Compositions, systems and methods for cancer treatment using alternating current electric fields and dendritic cells

Abstract

Compositions, systems, and methods for activating dendritic cells are disclosed. Also disclosed are compositions, systems, and methods for reducing the viability of cancer cells, treating cancer, and preventing tumor volume growth within a living organism, as well as methods for treating other diseases and infectious diseases. The systems and methods include applying an alternating current electric field in vivo or ex vivo to dendritic cell(s) and / or cancer cells isolated from a subject. The systems and methods may further include administering the activated dendritic cells to the subject. The composition includes a population of isolated dendritic cells that are activated by exposure to an alternating current electric field.

Description

[Technical Field]

[0001] Cross-Reference to Related Applications / Incorporation-by-Reference Statement This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 378,004, filed September 30, 2022, and U.S. Provisional Application No. 63 / 486,007, filed February 20, 2023. The entire contents of the above-referenced patent applications are expressly incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable [Background technology]

[0003] Tumor treating fields (TT fields) are low-intensity (e.g., 1-3 V / cm) alternating 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 are typically delivered through two pairs of transducer arrays, each pair consisting of an electrode array positioned on opposite sides of the body part being treated, that generate perpendicular electric fields within the tumor. Specifically, in the OPTUNE® system, one pair of electrodes is placed on the left and right (LR) sides of the tumor, and another pair 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.

[0004] 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.

[0005] 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. [Brief explanation of the drawings]

[0006] [Figure 1] A representative dendritic cell (DC) gating strategy utilized in accordance with the present disclosure is shown diagrammatically. Peripheral blood mononuclear cells (PBMCs) from control and 150 kHz TT field treatment groups were stained with the DC panel shown in Table 2, read by flow cytometry, and analyzed with FlowJo. The gating strategy is the same for all samples, as shown in the first two rows. Rows 3 and 4 display all viable DCs within three separate samples from the control and three separate samples from the TT field. [Figure 2]The graph shows the viability of dendritic cells in the various experimental groups. The mean viability is shown along with the standard error of the mean (SEM) for each DC subtype, based on eight experiments with a total of 15 technical replicates per group. [Figure 3] This graph shows DC maturation after TT field treatment according to the present disclosure. Three subtypes of live DCs (cDC1, cDC2, and pDC) were gated for two maturation markers, CD80 and CD83. From bottom to top of each 100% data bar, the individual segments of each data bar are as follows: (i) CD83+CD80+, (ii) CD83+, (iii) CD80+, and (iv) CD83-CD80-. The bars indicate the percentage of single-positive (positive for one activation marker but not the other, shown as the second and third segments of each bar for CD83+ and CD80+ cells, respectively) or double-positive (CD83+CD80+, shown as the bottom segment of each data bar), which represent fully mature DCs. Results represent the mean of eight experiments (15 technical replicates per group). SEM was added for the double-positive group. [Figure 4] The graph shows the percentage of double-positive (CD80+, CD83+) cDC1s in the control (shown as data bars on the left of each experiment) and 150 kHz TT field groups (shown as data bars on the right of each experiment) across eight experiments. Individual values ​​or the average of two to three replicates within a particular experiment are shown. The average double activation in the 150 kHz group was higher than in the control, although the degree of difference between experiments varied. [Figure 5] The graph shows the average cDC2 double positivity (CD80+ and CD83+) in the control (shown as data bars on the left of each experiment) and 150 kHz TT field-treated groups (shown as data bars on the right of each experiment) across the eight experiments performed. Shown are individual values ​​or the average of two to three replicates within a particular experiment. [Figure 6]The graph shows the average pDC double-positive (CD80+ and CD83+) fraction in the control (shown as data bars on the left side of each experiment) and 150 kHz TT field-treated groups (shown as data bars on the right side of each experiment) across all eight experiments. Shown are individual values ​​or the average of two to three replicates within a particular experiment. The pDC double-positive fraction is higher in the 150 kHz group than in the control samples (P=0.018). DETAILED DESCRIPTION OF THE INVENTION

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

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

[0012] 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.

[0013] 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.

[0014] 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).

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[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 effect.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Turning now to the inventive concept(s), disclosed herein are methods for maturing and activating dendritic cells, as well as the use of such activated dendritic cells in treating a subject. The methods include applying an alternating current electric field (e.g., a TT field) to immature dendritic cells or their precursor cells to mature / activate the dendritic cells. These methods may further include loading the dendritic cells with antigens from a particular source (including, but not limited to, cancer antigens, viral antigens, bacterial antigens, fungal antigens, etc.). The activated, antigen-loaded dendritic cells can then be administered to a subject for treatment of a condition, infection, or disease.

[0029] In certain (but non-limiting) embodiments, dendritic cells are loaded with antigens from cancer cells, including but not limited to, cancer cells that have been exposed to an alternating electric field (e.g., a TT field) either in vivo or ex vivo. Administration of activated, antigen-loaded dendritic cells to cancer patients provides synergistic results in the treatment of cancer.

[0030] The concepts of the present invention also encompass a combination cancer therapy that combines the steps of (i) generating alternating current electric field (e.g., TT field)-treated cancer cells by applying an alternating current electric field (e.g., TT field) ex vivo to a subject or cells isolated from the subject, (ii) activating dendritic cells using these alternating current electric field-treated cancer cells (i.e., loading dendritic cells ex vivo with antigens from the alternating current electric field-treated cancer cells), and (iii) administering to the subject at least one composition comprising the activated, antigen-loaded dendritic cells. The combination of an alternating current electric field and a composition(s) comprising dendritic cells activated by co-culture with alternating current electric field-treated cancer cells provides synergistic results in the treatment of cancer.

[0031] Certain non-limiting embodiments of the present disclosure relate to methods of activating dendritic cells, comprising applying an alternating current electric field in vitro to a composition comprising immature dendritic cells or their precursors for a time sufficient to generate activated dendritic cells. The method may further comprise contacting the activated dendritic cells with a source of antigen to generate antigen-bearing dendritic cells.

[0032] Certain non-limiting embodiments of the present disclosure relate to methods for preparing an immunogenic composition, comprising applying an alternating current electric field in vitro to a composition comprising immature dendritic cells or precursors thereof for a time sufficient to generate activated dendritic cells, contacting the activated dendritic cells with an antigen source to generate antigen-bearing dendritic cells, and isolating the antigen-bearing dendritic cells to form the immunogenic composition. The dendritic cells may be pulsed with an antigen (such as, but not limited to, a bacterial, viral, fungal, tumor, and / or cancer antigen) or co-cultured with an antigen source, for example (but not limited to), the dendritic cells may be co-cultured with at least one cancer cell isolated from a subject to generate the antigen-bearing dendritic cells.

[0033] Certain non-limiting embodiments of the present disclosure relate to methods for preparing an immunogenic composition, comprising: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells, (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to generate antigen-bearing dendritic cells, and (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell to form the immunogenic composition.

[0034] Certain non-limiting embodiments of the present disclosure relate to a method of treating cancer in a subject, comprising: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells, (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to generate antigen-bearing dendritic cells, (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell, and (4) administering the antigen-bearing dendritic cells to the subject.

[0035] Certain additional non-limiting embodiments of the present disclosure are directed to a method for reducing the volume of a tumor present in a living organism, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a tumor in a subject to generate antigen-bearing dendritic cells; (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell; and (4) administering the antigen-bearing dendritic cells to the subject.

[0036] Certain additional non-limiting embodiments of the present disclosure are directed to a method for preventing an increase in the volume of a tumor present in a living organism, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a tumor in a subject to generate antigen-bearing dendritic cells; (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell; and (4) administering the antigen-bearing dendritic cells to the subject.

[0037] In certain (but non-limiting) embodiments of the present disclosure, at least one cancer cell is also exposed to an AC electric field. This exposure may occur during the co-culture step. That is, at least a portion of steps (1) and (2) of any of the methods disclosed herein above or otherwise discussed herein can be performed simultaneously, whereby an AC electric field is also applied to at least one cancer cell during co-culture. Alternatively (and / or in addition), this exposure may occur prior to contact with the dendritic cells / progenitor cells. For example (but not limited to), an AC electric field may be applied to a target region of a subject prior to isolation of at least one cancer cell from the subject, and / or cancer cell(s) isolated from the subject may be exposed to an AC electric field ex vivo prior to co-culture.

[0038] Any of the methods disclosed or contemplated herein may, in certain non-limiting embodiments, further comprise a step (5) of applying an alternating electric field to a target area of ​​the subject after administration of the activated antigen-bearing dendritic cells.

[0039] Certain non-limiting embodiments of the present disclosure relate to a method of preparing an immunogenic composition, comprising co-culturing dendritic cells with at least one cancer cell isolated from a subject, wherein the at least one cancer cell has been exposed in vivo or ex vivo to an alternating electric field prior to co-culturing with the dendritic cells, to generate antigen-bearing dendritic cells, and isolating the population of antigen-bearing dendritic cells to form the immunogenic composition.

[0040] Certain non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition, comprising the steps of: (1) applying an alternating current electric field to a target site in a subject; (2) isolating cancer cells from the target site to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate activated antigen-bearing dendritic cells; and (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells contained therein to form the immunogenic composition.

[0041] Certain non-limiting embodiments of the present disclosure relate to a method of treating cancer in a subject, comprising the steps of: (1) applying an alternating current electric field to a target site in the subject; (2) isolating cancer cells from the target site to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate activated antigen-bearing dendritic cells; (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells contained therein; and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0042] Certain additional non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition, comprising: (1) isolating at least one cancer cell from a subject (such as, but not limited to, from at least a portion of a tumor in the subject), (2) applying an alternating current electric field ex vivo to the at least one isolated cancer cell, (3) co-culturing the at least one cancer cell that has been subjected to the alternating current electric field with dendritic cells to generate activated, antigen-bearing dendritic cells, and (4) isolating the activated, antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell contained therein to form the immunogenic composition.

[0043] Certain additional non-limiting embodiments of the present disclosure relate to a method of treating cancer in a subject, comprising the steps of: (1) isolating at least one cancer cell from the subject (such as, but not limited to, from at least a portion of a tumor in the subject), (2) applying an alternating current electric field ex vivo to the at least one isolated cancer cell, (3) co-culturing the at least one cancer cell that has been subjected to the alternating current electric field with dendritic cells to generate activated antigen-bearing dendritic cells, (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell contained therein, and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0044] Certain additional non-limiting embodiments of the present disclosure are directed to a method for reducing the volume of a tumor present in a living organism, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) applying an alternating current electric field to a target area of ​​a subject, the target area comprising the tumor; (2) isolating cancer cells from the target area to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate activated antigen-bearing dendritic cells; (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells present therein; and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0045] Certain additional non-limiting embodiments of the present disclosure are directed to a method for reducing the volume of a tumor present in a living organism, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) isolating at least one cancer cell from a subject (such as, but not limited to, from at least a portion of the subject's tumor), (2) applying an alternating current electric field ex vivo to the at least one isolated cancer cell, (3) co-culturing the at least one cancer cell that has been subjected to the alternating current electric field with dendritic cells to generate activated antigen-bearing dendritic cells, (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell contained therein, and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0046] Certain additional non-limiting embodiments of the present disclosure relate to a method for preventing an increase in the volume of a tumor present in a living body, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) applying an alternating current electric field to a target area of ​​a subject, the target area comprising the tumor; (2) isolating cancer cells from the target area to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate activated antigen-bearing dendritic cells; (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells present therein; and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0047] Certain additional non-limiting embodiments of the present disclosure are directed to a method for preventing an increase in the volume of a tumor present in a living organism, the tumor comprising a plurality of cancer cells, comprising the steps of: (1) isolating at least one cancer cell from a subject (such as, but not limited to, from at least a portion of the subject's tumor), (2) applying an alternating current electric field ex vivo to the at least one isolated cancer cell, (3) co-culturing the at least one cancer cell that has been subjected to the alternating current electric field with dendritic cells to generate activated antigen-bearing dendritic cells, (4) isolating the activated antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell contained therein, and (5) administering the activated antigen-bearing dendritic cells to the subject.

[0048] Any of the above methods disclosed or contemplated herein may, in certain non-limiting embodiments, further comprise a step (6) of applying an alternating electric field to a target area of ​​the subject after administration of the activated antigen-bearing dendritic cells.

[0049] After AC electric field treatment, the treated cancer cells utilized in the subsequent co-culture step can have any viability state, i.e., regardless of whether the cells are viable, apoptotic, and / or non-viable, the treated cancer cells used in the co-culture step are capable of inducing dendritic cell maturation in the co-culture step.

[0050] The utilized dendritic cells / progenitor cells thereof may be obtained from the subject or from another source, such as (but not limited to) an HLA-matched donor. For example (but not limited to), the method may further include isolating dendritic cells or progenitor cells thereof from the subject. When the method includes directly applying an AC electric field to the subject, the dendritic cells or progenitor cells thereof may be isolated from the subject before or after application of the AC electric field.

[0051] In another specific (but not limited to) embodiment, dendritic cells or their precursor cells are isolated from an HLA-matched donor. For example (but not limited to), if the patient has been treated with immunosuppressants or similar techniques (e.g., CRISPR technology to reduce the alloreactivity of mismatched donors), an HLA-matched donor can be used to isolate dendritic cells.

[0052] In certain (but non-limiting) embodiments, the method further comprises isolating immature monocytes (or other dendritic cell precursors) from the bloodstream of a subject or donor (such as, but not limited to, an HLA-matched donor) and generating immature dendritic cells from the immature monocytes / dendritic cell precursors.

[0053] In a specific (but non-limiting) embodiment, the composition comprising dendritic cells / their progenitors comprises peripheral blood mononuclear cells (PBMCs) isolated from the subject or an HLA-matched donor.

[0054] The co-culturing step can be performed under any conditions that allow dendritic cells to carry antigens from cancer cells. In certain (but not limited to) embodiments, the co-culturing step can be performed in the presence of at least one composition selected from the group consisting of cytokines, interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40 ligand (CD40L), Toll-like receptor (TLR) agonists, etc., and any combination thereof. The co-culturing step can also be performed with or without the application of an AC electric field.

[0055] Activated antigen-bearing dendritic cells can be isolated from the co-culture and the cancer cells contained therein using methods known in the art or methods discussed herein. Isolation of antigen-bearing dendritic cells can be performed in a single step or multiple steps. For example, but not limited to, cells can be first isolated from the co-culture by a general cell isolation method, and then a second specific isolation step (including, but not limited to, Percoll / Ficoll gradient, flow cytometry sorting, bead sorting, etc.) can be used to ensure that all cancer cells are removed and only antigen-bearing dendritic cells remain.

[0056] It should be noted that the isolated dendritic cells administered to a subject may contain antigen-loaded dendritic cells as well as unloaded cells, and therefore the composition administered to a subject may also be referred to herein as "co-cultured dendritic cells" or "antigen-experienced dendritic cells."

[0057] The compositions and methods of the present disclosure may be utilized with any type of cancer cell and / or to treat any type of cancer cell / cancer / tumor, such as, but not limited to, cancers that respond to alternating current electric fields and / or activated dendritic cell therapy. Non-limiting examples of cancer cells / cancers / tumors that may be utilized in accordance with the present disclosure include hepatocellular carcinoma / cancer cells, glioblastoma / glioblastoma cells, pleural mesothelioma / mesothelioma cells, differentiated thyroid cancer / cancer cells, advanced renal cell carcinoma / cancer cells, ovarian cancer / cancer cells, cervical cancer / cancer cells, breast cancer / cancer cells, pancreatic cancer / cancer cells, lung cancer / cancer cells (such as, but not limited to, non-small cell lung cancer / cancer cells), and the like, and any combination thereof.

[0058] In certain (but non-limiting) embodiments, the cancer cell(s) utilized in accordance with the present disclosure may be harvested from at least a portion of a tumor.

[0059] In certain (but non-limiting) embodiments, the cancer may be a solid tumor.

[0060] 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 AC electric fields in accordance with the present disclosure include those that function as part of AC electric field generation systems (i.e., TT field systems) described in, for example, but not limited to, 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.

[0061] The AC electric field can be generated at any frequency according to the present disclosure. For example (but not limited to), the AC electric field can be generated at about 50 kHz, about 60 kHz, about 70 kHz, about 75 kHz, about 80 kHz, about 90 kHz, about 100 kHz, about 105 kHz, about 110 kHz, about 115 kHz, about 120 kHz, about 125 kHz, about 130 kHz, about 135 kHz, about 140 kHz, about 145 kHz, about 150 kHz, about 155 kHz, about 160 kHz, about 165 kHz, about 170 kHz, z, approximately 175kHz, approximately 180kHz, approximately 185kHz, approximately 190kHz, approximately 195kHz, approximately 200kHz, approximately 225kHz, approximately 250kHz, approximately 275kHz, approximately 300kHz, approximately 325kHz, 350kHz, 375kHz, 400kHz, 425kHz, 450kHz, 475kHz, 500kHz, 550kHz, 600kHz, 650kHz, 700kHz, 75 The reference values ​​may be 0 kHz, about 800 kHz, about 850 kHz, about 900 kHz, about 950 kHz, about 1 MHz, etc., as well as ranges formed from any of the above values ​​(e.g., about 50 kHz to about 1 MHz, about 100 kHz to about 500 kHz, about 150 kHz to about 300 kHz, about 50 kHz to about 190 kHz, about 50 kHz to about 180 kHz, about 50 kHz to about 175 kHz, about 50 kHz to about 160 kHz, about 50 kHz to about 150 kHz, about 250 kHz to about 350 kHz, about 350 kHz to about 500 kHz, about 250 kHz to about 500 kHz, etc.), and ranges combining two integers between two of the above reference values ​​(e.g., about 122 kHz to about 313 kHz, about 78 kHz to about 298 kHz, etc.).

[0062] 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 ​​above, or a range formed from any of the values ​​above, or a range combining two integers between any two of the values ​​above.

[0063] The alternating electric field can have any electric field strength within the subject / cancer cells, so long as it is capable of functioning in accordance with the present disclosure. For example (but not limited to), the alternating electric field can have at least about 1 V / cm, about 1.5 V / cm, about 2 V / cm, about 2.1 V / cm, about 2.2 V / cm, about 2.3 V / cm, about 2.4 V / cm, about 2.5 V / cm, about 2.6 V / cm, about 2.7 V / cm, about 2.8 V / cm, about 2.9 V / cm, about 3 V / cm, about 3.5 V / cm, about 4 V / cm, Approx. 4.5V / cm, Approx. 5V / cm, Approx. 5.5V / cm, Approx. 6V / cm, Approx. 6.5V / cm, Approx. 7V / cm, Approx. 7.5V / cm, Approx. 8V / cm, Approx. 9V / cm, Approx. 9.5V / cm, approximately 10V / cm, approximately 10.5V / cm, approximately 11V / cm, approximately 11.5V / cm, approximately 12V / cm, approximately 12.5V / cm, approximately 13V / cm, approximately 13.5V / cm, approximately 1 The field strength may be about 4 V / cm, about 14.5 V / cm, about 15 V / cm, 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 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 ranges formed by combining two integers between the above two 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.).

[0064] The AC electric field can be applied in a single direction between a pair of arrays, or can be applied alternatingly in two (or more) directions (e.g., front-to-back and side-to-side) between two (or more) pairs of arrays. For example, certain TT field devices (such as the OPTUNE® system (Novocure Limited, St. Helier, Jerse)) operate in two directions to increase the likelihood that dividing cells will align with the electric field, allowing the electric field to exert its desired antimitotic effect. However, it is understood that the scope of the present invention also encompasses the application of a unidirectional AC electric field to achieve the immunogenic response described herein.

[0065] The AC electric field can be applied to the subject, dendritic cells, and / or cancer cells (or a co-culture comprising both dendritic cells and cancer cells) for any period of time disclosed herein or otherwise contemplated. For example, and without limitation, in certain non-limiting embodiments, the AC electric field is applied for a time sufficient to cause / promote maturation of dendritic cells and / or cause / promote presentation by dendritic cells of specific antigens from co-cultured cancer cells. For example, but not by way of limitation, the AC electric field may be applied for at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 76 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 109 hours, about 110 hours, about 111 hours, about 120 hours, about 121 hours, about 122 hours, about 123 hours, about 124 hours, about 125 hours, about 126 hours, about 127 hours, about 128 hours, about 129 hours, about 130 hours, about 1 The ranges that can be applied include 8 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., 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 that combine two integers between two of the above reference values ​​(e.g., a range of about 14 hours to about 68 hours, etc.).

[0066] In certain (but non-limiting) embodiments, the alternating electric field is applied for a period of at least about 24 hours.

[0067] Additionally, when an AC electric field is applied to a subject, the duration of application of the AC electric field can be continuous or cumulative. That is, the duration of application of the AC electric field can include a single session (i.e., continuous application) as well as multiple sessions with short breaks between sessions (i.e., continuous application over a cumulative period). For example, subjects are allowed to take breaks during treatment with the AC electric field device, and are expected to wear the device on their body and activate it for at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the total treatment period (e.g., 1 day, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, etc.).

[0068] Antigen-carrying dendritic cells (hereinafter "activated dendritic cells") can be placed and administered in any formulation known in the art or contemplated herein that allows the activated dendritic cells to exert a detrimental effect on a cancer present in a subject. For example, but not limited to, the activated dendritic cells can be administered in the form of a pharmaceutical composition comprising a combination of the activated dendritic cells and 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, conductive and non-conductive nanoparticles, 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).

[0069] In certain non-limiting embodiments, the pharmaceutical composition comprising activated dendritic cells can be further formulated as an immunogenic composition. The immunogenic composition can include the same components as the pharmaceutical composition described above (i.e., activated dendritic 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.

[0070] Additionally, any of the activated dendritic cell-containing compositions of the present disclosure may contain other agents that allow for administration of the composition via a particular route of administration. For example, but not limited to, the compositions may be formulated for administration via oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, and / or intravenous routes. Depending on the route of administration, the composition may also contain one or more additional components (i.e., an immunogenic composition and / or an additional therapeutic agent) in addition to the active agent. Examples of additional secondary compounds that may be present include, but are not limited to, carriers, gels, adhesives, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifiers, dispersing agents, and other substances well known in the art.

[0071] In certain (but non-limiting) embodiments, the composition comprising activated dendritic cells is administered to the subject intradermally, subcutaneously, intravenously, and / or intralymphatically.

[0072] In certain non-limiting embodiments, the method may further include one or more additional steps of applying an alternating current electric field to a target area of ​​a subject (1) after isolation of dendritic cells and / or their progenitor cells, (2) after isolation of cancer cells (and / or after tumor resection), and / or (3) before or after administration of an activated dendritic cell-containing composition. When additional alternating current electric field application steps are present, the alternating current electric field may be applied simultaneously with administration of the activated dendritic cell-containing composition, or sequentially, in whole or in part. In certain (but non-limiting) embodiments, the alternating current electric field may be applied after administration of the activated dendritic cell-containing composition. In other (but non-limiting) embodiments, the alternating current electric field may be applied simultaneously with or after administration of the activated dendritic cell-containing composition. In another (but non-limiting) embodiment, the activated dendritic cell-containing composition may be administered during application of the alternating current electric field (i.e., before the time during which the alternating current electric field is applied has elapsed).

[0073] For example (but not limited to), the activated dendritic cell-containing composition may be activated 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, or about 45 hours after the initiation of the additional application of the alternating current electric field. The administration time 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 a range formed from any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and a range 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.).

[0074] In other non-limiting examples, the activated dendritic cell-containing composition is maintained 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 The administration may be for up to 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 a range formed from any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and a range 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.).

[0075] In yet another non-limiting example, the activated dendritic cell-containing composition may be administered after the time period during which the additional alternating electric field is applied has elapsed, and ... It is administered within 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.

[0076] The activated dendritic cell-containing composition(s) can be administered to a subject at any concentration capable of inducing an inflammatory response to tumor or cancer cells. For example, but not limited to, activated dendritic 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 4 cells / kg body weight, approx. 10 5cells / 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 ~about 10 9 The dose may be in the range of 1000 to 15000 cells / kg body weight.

[0077] In certain (but non-limiting) embodiments, the method involves simultaneous treatment 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 activated dendritic cell-containing composition or as part of a second composition administered in whole or in part sequentially include, but are not limited to, lenvatinib, pembrolizumab, and other anti-PD-1 therapeutic agents, such as, but not limited to, tislelizumab, nivolumab, and cemiplimab; anti-LAG3 agents, such as, for example, 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.

[0078] When concurrent therapy is present, the concurrent therapy can be performed substantially simultaneously with the administration of the activated dendritic cell-containing composition, or completely 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).

[0079] When both the step of administering a second composition to a subject to which an activated dendritic cell-containing composition has been administered and the optional step of applying an alternating current electric field are present, the optional administration steps may be carried out before or after application of the alternating current electric field is initiated, and during 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 antigen-carrying dendritic cell-containing composition.

[0080] 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 a specific (but non-limiting) embodiment, the second composition is administered at least about 24 hours after application of the alternating electric field begins.

[0081] 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.

[0082] Further, for example (and not limitation), 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 administration of the activated dendritic cell-containing composition. The second composition may be administered for 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 a specific (but non-limiting) embodiment, the second composition is administered at least about 12 hours after administration of the composition containing antigen-carrying dendritic cells.

[0083] 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 TT field and / or activated dendritic 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.

[0084] In certain (but not limited to) 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 placement on the subject, and this repositioning of the array may further facilitate tumor / cancer treatment. Additionally, any of the administration steps (including the step of administering the dendritic 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.

[0085] While the methods described herein above relate to the use of activated dendritic cells in cancer treatment, it is understood that the scope of the present disclosure is not limited to use in cancer treatment. Rather, the present disclosure includes treating other related diseases, infections, or conditions for which dendritic cell therapy is beneficial by activating dendritic cells by exposure to an alternating electric field and then loading the activated dendritic cells with any desired antigen. For example, but not limited to, dendritic cells activated by exposure to an alternating electric field can be pulsed with (or co-cultured with) an antigen, including but not limited to, bacterial, viral, fungal, parasitic, tumor, or cancer antigens, and any combination thereof.

[0086] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions produced by any of the methods disclosed or contemplated herein.

[0087] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions comprising a population of isolated antigen-bearing dendritic cells, wherein the antigen-bearing dendritic cells are generated by co-culturing the dendritic cells with at least one cancer cell isolated from a subject to generate the antigen-bearing dendritic cells, and wherein the at least one cancer cell has been exposed in vivo or ex vivo to an alternating electric field prior to co-culturing with the dendritic cells.

[0088] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions comprising any population of isolated, antigen-loaded (and optionally exposed to an AC electric field) dendritic cells produced as described herein or otherwise contemplated. In certain (but non-limiting) embodiments, the dendritic cells are further co-cultured with at least one cancer cell isolated from a subject to generate activated, antigen-loaded dendritic cells. In certain (but non-limiting) embodiments, a TT field may be applied to the subject or either or both cell types prior to or during the co-culture step, and the dendritic cells and / or cancer cell(s) utilized in the co-culture are exposed ex vivo to the AC electric field. In yet another specific (but non-limiting) embodiment, the dendritic cells have been co-cultured or pulsed to load other types of antigens into the dendritic cells.

[0089] 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.

[0090] 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 (or other diseased, infected, or bacterial cells) within 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 leratimab (Bristol-Myers Squibb, New York, NY)), and combinations thereof.

[0091] The dendritic cells of the compositions and methods may include any dendritic cells known in the art or contemplated herein. For example, without limitation, the dendritic cells may include at least one of conventional DC1 (cDC1), cDC2, plasmacytoid DC (pDC), etc.

[0092] Specific non-limiting embodiments of the present disclosure include any component of an AC electric field generating system, such as one or more transducer arrays and / or one or more hydrogel compositions 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 a subject, dendritic cells (including, but not limited to, a dendritic cell precursor, a dendritic cell precursor, and / or a cancer cell), in combination with components, devices, and / or reagents for use in one or more of the following steps: isolating one or more cell types (e.g., dendritic cells or their progenitors and / or cancer cells), exposing the subject, dendritic cells (or their progenitors), and / or cancer cells to a TT field, co-culturing dendritic cells with cancer cells, pulsing the dendritic cells with an antigen, isolating activated dendritic cells, and / or formulating the activated dendritic cells for administration to a subject, according to 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 (including, 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) for use in one or more additional treatment steps.

[0093] In certain (but not limited to) 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 isolating one or more cell types and exposing a subject and / or cell culture to an AC electric field-generating system, instructions for isolating and formulating activated dendritic cells for administration to a subject, instructions for applying one or more components of an AC electric field-generating system to the skin of a subject, instructions for applying an AC electric field to a subject, instructions regarding when and how to administer the dendritic cell-containing composition(s) and, optionally, one or more optional additional compositions, and / or instructions regarding when to activate and turn off the AC electric field in connection with the administration of the dendritic cell-containing composition(s) and / or the administration of one or more optional compositions.

[0094] In addition to the components detailed above, the kits may further include 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 include: (i) components for preparing the skin before disposing the hydrogel composition and / or transducer array thereon (e.g., a razor, cleaning composition, 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 fractions, (v) components for isolating dendritic cells or their precursors, and / or (vi) components for maturing dendritic cells or their precursors. The nature of these additional component(s) / reagent(s) will depend on the particular treatment format, and their identification is within the skill of those of ordinary skill in the art, and therefore further description is not believed to be necessary. Additionally, the components / reagents in a kit may be in separate containers / compartments or the various ingredients / reagents may be combined in one or more containers / compartments depending on the sterility, cross-reactivity, and stability of the components / reagents.

[0095] 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.

[0096] Additionally, 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.

[0097] 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.

[0098] 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 No. 2018 / 0160933). The present invention relates to a system that includes one or more transducer arrays and / or one or more hydrogel compositions (such as, but not limited to, those disclosed in US Pat. Nos. 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016) in combination with at least one of any of the compositions comprising activated dendritic cells disclosed or contemplated herein. 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, co-culture, or administration steps, or optional additional treatment / therapy steps. Example

[0099] Examples are provided below. 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, the examples are provided merely as one of various embodiments, and are intended to be illustrative rather than exhaustive. Example 1

[0100] overview

[0101] Dendritic cells (DCs) are a key component of the cancer immune cycle, directing T cell activation or tolerance [Chen DS, Immunity, 2013]. The effects of TT fields on human DCs are still unclear, but the mechanism of action of TT fields may affect DC survival or activation. We tested the effects of TT fields on human monocyte-derived DCs (IL4+GMCSF-cultured monocytes) and then transitioned them to physiological blood-derived DCs: conventional DCs (cDCs), cDCs (cDCs), and plasmacytoid DCs (pDCs).

[0102] We tested the effects of TT fields on DC viability and their activation and maturation capacities. Because differences can be subtle and the INOVITRO™ system (Novocure GmbH, Root, Switzerland) can result in considerable intra-experimental variability, eight experiments were performed to confirm the results. The TT field conditions used were 150 kHz and 200 kHz, approved for the treatment of lung cancer and brain tumors, respectively. The field strength was 2.7 V / cm, and the exposure time was 48 hours (DCs change significantly after 2 days in culture). Freshly purified peripheral blood mononuclear cells (PBMCs) collected from healthy blood donors were used. PBMCs were evaluated on day 0 to define the DC baseline status (group 1). After 48 hours of culture, six additional groups were monitored. (Group 2) Control - TT field-naive DCs grown in INOVITRO™ dishes; (Group 3) Control + LPS + R848 (hereafter referred to as LPS) - fully activated under standard growth conditions; (Group 4) 150 kHz TT field - assessing the effect of the TT field on DC viability and maturation; (Group 5) 150 kHz TT field + LPS - assessing the effect of an activation signal under TT field conditions; (Group 6) 200 kHz TT field; and (Group 7) 200 kHz TT field + LPS.

[0103] After treatment, cells were stained with a 12-color DC panel to recognize all three physiological DC subsets, two maturation / activation markers, and cell viability. Analysis of eight experiments revealed a slight, but usually insignificant, decrease in viability (2-6%) under TT field conditions. Importantly, we repeatedly confirmed that a 150 kHz frequency induced potent DC activation compared with untreated controls in a 2d TT field. This activation was not induced by a 200 kHz TT field. Even under TT field conditions, activation with LPS, a potent DC activator, further activated DCs, demonstrating that physiological DCs can be effectively activated under TT field conditions.

[0104] These results indicate that physiological DCs of all subtypes retain viability and maturation capacity, two parameters important for effective DC function. These results also suggest that TT fields at specific frequencies may function as "physical adjuvants" that can promote DC maturation.

[0105] Experimental Design: To evaluate the effects of TT field treatment on the viability and activation potential of physiological dendritic cells in the blood, a series of eight experiments (a total of 15 technical repeats per group) was performed. The TT field frequencies used were 150 kHz and 200 kHz, and the field strength was 2.7 V / cm for 48 h. Fresh PBMCs from healthy donors were divided into seven groups as shown in Table 1. DC activation was induced by exposing cultures to 1 μg / mL lipopolysaccharide derived from E. coli strain O111:B4 plus 2.5 μg / mL R848 (Resiquimod) (denoted as LPS). After 48 h of treatment, cells were stained with a 12-color flow cytometry panel (Table 2). Nine channels were used to identify three DC subtypes, one channel was used to distinguish live from dead cells, and two channels were used to quantify the expression of DC maturation / activation markers CD80 and CD83. Stained cells were read on a 4-laser BD FACSAria-Fusion flow cytometer and analyzed using the FlowJo package. Treatment groups in the experiments conducted [Table 1] List of markers and clones in the DC flow cytometry panel [Table 2]

[0106] A gating strategy aimed at assessing the viability and maturity of three blood DC subtypes was devised (Figure 1). This figure shows the complete gating of the control, and the bottom two rows show the viability and activation of the control and TT field 150 kHz groups, with three DC subtypes shown in each row.

[0107] For most experiments performed, technical replicates were performed (15 replicates per 8 experiments). This was done because the INOVITRO™ system is noisy, has a low cell frequency (0.03%-0.5%), and is sensitive to slight changes during culture, especially when querying cells from PBMCs of different donors. The results presented per experiment are the average of the corresponding experiments (1-3 technical replicates per group). In each experiment, approximately 3.5 x 10 replicates were obtained per replicate. 6 PBMCs were cultured. Paired t-tests were used to test significance for each of the eight total experiments.

[0108] result

[0109] DC viability was largely maintained in the TT field.

[0110] Figure 2 shows the cross-sectional mean viability ± SEM for various groups. The bar graph scale ranges from 80 to 100%, revealing only minor differences in viability. Overall, the difference in viability between the control and non-LPS-treated groups on day 2 ranged from 2 to 6%. cDC1-4% showed a mean decrease in viability in the TT field (NS), cDC2-4% showed a mean decrease in viability at 150 kHz (P = 0.03), and 6% showed a mean decrease in viability at 200 kHz (NS). pDCs treated with 150 kHz showed a 2% decrease in viability (P = 0.01). While some mean values ​​were significantly decreased, the effect sizes in all groups were very small. LPS reduced DC viability in all groups, but there was no statistical difference between the TT field + LPS group and the control + LPS group.

[0111] Effects of TT fields on DC activation and maturation

[0112] Next, we tested the effect of the TT field on DC maturation and their ability to undergo activation by TLR receptor agonists. The combination of R848 and LPS, a TLR4 agonist, potently stimulated all physiological DCs [Lovgren T, Cancer Imm. Immunother, 2017]. Figure 3 shows the mean ± SEM of two monitored maturation markers, CD80 (B7.1) and CD83, either expressed alone (e.g., CD83+CD80-) or co-expressed (e.g., CD83+CD80+). Double-positive cells represent fully mature DCs [Dudek AM, Front Immu 2013].

[0113] In all DC types, maturation was nearly zero on day 0. By day 2 (48 hours), two cDC subtypes and, to a lesser extent, the pDC subtype, showed increased expression of CD80 and / or CD83. The increase in single-positive DCs in the cDC population was primarily driven by increased expression of CD83. In all DC subtypes, exposure to LPS increased the proportion of double-positive DCs.

[0114] Effects of TT field on cDC1 dendritic cells

[0115] CDC1s are the rarest dendritic cells in humans (3%–5% of dendritic cells) but are a critical component in tumor immune rejection. Uniquely, they can collect dead cell material and cross-present it on MHC-I to cytotoxic T cells [Wculek SK, Nat Rev Immunol 2020, Volovitz I, 2016 Int Rev Immunol]. Regarding cDC1 activation (Table 3), all treatment groups except 200 kHz induced significantly higher percentages of fully activated (double-positive) DCs. This increase in double-positive DCs was also detected in the 150 kHz treatment group without LPS (P=0.003). 200 kHz treatment did not induce an increase in the double-positive DC fraction compared to the control. 150+LPS and 200+LPS treatments showed similar double-positive fractions to the control (all in the range of 63%–70%), indicating that cDC1s are effectively activated under TT fields. 200kHz+LPS was significantly higher than 200kHz in double-positive cases, whereas 150kHz+LPS was only slightly higher (NS) than 150kHz. Of the total increase in double-positive cDC1 (total increase = control minus 150kHz+LPS on day 2), 93% was achieved by exposing cDC1 to a 150kHz TT field. Individual and combined results of double-positive cDC1 Fully mature DC fraction (CD80+, CD83+) [Table 3] Percentage of double-positive DCs at day 0 and after 48 hours of culture. Shown are individual percentages or the average of two to three technical repeats per treatment group. Average values ​​for each of eight experiments are shown, with two rows of statistical comparisons below. The top row compares all groups to the control (TT field untreated) on day 2. The bottom row compares each treatment group to the same group treated with LPS (e.g., 150 kHz vs. 150 kHz + LPS). NA in the percentage of responding cells indicates experiments in which specific DC subsets could not be clearly identified using the gating strategy.

[0116] To illustrate the effect of 150 kHz on cDC1s, Figure 4 shows a comparison of cDC1 double-positive cells (CD80+ and CD83+) in the control and 150 kHz groups in all eight experiments.

[0117] Effect of TTF on cDC2-type dendritic cells

[0118] CDC2s are the most common conventional dendritic cells and the only dendritic cell subtype found in significant numbers in glioblastomas. CDC2s can effectively activate helper T cells. CDC2s secrete more inflammatory cytokines (IL1β, IL6, TNFα, and IL8) than cDC1s. These may play tumor-promoting or anti-tumor roles within tumors, depending on their activation context and maturation state [Wculek SK, Nat Rev Immunol 2020, Volovitz I, Int Rev Immunol, 2016].

[0119] Table 4 shows that cDC2 activation mirrors cDC1 activation in many ways. Again, in all treated groups, 200 kHz resulted in significantly higher double-positive fully activated DCs than control on day 2. Exposure of cDC2 to 150 kHz without LPS significantly increased the percentage of double-positive cDC2 (P = 0.006), whereas 200 kHz had no effect. 150 kHz + LPS and 200 kHz + LPS showed similar double-positive rates to the control (all in the range of 48%-50%), indicating that cDC2 are effectively activated under TT fields. 200 kHz + LPS had a significantly higher double-positive rate than 200 kHz, whereas 150 kHz + LPS was only slightly higher than 150 kHz (NS). The 150 kHz frequency induced 72% of the total achievable increase in double-positive cDC2 (control minus 150 kHz + LPS on day 2). Individual and combined results of cDC2 double-positive fully mature DC fraction (CD80+, CD83+) [Table 4] Percentage of double-positive cDC2s on day 0 and after 48 hours of culture. Shown are individual percentages or the average of two to three technical repeats for each treatment group. Average values ​​for each of eight experiments are shown, with two rows of statistical comparisons below. The top row compares all groups to the control (TT field untreated) on day 2. The bottom row compares each treatment group to the same group treated with LPS (e.g., 150 kHz vs. 150 kHz + LPS). NA in the percentage of responder cells indicates experiments in which specific DC subsets could not be clearly identified using the gating strategy.

[0120] To illustrate the effect of 150 kHz TTF on cDC2 activation, Figure 5 shows a comparison of control (day 2) and 150 kHz double-positive cells.

[0121] Effect of TTF on pDCs

[0122] PDCs are the primary cells producing type 1 IFN (IFN-α / β) in the initial response to viruses, bacteria, or self-nucleic acids. IFN-α is an important antiviral and antitumor immune factor. [Wculek SK, Nat Rev Immunol 2020, Volovitz I, 2016 Int Rev Immunol]. Table 5 shows that the TT field had a relatively small effect on the percentage of double-positive pDCs compared with cDCs. At 150 kHz, the mean percentage of double-positive cells was significantly higher than in controls (P = 0.018), but only 11% of double-positive cells were in this group compared with 2% in controls (a 9% difference). Again, 200 kHz did not affect DC maturation. Activation to LPS was consistently higher without the TT field (mean 35%) than at 150 kHz (mean 18%) or 200 kHz (mean 12%), but these differences did not reach statistical significance. Again, 200 kHz + LPS was significantly higher than 200 kHz, and 150 kHz + LPS was non-significantly higher than 150 kHz, where only 56% of the increase in double-positive pDCs was induced by 150 kHz treatment. Individual and combined results of pDC double positives Fully mature DC fraction (CD80+, CD83+) [Table 5] Percentage of double-positive pDCs on day 0 and after 48 hours of culture. Shown are individual percentages or the average of two to three technical repeats per treatment group. Average values ​​for each of eight experiments are shown, with two rows of statistical comparisons below. The top row compares all groups to the control (TT field untreated) on day 2. The bottom row compares each treatment group to the same group treated with LPS (e.g., 150 kHz vs. 150 kHz + LPS). NA in the percentage of responding cells indicates experiments in which specific DC subsets could not be clearly identified using the gating strategy.

[0123] To illustrate the effect of 150 kHz TTF on pDC double-positive cells, the mean cell frequencies of control and matched 150 kHz samples are shown in Figure 6. Although the scale of pDC activation is lower than that of cDC, consistent pDC activation driven by 150 kHz is evident in all experiments.

[0124] conclusion

[0125] Only slight differences were observed in the viability of DCs treated with TT field, and no significant differences were observed in the percentage of double-positive (CD80+CD83+) DCs between control + LPS and TT field-treated samples + LPS, indicating that TT field does not inhibit DC maturation induced by activating agents such as LPS + R848.

[0126] Data from eight experiments demonstrate that physiological DCs, cDC1, cDC2, and pDC, are activated by 150 kHz TT field treatment. Indeed, for all DC subtypes, much of the maturation achievable by exposure to potent activators such as LPS can be achieved by simply exposing PBMCs to a 150 kHz TT field. These results suggest that the TT field may function as a "physical adjuvant" to enhance the maturation state of DCs in an antigen-nonspecific manner.

[0127] DCs play a critical role within tumors by attracting T cells to and within the tumor area and reactivating these T cells to enable effective antitumor responses [Wculek SK, Nat Rev Immunol 2020]. The immunostimulatory effects of 150 kHz can fully mature tumor-associated DCs after just 48 hours of exposure. DC maturation is a critical parameter for DCs' ability to induce potent antitumor responses. Differences between 150 kHz and 200 kHz TT field treatment may not only affect their ability to kill specific tumor cells. As previously shown, they may affect the viability and function of tumor-infiltrating T cells [Diamant G, 2021, J Immunol] or the maturation state or function of DCs within the therapeutic range. Example 2

[0128] 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.

[0129] To support the rationale for using TT fields as immunomodulatory agents, mice are treated with TT fields for 72 hours using the INOVITRO™ system (Novocure GmbH, Root, Switzerland). Cancer cells are then isolated from the mice. PBMCs are also isolated from the mice before or after TT field exposure, or from HLA-matched donors. The PBMCs are co-cultured with cancer cells and activated to load neoantigens into dendritic cells. Activated, antigen-loaded dendritic cells are isolated from the co-culture and cancer cells and administered to mice as a vaccine to elicit an immune response against cancer development.

[0130] 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 personalized activated dendritic cell-containing composition(s) provides a synergistic effect compared to either treatment alone, initiating a patient's immune response and enabling the immune system to eliminate cancer cells. Example 3

[0131] Dendritic cells were activated by exposure to a TT field as in Example 1. The TT field-exposed dendritic cells were then loaded with an antigen by pulse injection of the antigen of interest or by co-culturing with an antigen source (cancer cells, bacterial cells, virus-infected cells, fungus-infected cells, etc.). The activated, antigen-loaded dendritic cells were isolated from the culture and administered to the same subject or allogeneic subjects as an immunomodulator or vaccine to elicit an immune response. In this way, TT field treatment functions as a physical adjuvant. Non-Limiting Exemplary Embodiments of the Inventive Concept

[0132] Exemplary Embodiment 1. A method of activating dendritic cells, the method comprising applying an alternating electric field in vitro to a composition comprising immature dendritic cells or precursors thereof for a time sufficient to generate activated dendritic cells.

[0133] Exemplary Embodiment 2 The method of exemplary embodiment 1, further comprising contacting the activated dendritic cells with an antigen source to generate antigen-bearing dendritic cells.

[0134] Exemplary embodiment 3. A method of preparing an immunogenic composition, the method comprising applying an alternating current electric field in vitro to a composition comprising immature dendritic cells or their precursors for a time sufficient to generate activated dendritic cells, contacting the activated dendritic cells with an antigen source to generate antigen-bearing dendritic cells, and isolating the antigen-bearing dendritic cells to form the immunogenic composition.

[0135] Exemplary Embodiment 4 The method of exemplary embodiment 2 or 3, wherein the dendritic cells are pulsed with an antigen.

[0136] Exemplary Embodiment 5 The method of exemplary embodiment 2 or 3, wherein the dendritic cells are co-cultured with a source of antigen.

[0137] Exemplary Embodiment 6 The method of Exemplary Embodiment 5, wherein the contacting step is further defined as co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to generate antigen-bearing dendritic cells.

[0138] Exemplary Embodiment 7 The method of any of Exemplary Embodiments 2-6, wherein the antigen is selected from the group consisting of a bacterial antigen, a viral antigen, a fungal antigen, a tumor antigen, and a cancer antigen.

[0139] Exemplary embodiment 8. A method for preparing an immunogenic composition, the method comprising: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to generate antigen-bearing dendritic cells; and (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell to form the immunogenic composition.

[0140] Exemplary embodiment 9. A method of treating cancer in a subject, the method comprising: (1) applying an alternating current electric field ex vivo to a composition comprising immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to generate antigen-bearing dendritic cells; (3) isolating the antigen-bearing dendritic cells from the co-culture of (2) and the at least one cancer cell; and (4) administering the antigen-bearing dendritic cells to the subject.

[0141] Exemplary Embodiment 10 The method of exemplary embodiment 8 or 9, wherein at least a portion of steps (1) and (2) are performed simultaneously, whereby an alternating electric field is also applied to the at least one cancer cell during co-culture.

[0142] Exemplary Embodiment 11 The method of any of Exemplary Embodiments 8-10, wherein the at least one isolated cancer cell is exposed to an alternating electric field prior to step (2).

[0143] Exemplary Embodiment 12 The method of exemplary embodiment 11, wherein an alternating electric field is applied to the target region of the subject prior to isolating at least one cancer cell from the subject.

[0144] Exemplary Embodiment 13 The method of exemplary embodiment 11 or 12, wherein at least one cancer cell is exposed to an alternating electric field ex vivo prior to co-culturing.

[0145] Exemplary Embodiment 14 The method of any of Exemplary Embodiments 8-13, comprising isolating the composition from the subject prior to step (1).

[0146] Exemplary Embodiment 15 The method of exemplary embodiment 14, wherein the composition comprises peripheral blood mononuclear cells (PBMCs).

[0147] Exemplary Embodiment 16 The method of exemplary embodiment 14 or 15, wherein isolating the composition is further defined as comprising isolating immature monocytes (dendritic cell precursors) from the subject's bloodstream and generating immature dendritic cells from the immature monocytes / dendritic cell precursors.

[0148] Exemplary Embodiment 17 The method of any of Exemplary Embodiments 8-16, wherein the composition of (1) comprises PBMCs isolated from an HLA-matched donor.

[0149] Exemplary Embodiment 18 The method of any of Exemplary Embodiments 8-17, wherein the at least one cancer cell is further defined as at least part of a solid tumor.

[0150] Exemplary Embodiment 19. The method of any of Exemplary Embodiments 8-18, wherein step (2) is carried out in the presence of at least one composition selected from the group consisting of a cytokine, an interferon, a granulocyte-macrophage colony-stimulating factor (GM-CSF), a CD40 ligand (CD40L), a Toll-like receptor (TLR) agonist, and combinations thereof.

[0151] Exemplary Embodiment 20 The method of any of Exemplary Embodiments 9-19, further comprising the step of (5) applying an alternating electric field to the target region of the subject.

[0152] Exemplary Embodiment 21. The method of any of Exemplary Embodiments 9-20, further defined as a method of 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, and at least one cancer cell is isolated from the tumor prior to step (2).

[0153] Exemplary Embodiment 22 The method of exemplary embodiment 21, further comprising applying an alternating electric field to a target region of the subject prior to isolating the at least one cancer cell, wherein the target region comprises a tumor.

[0154] Exemplary embodiment 23. An immunogenic composition comprising a population of antigen-bearing isolated dendritic cells, wherein the antigen-bearing dendritic cells are generated by co-culturing the dendritic cells with at least one cancer cell isolated from a subject to generate the antigen-bearing dendritic cells, wherein the at least one cancer cell has been exposed in vivo or ex vivo to an alternating electric field prior to co-culturing with the dendritic cells.

[0155] Exemplary embodiment 24. A method of preparing an immunogenic composition, the method comprising: co-culturing at least one cancer cell isolated from a subject with dendritic cells to generate antigen-bearing dendritic cells, wherein the at least one cancer cell is exposed in vivo or ex vivo to an alternating electric field prior to co-culturing with the dendritic cells; and isolating the population of antigen-bearing dendritic cells to form the immunogenic composition.

[0156] Exemplary Embodiment 25. A method of preparing an immunogenic composition, the method comprising: (1) applying an alternating current electric field to a target site in a subject; (2) isolating cancer cells from the target site to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate antigen-bearing dendritic cells; and (4) isolating the antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells to form the immunogenic composition.

[0157] Exemplary embodiment 26. A method of treating cancer in a subject, the method comprising the steps of: (1) applying an alternating current electric field to a target site in the subject; (2) isolating cancer cells from the target site to which the alternating current electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating the antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells; and (5) administering the antigen-bearing dendritic cells to the subject.

[0158] Exemplary Embodiment 27 The method of exemplary embodiment 25 or 26, further comprising, prior to step (1), isolating from the subject the dendritic cells or precursor cells thereof utilized in step (3).

[0159] Exemplary Embodiment 28. The method of Exemplary Embodiment 27, wherein isolating dendritic cells is further defined as comprising the steps of isolating immature monocytes (dendritic cell precursors) from the subject's bloodstream, generating immature dendritic cells from the immature monocytes / dendritic cell precursors, and culturing the dendritic cell precursors to induce differentiation into mature dendritic cells.

[0160] Exemplary Embodiment 29 The method of any of Exemplary Embodiments 25-28, further comprising the step of isolating dendritic cells or precursor cells thereof from the subject after step (1).

[0161] Exemplary Embodiment 30 The method of any of Exemplary Embodiments 25-29, wherein the dendritic cells are isolated from an HLA-matched donor.

[0162] Exemplary Embodiment 31. The method of any of Exemplary Embodiments 25-30, wherein step (3) is carried out in the presence of at least one composition selected from the group consisting of a cytokine, an interferon, a granulocyte-macrophage colony-stimulating factor (GM-CSF), a CD40 ligand (CD40L), a Toll-like receptor (TLR) agonist, and combinations thereof.

[0163] Exemplary Embodiment 32 The method of any of Exemplary Embodiments 26-31, further comprising the step of (6) applying an alternating electric field to the target region of the subject.

[0164] Exemplary Embodiment 33. The method of any of Exemplary Embodiments 26-32, further defined as a method for reducing tumor volume and / or preventing tumor volume increase, wherein the tumor is present within a living organism and comprises a plurality of cancer cells, and step (1) is further defined as applying an alternating electric field to a target region of the subject, wherein the target region comprises the tumor.

[0165] Exemplary Embodiment 34. A method for preparing an immunogenic composition, the method comprising: (1) isolating at least one cancer cell from a subject; (2) applying an alternating current electric field ex vivo to the isolated at least one cancer cell; (3) co-culturing the isolated at least one cancer cell that has been subjected to the alternating current electric field with dendritic cells to generate antigen-bearing dendritic cells; and (4) isolating the antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell to form the immunogenic composition.

[0166] Exemplary embodiment 35. A method of treating cancer in a subject, the method comprising: (1) isolating at least one cancer cell from the subject; (2) applying an alternating current electric field ex vivo to the isolated at least one cancer cell; (3) co-culturing the isolated at least one cancer cell to which the alternating current electric field has been applied with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating the antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell; and (5) administering the antigen-bearing dendritic cells to the subject.

[0167] Exemplary Embodiment 36 The method of exemplary embodiment 34 or 35, wherein the at least one cancer cell is further defined as at least part of a tumor.

[0168] Exemplary Embodiment 37 The method of any of Exemplary Embodiments 34-36, wherein the dendritic cells are isolated from the subject and / or an HLA-matched donor.

[0169] Exemplary Embodiment 38. The method of any of Exemplary Embodiments 34-37, wherein step (3) is carried out in the presence of at least one composition selected from the group consisting of a cytokine, an interferon, a granulocyte-macrophage colony-stimulating factor (GM-CSF), a CD40 ligand (CD40L), a Toll-like receptor (TLR) agonist, and combinations thereof.

[0170] Exemplary Embodiment 39. The method of any of Exemplary Embodiments 35-38, further comprising the step of (6) applying an alternating electric field to the target region of the subject.

[0171] Exemplary Embodiment 40. The method of any of Exemplary Embodiments 35-39, further defined as 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, and steps (1)-(3) are further defined as: (1) excising at least a portion of the tumor from the subject; (2) applying an alternating current electric field ex vivo to at least a portion of the excised tumor; and (3) applying the alternating current electric field to at least a portion of the excised tumor and co-culturing it with dendritic cells to generate antigen-bearing dendritic cells.

[0172] Exemplary Embodiment 41. The method of any of Exemplary Embodiments 6-40, wherein the at least one cancer cell is selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, cervical cancer cells, breast cancer cells, and combinations thereof.

[0173] Exemplary Embodiment 42 The method of any of exemplary embodiments 9-22, 26-33, and 35-41, wherein the antigen-bearing dendritic cells are administered intradermally, subcutaneously, intravenously, and / or intralymphatically.

[0174] Exemplary Embodiment 43. The method of any of Exemplary Embodiments 9-22, 26-33, and 35-42, wherein the antigen-bearing dendritic 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 compound 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 (such as, but not limited to, OPDUALAG™ and / or leratimab (Bristol-Myers Squibb, New York, NY)), and combinations thereof.

[0175] Exemplary Embodiment 44. An immunogenic composition comprising a population of isolated antigen-bearing dendritic cells produced by the method of any of exemplary embodiments 3-8, 10-19, 24-25, 27-31, 34, 36-38, and 41.

[0176] Exemplary Embodiment 45. The method or immunogenic composition of any of Exemplary Embodiments 1-44, wherein the alternating electric field is applied at a frequency ranging from 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 cancer cells, and the alternating electric field is applied for a time period of at least about 24 hours.

[0177] Exemplary Embodiment 46. The method or immunogenic composition of exemplary embodiment 45, wherein the alternating electric field is applied at a frequency in the range of about 50 kHz to about 500 kHz, or in the range of about 50 kHz to about 190 kHz, or in the range of about 50 kHz to about 180 kHz, or in the range of about 50 kHz to about 175 kHz, or in the range of about 50 kHz to about 160 kHz, or in the range of about 50 kHz to about 150 kHz.

[0178] Exemplary Embodiment 47 The method or immunogenic composition of exemplary embodiment 46, wherein the alternating electric field is applied at a frequency of about 150 kHz and a field strength of about 2.7 V / cm for about 48 hours.

[0179] Exemplary Embodiment 48 The immunogenic composition of any of exemplary embodiments 23 and 44-47, further comprising a pharmaceutically acceptable carrier.

[0180] Exemplary Embodiment 49 The immunogenic composition of any of exemplary embodiments 23 and 44-48, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

[0181] Exemplary Embodiment 50. The immunogenic composition of any of exemplary embodiments 23 and 44-49, 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.

[0182] Exemplary Embodiment 51 The method or immunogenic composition of any of Exemplary Embodiments 1-50, wherein the dendritic cells comprise at least one of conventional DC1 (cDC1), cDC2, and plasmacytoid DC (pDC).

[0183] Exemplary embodiment 52. Use of the immunogenic composition of any of exemplary embodiments 23 and 44-51 in a method for treating cancer.

[0184] Exemplary embodiment 53. Use of an immunogenic composition in a method for treating cancer, including any of the methods of exemplary embodiments 9-22, 26-33, 35-43, 45-47, and 51.

[0185] 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 for activating dendritic cells, the method comprising: A method comprising applying an alternating electric field in vitro to a composition comprising immature dendritic cells or precursor cells thereof for a time sufficient to generate activated dendritic cells.

2. 10. The method of claim 1, further comprising contacting the activated dendritic cells with an antigen source to generate antigen-bearing dendritic cells.

3. 1. A method of preparing an immunogenic composition, said method comprising: applying an alternating electric field in vitro to a composition comprising immature dendritic cells or their precursors for a time sufficient to generate activated dendritic cells; contacting the activated dendritic cells with an antigen source to generate antigen-bearing dendritic cells; and isolating the antigen-bearing dendritic cells to form an immunogenic composition.

4. The method according to claim 2 or 3, wherein dendritic cells are pulsed with an antigen.

5. The method of claim 2 or 3, wherein the dendritic cells are co-cultured with a source of antigen.

6. The method of any one of claims 2 to 5, wherein the antigen is selected from the group consisting of a bacterial antigen, a viral antigen, a fungal antigen, a tumor antigen, and a cancer antigen.

7. 1. A method of preparing an immunogenic composition, said method comprising: (1) applying an AC electric field ex vivo to a composition containing immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to generate antigen-bearing dendritic cells; (3) isolating antigen-bearing dendritic cells from the co-culture of (2) and at least one cancer cell to form an immunogenic composition.

8. 1. A method of treating cancer in a subject, the method comprising: (1) applying an AC electric field ex vivo to a composition containing immature dendritic cells and / or dendritic cell precursors to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to generate antigen-bearing dendritic cells; (3) isolating antigen-bearing dendritic cells from the co-culture of (2) and at least one cancer cell; (4) administering the antigen-carrying dendritic cells to the subject.

9. 9. The method of claim 7 or 8, wherein at least a portion of steps (1) and (2) are performed simultaneously, whereby an alternating electric field is also applied to at least one cancer cell during co-culture.

10. 10. The method of any one of claims 7 to 9, wherein the at least one isolated cancer cell is exposed to an alternating electric field prior to step (2).

11. 11. The method of claim 10, wherein an alternating electric field is applied to a target region of the subject prior to isolating at least one cancer cell from the subject.

12. 12. The method of claim 10 or 11, wherein the at least one cancer cell is exposed to an alternating electric field ex vivo prior to co-culturing.

13. 13. The method of any one of claims 7 to 12, wherein the method comprises isolating the composition from the subject prior to step (1), and the composition comprises peripheral blood mononuclear cells (PBMCs).

14. The method according to any one of claims 7 to 13, wherein the composition (1) comprises PBMCs isolated from an HLA-matched donor.

15. 15. The method of any one of claims 7 to 14, wherein the at least one cancer cell is further defined as at least part of a solid tumor.

16. The method of any one of claims 7 to 15, wherein step (2) is carried out in the presence of at least one composition selected from the group consisting of cytokines, interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40 ligand (CD40L), Toll-like receptor (TLR) agonists, and combinations thereof.

17. moreover, (5) The method according to any one of claims 8 to 16, further comprising the step of applying the alternating electric field to a target site of the subject.

18. 1. An immunogenic composition comprising: An immunogenic composition comprising a population of antigen-bearing isolated dendritic cells, the antigen-bearing dendritic cells being generated by co-culturing at least one cancer cell isolated from a subject with dendritic cells to generate the antigen-bearing dendritic cells, the at least one cancer cell being exposed to the alternating electric field in vivo or ex vivo prior to co-culturing with the dendritic cells.

19. 1. A method of preparing an immunogenic composition, said method comprising: co-culturing at least one cancer cell isolated from a subject with dendritic cells to generate antigen-bearing dendritic cells, wherein the at least one cancer cell has been exposed in vivo or ex vivo to an alternating electric field prior to co-culturing with the dendritic cells; and isolating the population of antigen-bearing dendritic cells to form an immunogenic composition.

20. 1. A method of preparing an immunogenic composition, said method comprising: (1) applying an alternating electric field to a target area of ​​the subject; (2) isolating cancer cells from the target area to which the AC electric field is applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells to form an immunogenic composition.

21. 1. A method of treating cancer in a subject, the method comprising: (1) applying an alternating electric field to a target area of ​​the subject; (2) isolating the cancer cells from the target area to which the AC electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating antigen-bearing dendritic cells from the co-culture of (3) and the cancer cells; (5) administering the antigen-carrying dendritic cells to a subject.

22. The method of claim 20 or 21, further comprising, prior to step (1), isolating dendritic cells or precursor cells thereof to be used in step (3) from the subject.

23. The method of any one of claims 20 to 22, wherein the dendritic cells are isolated from an HLA-matched donor.

24. The method of any one of claims 20 to 23, wherein step (3) is carried out in the presence of at least one composition selected from the group consisting of cytokines, interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40 ligand (CD40L), Toll-like receptor (TLR) agonists, and combinations thereof.

25. moreover, (6) The method of any one of claims 21 to 24, comprising the step of applying an alternating electric field to a target region of the subject.

26. 1. A method of preparing an immunogenic composition, said method comprising: (1) isolating at least one cancer cell from a subject; (2) applying an alternating current electric field to the isolated at least one cancer cell ex vivo; (3) co-culturing the at least one cancer cell isolated by applying the AC electric field with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell to form an immunogenic composition.

27. 1. A method of treating cancer in a subject, the method comprising: (1) isolating at least one cancer cell from the subject; (2) applying an alternating current electric field to the isolated at least one cancer cell ex vivo; (3) co-culturing the at least one cancer cell isolated by applying the AC electric field with dendritic cells to generate antigen-bearing dendritic cells; (4) isolating antigen-bearing dendritic cells from the co-culture of (3) and the at least one cancer cell; (5) administering the antigen-carrying dendritic cells to the subject.

28. 28. The method of claim 26 or 27, wherein the at least one cancer cell is further defined as at least part of a tumor.

29. The method of any one of claims 26 to 28, wherein the dendritic cells are isolated from the subject and / or an HLA-matched donor.

30. 30. The method of any one of claims 26 to 29, wherein step (3) is carried out in the presence of at least one composition selected from the group consisting of cytokines, interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40 ligand (CD40L), Toll-like receptor (TLR) agonists, and combinations thereof.

31. The method of any one of claims 27 to 30, further comprising the step of (6) applying an alternating current electric field to the target site of the subject.

32. 32. The method of any one of claims 7 to 31, wherein the at least one cancer cell is selected from the group consisting of hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, cervical cancer cells, breast cancer cells, and combinations thereof.

33. The method of any one of claims 8 to 17, 21 to 25, and 27 to 32, wherein the antigen-bearing dendritic cells are administered intradermally, subcutaneously, intravenously, and / or intralymphatically.

34. 34. The method of any one of claims 8-17, 21-25, and 27-33, wherein the antigen-bearing dendritic cells are administered to the subject in the form of the at least one immunogenic composition, wherein the at least one immunogenic composition further comprises at least one compound 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 (such as, but not limited to, OPDUALAG™ and / or leratimab (Bristol-Myers Squibb, New York, NY)), and combinations thereof.

35. 1. An immunogenic composition comprising: A population of isolated antigen-bearing dendritic cells produced by the method of any one of claims 3-7, 9-16, 19-20, 22-24, 26, 28-30, and 32.

36. 36. The method or immunogenic composition of any one of claims 1-35, wherein 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 cancer cells, and the duration for which the alternating electric field is applied is at least about 24 hours.

37. 37. The method or immunogenic composition of claim 36, wherein the alternating electric field is applied at a frequency in the range of about 50 kHz to about 500 kHz, or in the range of about 50 kHz to about 190 kHz, or in the range of about 50 kHz to about 180 kHz, or in the range of about 50 kHz to about 175 kHz, or in the range of about 50 kHz to about 160 kHz, or in the range of about 50 kHz to about 150 kHz.

38. 38. The method or immunogenic composition of claim 37, wherein the alternating electric field is applied at a frequency of about 150 kHz and a field strength of about 2.7 V / cm for about 48 hours.

39. The immunogenic composition of any one of claims 18 and 35 to 38, further comprising a pharmaceutically acceptable carrier.

40. The immunogenic composition of any one of claims 18 and 35 to 39, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous, and / or intralymphatic administration.

41. 41. The immunogenic composition of any one of claims 18 and 35-40, 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.

42. 42. The method or immunogenic composition of any one of claims 1 to 41, wherein the dendritic cells comprise at least one of conventional DC1 (cDC1), cDC2, and plasmacytoid DC (pDC).

43. Use of the immunogenic composition of any one of claims 18 and 35 to 41 in a method for treating cancer.

44. 19. Use of an immunogenic composition in a method for treating cancer, the use comprising the method of any one of claims 8 to 17, 21 to 25, 27 to 34, 36 to 38, and 42.

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