Compositions, systems and methods for cancer treatment using alternating electric fields and apoptotic cancer cell vaccines

Abstract

Immunogenic compositions and systems, as well as methods for producing immunogenic compositions, are provided. Also disclosed are compositions, systems, and methods for reducing cancer cell viability and treating cancer, as well as methods for reducing tumor volume and / or preventing tumor volume growth within a living organism. The systems and methods include applying an alternating current electric field to cancer cell(s) (before or after isolation from a target area of ​​a subject) and irradiating the isolated and treated cancer cell(s). The methods may further include re-administering the irradiated, treated cancer cells to the subject from whom the cancer cell(s) were isolated and / or applying an alternating current electric field to the subject. The immunogenic composition comprises a population of isolated apoptotic cancer cells.

Description

Cross-Reference / Incorporation-by-Reference Statement of Related Applications

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 377,951, filed September 30, 2022, and U.S. Provisional Application No. 63 / 496,831, filed April 18, 2023. The entire contents of the above-referenced patent applications are expressly incorporated herein by reference. [Background technology]

[0002] Tumor treating fields (TT fields) are low-intensity (e.g., 1-3 V / cm) alternating current electric fields in the mid-frequency range (e.g., 100-500 kHz) that target solid tumors by inhibiting mitosis. This non-invasive treatment is targeted to solid tumors and is described, for example, 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. TT fields (and similar AC electric fields used to treat other conditions) are typically delivered through two pairs of transducer arrays that generate perpendicular electric fields within the tumor to be treated; the electrode arrays that make up each pair are positioned on opposite sides of the body part to be treated. Specifically, in the OPTUNE® system, one pair of electrodes is placed on the left and right (LR) sides of the tumor, and another pair of electrodes is placed on the anterior-posterior (AP) sides of the tumor. TT fields are approved for the treatment of glioblastoma multiforme (GBM) and can be delivered, for example, via the OPTUNE® system (Novocure Limited, St. Helier, Jersey), which includes a transducer array placed on the patient's shaved head.

[0003] Each transducer array used to deliver TT fields in the OPTUNE® device consists of a series of ceramic disc electrodes coupled to the patient's skin (such as a patient's shaved head for the treatment of GBM) via a layer of conductive medical gel. The purpose of the medical gel is to conform to the contours of the body and provide good electrical contact between the array and the skin, so the gel interface bridges the skin and reduces interference. The device is intended to be worn continuously by the patient for 2-4 days for hygiene purposes and re-shaving (if necessary), after which a new set of arrays is reapplied. Thus, the medical gel remains in substantially continuous contact with an area of ​​the patient's skin for 2-4 days at a time, leaving only a short period of time for that area to be uncovered and exposed to the environment before further application of medical gel is required. Another cancer treatment is cancer immunotherapy. The main goal of cancer immunotherapy is to activate the existing endogenous immune response in cancer patients. Potential targets for cancer immunotherapy include tumor-specific antigens (neoantigens) derived from mutations, which are not present on normal cells and are recognized by the immune system, making them specific targets for antitumor therapy. Although significant progress has been made in this field, therapeutic efficacy still needs to be improved. DETAILED DESCRIPTION OF THE INVENTION

[0004] Before describing at least one embodiment of the inventive concept(s) in detail using illustrative language and results, it should be understood that application of the inventive concept(s) is not limited to the details of construction and the arrangement of components set forth in the following description. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. Accordingly, the language used herein is intended to be accorded the broadest possible scope and meaning, and the embodiments are intended to be illustrative and not exhaustive. It should also be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0005] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concept(s) shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and described throughout the specification. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0006] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the presently disclosed inventive concept(s) pertain. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated by reference herein in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0007] All of the compositions, assemblies, systems, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, assemblies, systems, kits, and methods of the inventive concept(s) have been described with reference to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods, and to the steps or sequence of steps of the methods described herein without departing from the concept(s), spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept(s) of the inventive concept(s) as defined by the appended claims.

[0008] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0009] The use of the terms "a" or "an" in the claims and / or specification when used in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," or "one or more." Thus, the terms "a," "an," and "the" encompass the plural meaning unless the context clearly indicates otherwise. Thus, for example, the phrase "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or an even greater number of compounds. The term "plurality" refers to two or more.

[0010] Use of the term "at least one" is understood to encompass quantities greater than one, including but not limited to 1, as well as 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is attached, and quantities of 100 / 1000 are not considered limiting, and higher limits may also be satisfactory. Additionally, use of the term "at least one of X, Y, Z" is understood to encompass X alone, Y alone, Z alone, and any combination of X, Y, and Z, and the use of ordinal terms (e.g., "first," "second," "third," "fourth," etc.) is intended only to distinguish between two or more items and does not imply any order of superiority or importance of one item over another, or any additional order.

[0011] The use of the word "or" in the 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" can be satisfied by any of the following: A being true (or existing) and B being false (or not existing), A being false (or existing) and B being true (or existing), and both A and B being true (or existing).

[0012] As used herein, references to "one embodiment," "embodiment," "some embodiments," "one example," "for example," or "one example" mean that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "in one example" in various places in the specification do not necessarily all refer to the same embodiment. Moreover, all references to one or more embodiments or examples should not be construed as limiting the claims.

[0013] Throughout this application, the term "about" is used to indicate that a value encompasses the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists between study subjects. For example, and without limitation, when the term "about" is used, the specified value may vary from the specified value by plus or minus 20 percent, 15 percent, 12 percent, 11 percent, 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent, as appropriate for performing the disclosed methods and as understood by one of ordinary skill in the art.

[0014] As used in this specification and claim(s), the words "comprising" (and all forms of "including", such as "comprise" and "comprises"), "having" (and all forms of "having", such as "have" and "has"), "including" (and all forms of "including", such as "includes" and "include") or "containing" (and all forms of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0015] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before 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 order is important in a particular context, also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there is typically no limit to the number of items or terms that may be combined unless otherwise apparent from the context.

[0016] As used herein, the term "substantially" means that a described event or circumstance occurs entirely, or that a described event or circumstance occurs to a significant extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, at least 85% of the time, at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" can mean that two items are 100% adjacent to each other, that two items are very close to each other but not 100% adjacent, or that a portion of one of two items is not 100% adjacent to the other item but is very close to the other item.

[0017] The term "pharmaceutically acceptable" refers to compounds and compositions that are suitable for administration to humans and / or animals without undue side effects, such as toxicity, irritation, and / or allergic response, commensurate with a reasonable benefit / risk ratio.

[0018] As used herein, the term "patient" or "subject" includes human and veterinary subjects. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including, but not limited to, humans, domestic animals, farm animals, non-human primates, and other animals with mammary tissue.

[0019] The term "treatment" refers to both therapeutic and prophylactic measures. Those in need of treatment include, but are not limited to, those who already have a particular condition / disease / infection, as well as those who are at risk of contracting a particular condition / disease / infection (such as those who require prophylactic measures). The term "treating" refers to the administration of an agent / substance / method to a patient for therapeutic and / or prophylactic purposes.

[0020] 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 effect.

[0021] Administration of a therapeutically or prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific therapeutically effective amount can be readily determined by a practitioner of ordinary skill and may vary depending on factors known in the art, including, but not limited to, the type of condition / disease / infection, the patient's medical history and age, the stage of the condition / disease / infection, and the use of other drugs.

[0022] The term "effective amount," when used in accordance with the concept(s) of the present invention, refers to an amount of a biologically active molecule or its conjugate or derivative, or a treatment protocol (i.e., an alternating electric field), sufficient to exert a detectable therapeutic effect without undue side effects (such as, but not limited to, toxicity, irritation, allergic reaction, etc.) commensurate with a reasonable benefit / risk ratio. The therapeutic effect may include, for example, but is not limited to, preventing, inhibiting, or alleviating the occurrence of at least one condition, disease, and / or infection. The effective amount for a subject will vary depending on the type of subject, the subject's size and health, the nature and severity of the symptom / disease / infection being treated, the method of administration, the duration of treatment, the nature of concomitant therapy (if any), the particular formulation used, and the like. Thus, an exact effective amount cannot be specified in advance. However, an effective amount for a particular situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0023] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjunctive therapy" and is understood to mean that a patient in need of treatment is treated with or administered another agent for a condition / disease / infection in conjunction with the treatment of the present disclosure. This concurrent therapy can be sequential therapy, where the patient is treated first with one therapeutic protocol / pharmaceutical composition and then with another therapeutic protocol / pharmaceutical composition, or where two therapeutic protocols / pharmaceutical compositions are administered simultaneously.

[0024] As used herein, the terms "administration" and "administering" are understood to encompass 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 to encompass both local and systemic application. In addition, the compositions of the present disclosure (and / or methods of administration thereof) may be designed to provide delayed-, controlled-, or sustained-release using formulation techniques well known in the art.

[0025] Turning now to the concept(s) of the present invention, a combination therapy for cancer is disclosed herein. The combination therapy combines (i) generating AC electric field-treated apoptotic cancer cells (e.g., TT field) by applying an AC electric field, (ii) irradiating the TT field-treated apoptotic cancer cells, (iii) administering the AC electric field-treated apoptotic cancer cells to a subject, and (iv) applying an AC electric field (e.g., TT field) to a target region of the subject. Combining AC electric field treatment with apoptotic cancer cell vaccination provides a synergistic effect in cancer treatment.

[0026] Certain non-limiting embodiments of the present disclosure relate to methods for treating cancer in a subject, and / or for reducing tumor volume in a subject, and / or for preventing an increase in tumor volume in a subject, comprising the steps of: (1) isolating cancer cells from a target area of ​​the subject, (2) applying an alternating current electric field to the isolated cancer cells, (3) irradiating the isolated cancer cells that have been subjected to the alternating current electric field, (4) administering the isolated and irradiated cancer cells to the subject, and (5) applying the alternating current electric field to the target area of ​​the subject.

[0027] Certain non-limiting embodiments of the present disclosure are directed to other similar methods of treating cancer in a subject (and / or methods of reducing the volume of a tumor in a subject and / or methods of preventing an increase in the volume of a tumor present in a subject), except that an alternating current electric field is applied to a target region of the subject (such as, but not limited to, to induce an immunogenic response) prior to isolating cancer cells from the target region of the subject. In other words, the methods of the present disclosure involve irradiating the cancer cells that have been treated with the alternating current electric field and applying an alternating current electric field to the cancer cells both in vivo and ex vivo prior to administering the cells to a subject.

[0028] The methods of the present disclosure can be utilized to treat any type of cancer cell / cancer / tumor that responds to AC electric fields / TT fields and / or apoptotic cancer cell therapy. Non-limiting examples of cancer cells / cancers / tumors that can be treated according to the present disclosure can 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), and the like, and any combination thereof.

[0029] Any type of conductive or non-conductive electrode(s) and / or transducer array(s) known in the art or contemplated herein that can be utilized to generate an alternating electric field can be utilized to generate an alternating electric field in accordance with the methods of the present disclosure. Non-limiting examples of electrodes and transducer arrays that can be utilized to generate alternating electric fields in accordance with the present disclosure include, but are not limited to, those that function as part of the TT field systems described 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. 2018 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016.

[0030] According to the present invention, the AC electric field can be generated at any frequency, for example (but not limited to) 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, about 850 kHz, about 900 kHz, about 950 kHz, about 1 MHz, and the like. Hz, 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., as well as ranges formed from any of the above values ​​(e.g., a range of about 50 kHz to about 10 MHz, a range of about 50 kHz to about 1 MHz, a range of about 100 kHz to about 500 kHz, a range of about 150 kHz to about 300 kHz, etc.), and ranges formed by combining two integers between two of the above reference values ​​(e.g., a range of about 122 kHz to about 313 kHz, a range of about 78 kHz to about 298 kHz, etc.).

[0031] In certain (but non-limiting) embodiments, the alternating electric field may be applied at two or more different frequencies, where two or more frequencies are present, each frequency being selected from any of the values ​​listed above, or from a range formed from any of the values ​​listed above, or from a range combining two integers between any two of the values ​​listed above.

[0032] The alternating electric field can have any electric field strength within the subject / cancer cells as long as it is capable of functioning in accordance with the present disclosure. For example (but not limited to), the alternating electric field may be 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 15.5 V / cm, about 16 V / cm The electric field strength may be 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 ranges formed from any of the above values ​​(e.g., 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 formed by combining two integers between two of the above reference values ​​(e.g., 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.).

[0033] The alternating electric field can be applied for any period of time sufficient to achieve a desired result in accordance with the present disclosure (such as, but not limited to, generating an immune response, altering cancer cells to induce an immunogenic response, reducing cancer cell viability, and / or reducing tumor volume (and / or preventing tumor volume from increasing)). For example, but not limited to, the alternating electric field can be applied for a period of time sufficient to achieve a desired result in accordance with the present disclosure (such as, but not limited to, generating an immune response, alternating cancer cells to induce an immunogenic response, reducing cancer cell viability, and / or reducing tumor volume (and / or preventing tumor volume from increasing)). For example, but not limited to, the alternating electric field can be applied for a period of time sufficient to achieve a desired result in accordance with the present disclosure (such as, but not limited to, generating an immune response, alternating cancer cells to induce an immunogenic response, reducing cancer cell viability, and / or reducing tumor volume (and / or preventing tumor volume from increasing)). For example, but not limited to, the alternating electric field can be applied for a period of time sufficient to achieve a desired result in accordance with the present disclosure (such as, but not limited to, generating an immune response, alternating cancer cells to induce an immunogenic response, reducing cancer cell viability, and / or preventing tumor volume from increasing). hours, 69 hours, 72 hours, 75 hours, 78 hours, 81 hours, 84 hours, 87 hours, 90 hours, 93 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc., as well as ranges formed from any of the above values ​​(e.g., a range of about 1 minute to about 12 hours, a range of about 1 minute to about 1 hour, a range of about 1 hour to about 7 days, a range of about 24 hours to about 72 hours, etc.), and ranges formed by combining two integers between two of the above values ​​(e.g., a range of about 14 hours to about 68 hours, etc.).

[0034] In certain (but non-limiting) embodiments, the alternating electric field is applied in vivo at a frequency in the range of about 50 kHz to about 1 MHz, at 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 for a duration of at least about 24 hours.

[0035] In certain (but non-limiting) embodiments, the alternating electric field is applied ex vivo at a frequency ranging from about 50 kHz to about 1 MHz, at 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 for a duration ranging from about 1 hour to about 7 days.

[0036] Any method of irradiating cancer cells known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, so long as the cancer cells are irradiated at a radiation intensity sufficient and for a sufficient time to render them non-viable / proliferatively incapable. For example, but not limited to, cancer cells can be irradiated at an intensity of at least about 1 Gray (Gy). The ability to select a sufficient radiation intensity and a sufficient time is within the knowledge of one of ordinary skill in the art, and therefore further explanation is not believed necessary.

[0037] The cancer cells treated with an AC electric field are irradiated for a sufficient time to achieve a state of apoptosis and render the cancer cells substantially non-viable.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., as well as ranges formed from any of the above values ​​(for example, a range of about 30 minutes to about 24 hours, etc.), and ranges formed by combining two integers between two of the above reference values ​​(for example, a range of about 45 minutes to about 20.5 hours, etc.).

[0038] In certain (but non-limiting) embodiments, the time period for which the cancer cells treated with the AC electric field are irradiated ranges from about 24 hours to about 96 hours.

[0039] In certain (but non-limiting) embodiments, the irradiated non-viable cancer cells are isolated prior to administration to the subject (i.e., between steps (3) and (4) of the above method). This optional isolation step ensures that the composition administered to the subject does not contain cancer cells that may not have been sufficiently irradiated and are still viable. For example, but not by way of limitation, the cells may be subjected to one or more sorting steps to isolate non-viable cells and confirm the absence of viable cells among the sorted cells.

[0040] Alternatively, the radiation conditions can be sufficiently stringent (i.e., via long duration and / or high intensity) to ensure that cells present within the irradiated sample are not yet viable, in which case the irradiated, nonviable cancer cells can be administered directly to the subject.

[0041] The alternating current electric field-treated and irradiated cancer cells can be placed and administered in any formulation known in the art or contemplated herein that allows the apoptotic cancer cells to have a detrimental effect on cancer cells present in a subject. For example, but not limited to, the alternating current electric field-treated and irradiated cancer cells can be administered in the form of a pharmaceutical composition comprising the cells in combination with at least one pharmaceutically acceptable carrier. Non-limiting examples of suitable pharmaceutically acceptable carriers that can be utilized in accordance with the present disclosure include water, saline, dextrose solution, fructose or mannitol, calcium carbonate, cellulose, ethanol, oils of animal, vegetable, or synthetic origin, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, detergents, liposome carriers, sodium chloride, buffer solutions such as saline and phosphate-buffered saline, and / or other substances, diluents, excipients such as polyethylene glycol (PEG), or any combination thereof that are physiologically acceptable and / or safe to use. Suitable pharmaceutically acceptable carriers for pharmaceutical formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd ed (2020).

[0042] In certain non-limiting embodiments, the pharmaceutical composition comprising the cancer cells treated with an alternating electric field and irradiated can be further formulated as an immunogenic composition. The immunogenic composition can include the same components as the pharmaceutical composition described above (i.e., apoptotic cancer cells and a pharmaceutically acceptable carrier). In certain (but non-limiting) embodiments, the immunogenic composition can further include at least one additional agent. Non-limiting examples of agents that can be included as part of the immunogenic composition can 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 (CLRs), anti-LAG3 agents (such as, but not limited to, OPDUALAG™ and / or Relatimab (Bristol-Myers Squibb, New York, NY)), other active agents, and the like, as well as any combination thereof.

[0043] Additionally, any of the apoptotic cancer cell-containing compositions of the present disclosure may contain other agents that allow for administration of the composition by a particular route of administration. For example, without limitation, the compositions may be formulated for administration by oral, topical, transdermal, intradermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, intravenous, and / or intralymphatic routes. Based on the route of administration, the composition may contain, in addition to the active agent, one or more additional components (e.g., apoptotic cancer cells, immunogenic composition, and / or additional treatment(s)). Examples of additional secondary compounds that may be present may include, but are not limited to, fillers, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifiers, dispersing agents, and other substances well known in the art.

[0044] In certain (but non-limiting) embodiments, the composition comprising apoptotic cancer cells is administered to the subject intradermally, subcutaneously, intravenously, and / or intralymphatically.

[0045] In certain non-limiting embodiments, the method may further comprise the additional step of applying an alternating electric field to the target area of ​​the subject after isolation of the cancer cells (and / or resection of the tumor) and before or after administration of the apoptotic cancer cell-containing composition. If this additional step of applying an alternating electric field is present, the alternating electric field may be applied simultaneously with administration of the apoptotic cancer cell-containing composition, or sequentially, in whole or in part. In certain (but non-limiting) embodiments, the alternating electric field may be applied after administration of the apoptotic cancer cell-containing composition. In other certain (but non-limiting) embodiments, the alternating electric field may be applied simultaneously with or after administration of the apoptotic cancer cell-containing composition. In another specific (but non-limiting) embodiment, the apoptotic cancer cell-containing composition may be administered during application of the alternating electric field (i.e., before the period during which the alternating electric field is applied has elapsed).

[0046] For example, but not limited to, the apoptotic cancer cell-containing composition may be maintained 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 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 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 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, The range may be up to 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 from any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and ranges formed by combining two integers between two of the above reference values ​​(e.g., a range of about 14 minutes to about 94 hours, etc.).

[0047] In other non-limiting examples, the apoptotic cancer cell-containing composition remains viable 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 50 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 72 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 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about The range may be up to 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 from any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and ranges formed by combining two integers between two of the above reference values ​​(e.g., a range of about 14 minutes to about 94 hours, etc.).

[0048] In yet other non-limiting examples, the apoptotic cancer cell-containing composition may be administered after a period of time during which an additional alternating electric field is applied, and the apoptotic cancer cell-containing composition may be administered after a period of time during which the additional alternating electric field is applied, such as 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 20 hours, about 30 hours, about 45 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, about 100 hours, about 120 hours, about 15 ... 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.

[0049] The apoptotic cancer cell-containing composition(s) can be administered to a subject at any concentration capable of inducing an inflammatory response to the tumor or cancer cells. For example, but not limited to, the apoptotic cancer cells can be administered at a concentration of about 10 cells / kg body weight, about 100 cells / kg body weight, about 1000 cells / kg body weight, about 10 4cells / kg body weight, approx. 10 5 cells / kg body weight, approx. 10 6 cells / kg body weight, approx. 10 7 cells / kg body weight, approx. 10 8 cells / kg body weight, approx. 10 9 cells / kg body weight, approx. 10 10 cells / kg body weight, approx. 10 11 cells / kg body weight, approx. 10 12 cells / kg body weight, approx. 10 13 cells / kg body weight, approx. 10 14 cells / kg body weight, approx. 10 15 cells / kg body weight, or more, as well as ranges formed from any of the above values ​​(e.g., about 10 4 from about 10 9 The dose may be in the range of 1000 to 15000 cells / kg body weight.

[0050] In certain (but non-limiting) embodiments, the method further comprises simultaneous therapy with two or more compositions. Thus, the method may include the additional step of administering at least a second composition to the subject. Additional non-limiting examples of therapeutic agents that may be utilized simultaneously with the apoptotic cancer cell-containing composition or as part of a second composition administered in whole or in part sequentially 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 OPDUALAG™ and / or leratimab (Bristol-Myers Squibb, New York, NY), anti-PD-L1 therapeutic agents, for example, but not limited to, atezolizumab, avelumab, and durvalumab, anti-CTLA-4 therapeutic agents, for example, but not limited to, ipilimumab, chemotherapeutic agents such as paclitaxel, docetaxel, ifosamide, etoposide (bepesid), gemcitabine, lomustine, nab-paclitaxel, temozolomide, carboplatin, etc., TKI inhibitors such as 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, pembrolizumab), etc., and any combination thereof.

[0051] When concurrent therapy is present, the concurrent therapy can be performed substantially simultaneously with the administration of the apoptotic cancer cell-containing composition, or wholly or partially sequentially. Furthermore, the two compositions can be administered by the same route (e.g., both orally or by injection), or the two compositions can be administered by different routes (e.g., one composition administered orally and another composition administered intravenously).

[0052] When both a step of administering a second composition to a subject to which an apoptotic cancer cell-containing composition has been administered and a step of applying an alternating current electric field are present, the optional administration step may be performed before or after application of the alternating current electric field is initiated, during application of the alternating current electric field, and / or after application of the alternating current electric field has elapsed, in the same manner(s) and time frame(s) as described above for the apoptotic cancer cell-containing composition.

[0053] That is, for example (but not limited to), the second composition may be applied for at least about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, 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., from the start of application of the AC electric field, as well as a range formed from any of the above values ​​(e.g., a range of about 24 hours to about 96 hours), and a range combining two integers between the above two values ​​(e.g., a range of about 14 hours to about 94 hours). In certain (but non-limiting) embodiments, the second composition is administered at least about 24 hours after application of the alternating electric field begins.

[0054] In other non-limiting examples, the second composition may be administered after the period of time during which the alternating electric field has been applied has elapsed, in which case the second composition may be 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 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. In certain (but non-limiting) embodiments, the second composition is administered within about 96 hours after the period of time has elapsed.

[0055] Further, for example (but not limited to), the second composition may be administered at least about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, or about 66 hours after the apoptotic cancer cell-containing composition is administered. The administration time may be up to 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 from any of the above values ​​(e.g., a range of about 24 hours to about 96 hours, etc.), and ranges formed by combining two integers between two of the above reference values ​​(e.g., a range of about 14 hours to about 94 hours, etc.). In certain (but non-limiting) embodiments, the second composition is administered at least about 12 hours after the administration of the apoptotic cancer cell-containing composition.

[0056] In certain (but not limited to) embodiments, the method may further comprise administering at least one additional treatment to the subject. Any therapy known in the art or contemplated herein for use in alternating current electric field and / or apoptotic cancer cell therapy may be utilized in accordance with the methods of the present disclosure. Non-limiting examples of additional treatments that may be utilized include radiation therapy, photodynamic therapy, transarterial chemoembolization (TACE), or a combination thereof.

[0057] In certain (but non-limiting) embodiments, the method includes one or more additional steps. For example, but not limited to, the method may further include repeating any of the steps one or more times. Each step can be repeated as many times as necessary. Repeated application of the alternating electric field may position the transducer array slightly differently from its original position on the subject, and this repositioning of the array may further facilitate tumor / cancer treatment. Additionally, any of the administration steps (including administering the apoptotic cancer cell-containing composition and any administration steps) may be repeated various times and at various intervals according to known and / or commonly accepted dosage / treatment regimens for the composition(s) / therapy.

[0058] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions comprising any population of isolated, irradiated apoptotic cancer cells produced as described herein or otherwise contemplated. In certain (but non-limiting) embodiments, the apoptotic cancer cells are produced by exposing isolated cancer cells to an alternating electric field (either in vivo or ex vivo) and then irradiating the cancer cells to which the alternating electric field has been applied.

[0059] The immunogenic compositions may be formulated to be administered by any of the routes of administration disclosed herein or otherwise contemplated, hi certain (but non-limiting) embodiments, the immunogenic compositions are formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

[0060] In certain (but non-limiting) embodiments, the immunogenic composition may further include one or more additional active agents that further assist in stimulating the immune system to recognize and attack cancer cells in the subject. Non-limiting examples of additional agents that may be present in the immunogenic composition may 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 (CLRs), anti-LAG3 agents (such as, but not limited to, OPDUALAG™ and / or relatimab (Bristol-Myers Squibb, New York, NY)), and combinations thereof.

[0061] Certain non-limiting embodiments of the present disclosure may include any component of an AC electric field generation system, such as one or more transformers 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, and U.S. Patent Application Nos. 2018 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016. The present invention relates to kits that include apoptotic cancer cells (such as, but not limited to, a laser array and / or one or more hydrogel compositions) in combination with components, devices, and / or reagents used in one or more of the method steps, including isolating cancer cells, irradiating the cancer cells, isolating the irradiated and treated apoptotic cancer cells, and / or preparing the isolated apoptotic cancer cells for administration to a subject, according to one or more of the methods disclosed herein or otherwise discussed. The kits may optionally further include one or more of any of the compositions disclosed herein or otherwise discussed (such as, but not limited to, one or more compositions utilized in any concurrent treatment step(s)). The kits may optionally further include one or more devices (or one or more components of a device) used in one or more additional treatment steps.

[0062] In certain (but non-limiting) embodiments, the kit may further include instructions for carrying out any of the methods disclosed or contemplated herein. For example (but not limited to), the kit may include instructions for applying one or more components of an AC electric field generating system to the patient's skin, instructions for applying an AC electric field to the patient, instructions regarding when and how to administer the apoptotic cancer cell-containing composition(s) and, optionally, how to administer one or more optional additional compositions, and / or instructions regarding when to activate and deactivate the AC electric field in connection with the administration of the apoptotic cancer cell-containing composition(s) and / or the administration of one or more optional compositions.

[0063] In addition to the components detailed above, the kits may further contain other component(s) / reagent(s) for carrying out any of the specific methods described herein or otherwise discussed. For example (but not limited to), the kits may further contain: (i) components for preparing the skin before disposing the hydrogel composition and / or transducer array thereon (i.e., razors, cleaning compositions, or wipes / towels, etc.); (ii) components for removing the gel / transducer array(s); (iii) components for cleaning the skin after removing the gel / transducer array(s); (iv) components for isolating cancer cells / tumor portions; (v) components for irradiating the isolated and AC field-treated cancer cells / tumor portions; and / or (vi) components for isolating the irradiated and AC field-treated cancer cells. The nature of these additional component(s) / reagent(s) will depend on the particular treatment format, and their identification is within the skill of one of ordinary skill in the art, and therefore further description is not believed necessary. Additionally, the components / reagents in a kit may be contained in separate containers / compartments or various components / reagents may be combined in one or more containers / compartments depending on the sterility, cross-reactivity, and stability of the components / reagents.

[0064] The kit can be disposed in any packaging that enables the components contained therein to function according to the present disclosure. In certain non-limiting embodiments, the kit further comprises a sealed package into which the components are disposed. In certain (but non-limiting) embodiments, the sealed package is substantially impermeable to air and / or substantially impermeable to light.

[0065] In addition, the kits may also include a set of written instructions that describe how to use one or more components of the kit. Such kits can be used in any of the methods described herein or otherwise discussed.

[0066] 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.

[0067] Certain non-limiting embodiments of the present disclosure may include any component of an AC electric field generation system (see 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. 2018 / 0160933, 2019 / 0160933, 2019 / 0160936, and 2019 / 0160937). The present invention relates to a system comprising a therapeutic agent (such as, but not limited to, one or more transducer arrays and / or one or more hydrogel compositions disclosed in US Pat. Nos. 17956, 2019 / 0307781, and 2019 / 0308016) in combination with at least one of any of the compositions comprising apoptotic cancer cells generated as disclosed herein or otherwise contemplated. The system may optionally further include one or more of any of the compositions disclosed or contemplated herein. The system may optionally further include one or more devices (or one or more components of a device) used in various isolation, treatment, irradiation, or administration steps, or optional additional treatment / therapy steps. Example

[0068] The following examples are provided. However, it should be understood that the present disclosure is not limited to the application of the specific experiments, results, and experimental procedures disclosed herein below. Rather, this example is provided merely as one of various embodiments, and is intended to be illustrative rather than exhaustive.

[0069] The primary goal of cancer immunotherapy is to activate existing endogenous immune responses in cancer patients. Although significant progress has been made in this field, therapeutic efficacy still has room for improvement. Personalized cancer vaccines are a promising strategy to enhance antitumor immune responses through immunogenic apoptosis, also known as immunogenic cell death (ICD). ICD is characterized by the release of danger-associated molecular patterns, which attract immune cells to the tumor site. Previous studies have shown that TT field treatment promotes immunogenic cell death in cancer cells, ultimately engulfing them and stimulating the immune response. Then, neoantigens are presented to further initiate adaptive immunity.

[0070] To support the rationale for using TT fields as immunomodulatory agents, mice were treated with TT fields for 72 hours using the INOVITRO™ system (Novocure GmbH, Root, Switzerland). Cancer cells were then isolated from the mice and irradiated. The irradiated, apoptotic cancer cells were isolated from the culture and administered to mice as a vaccine to elicit an immune response against cancer progression. In this way, a method for enhancing immunity against cancer cells is combined with TT field therapy. The combination of TT field therapy and administration of a personalized apoptotic cancer cell-containing composition provides a synergistic effect compared to either treatment alone, initiating an immune response in the patient's body and enabling the immune system to eliminate cancer cells. Non-Limiting Exemplary Embodiments of the Inventive Concept

[0071] Exemplary Embodiment 1. A method of 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 current electric field to the isolated cancer cells; and (3) irradiating the isolated cancer cells to which the alternating current electric field has been applied to form an immunogenic composition.

[0072] Exemplary Embodiment 1a. A method of preparing an immunogenic composition, the method comprising: (a) applying an alternating current electric field to cancer cells isolated from a target area of ​​a subject; and (b) irradiating the isolated cancer cells to which the alternating current electric field has been applied to form an immunogenic composition.

[0073] Exemplary Embodiment 2. A method of preparing an immunogenic composition, the method comprising the steps of: (i) applying an alternating current electric field to a target area of ​​a subject; (2) isolating cancer cells from the target area of ​​the subject to which the alternating current electric field has been applied; and (3) irradiating the isolated cancer cells to which the alternating current electric field has been applied to form the immunogenic composition.

[0074] Exemplary Embodiment 2a. A method of preparing an immunogenic composition, the method comprising: (a) isolating cancer cells from a target area of ​​a subject, wherein an alternating electric field is applied to the target area of ​​the subject prior to isolating the cancer cells; and (b) irradiating the isolated cancer cells that have been subjected to the alternating electric field to form an immunogenic composition.

[0075] Exemplary Embodiment 3. 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 current electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells with the applied alternating current electric field; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying the alternating current electric field to the target area of ​​the subject.

[0076] Exemplary Embodiment 3a. A method of treating cancer in a subject, the method comprising: (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 is 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.

[0077] Exemplary Embodiment 4. A method of treating cancer in a subject, the method comprising the steps of: (i) applying an alternating current electric field to a target site in the subject; (2) isolating cancer cells from the target site in the subject that have had the alternating current electric field applied to them; (3) irradiating the isolated cancer cells that have had the alternating current electric field applied to them; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying the alternating current electric field to the target site in the subject.

[0078] Exemplary Embodiment 4a. A method of treating cancer in a subject, the method comprising: (a) isolating cancer cells from a target area of ​​the subject, wherein an alternating current electric field is applied to the target area of ​​the subject prior to isolating the cancer cells; (b) irradiating the isolated cancer cells to which the alternating current electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying an alternating current electric field to the target area of ​​the subject.

[0079] Exemplary embodiment 5. A method for reducing tumor volume and / or preventing tumor volume increase, wherein the tumor is present in a living organism and comprises a plurality of cancer cells, the method comprising: (1) isolating cancer cells from a target area of ​​a subject; (2) applying an alternating current electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells with the applied alternating current electric field; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying the alternating current electric field to the target area of ​​the subject.

[0080] Exemplary Embodiment 5a. A method for reducing tumor volume and / or preventing tumor volume increase, wherein the tumor is present in a living organism and comprises a plurality of cancer cells, the method comprising: (a) applying an alternating current electric field to cancer cells isolated from a target area of ​​a subject; (b) irradiating the isolated cancer cells to which the alternating current electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying the alternating current electric field to the target area of ​​the subject.

[0081] Exemplary embodiment 6. A method for reducing tumor volume and / or preventing tumor volume increase, wherein the tumor is present in a living organism and comprises a plurality of cancer cells, the method comprising: (i) applying an alternating current electric field to a target site in a subject; (2) isolating the alternating current electric field-applied cancer cells from the target site in the subject; (3) irradiating the isolated alternating current electric field-applied cancer cells; (4) administering the isolated and irradiated cancer cells to the subject; and (5) applying the alternating current electric field to the target site in the subject.

[0082] Exemplary embodiment 6a. A method for reducing tumor volume and / or preventing tumor volume increase, wherein the tumor is present in a living organism and comprises a plurality of cancer cells, the method comprising: (a) isolating cancer cells from a target area of ​​a subject, wherein an alternating current electric field is applied to the target area of ​​the subject prior to isolating the cancer cells; (b) irradiating the isolated cancer cells to which the alternating current electric field has been applied; (c) administering the isolated and irradiated cancer cells to the subject; and (d) applying an alternating current electric field to the target area of ​​the subject.

[0083] Exemplary Embodiment 7. The method of any of Exemplary 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 cells, cervical cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

[0084] Exemplary Embodiment 8. The method of any of Exemplary Embodiments 1-7, comprising at least one of: the alternating electric field is applied at a frequency ranging from about 50 kHz to about 1 MHz; the alternating electric field has an electric field strength of at least about 1 V / cm in at least a portion of the isolated cancer cells; and the duration of time for which the alternating electric field is applied is at least about 24 hours.

[0085] Exemplary Embodiment 9. The method of any one of exemplary embodiments 1-8, wherein the cells are irradiated for a time sufficient to ensure that the cells are non-viable.

[0086] Exemplary Embodiment 10. The method of exemplary embodiment 9, wherein the cells are irradiated for a period ranging from about 24 hours to about 96 hours.

[0087] Exemplary Embodiment 11. The method of any one of Exemplary Embodiments 1-2a, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

[0088] Exemplary Embodiment 12. The method of any one of Exemplary Embodiments 1-2a or 11, further comprising adding at least one additional composition to the immunogenic composition, wherein the at least one additional composition is 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.

[0089] Exemplary Embodiment 13. The method of any one of exemplary embodiments 3-10, wherein the isolated and irradiated cancer cells are administered intradermally, subcutaneously, intravenously, and / or intralymphatically.

[0090] Exemplary Embodiment 14. The method of any one of Exemplary Embodiments 3-10 and 13, wherein the isolated, irradiated cancer cells are administered to the subject in the form of at least one immunogenic composition, 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.

[0091] Exemplary Embodiment 15. The method of any one of Exemplary Embodiments 1-14, further comprising sorting out irradiated non-viable cancer cells to ensure that irradiated but viable cancer cells are not substantially administered to the subject (i.e., are present in the immunogenic composition).

[0092] Exemplary Embodiment 16. An immunogenic composition prepared by the method of any one of exemplary embodiments 1-2a, 7-12, and 15.

[0093] Exemplary embodiment 17. An immunogenic composition comprising a population of cancer cells isolated from a target region of a subject, treated ex vivo with an alternating electric field, and then irradiated.

[0094] Exemplary embodiment 18. An immunogenic composition comprising a population of cancer cells generated 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.

[0095] Exemplary Embodiment 19 The immunogenic composition of exemplary 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 cells, cervical cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

[0096] Exemplary Embodiment 20. The immunogenic composition of any one of exemplary embodiments 17-19, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

[0097] Exemplary Embodiment 21. The immunogenic composition of any one of claims 17-20, 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 combinations thereof.

[0098] Exemplary Embodiment 22 The immunogenic composition of any one of exemplary embodiments 17-21, wherein the irradiated cancer cells are non-viable.

[0099] While the above disclosure, in conjunction with specific experiments, results, and written language, describes the inventive concept(s), it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. 1. A method of preparing an immunogenic composition, said method comprising: (1) isolating cancer cells from a target area of ​​a subject; (2) applying an alternating current electric field to the isolated cancer cells; (3) irradiating the isolated cancer cells to which the AC electric field has been applied to obtain an immunogenic composition.

2. 2. The method of 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 cells, breast cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

3. In the step (2), the alternating electric field is applied at a frequency ranging from about 50 kHz to about 1 MHz; the alternating electric field has an electric field strength of at least about 1 V / cm in at least a portion of the isolated cancer cells; and 3. The method of claim 1, wherein the time period during which the alternating electric field is applied is at least about 24 hours.

4. 4. The method according to claim 1, wherein in step (3), the cells are irradiated for a time sufficient to ensure that the cells are non-viable.

5. 5. The method of any one of claims 1 to 4, further comprising the step of selecting non-viable irradiated cancer cells to ensure that the immunogenic composition is substantially free of viable irradiated cancer cells.

6. The method of any one of claims 1 to 5, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

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

8. 8. The method of 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 cells, cervical cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

9. In the step (2), the alternating electric field is applied at a frequency ranging from about 50 kHz to about 1 MHz; the alternating electric field has an electric field strength of at least about 1 V / cm in at least a portion of the isolated cancer cells; and 9. The method of claim 7 or 8, wherein the time period for which the alternating electric field is applied is at least about 24 hours.

10. 10. The method according to any one of claims 7 to 9, wherein in step (3), the cells are irradiated for a time sufficient to ensure that the cells are non-viable.

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

12. 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 intralymphatically.

13. 13. The method of 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 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. In the step (5), the alternating electric field is applied at a frequency ranging from about 50 kHz to about 1 MHz; the alternating electric field has an electric field strength of at least about 1 V / cm in at least a portion of the target area of ​​the subject; and The method according to any one of claims 7 to 13, wherein the time period during which the AC electric field is applied is at least about 24 hours.

15. 15. The method of any one of claims 7 to 14, further defined as a method for reducing the volume of a tumor and / or preventing an increase in the volume of the tumor, wherein the tumor is present in a living organism and comprises a plurality of cancer cells.

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

17. 17. The immunogenic composition of claim 16, 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 cells, cervical cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

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

19. 19. The immunogenic composition of any one of claims 16 to 18, further comprising 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.

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

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