] Alternatively, the irradiation conditions can be sufficiently stringent (ie, by prolonged time and / or high intensity) to ensure that no viable cells remain in the irradiated sample. In this case, the non-viable, irradiated cancer cells can be directly administered to the subject.

[0044] The irradiated cancer cells treated with an alternating electric field may be treated and administered in any formulation known in the art or otherwise contemplated herein such that the apoptotic cancer cells have a deleterious effect on existing cancer in the subject. For example, but not by way of limitation, the irradiated cancer cells treated with an alternating electric field may be administered in the form of a pharmaceutical composition comprising cells in combination with at least one pharmaceutically acceptable carrier. Non-limiting examples of suitable pharmaceutically acceptable carriers that may be used in accordance with the present disclosure include water; saline; glucose solution; fructose or mannitol; calcium carbonate; cellulose; ethanol; oils of animal, plant or synthetic origin; carbohydrates such as glucose, sucrose or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposomal carriers; buffer solutions such as sodium chloride, saline, phosphate buffered saline and / or other substances that are physiologically acceptable and / or safe to use; diluents; excipients such as polyethylene glycol (PEG); or any combination thereof. Suitable pharmaceutically acceptable carriers for use in pharmaceutical formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd Edition (2020).

[0045] In certain non-limiting embodiments, the pharmaceutical composition containing irradiated cancer cells treated with an alternating electric field can be further formulated into an immunogenic composition. The immunogenic composition can contain the same components as the above-mentioned pharmaceutical composition (i.e., apoptotic cancer cells plus a pharmaceutically acceptable carrier). In certain specific (but non-limiting) embodiments, the immunogenic composition can also include at least one additional agent. Non-limiting examples of agents that can be included as part of the immunogenic composition include adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG3 agents (such as but not limited to OPDUALAG), TM and / or relalizumab (Bristol-Myers Squibb, New York, NY)), other active agents, etc., and any combination thereof.

[0046] In addition, any composition containing apoptotic cancer cells disclosed herein may contain other agents that allow the composition to be administered via a specific route of administration. For example, but not limited to, the composition may be formulated for administration via oral, topical, transdermal, intradermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, intravenous and / or intranodal routes of administration. Based on the route of administration, the composition may contain one or more additional components (e.g., apoptotic cancer cells, immunogenic compositions and / or additional therapeutic agents) in addition to the active agent. Examples of other auxiliary compounds that may be present include, but are not limited to, fillers, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifiers, dispersants, and other materials well known in the art.

[0047] In a specific (but non-limiting) embodiment, the composition comprising apoptotic cancer cells is administered to a subject intradermally, subcutaneously, intravenously, and / or intranodally.

[0048] In certain non-limiting embodiments, the method may further include an additional step of applying an alternating electric field to the target area of ​​the subject after separating the cancer cells (and / or removing the tumor), and before or after administering the composition containing apoptotic cancer cells. When this additional alternating electric field application step is present, the application of the alternating electric field may be performed simultaneously with the administration of the composition containing apoptotic cancer cells, or in whole or in part sequentially. In certain specific (but non-limiting) embodiments, the alternating electric field may be applied after the administration of the composition containing apoptotic cancer cells. In other specific (but non-limiting) embodiments, the alternating electric field may be applied while or after the administration of the composition containing apoptotic cancer cells. In another specific (but non-limiting) embodiment, the composition containing apoptotic cancer cells may be administered during the application of the alternating electric field (i.e., before the time period for applying the alternating electric field has passed).

[0049] For example, but not by way of limitation, the additional application of the alternating electric field may have begun for at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, 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 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, etc., as well as ranges formed by any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and ranges combining two integers falling between two of the above values ​​(e.g., a range of about 14 minutes to about 94 hours, etc.).

[0050] In other non-limiting embodiments, the additional application of the alternating electric field may be started for at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, 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 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. The composition containing apoptotic cancer cells is administered after about 1 minute, 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, etc., as well as ranges formed by any of the above values ​​(e.g., a range from about 1 minute to about 24 hours, etc.), and a range combining two integers falling between two of the above values ​​(e.g., a range from about 14 minutes to about 94 hours, etc.).

[0051] In yet another non-limiting embodiment, the composition comprising apoptotic cancer cells can be administered after the time period during which the additional alternating electric field is applied has elapsed, wherein the composition comprising apoptotic cancer cells is administered about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, 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 12 hours, about 15 minutes, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 48 hours, about 49 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours 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, etc.

[0052] The composition containing apoptotic cancer cells can be administered to a subject at any concentration that can induce an inflammatory response in a tumor or cancer cell. For example, but not limited to, the amount of apoptotic cancer cells administered can be about 10 cells / kg (body weight), about 100 cells / kg (body weight), about 1000 cells / kg (body weight), about 10 4 cells / kg (body weight), about 10 5 cells / kg (body weight), about 10 6 cells / kg (body weight), about 10 7 cells / kg (body weight), about 10 8 cells / kg (body weight), about 10 9 cells / kg (body weight), about 10 10 cells / kg (body weight), about 10 11 cells / kg (body weight), about 10 12 cells / kg (body weight), about 10 13 cells / kg (body weight), about 10 14 cells / kg (body weight), about 10 15 cells / kg (body weight) or more, and ranges formed by any of the above values ​​(e.g., about 10 4 cells / kg (body weight) to about 10 9 cells / kg (body weight) range).

[0053] In certain specific (but non-limiting) embodiments, the method also involves simultaneous therapy using two or more compositions. Thus, the method may include an additional step of administering at least a second composition to the subject. Other non-limiting examples of therapeutic agents that can be used as part of a second composition administered simultaneously or in whole or in part sequentially with a composition containing apoptotic cancer cells include Lenvatinib, Pembrolizumab, and other anti-PD-1 therapeutic agents, such as (but not limited to) Tislelizumab, Nivolumab, and Cemiplimab; anti-LAG3 agents, such as (but not limited to) OPDUALAG TM and / or relalizumab (Bristol-Myers Squibb, New York, NY); anti-PD-L1 therapeutics such as, but not limited to, atezolizumab, avelumab, and durvalumab; anti-CTLA-4 therapeutics such as, but not limited to, ipilimumab; chemotherapeutic agents such as, but not limited to, paclitaxel, docetaxel, ifosamide, etoposide (Vepesid), gemcitabine, lomustine, nab-paclitaxel (Nab Paclitaxel), Temozolomide and Carboplatin; TKI inhibitors such as (but not limited to) Everolimus; mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; VEGF inhibitors; FGF inhibitors; aromatase inhibitors such as (but not limited to) Letrozole; biologics such as monoclonal antibodies (such as but not limited to Denosumab and Pembrolizumab), etc. and any combination thereof.

[0054] When this occurs, simultaneous treatment can be performed substantially simultaneously or in whole or in part sequentially with the administration of the composition containing apoptotic cancer cells. In addition, the two compositions can be administered by the same route (e.g., oral administration or injection), or the two compositions can be administered by different routes (e.g., one composition is administered orally and the other composition is administered intravenously).

[0055] When both the steps of administering the second composition and applying an alternating electric field to a subject to which the composition containing apoptotic cancer cells has been administered are present, the optional administration step can be performed in the same manner and time range as described above for the composition containing apoptotic cancer cells, before or after the application of the alternating electric field begins, as well as during the application of the alternating electric field and / or after the application of the alternating electric field has elapsed.

[0056] That is, for example (but not by way of limitation), the second composition may be applied 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, about 93 hours, about 96 hours, etc., as well as ranges formed by any of the above values ​​(e.g., a range of about 24 hours to about 96 hours, etc.), and a range combining two integers falling between two of the above values ​​(e.g., a range of about 14 hours to about 94 hours, etc.). In specific (but non-limiting) embodiments, the second composition is applied at least about 24 hours after application of the alternating electric field is initiated.

[0057] In other non-limiting examples, the second composition can be applied after the time period for applying the alternating electric field has passed, wherein the second composition is applied 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 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, etc. after the time period has passed. In a specific (but non-limiting) embodiment, the second composition is administered within about 96 hours after the time period has passed.

[0058] In addition, for example (but not by way of limitation), the composition comprising apoptotic cancer cells may be administered for at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about The second composition is administered at least about 12 hours after administration of the composition containing apoptotic cancer cells, and the second composition is administered at least about 12 hours after administration of the composition containing apoptotic cancer cells.

[0059] In certain specific (but non-limiting) embodiments, the method may further include the step of administering at least one additional therapy to the subject. According to the methods of the present disclosure, any therapy known in the art or otherwise contemplated herein for use with alternating electric fields and / or apoptotic cancer cell therapy may be used. Non-limiting examples of additional therapies that may be used include radiotherapy, photodynamic therapy, transarterial chemoembolization (TACE), or a combination thereof.

[0060] In certain specific (but non-limiting) embodiments, the method includes one or more additional steps. For example (but not by way of limitation), the method may also include repeating any step once or multiple times. Each step may be repeated as many times as desired. When the alternating electric field is repeatedly applied, the transducer array may be placed in a slightly different position from the original position on the subject; repositioning the array in this way may also help treat tumors / cancers. In addition, any administration step (including the step of administering a composition containing apoptotic cancer cells and any optional administration step) may be repeated at different times and at different intervals according to any known and / or generally accepted dosage / treatment regimen of the composition / therapy.

[0061] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions comprising any isolated and irradiated apoptotic cancer cell population produced as described herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, apoptotic cancer cells are produced by exposing isolated cancer cells to an alternating electric field (in vivo or in vitro) and then irradiating the cancer cells to which the alternating electric field has been applied.

[0062] The immunogenic composition may be formulated for administration by any route of administration disclosed herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

[0063] In certain specific (but non-limiting) embodiments, the immunogenic composition may also include one or more additional active agents that further help stimulate the immune system to recognize and attack cancer cells in the subject. Non-limiting examples of other agents that may be present in the immunogenic composition include adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptors (CLR), anti-LAG3 agents (such as but not limited to OPDUALAG), and anti-LAG3 agents (such as but not limited to OPDUALAG). TM and / or relalizumab (Bristol-Myers Squibb, New York, NY)) and any combination thereof.

[0064] Certain non-limiting embodiments of the present disclosure are directed to kits comprising any components of an alternating electric field generating system (such as, but not limited to, one or more transducer arrays and / or one or more hydrogel compositions, as disclosed in U.S. Pat. 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 No.; and U.S. Patent Application Nos. US2018 / 0160933, US2019 / 0117956, US2019 / 0307781, and US2019 / 0308016) are combined with one or more components, devices, and / or reagents used in one or more method steps, which method steps include separating cancer cells, irradiating cancer cells, separating irradiated and treated apoptotic cancer cells, and / or preparing the separated apoptotic cancer cells for administration to a subject according to the methods disclosed herein or otherwise contemplated. The kit may optionally also include one or more of any optional compositions disclosed herein or otherwise contemplated (such as, but not limited to, one or more compositions for optional simultaneous therapy steps). The kit may optionally also include one or more devices (or one or more components of a device) used in one or more additional therapy steps.

[0065] In a specific (but non-limiting) embodiment, the kit may also include instructions for practicing any method disclosed herein or otherwise contemplated. For example (but not by way of limitation), the kit may include instructions for applying one or more components of the alternating electric field generating system to the patient's skin, instructions for applying the alternating electric field to the patient, instructions for when and how to apply the composition containing apoptotic cancer cells and optionally how to apply one or more optional additional compositions, and / or instructions for when to activate and deactivate the alternating electric field relative to applying the composition containing apoptotic cancer cells and / or applying one or more optional compositions.

[0066] In addition to the components described in detail above, the kit may contain other components / reagents for practicing any specific method described herein or otherwise contemplated. For example (but not by way of limitation), the kit may further include: (i) components for preparing the skin prior to treating the hydrogel composition and / or transducer array thereon (i.e., a razor, a cleansing composition or a wipe / towel, etc.); (ii) components for removing the gel / transducer array; (iii) components for cleaning the skin after removing the gel / transducer array; (iv) components for separating cancer cells / tumor portions; (v) components for irradiating the separated and alternating electric field treated cancer cells / tumor portions; and / or (vi) components for separating irradiated and alternating electric field treated cancer cells. The nature of these additional components / reagents will depend on the specific treatment modality, and their identification is well within the skill of a person of ordinary skill in the art; therefore, no further description thereof is required. Furthermore, the components / reagents present in the kit may each be in separate containers / compartments, or the various components / reagents may be combined in one or more containers / compartments, depending on the sterility, cross-reactivity and stability of the components / reagents.

[0067] The test kit can be arranged in any package that allows the components present therein to function according to the present disclosure. In certain non-limiting embodiments, the test kit also includes a sealed package in which the components are placed. In certain specific (but non-limiting) embodiments, the sealed package is substantially airtight and / or substantially opaque to light.

[0068] In addition, the kit may also include a set of written instructions explaining how to use one or more components of the kit. Kits of this nature can be used in any method described or contemplated herein.

[0069] In certain non-limiting embodiments, the kit has a shelf life of at least about 6 months, such as, but not limited to, at least about 9 months, or at least about 12 months.

[0070] Certain non-limiting embodiments of the present disclosure relate to systems comprising any components of an alternating electric field generating system (such as, but not limited to, one or more transducer arrays and / or one or more hydrogel compositions, as disclosed in: U.S. Patent Nos. 7,016,725; 7,089,054; 7,333,852; 7,565,205; 8,244,345; 8,715,203; 8,764,675; 10,188,851; and 10,441,776; and U.S. Patent Application Nos. US2018 / 0160933; US2019 / 0117956; US2019 / 0307781; and US2019 / 0308016) combined with at least one of any compositions comprising apoptotic cancer cells produced according to the methods disclosed herein or otherwise contemplated. The system may optionally further include one or more of any optional compositions disclosed herein or otherwise contemplated. The system may optionally further include one or more devices (or one or more components of a device) used in various separation, treatment, irradiation or administration steps or optional additional treatment / therapy steps.

[0071] Example

[0072] Examples are provided below. However, the present disclosure should be understood to be not limited in its application to the specific experiments, results and laboratory procedures disclosed below. On the contrary, the examples are provided only as one of the various embodiments and are exemplary, not exhaustive.

[0073] The main goal of cancer immunotherapy is to activate pre-existing endogenous immune responses in cancer patients. Although significant progress has been made in this field, the effectiveness of treatment still needs to be improved. Personalized cancer vaccines are a promising strategy to enhance anti-tumor immune responses through immunogenic forms of apoptosis, also known as immunogenic cell death (ICD). ICD is characterized by the release of danger-associated molecular patterns that are used to recruit immune cells to tumor sites. Previous studies have shown that TTField treatment can enhance immunogenic cell death of cancer cells, ultimately stimulating immune responses by phagocytosis of cancer cells. In turn, it can also present new antigens to further initiate adaptive immunity.

[0074] To justify the use of TTField as an immunomodulator, INOVITRO TM The mice were treated with the TTField system (Novocure GmbH, Root, Switzerland) for 72 hours. Cancer cells were then isolated from the mice and irradiated. The irradiated apoptotic cancer cells were isolated from the culture medium and then injected into the mice as a vaccine to elicit an immune response to the cancer development.

[0075] In this way, a method to enhance cancer cell immunity is combined with TTField therapy. Combining TTField therapy with the administration of a personalized composition containing apoptotic cancer cells has a more synergistic effect than either treatment alone and triggers an immune response in the patient, enabling the immune system to eliminate the cancer cells.

[0076] Non-limiting illustrative embodiments of the present inventive concept

[0077] Illustrative embodiment 1: A method for preparing an immunogenic composition, the method comprising the steps of: (1) isolating cancer cells from a target area of ​​a subject; (2) applying an alternating electric field to the isolated cancer cells; and (3) irradiating the isolated cancer cells to which the alternating electric field has been applied to form the immunogenic composition.

[0078] Illustrative embodiment 1a: A method for preparing an immunogenic composition, the method comprising the steps of: (a) applying an alternating electric field to cancer cells isolated from a target area of ​​a subject; and (3) irradiating the isolated cancer cells to which the alternating electric field has been applied to form the immunogenic composition.

[0079] Illustrative embodiment 2: A method for preparing an immunogenic composition, the method comprising the steps of: (i) applying an alternating electric field to a target area of ​​a subject; (2) isolating cancer cells to which the alternating electric field has been applied from the target area of ​​the subject; and (3) irradiating the isolated cancer cells to which the alternating electric field has been applied to form the immunogenic composition.

[0080] Illustrative embodiment 2a: A method for preparing an immunogenic composition, the method comprising the following steps: (a) isolating cancer cells from a target area of ​​a subject, wherein an alternating electric field has been applied to the target area of ​​the subject before isolating the cancer cells; and (b) irradiating the isolated cancer cells to which the alternating electric field has been applied to form the immunogenic composition.

[0081] Illustrative embodiment 3: A method for treating cancer in a subject, the method comprising the steps of: (1) isolating cancer cells from a target area of ​​the subject; (2) applying an alternating electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells to which the alternating electric field has been applied; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying an alternating electric field to the target area of ​​the subject.

[0082] Illustrative embodiment 3a: A method for treating cancer in a subject, the method comprising the steps of: (a) applying an alternating electric field to cancer cells isolated from a target area of ​​the subject; (b) irradiating the isolated cancer cells to which the alternating electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying the alternating electric field to the target area of ​​the subject.

[0083] Illustrative embodiment 4: A method for treating cancer in a subject, the method comprising the following steps: (i) applying an alternating electric field to a target area of ​​the subject; (2) separating cancer cells to which the alternating electric field has been applied from the target area of ​​the subject; (3) irradiating the separated cancer cells to which the alternating electric field has been applied; (4) administering the separated and irradiated cancer cells to the subject; and (5) applying an alternating electric field to the target area of ​​the subject.

[0084] Illustrative embodiment 4a: A method for treating cancer in a subject, the method comprising the following steps: (a) isolating cancer cells from a target area of ​​the subject, wherein an alternating electric field has been applied to the target area of ​​the subject prior to isolating the cancer cells; (b) irradiating the isolated cancer cells to which the alternating electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying the alternating electric field to the target area of ​​the subject.

[0085] Illustrative embodiment 5: A method for reducing tumor volume and / or preventing the tumor volume from increasing, wherein the tumor is present in a living subject and includes a plurality of cancer cells, the method comprising the following steps: (1) isolating cancer cells from a target area of ​​the subject; (2) applying an alternating electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells to which the alternating electric field has been applied; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying an alternating electric field to the target area of ​​the subject.

[0086] Illustrative embodiment 5a: A method for reducing the volume of a tumor and / or preventing the volume of the tumor from increasing, wherein the tumor is present in a living subject and includes a plurality of cancer cells, the method comprising the following steps: (a) applying an alternating electric field to cancer cells separated from a target area of ​​the subject; (b) irradiating the separated cancer cells to which the alternating electric field has been applied; (c) administering the separated and irradiated cancer cells to the subject; and (d) applying an alternating electric field to the target area of ​​the subject.

[0087] Illustrative embodiment 6: A method for reducing tumor volume and / or preventing the tumor volume from increasing, wherein the tumor is present in a living subject and includes a plurality of cancer cells, the method comprising the following steps: (i) applying an alternating electric field to a target area of ​​the subject; (2) separating the cancer cells to which the alternating electric field has been applied from the target area of ​​the subject; (3) irradiating the separated cancer cells to which the alternating electric field has been applied; (4) administering the separated and irradiated cancer cells to the subject; and (5) applying an alternating electric field to the target area of ​​the subject.

[0088] Illustrative embodiment 6a: A method for reducing tumor volume and / or preventing the tumor volume from increasing, wherein the tumor is present in a living subject and includes a plurality of cancer cells, the method comprising the following steps: (a) isolating cancer cells from a target area of ​​the subject, wherein an alternating electric field has been applied to the target area of ​​the subject before isolating the cancer cells; (b) irradiating the isolated cancer cells to which the alternating electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying an alternating electric field to the target area of ​​the subject.

[0089] Illustrative embodiment 7: The method according to any one of illustrative embodiments 1-6a, wherein the cancer cells are selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer and combinations thereof.

[0090] Illustrative embodiment 8: The method according to any one of 1-7, wherein at least one of the following is achieved: 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 a portion of the separated cancer cells; and the alternating electric field is applied for a period of at least about 24 hours. Illustrative embodiment

[0091] Illustrative Embodiment 9: The method according to any one of Illustrative Embodiments 1-8, wherein the cells are irradiated for a period of time sufficient to ensure that the cells are inactivated.

[0092] Illustrative embodiment 10: The method according to illustrative embodiment 9, wherein the cells are irradiated for a period of time ranging from about 24 hours to about 96 hours.

[0093] Illustrative Embodiment 11: The method according to any one of Illustrative Embodiments 1-2a, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

[0094] Illustrative embodiment 12: According to the method according to any one of illustrative embodiments 1-2a or 11, the method further includes: a step of adding at least one additional composition to the immunogenic composition, wherein the at least one additional composition is selected from the group consisting of: adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG3 agents and combinations thereof.

[0095] Illustrative Embodiment 13: The method according to any one of Illustrative Embodiments 3-10, wherein the isolated and irradiated cancer cells are administered intradermally, subcutaneously, intravenously and / or intranodally.

[0096] Illustrative embodiment 14: The method according to any one of illustrative embodiments 3-10 and 13, wherein the isolated and irradiated cancer cells are administered to the subject in the form of at least one immunogenic composition, and wherein the at least one immunogenic composition further comprises at least one composition selected from the group consisting of an adjuvant, a cytokine, an interferon, a TLR agonist, a STING (stimulator of interferon genes) agonist, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), a C-type lectin receptor (CLR), an anti-LAG3 agent, and combinations thereof.

[0097] Illustrative Embodiment 15: The method according to any one of Illustrative Embodiments 1-14, further comprising: a step of sorting the inactivated, irradiated cancer cells to ensure that substantially no irradiated but still active cancer cells are administered to the subject (i.e., present in the immunogenic composition).

[0098] Illustrative Embodiment 16: An immunogenic composition prepared by the method according to any one of Illustrative Embodiments 1-2a, 7-12 and 15.

[0099] Illustrative Embodiment 17: An immunogenic composition comprising: a population of cancer cells isolated from a target area of ​​a subject, treated in vitro with an alternating electric field, and then irradiated.

[0100] Illustrative Embodiment 18: An immunogenic composition comprising: a population of cancer cells produced by treating a target area of ​​a subject in vivo with an alternating electric field, isolating cancer cells from the treated target area of ​​the subject, and irradiating the isolated, treated cancer cells.

[0101] Illustrative embodiment 19: The immunogenic composition according to illustrative embodiment 17 or 18, wherein the cancer cells are selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer and combinations thereof.

[0102] Illustrative Embodiment 20: The immunogenic composition according to any one of Illustrative Embodiments 17-19, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

[0103] Illustrative embodiment 21: The immunogenic composition according to any one of illustrative embodiments 17-20, wherein the immunogenic composition further comprises at least one composition selected from the group consisting of: adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG3 agents, and combinations thereof.

[0104] Illustrative Embodiment 22: The immunogenic composition according to any one of Illustrative Embodiments 17-21, wherein the irradiated cancer cells are inactivated.

[0105] Although the above disclosure describes the inventive concept in conjunction with the specific experiments, results and language of elaboration, it is obvious that many substitutions, modifications and variations are apparent to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A method for preparing an immunogenic composition, the method comprising the following steps: (1) isolating cancer cells from a target area of ​​a subject; (2) applying an alternating electric field to the separated cancer cells; as well as (3) irradiating the isolated cancer cells to which the alternating electric field has been applied to provide the immunogenic composition.

2. The method according to claim 1, wherein in step (1), the cancer cells are selected from the group consisting of: hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, cervical cancer, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer and combinations thereof.

3. The method according to claim 1 or 2, wherein in step (2), at least one of the following is achieved: 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 a portion of the isolated cancer cells; and The alternating electric field is applied for a period of at least about 24 hours.

4. The method according to any one of claims 1 to 3, wherein in step (3), the cells are irradiated for a period of time long enough to ensure that the cells lose their viability.

5. The method according to any one of claims 1 to 4, further comprising: The step of sorting the inactivated irradiated cancer cells ensures that irradiated but still viable cancer cells are substantially absent from the immunogenic composition.

6. The method of any one of claims 1-4, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

7. A method of treating cancer in a subject, the method comprising the steps of: (1) isolating cancer cells from a target area of ​​the subject; (2) applying an alternating electric field to the separated cancer cells; (3) irradiating the isolated cancer cells to which the alternating electric field has been applied; (4) administering the isolated and irradiated cancer cells to the subject; as well as (5) Applying the alternating electric field to the target area of ​​the subject.

8. The method according to claim 7, wherein in step (1), the cancer cells are selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer and combinations thereof.

9. The method according to claim 7 or 8, wherein in step (2), at least one of the following is achieved: 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 a portion of the isolated cancer cells; and The alternating electric field is applied for a period of at least about 24 hours.

10. The method according to any one of claims 7 to 9, wherein in step (3), the cells are irradiated for a period of time long enough to ensure that the cells are inactivated.

11. The method of claim 10, wherein the cells are irradiated for a period of time ranging from about 24 hours to about 96 hours.

12. The method according to any one of claims 7 to 11, wherein in step (4), the isolated and irradiated cancer cells are administered intradermally, subcutaneously, intravenously and / or intranodally.

13. The method according to any one of claims 7 to 12, wherein in step (4), the isolated and irradiated cancer cells are administered to the subject in the form of at least one immunogenic composition, and wherein the at least one immunogenic composition further comprises at least one composition selected from the group consisting of an adjuvant, a cytokine, an interferon, a TLR agonist, a STING (stimulator of interferon genes) agonist, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), a C-type lectin receptor (CLR), an anti-LAG3 agent, and a combination thereof.

14. The method according to any one of claims 7 to 13, wherein in step (5), at least one of the following is implemented: 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 a portion of the target area of ​​the subject; and The period of time during which the alternating electric field is applied is at least about 24 hours.

15. The method according to any one of claims 7-14, further defined as a method of reducing the volume of a tumor and / or preventing the volume of the tumor from increasing, wherein the tumor is present in a living subject and comprises a plurality of cancer cells.

16. An immunogenic composition, comprising: A population of cancer cells isolated from a target area of ​​a subject, treated ex vivo with an alternating electric field, and then irradiated.

17. The immunogenic composition of claim 16, wherein the cancer cell is selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid carcinoma cells, advanced renal cell carcinoma cells, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, lung cancer, and combinations thereof.

18. The immunogenic composition of claim 16 or 17, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

19. The immunogenic composition of any one of claims 16-18, wherein the immunogenic composition further comprises at least one composition selected from the group consisting of an adjuvant, a cytokine, an interferon, a TLR agonist, a STING (stimulator of interferon genes) agonist, GM-CSF, CD40L, Fms-related tyrosine kinase 3 ligand (FLT3L), a C-type lectin receptor (CLR), an anti-LAG3 agent, and a combination thereof.

20. The immunogenic composition of any one of claims 16-19, wherein the irradiated cancer cells are inactivated.