Compositions, systems and methods for treating cancer using alternating electric field and dendritic cells

Abstract

Compositions, systems, and methods for activating dendritic cells are disclosed. Also disclosed are compositions, systems, and methods for reducing cancer cell activity and treating cancer, as well as preventing an increase in the volume of a tumor present in a living subject, as well as methods of treating other diseases and infections. The systems and methods involve applying an alternating electric field in vivo or in vitro to dendritic cells and / or to a subject or cancer cells isolated from the subject. The systems and methods may further include administering the activated dendritic cells to the subject. The composition comprises a population of isolated dendritic cells activated by exposure to an alternating electric field.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS / INCORPORATION BY REFERENCE STATEMENT

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 378,004, filed on September 30, 2022, and U.S. Provisional Application No. 63 / 486,007, filed on February 20, 2023, under 35 U.S.C. § 119(e). The entire contents of the above-referenced patent applications are hereby incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] not applicable. Background Art

[0005] Tumor Treatment Fields (TTFields) are low intensity (e.g., 1-3 V / cm) alternating electric fields in the mid-frequency range (such as, but not limited to, 100-500 kHz) that target solid tumors by disrupting mitosis. This non-invasive treatment targets solid tumors and is described, for example, in U.S. Patents 7,016,725; 7,089,054; 7,333,852; 7,565,205; 8,244,345; 8,715,203; 8,764,675; 10,188,851; and 10,441,776. TTFields are typically delivered via two pairs of transducer arrays that generate perpendicular fields within the tumor being treated; the electrode arrays that make up each of these pairs are positioned on opposite sides of the body part being treated. More specifically, for In the TTField system, one pair of electrodes is located at the left and right sides (LR) of the tumor, and the other pair of electrodes is located at the anterior and posterior sides (AP) of the tumor. TTField is approved for the treatment of glioblastoma multiforme (GBM) and can be used, for example, by system (Novocure Limited, St. Helier, Jersey), The system includes a transducer array placed on a light head over the patient.

[0006] Used in Each transducer array in the device that delivers TTFields includes a set of ceramic disc electrodes that are coupled to the patient's skin (such as, but not limited to, a shaved head for a patient being treated for GBM) via a layer of conductive medical gel. The medical gel is intended to deform to match the contours of the body and provide good electrical contact between the array and the skin; thus, the gel interface bridges the skin and reduces interference. The device is intended to be worn continuously by the patient for 2 to 4 days, then removed, allowing the patient to perform hygiene care and re-shave the head (if necessary), followed by reapplication of a new set of arrays. In this way, the medical gel remains in essentially continuous contact with the patient's skin area for 2-4 days at a time, and the skin area is only uncovered and exposed to the environment for a brief period of time before more medical gel is applied to the skin.

[0007] Another form of cancer treatment is cancer immunotherapy. The main goal of cancer immunotherapy is to activate pre-existing endogenous immune responses in cancer patients. Some possible targets for cancer immunotherapy include tumor-specific antigens derived from mutations, or neoantigens, which are not present in normal cells and can be recognized by the immune system, thus providing specific targets for anti-tumor therapy. Although significant progress has been made in this field, the effectiveness of treatment still needs to be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A representative dendritic cell (DC) gating strategy utilized in accordance with the present disclosure is depicted in graphical form. Peripheral blood mononuclear cells (PBMCs) from the control and 150kHz TTField treated groups were stained with the DC panel provided in Table 2, read by flow cytometry, and analyzed by FlowJo. The same gating strategy was used for all samples shown in the first two rows. The third and fourth rows show all active DCs in 3 independent samples of the control group and 3 independent samples of the TTField group.

[0009] Figure 2 The activity of dendritic cells in different experimental groups is depicted in a graph. The average activity of each DC subset is presented as standard error of the mean (SEM) based on 8 experiments with a total of 15 technical replicates per group.

[0010] Figure 3The maturation of DCs after TTField treatment according to the present disclosure is depicted in a graphical form. Live DCs of three subtypes (cDC1, cDC2, and pDC) were gated by two maturation markers, CD80 and CD83. From the bottom to the top of each 100% data bar, the various parts of the data bar are as follows: (i) CD83+CD80+; (ii) CD83+; (iii) CD80+; and (iv) CD83-CD80-. These data bars depict the percentage of cells that are single positive (positive for only one activation marker but not for the other, shown as the second and third parts of each data bar for CD83+ and CD80+ cells, respectively) or double positive (CD83+CD80+, shown as the bottom part of each data bar), and double positive represents fully mature DCs. The results are the average of 8 experiments (15 technical replicates per group). SEM is added to the double positive group.

[0011] Figure 4 The proportion of double positive (CD80+, CD83+) cDC1 in the control group (left data bar of each experiment) and the 150kHz TTField treated group (right data bar of each experiment) in 8 experiments is depicted in a graph. Single values ​​or the average of 2-3 replicates in a particular experiment are shown. The average of double activation in the 150kHz group was higher than that in the control group, but the degree of difference varied between experiments.

[0012] Figure 5 The average values ​​of cDC2 double positive (CD80+ and CD83+) in the control group (presented as the left data bar of each experiment) and the 150kHz TTField treated group (presented as the right data bar of each experiment) in 8 experiments performed are depicted in a graph. Single values ​​or the average values ​​of 2-3 replicates in a specific experiment are shown.

[0013] Figure 6 The average values ​​of pDC double positive (CD80+ and CD83+) in the control group (presented as the left data bar of each experiment) and the 150kHz TTField treated group (presented as the right data bar of each experiment) in 8 experiments performed are depicted in a graph. Single values ​​or the average of 2-3 replicates in a specific experiment are shown. The proportion of pDC double positive in the 150kHz group was higher than that in the control samples (P=0.018). DETAILED DESCRIPTION

[0014] Before explaining at least one embodiment of the inventive concept in detail through exemplary language and results, it should be understood that the application of the inventive concept is not limited to the details of the construction and arrangement of the components set forth in the following description. The inventive concept can have other embodiments or be practiced or executed in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning; and these embodiments are intended to be exemplary rather than exhaustive. In addition, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0015] Unless otherwise defined herein, scientific and technical terms used in conjunction with the inventive concepts disclosed herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification. Terms and laboratory procedures and techniques associated with analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry as described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0016] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the technical level of those skilled in the art to which the inventive concepts disclosed in the present invention pertain. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, as if each individual patent or publication was specifically and individually indicated to be incorporated herein by reference.

[0017] All compositions, assemblies, systems, kits and / or methods disclosed herein can be prepared and performed according to the present disclosure without undue experimentation. Although the compositions, assemblies, systems, kits and methods of the present invention have been described according to specific embodiments, it will be understood by those skilled in the art that the steps or step sequences of the compositions and / or methods and methods described herein can be changed without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0018] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0019] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a" or "an" may mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Thus, unless the context clearly indicates otherwise, the terms "a", "an", and "the" include plural referents. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or an increased number of compounds. The term "plurality" means "two or more".

[0020] The use of the term "at least one" should be understood to include one as well as any amount more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, quantities of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. Furthermore, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used solely for the purpose of distinguishing two or more items and is not meant to imply, for example, any order or sequence or importance of one item relative to another or any order of addition.

[0021] The term "or" as used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" may be satisfied by any of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exists).

[0022] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the appearance of the phrase "in some embodiments" or "an example" in different places in the specification does not necessarily refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0023] In this application, the term "about" is used to indicate that a value includes the inherent error variation of the composition / device / apparatus, the method used to determine the value, or the variation that exists in the subject matter. For example, but not as a limitation, when the term "about" is used, the specified value can vary within plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent of the specified value, as such variations are suitable for performing the disclosed methods and as understood by those of ordinary skill in the art.

[0024] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of containing, such as "includes" and "include"), or "containing" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0025] As used herein. The term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will appreciate that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0026] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a large extent. For example, when related to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that the two items are very close to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0027] The term "pharmaceutically acceptable" refers to compounds and compositions that are suitable for administration to humans and / or animals without excessive adverse side effects (such as (but not limited to) toxicity, irritation and / or allergic response) and that are commensurate with a reasonable benefit / risk ratio.

[0028] As used herein, the term "patient" or "subject" includes both human and veterinary subjects. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including but not limited to humans, domestic and farm animals, non-human primates, and any other animal with mammary tissue.

[0029] The term "treatment" refers to therapeutic treatment as well as prophylactic or preventive measures. Those in need of treatment include, but are not limited to, individuals who already have a particular condition / disease / infection and individuals at risk of acquiring a particular condition / disease / infection (e.g., individuals in need of prophylactic / preventive measures). The term "treatment" refers to the administration of an agent / element / method to a patient for therapeutic and / or prophylactic / preventive purposes.

[0030] As used herein, the term "therapeutic composition" or "pharmaceutical composition" refers to an agent that can be administered in vivo to produce a therapeutic and / or prophylactic / preventive effect.

[0031] Administration of a therapeutically effective amount or a prophylactically effective amount is to provide a therapeutic benefit in the treatment, prevention and / or management of a disease, disorder and / or infection. The specific amount that is therapeutically effective can be readily determined by a general practitioner and can vary according to factors known in the art, such as, but not limited to, the type of disorder / disease / infection, the patient's medical history and age, the stage of the disorder / disease / infection, and the combined administration of other agents.

[0032] The term "effective amount" refers to the amount of a bioactive molecule or its conjugate or derivative, or the amount of a treatment regimen (i.e., an alternating electric field), which, when used in the manner conceived by the present invention, is sufficient to show a detectable therapeutic effect without excessive adverse side effects (such as, but not limited to, toxicity, irritation, and allergic reactions), and the adverse side effects are comparable to a reasonable benefit / risk ratio. The therapeutic effect may include, for example, but not limited to, preventing, inhibiting, or reducing the occurrence of at least one illness, disease, and / or infection. The effective amount of the subject will depend on the type of the subject, the size and health of the subject, the nature and severity of the illness / disease / infection to be treated, the method of administration, the duration of treatment, the nature of simultaneous therapy (if any), the specific preparation used, etc. Therefore, it is impossible to predetermine an accurate effective amount. However, the effective amount for a given situation can be determined by a person of ordinary skill in the art using routine experiments based on the information provided herein.

[0033] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjuvant therapy" and will be understood to mean that a patient in need of treatment is treated with or administered another drug for the condition / disease / infection in combination with the treatment of the present disclosure. Concurrent therapy can be sequential therapy, in which the patient is first treated with one therapeutic regimen / drug composition and then with another therapeutic regimen / drug composition, or both therapeutic regimens / drug compositions are administered simultaneously.

[0034] The terms "administration" and "administering" as used herein are understood to include all routes of administration known in the art, including but not limited to oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal and intravenous routes, and include local and systemic administration. In addition, the compositions of the present disclosure (and / or methods of administration thereof) can be designed to provide delayed, controlled or sustained release using formulation techniques well known in the art.

[0035] Turning now to the inventive concept, disclosed herein is a method for maturing and activating dendritic cells, and the use of such activated dendritic cells for treating a subject. The method involves applying an alternating electric field (such as but not limited to TTField) to immature dendritic cells or their precursors to mature / activate dendritic cells. The method may further include the following steps: antigens from a specific source (such as but not limited to cancer antigens, viral antigens, bacterial antigens, fungal antigens, etc.) are loaded onto dendritic cells. Then, activated and antigen-loaded dendritic cells can be administered to a subject for the treatment of a certain condition, infection or disease.

[0036] In a specific (but non-limiting) embodiment, the dendritic cells are loaded with antigens from cancer cells, including but not limited to cancer cells that have also been exposed to alternating electric fields (such as but not limited to TTFields) in vivo or in vitro. Administration of activated and antigen-loaded dendritic cells to a subject with cancer can produce a synergistic effect in cancer treatment.

[0037] The present invention also contemplates a combination therapy for cancer that combines the following steps: (i) generating cancer cells treated with an alternating electric field (such as but not limited to TTField) by applying an alternating electric field (such as but not limited to TTField) to a subject or to cells isolated from a subject in vitro; (ii) using these alternating electric field-treated cancer cells to activate dendritic cells (i.e., antigens from cancer cells treated with alternating electric fields are loaded onto dendritic cells in vitro); and (iii) administering at least one composition containing activated and antigen-loaded dendritic cells to the subject. The combination of an alternating electric field and a composition comprising dendritic cells activated by co-culture with cancer cells treated with alternating electric fields can produce a synergistic effect in cancer treatment.

[0038] Certain non-limiting embodiments of the present disclosure relate to a method for activating dendritic cells, the method comprising: applying an alternating electric field to a composition comprising immature dendritic cells or precursors thereof in vivo for a sufficient period of time to produce activated dendritic cells. The method may further comprise the steps of: contacting the activated dendritic cells with an antigen source to produce antigen-loaded dendritic cells.

[0039] Certain non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition, wherein the method comprises the following steps: applying an alternating electric field to a composition comprising immature dendritic cells or their precursors in vivo for a sufficient time to produce activated dendritic cells; contacting the activated dendritic cells with an antigen source to produce antigen-loaded dendritic cells; and separating the antigen-loaded dendritic cells to form the immunogenic composition. The dendritic cells can be impacted by antigens (such as, but not limited to, bacterial antigens, viral antigens, fungal antigens, tumor antigens, and / or cancer antigens), or co-cultured with an antigen source; for example (but not limited to), the dendritic cells can be co-cultured with at least one cancer cell separated from a subject to produce antigen-loaded dendritic cells.

[0040] Certain non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition. The method comprises the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells; and (3) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell in (2) to form the immunogenic composition.

[0041] Certain non-limiting embodiments of the present disclosure relate to a method for treating cancer in a subject. The method comprises the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture described in (2) and the at least one cancer cell; and (4) administering the antigen-loaded dendritic cells to the subject.

[0042] Certain additional non-limiting embodiments of the present disclosure relate to a method for reducing the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture described in (2) and the at least one cancer cell; and (4) administering the antigen-loaded dendritic cells to the subject.

[0043] Certain additional non-limiting embodiments of the present disclosure relate to a method for preventing the increase in the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture described in (2) and the at least one cancer cell; and (4) administering the antigen-loaded dendritic cells to the subject.

[0044] In certain specific (but non-limiting) embodiments of the present disclosure, the at least one cancer cell is also exposed to an alternating electric field. This exposure can occur during the co-culture step; that is, at least a portion of steps (1) and (2) in any of the methods disclosed above or otherwise contemplated herein can be performed simultaneously, so that during the co-culture, the alternating electric field is also applied to the at least one cancer cell. In addition (and / or in addition), this exposure can also occur before contact with the dendritic cells / precursors. For example (but not by way of limitation), before separating the at least one cancer cell from the subject, an alternating electric field can be applied to the target area of ​​the subject; and / or, before co-culture, the cancer cell separated from the subject can also be exposed to an alternating electric field in vitro.

[0045] Any of the methods disclosed herein or otherwise contemplated may, in certain non-limiting embodiments, further comprise the following step: (5) applying the alternating electric field to the target area of ​​the subject after administering the activated and antigen-loaded dendritic cells.

[0046] Certain non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition. The method comprises the following steps: co-culturing dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells, wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro before co-culturing with the dendritic cells; and isolating a population of antigen-loaded dendritic cells to form the immunogenic composition.

[0047] Certain non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition. The method comprises the following steps: (1) applying an alternating electric field to a target area of ​​a subject; (2) isolating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to produce activated and antigen-loaded dendritic cells; and (4) isolating the activated and antigen-loaded dendritic cells from the co-culture described in (3) and the cancer cells present therein to form the immunogenic composition.

[0048] Certain non-limiting embodiments of the present disclosure relate to a method for treating cancer in a subject. The method comprises the following steps: (1) applying an alternating electric field to a target area of ​​the subject; (2) separating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the separated cancer cells with dendritic cells to produce activated and antigen-loaded dendritic cells; (4) separating the activated and antigen-loaded dendritic cells from the co-culture described in (3) and the cancer cells present therein; and (5) administering the activated and antigen-loaded dendritic cells to the subject.

[0049] Certain additional non-limiting embodiments of the present disclosure relate to a method for preparing an immunogenic composition. The method comprises the following steps: (1) isolating at least one cancer cell from a subject (such as but not limited to at least a portion of a tumor of the subject); (2) applying an alternating electric field to the at least one cancer cell isolated in vitro; (3) co-culturing the at least one cancer cell isolated to which the alternating electric field has been applied with a dendritic cell to produce an activated and antigen-loaded dendritic cell; and (4) isolating the activated and antigen-loaded dendritic cell from the co-culture described in (3) and the at least one cancer cell present therein to form the immunogenic composition.

[0050] Certain additional non-limiting embodiments of the present disclosure relate to a method for treating cancer in a subject. The method comprises the following steps: (1) isolating at least one cancer cell from the subject (such as but not limited to at least a portion of a tumor of the subject); (2) applying an alternating electric field to the at least one cancer cell separated in vitro; (3) co-culturing the at least one cancer cell separated to which the alternating electric field has been applied with a dendritic cell to produce an activated and antigen-loaded dendritic cell; (4) isolating the activated and antigen-loaded dendritic cell from the co-culture described in (3) and the at least one cancer cell present therein; and (5) administering the activated and antigen-loaded dendritic cell to the subject.

[0051] Certain additional non-limiting embodiments of the present disclosure relate to a method for reducing the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) applying an alternating electric field to a target area of ​​the subject, wherein the target area comprises the tumor; (2) separating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the separated cancer cells with dendritic cells to produce activated and antigen-loaded dendritic cells; (4) separating the activated and antigen-loaded dendritic cells from the co-culture described in (3) and the cancer cells present therein; and (5) administering the activated and antigen-loaded dendritic cells to the subject.

[0052] Certain additional non-limiting embodiments of the present disclosure relate to a method for reducing the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) isolating at least one cancer cell from the subject (such as but not limited to at least a portion of the tumor from the subject); (2) applying an alternating electric field to the at least one cancer cell separated in vitro; (3) co-culturing the at least one cancer cell separated to which the alternating electric field has been applied with a dendritic cell to produce an activated and antigen-loaded dendritic cell; (4) isolating the activated and antigen-loaded dendritic cell from the co-culture described in (3) and the at least one cancer cell present therein; and (5) administering the activated and antigen-loaded dendritic cell to the subject.

[0053] Certain additional non-limiting embodiments of the present disclosure relate to a method for preventing the increase in the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) applying an alternating electric field to a target area of ​​the subject, wherein the target area comprises the tumor; (2) separating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the separated cancer cells with dendritic cells to produce activated and antigen-loaded dendritic cells; (4) separating the activated and antigen-loaded dendritic cells from the co-culture described in (3) and the cancer cells present therein; and (5) administering the activated and antigen-loaded dendritic cells to the subject.

[0054] Certain additional non-limiting embodiments of the present disclosure relate to a method for preventing the increase in the volume of a tumor present in a living subject, wherein the tumor comprises a plurality of cancer cells. The method comprises the following steps: (1) isolating at least one cancer cell from the subject (such as but not limited to at least a portion of the tumor from the subject); (2) applying an alternating electric field to the at least one cancer cell separated in vitro; (3) co-culturing the at least one cancer cell separated to which the alternating electric field has been applied with a dendritic cell to produce an activated and antigen-loaded dendritic cell; (4) isolating the activated and antigen-loaded dendritic cell from the co-culture described in (3) and the at least one cancer cell present therein; and (5) administering the activated and antigen-loaded dendritic cell to the subject.

[0055] Any of the above methods disclosed herein or otherwise contemplated may, in certain non-limiting embodiments, further comprise the following step: (6) applying an alternating electric field to the target area of ​​the subject after administering the activated and antigen-loaded dendritic cells.

[0056] After the alternating electric field treatment, the treated cancer cells subsequently used in the co-culture step may have any activity state. That is, whether the cells are active, apoptotic, or inactive, the treated cancer cells used in the co-culture step will be able to trigger the maturation of dendritic cells in the co-culture step.

[0057] The dendritic cells or their precursors used may be obtained from the subject or other sources, such as (but not limited to) HLA-matched donors. For example (but not limited to), the method may further include the following steps: isolating dendritic cells or their precursors from the subject. When the method includes applying an alternating electric field directly to the subject, the dendritic cells or their precursors may be isolated from the subject before or after applying the alternating electric field.

[0058] In another specific (but non-limiting) embodiment, the dendritic cell or its precursor is isolated from an HLA-matched donor. For example (but not by way of limitation), when a patient is treated with an immunosuppressant or similar technology (e.g., CRISPR technology for reducing allogeneic reactions of non-matched donors), an HLA-matched donor can be used for dendritic cell isolation.

[0059] In specific (but non-limiting) embodiments, the method further comprises the steps of: isolating immature monocytes (or other dendritic cell precursors) from the bloodstream of the subject or donor (such as but not limited to, an HLA-matched donor); and generating immature dendritic cells from the immature monocyte / dendritic cell precursors.

[0060] In specific (but non-limiting) embodiments, the composition containing dendritic cells or precursors thereof comprises peripheral blood mononuclear cells (PBMCs) isolated from the subject or an HLA-matched donor.

[0061] The co-cultivation step can be carried out under any conditions that allow the antigen from the cancer cell to be loaded into the dendritic cell. In certain specific (but non-limiting) embodiments, the co-cultivation step is carried out in the presence of at least one composition, the composition being selected from the group consisting of the following: cytokines, interferons, granulocyte-macrophage colony stimulating factor (GM-CSF), CD40 ligand (CD40L), Toll-like receptor (TLR) agonists, etc. and any combination thereof. In addition, the co-cultivation step can be carried out in the presence or absence of an alternating electric field application.

[0062] Using any method known in the art or otherwise contemplated herein, activated and antigen-loaded dendritic cells can be isolated from the co-culture and the cancer cells present therein. The separation of the antigen-loaded dendritic cells can be completed by a single step or multiple steps. For example (but not limited to), cells can be first separated from the co-culture by a general cell separation method, and then a second specific separation step (such as but not limited to, Percoll / Ficoll gradient separation, flow cytometry sorting, bead sorting, etc.) can be used to ensure that all cancer cells are removed, leaving only antigen-loaded dendritic cells.

[0063] It should be noted that the isolated dendritic cells administered to the subject may contain unloaded cells in addition to the antigen-loaded dendritic cells. Therefore, in this article, the composition administered to the subject may also be referred to as "co-cultured dendritic cells" or "antigen-encountered dendritic cells".

[0064] The compositions and methods of the present disclosure can be used with any type of cancer cell and / or can be used to treat any type of cancer cell / cancer / tumor, such as (but not limited to) cancers that respond to alternating electric field and / or activated dendritic cell therapy. Non-limiting examples of cancer cells / cancers / tumors used according to the present disclosure include hepatocellular carcinoma cells, glioblastoma cells, pleural mesothelioma cells, differentiated thyroid cancer cells, advanced renal cell carcinoma cells, ovarian cancer cells, cervical cancer cells, breast cancer cells, pancreatic cancer cells, lung cancer cells (such as but not limited to, non-small cell lung cancer cells), etc., and any combination thereof.

[0065] In certain (but non-limiting) embodiments, cancer cells used in accordance with the present disclosure may be taken from at least a portion of a tumor.

[0066] In specific (but non-limiting) embodiments, the cancer may be a solid tumor.

[0067] According to the methods of the present disclosure, the alternating electric field may be generated using any type of conductive or non-conductive electrodes and / or transducer arrays known in the art or otherwise contemplated herein that can be used to generate an alternating electric field. Non-limiting examples of electrodes and transducer arrays that can be used to generate alternating electric fields in accordance with the present disclosure include those electrodes and transducer arrays that are part of an alternating electric field generating system (e.g., a TTField system), such as, but not limited to, those described in: U.S. Patent Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776; and U.S. Patent Application Nos. US2018 / 0160933, US2019 / 0117956, US2019 / 0307781, and US2019 / 0308016.

[0068] According to the present disclosure, the alternating electric field can be generated at any frequency. For example (but not by way of limitation), the alternating electric field can have a frequency of about 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, about 175 kHz, about 180 kHz, about 186 kHz, about 187 kHz, about 190 kHz, about 201 kHz, about 202 kHz, about 203 kHz, about 204 kHz, about 205 kHz, about 206 kHz, about 207 kHz, about 208 kHz, about 209 kHz, about 210 kHz, about 211 kHz, about 212 kHz, about 213 kHz, about 214 kHz, about 215 kHz, about 216 70kHz, about 175kHz, about 180kHz, about 185kHz, about 190kHz, about 195kHz, about 200kHz, about 225kHz, about 250kHz, about 275kHz, about 300kHz, about 325kHz, about 350kHz, about 375kHz, about 400kHz, about 425kHz, about 450kHz, about 475kHz, about 500kHz, about 550kHz, about 600kHz, about 650kHz, about The present invention relates to frequencies of about 700 kHz, about 750 kHz, about 800 kHz, about 850 kHz, about 900 kHz, about 950 kHz, about 1 MHz, and the like, as well as frequencies within a range formed by any of the above values ​​(e.g., a range of about 50 kHz to about 1 MHz, a range of about 100 kHz to about 500 kHz, a range of about 150 kHz to about 300 kHz, a range of about 50 kHz to about 190 kHz, a range of about 50 kHz to about 180 kHz, a range of about 50 kHz to about 175 kHz, a range of about 50 kHz to about 160 kHz, a range of about 50 kHz to about 150 kHz, a range of about 250 kHz to about 350 kHz, a range of about 350 kHz to about 500 kHz, a range of about 250 kHz to about 500 kHz, and the like), and frequencies within a range combining two integers falling between two of the above values ​​(e.g., a range of about 122 kHz to about 313 kHz, a range of about 78 kHz to about 298 kHz, and the like).

[0069] In certain specific (but non-limiting) embodiments, the alternating electric field can be applied at two or more different frequencies. When there are two or more frequencies, each frequency is selected from any of the above values, or a range formed by any of the above values, or a range combining two integers falling between two of the above values.

[0070] The alternating electric field can have any field strength in the subject / cancer cell as long as the alternating electric field can function according to the present disclosure. For example (but not by way of limitation), 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, about 5 V / cm, about 6 V / cm, about 7 V / cm, about 8 V / cm, about 9 V / cm, about 10 V / cm, about 11 V / cm, about 12 V / cm, about 13 V / cm, about 14 V / cm, about 15 V / cm, about 16 V / cm, about 17 V / cm, about 18 V / cm, about 19 V / cm, about 20 V / cm, about 21 V / cm, about 22 V / cm, about 23 V / cm, about 24 V / cm, about 25 V / cm, about 26 V / cm, about 27 V / cm, about 28 V / cm, about 29 V / cm, about 30 V / cm, about 31 V / cm, about 32 V / cm, about 34 V / cm, about 36 V / cm, about 37 V / cm, about 38 V / cm, about 39 V / cm, about 40 V / cm, about 41 V / cm, about 42 V / cm, about 43 V / cm, about 44 V / cm, about 45 V / cm, about 46 V / cm, about 47 V / cm, about 48 V / about 4.5V / cm, about 5V / cm, about 5.5V / cm, about 6V / cm, about 6.5V / cm, about 7V / cm, about 7.5V / cm, about 8V / cm, about 9V / cm, about 9.5V / cm, about 10V / cm, about 10.5V / cm, about 11V / cm, about 11.5V / cm, about 12V / cm, about 12.5V / cm, about 13V / cm, about 13.5V / cm, about 14 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, and the like, as well as field intensities within a range formed by 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 field intensities within a range combining two integers falling between two of the above 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.).

[0071] The alternating electric field may be applied in a single direction between a pair of arrays, or may alternate in two (or more) directions (e.g., front-to-back and side-to-side) between two (or more) pairs of arrays. For example, some TTField devices (such as, but not limited to, The system (Novocure Limited, St. Helier, Jersey) operates in two directions to increase the chance that dividing cells will align with the electric field so that the electric field can have the desired anti-mitotic effect. However, it should be understood that the scope of the present disclosure also includes applying an alternating electric field in a single direction to achieve the immunogenic response described herein.

[0072] The alternating electric field can be applied to the subject, the dendritic cells and / or the cancer cells (or a co-culture containing both the dendritic cells and the cancer cells) for any period of time disclosed herein or otherwise contemplated. For example, but not by way of limitation, in certain non-limiting embodiments, the alternating electric field is applied for a sufficient time to cause / help the dendritic cells to mature and / or cause / help the dendritic cells to present certain antigens in the co-cultured cancer cells. For example, but not by way of limitation, the alternating electric field can 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 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 About 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, etc., and ranges formed by any of the above values ​​(a range from about 1 minute to about 12 hours, a range from about 1 minute to about 1 hour, a range from about 1 hour to about 7 days, a range from about 24 hours to about 72 hours, etc.), and a range combining two integers falling between two of the above values ​​(for example, a range from about 14 hours to about 68 hours, etc.).

[0073] In certain (but non-limiting) embodiments, the alternating electric field is applied for a period of time of at least about 24 hours.

[0074] In addition, when the alternating electric field is applied to the subject, the time period for applying the alternating electric field may be a continuous time period or a cumulative time period. That is, the time period for applying the alternating electric field may include a single round (i.e., continuous application) and multiple rounds with short breaks between each round (i.e., continuous application cumulative time period). For example, the subject is allowed to rest during treatment with the alternating electric field device, and it is expected that the device will only be placed on the body and operated 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., during a day, a week, two weeks, a month, two months, three months, four months, five months, etc.).

[0075] Antigen-loaded dendritic cells (hereinafter referred to as "activated dendritic cells") can be treated and administered in any formulation known in the art or otherwise contemplated herein so that the activated dendritic cells have a deleterious effect on the cancer present in the subject. For example, but not by way of limitation, the activated dendritic cells can be administered in the form of a pharmaceutical composition comprising activated dendritic cells in combination with at least one pharmaceutically acceptable carrier. Non-limiting examples of suitable pharmaceutically acceptable carriers that can be used according to the present disclosure include water; saline; glucose solution; fructose or mannitol; calcium carbonate; cellulose; ethanol; oils of animal, plant or synthetic origin; carbohydrates such as glucose, sucrose or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposome carriers; conductive and non-conductive nanoparticles; buffer solutions such as sodium chloride, saline, phosphate buffered saline and / or other substances that are physiologically acceptable and / or safe to use; diluents; excipients such as polyethylene glycol (PEG); or any combination thereof. Suitable pharmaceutically acceptable carriers for pharmaceutical formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd Edition (2020).

[0076] In certain non-limiting embodiments, the pharmaceutical composition containing activated dendritic cells can be further formulated as an immunogenic composition. The immunogenic composition can contain the same components as the above-mentioned pharmaceutical composition (i.e., activated dendritic cells and a pharmaceutically acceptable carrier). In certain specific (but non-limiting) embodiments, the immunogenic composition can further comprise at least one additional agent. Non-limiting examples of agents that can be included as part of the immunogenic composition include adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, FMS-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG-3 agents (such as but not limited to OPDUALAG), and anti-LAG-3 agents (such as but not limited to OPDUALAG). TM and / or relalizumab (Bristol-Myers Squibb, New York, NY), other active agents, and the like, and any combination thereof.

[0077] In addition, any composition containing activated dendritic cells disclosed herein may contain other agents that allow the composition to be administered via a specific route of administration. For example, but not limited to, the composition may be formulated for administration by oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal and / or intravenous routes. Based on the route of administration, the composition may also contain one or more additional components in addition to the active agent (). The example of the additional second compound that may exist includes, but is not limited to, fillers, gels, adhesives, salts, buffers, preservatives, stabilizers, solubilizers, wetting agents, emulsifiers, dispersants and other materials well known in the art.

[0078] In certain (but non-limiting) embodiments, the composition comprising activated dendritic cells can be administered to a subject intradermally, subcutaneously, intravenously, and / or intranodally.

[0079] In certain non-limiting embodiments, the method may further include one or more additional steps of applying an alternating electric field to the target area of ​​the subject: (1) after isolating dendritic cells and / or their precursors; (2) after isolating cancer cells (and / or removing tumors); and / or (3) before or after administering a composition containing activated dendritic cells. When there is an additional alternating electric field application step, the application of the alternating electric field can be performed simultaneously with the administration of the composition containing activated dendritic cells or in whole or in part sequentially. In certain specific (but non-limiting) embodiments, the alternating electric field can be applied after the administration of the composition containing activated dendritic cells. In other specific (but non-limiting) embodiments, the alternating electric field can be applied while or after the administration of the composition containing activated dendritic cells. In another specific (but non-limiting) embodiment, the composition containing activated dendritic cells can be administered during the application of the alternating electric field (i.e., before the time period for applying the alternating electric field has passed).

[0080] For example (but not by way of limitation), the additional application of the alternating electric field may be preceded by about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours The composition containing activated dendritic cells is administered for a period of time such as about 1 minute, 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 period of time within a range formed by any of the above values ​​(e.g., a range of about 1 minute to about 24 hours, etc.), and a period of time within a range combining two integers falling between two of the above values ​​(e.g., a range of about 14 minutes to about 94 hours, etc.).

[0081] In other non-limiting examples, the additional application of the alternating electric field may be performed at about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about The composition containing activated dendritic cells is administered for a period of time such as about 1 minute, 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, and the like, as well as a period of time within a range formed by any of the above values ​​(e.g., a range of about 1 minute to 24 hours, etc.), and a period of time within a range combining two integers falling between two of the above values ​​(e.g., a range of about 14 minutes to about 94 hours, etc.).

[0082] However, in yet another non-limiting example, the composition containing activated dendritic cells can be administered after the time period during which the additional alternating electric field is applied has passed, wherein the composition containing activated dendritic cells is administered about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours Administration within 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.

[0083] The composition containing activated dendritic cells can be administered to the subject at any concentration that can induce an inflammatory response to a tumor or cancer cell. For example, but not limited to, about 10 cells / kg body weight, about 100 cells / kg body weight, about 1000 cells / kg body weight, about 10 4 cells / kg body weight, about 10 5 cells / kg body weight, about 10 6 cells / kg body weight, about 10 7 cells / kg body weight, about 10 8 cells / kg body weight, about 10 9 cells / kg body weight, about 10 10 cells / kg body weight, about 10 11 cells / kg body weight, about 10 12 cells / kg body weight, about 10 13 cells / kg body weight, about 10 14 cells / kg body weight, about 10 15 cells / kg body weight or more, and ranges formed by any of the above values ​​(e.g., about 10 4 to about 10 9 The activated dendritic cells are administered in the range of 10 cells / kg body weight, etc.

[0084] In certain specific (but non-limiting) embodiments, the method also involves simultaneous therapy using two or more compositions. Therefore, the method may include an additional step of administering at least a second composition to the subject. Additional non-limiting examples of therapeutic agents that can be used as part of a second composition administered simultaneously or in whole or in part sequentially with a composition containing activated dendritic cells include Lenvatinib, Pembrolizumab and other anti-PD-1 therapeutic agents, such as (but not limited to) Tislelizumab, Nivolumab and Cemiplimab; anti-LAG3 agents, such as (but not limited to) OPDUALAG TM and / or relalizumab (Bristol-Myers Squibb, New York, NY); anti-PD-L1 therapeutics such as, but not limited to, atezolizumab, avelumab, and durvalumab; anti-CTLA-4 therapeutics such as, but not limited to, ipilimumab; chemotherapeutic agents such as, but not limited to, paclitaxel, docetaxel, ifosamide, etoposide (Vepesid), gemcitabine, lomustine, nab-paclitaxel (Nab Paclitaxel), Temozolomide and Carboplatin; TKI inhibitors such as (but not limited to) Everolimus; mTOR inhibitors; Akt inhibitors; PI3K inhibitors; PARP inhibitors; VEGF inhibitors; FGF inhibitors; aromatase inhibitors such as (but not limited to) Letrozole; biologics such as monoclonal antibodies (such as but not limited to Denosumab and Pembrolizumab), etc. and any combination thereof.

[0085] When there is concurrent treatment, the concurrent treatment can be performed substantially simultaneously or in whole or in part sequentially with the administration of the composition containing activated dendritic cells. In addition, the two compositions can be administered by the same route (e.g., oral administration or injection), or the two compositions can be administered by different routes (e.g., one composition is administered orally and the other composition is administered intravenously).

[0086] When both optional steps of administering the second composition and applying an alternating electric field to a subject to which the composition containing activated dendritic cells has been administered are present, the optional administration step can be performed in the same manner and time frame as described above for the composition containing antigen-loaded dendritic cells, before or after the start of application of the alternating electric field, as well as during application of the alternating electric field and / or after application of the alternating electric field has elapsed.

[0087] That is, for example (but not as a limitation), the second composition can be applied for a period of time of about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, etc. after the start of application of the alternating electric field, as well as a period of time within a range formed by any of the above values ​​(for example, a range of about 24 hours to about 96 hours, etc., and a range combining two integers falling between two of the above values ​​(a range of about 14 hours to about 94 hours, etc.). In specific (but non-limiting) embodiments, the second composition is applied at least about 24 hours after application of the alternating electric field is initiated.

[0088] In other non-limiting examples, the second composition can be applied after the time period for applying the alternating electric field has passed, wherein the second composition is applied within about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, etc. after the time period has passed. In a specific (but non-limiting) embodiment, the second composition is administered within about 96 hours after the time period has passed.

[0089] In addition, for example (but not by way of limitation), the activated dendritic cells may be activated at 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 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours The second composition is administered at least about 12 hours after administration of the composition containing the antigen-loaded dendritic cells.

[0090] In certain specific (but non-limiting) embodiments, the method may further comprise the steps of administering at least one additional therapy to the subject. According to the methods disclosed herein, any therapy known in the art or otherwise contemplated herein for use with TTField and / or activated dendritic cell therapy may be used. Non-limiting examples of additional therapies that may be used include radiotherapy, photodynamic therapy, transarterial chemoembolization (TACE), or a combination thereof.

[0091] In certain specific (but non-limiting) embodiments, the method includes one or more additional steps. For example (but not by way of limitation), the method may further include repeating any step once or multiple times. Each step may be repeated multiple times as needed. When the alternating electric field is repeatedly applied, the transducer array may be placed in a slightly different position on the subject than its original placement; repositioning the array in this way may further help treat tumors / cancers. In addition, any administration step (including the step of administering a composition containing dendritic cells and any optional administration step) can be repeated at different times and at different intervals according to any known and / or generally accepted dosage / treatment regimen of the composition / therapy.

[0092] Although the above methods involve the use of activated dendritic cells to treat cancer, it should be understood that the scope of the present disclosure is not limited to cancer treatment. Rather, the present disclosure encompasses activation of dendritic cells by exposure to an alternating electric field, followed by loading of any desired antigens onto the subsequently activated dendritic cells for the treatment of any other related disease, infection, or condition for which dendritic cell therapy is beneficial. For example, but not by way of limitation, dendritic cells activated by exposure to an alternating electric field can be impacted with antigens (or co-cultured with an antigen source), including (but not limited to) bacterial antigens, viral antigens, fungal antigens, parasitic antigens, tumor antigens, cancer antigens, and the like, and any combination thereof.

[0093] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions produced by any method disclosed or otherwise contemplated herein.

[0094] Certain non-limiting embodiments of the present disclosure are directed to an immunogenic composition comprising a population of isolated and antigen-loaded dendritic cells, wherein the antigen-loaded dendritic cells are produced by co-culturing dendritic cells with at least one cancer cell isolated from a subject, and wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro prior to co-culturing with the dendritic cells.

[0095] Certain non-limiting embodiments of the present disclosure relate to immunogenic compositions comprising a population of any isolated and antigen-loaded (and optionally exposed to an alternating electric field) dendritic cells produced as described herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, the dendritic cells are further co-cultured with at least one cancer cell separated from a subject to produce activated and antigen-loaded dendritic cells. In specific (but non-limiting) embodiments, TTFields may be applied to a subject or to either or both cell types before or during the co-culture step, so that the dendritic cells and / or cancer cells used in the co-culture have been exposed to an alternating electric field in vitro. In yet another specific (but non-limiting) embodiment, the dendritic cells have been co-cultured or impacted to load other types of antigens in the dendritic cells.

[0096] The immunogenic composition can be formulated for administration by any route of administration disclosed herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

[0097] In certain specific (but non-limiting) embodiments, the immunogenic composition may further include one or more additional active agents that further help stimulate the immune system to recognize and attack the cancer cells (or other diseased cells, infected cells, or bacterial cells) of the subject. Non-limiting examples of additional agents that may be present in the immunogenic composition include adjuvants, cytokines, interferons, TLR agonists, STING (stimulator of interferon genes) agonists, GM-CSF, CD40L, FMS-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG-3 agents (such as but not limited to OPDUALAG), TM and / or relalizumab (Bristol-Myers Squibb, New York, NY)) and combinations thereof.

[0098] The dendritic cells in the compositions and methods may include any dendritic cells known in the art or otherwise contemplated herein. For example, but not limited to, the dendritic cells may include at least one of conventional DC1 (cDC1), cDC2, plasmacytoid DC (pDC), etc.

[0099] Certain non-limiting embodiments of the present disclosure relate to kits comprising any components of an alternating electric field generating system (such as, but not limited to, one or more transducer arrays and / or one or more hydrogel compositions, as disclosed in: U.S. Pat. Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776; and U.S. Patent Application Nos. US2018 / 0160933, US2019 / 0117956, US2019 / 0307781 and US2019 / 0308016) are combined with any components, devices and / or reagents used in one or more steps, the steps being as follows: isolating one or more cell types (e.g., dendritic cells or their precursors and / or cancer cells), exposing the subject, dendritic cells (or their precursors) and / or cancer cells to TTFields, co-culturing dendritic cells with cancer cells, pulsing dendritic cells with antigens, isolating activated dendritic cells and / or formulating activated dendritic cells for administration to the subject. The kit may optionally further include one or more of any optional compositions disclosed or otherwise contemplated herein (such as, but not limited to, one or more compositions for optional simultaneous therapy steps). The kit may optionally further include one or more devices (or one or more components of a device) used in one or more additional therapy steps.

[0100] In a specific (but non-limiting) embodiment, the kit may further include instructions for implementing any method disclosed herein or otherwise envisioned. For example (but not limited to), the kit may include instructions for separating one or more cell types, exposing a subject and / or cell culture to an alternating electric field generating system, instructions for separating activated dendritic cells and preparing to be applied to a subject, instructions for applying one or more components of an alternating electric field generating system to the subject's skin, instructions for applying an alternating electric field to a subject, instructions for when and how to apply a composition containing dendritic cells and optionally how to apply one or more optional additional compositions, and / or instructions for when to activate and close an alternating electric field relative to applying a composition containing dendritic cells and / or applying one or more optional compositions.

[0101] In addition to the components described in detail above, the kit may further contain other components / reagents for implementing any specific method described herein or otherwise contemplated. For example (but not by way of limitation), the kit may additionally include: (i) components for preparing the skin before treating the hydrogel composition and / or the transducer array thereon (i.e., razors, cleaning compositions or wipes / towels, etc.); (ii) components for removing the gel / transducer array; (iii) components for cleaning the skin after removing the gel / transducer array; (iv) components for separating cancer cells / tumor parts; (v) components for separating dendritic cells or their precursors; and / or (vi) components for maturing dendritic cells or their precursors. The nature of these additional components / reagents will depend on the specific treatment modality, and their identification is well within the skill of a person of ordinary skill in the art; therefore, no further description thereof is required. In addition, the components / reagents present in the kit may each be in a separate container / compartment, or the various components / reagents may be combined in one or more containers / compartments, depending on the sterility, cross-reactivity and stability of the components / reagents.

[0102] The test kit can be arranged in any packaging that allows the components present therein to function according to the present disclosure. In certain non-limiting embodiments, the test kit also includes a sealed package in which the components are placed. In certain specific (but non-limiting) embodiments, the sealed package is substantially airtight and / or substantially opaque.

[0103] In addition, the kit may also include a set of written instructions explaining how to use one or more components of the kit. Kits of this nature can be used in any method described herein or otherwise contemplated.

[0104] 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.

[0105] Certain non-limiting embodiments of the present disclosure are directed to systems that include any components of an alternating electric field generating system (such as, but not limited to, one or more transducer arrays and / or one or more hydrogel compositions, as disclosed in U.S. Pat. Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764, 675, 10,188,851 and 10,441,776; and U.S. Patent Application Nos. US2018 / 0160933, US2019 / 0117956, US2019 / 0307781 and US2019 / 0308016) in combination with at least one immunogenic composition of any immunogenic composition comprising activated dendritic cells disclosed or otherwise contemplated herein. The system may optionally further include one or more of any optional compositions disclosed or otherwise contemplated herein. The system may optionally further include one or more devices (or one or more components of a device) used in various separation, co-cultivation, administration steps or optional additional treatment / therapy steps.

[0106] Example

[0107] Examples are provided below. However, the present disclosure should be understood to be not limited in its application to the specific experiments, results and laboratory procedures disclosed below. On the contrary, the examples are provided only as one of the various embodiments and are exemplary, not exhaustive.

[0108] Example 1

[0109] Overview:

[0110] Dendritic cells (DCs) are key components in the cancer immunity cycle, driving T cell activation or tolerance [Chen DS, Immunity, 2013]. The effects of TTFields on human DCs are not yet clear, but the mode of action of TTFields may affect the activity of DCs or their ability to activate. After we tested the effects of TTFields on human monocyte-derived DCs (monocytes cultured in IL4+GMCSF), we turned to physiological blood-derived DCs: conventional DC1 (cDC1), cDC2, and plasmacytoid DC (pDC).

[0111] We tested the effects of TTFields on DC activity and their ability to undergo activation and maturation. TM The INOVITRO system (Novocure GmbH, Root, Switzerland) may introduce considerable intra-experimental variability, and we performed eight experiments to confirm the results. The TTField conditions used were 150 kHz and 200 kHz, which have been approved for the treatment of lung cancer and brain cancer, respectively. The field strength was 2.7 V / cm and the exposure time was 48 hours (DCs changed significantly after more than 2 days in culture). We used freshly purified peripheral blood mononuclear cells (PBMCs) obtained from healthy blood donors. PBMCs were assessed on day 0 to define the baseline status of DCs (Group 1). After 48 hours of culture, an additional 6 groups were monitored: (Group 2) Control group: DCs not treated with TTFields in the INOVITRO TM (3) Control group + LPS + R848 (hereinafter referred to as LPS): fully activated under standard culture conditions; (4) 150kHz TTField: evaluate the effect of TTField on DC activity and maturation; (5) 150kHz TTField + LPS: evaluate the effect of activation signal under TTField conditions; (6) 200kHz TTField; and (7) 200kHz TTField + LPS.

[0112] After treatment, cells were stained using a 12-color DC panel to identify all 3 physiological DC subsets, 2 maturation / activation markers, and cell activity. Analysis of 8 experiments showed a slight, generally non-significant decrease in activity (2%-6%) induced under TTField conditions. Importantly, it was repeatedly observed that the 150kHz frequency drove a potent activation of DCs compared to the control group without TTField treatment for 2 days. TTField 200kHz frequency did not induce this activation. Activation with the potent DC activator LPS further activated DCs even under TTField conditions, indicating that physiological DCs can still be effectively activated under TTField.

[0113] These results indicate that physiological DCs of all subtypes not only retain their activity but also their ability to mature, two factors that are essential for effective DC function. These results also suggest that TTFields at specific frequencies can act as a "physical adjuvant" that can promote the maturation of DCs.

[0114] Experimental design: To evaluate the effect of TTField treatment on the activity and activation potential of physiological blood-derived dendritic cells, 8 experiments (15 technical replicates per group) were performed. The TTField frequencies used were 150kHz and 200kHz, the field strength was 2.7V / cm, and the duration was 48 hours. Fresh PBMCs were from healthy donors and divided into 7 groups as described in Table 1. DC activation was induced by exposing the culture to 1ug / mL lipopolysaccharide and 2.5ug / mL R848 (Resiquimod) (expressed as LPS) derived from Escherichia coli strain O111:B4. After 48 hours of treatment, the cells were stained with a 12-color flow cytometry panel (see Table 2). Among them, 9 channels were used to identify three DC subtypes, 1 channel was used to distinguish between live and dead cells, and 2 channels were used to quantify the expression of two DC maturation or activation markers, CD80 and CD83. The stained cells were read on a 4-laser BD FACSAria Fusion flow cytometer and analyzed using the FlowJo software package.

[0115] Table 1 Treatment groups in the experiments performed

[0116]

[0117]

[0118] Table 2 List of markers and clones in DC flow cytometry panel

[0119]

[0120] A gating strategy was designed to assess the activity and maturation of three blood DC subsets ( Figure 1 ). The figure shows the complete gating of the control group, and the bottom two rows show the activity and activation of the control group and the 150kHz TTField group, with each row depicting the three DC subtypes.

[0121] Technical replicates were performed in most experiments (15 replicates per 8 experiments). TM The system is noisy, especially when interrogating cells from PBMCs of different donors, which are low in proportion (0.03%-0.5%) and sensitive to small changes in the culture environment. The results presented for each experiment are the average of the corresponding experiments (1-3 technical replicates per group). In each experiment, each replicate cultured approximately 3.5x10 6 Paired t-test was used to test the significance of 8 pooled experiments.

[0122] result:

[0123] Under TTField, the activity of DCs was basically maintained.

[0124] Figure 2 Mean activity ± SEM of each group in different experiments is shown. Only slight differences in activity were observed within the range of 80%-100% of the bar graph scale. Overall, the difference in activity between the groups not treated with LPS and the control group on day 2 was between 2% and 6%: the cDC1 group had an average decrease in activity of 4% (NS) under TTField, the cDC2 group had an average decrease in activity of 4% (P=0.03), and the activity at 200kHz had an average decrease of 6% (NS). For the pDC group treated with 150kHz, the activity decreased by 2% (P=0.01). Although some means showed significant decreases, the effect sizes were very small in all groups. In all groups, LPS reduced the activity of DCs, but no statistical difference was seen between the TTField+LPS group and the control+LPS group.

[0125] Effects of TTFields on DC activation and maturation

[0126] Then, the effects of TTFields on DC maturation and on the ability of DC to undergo activation driven by TLR receptor agonists were tested: TLR4 agonist - LPS and TLR7 / 8 agonist - R848. The combination of R848 and LPS has a strong stimulation on all physiological DCs [Lovgren T, Cancer Imm. Immunother, 2017]. Figure 3 Depicted are the mean ± SEM of two monitored maturation markers, CD80 (B7.1) and CD83, presented in the form of single expression (e.g., CD83+CD80-) or co-expression (CD83+CD80+). Double-positive cells represent fully mature DCs [Dudek AM, Front Immuno, 2013].

[0127] For all DC types, maturation was close to zero on day 0. By day 2 (48 hours), both cDC subtypes and, to a lesser extent, pDC subtypes showed increased expression of CD80 and / or CD83. The increase in single positives in the cDC group was primarily driven by increased expression of CD83. For all DC subtypes, exposure to LPS increased the proportion of double positive DCs.

[0128] Effects of TTField on cDC1 dendritic cells

[0129] cDC1 is the rarest dendritic cell in the human body (accounting for 3%-5% of DC), but they are a key component of tumor immune rejection. Uniquely, they are able to collect dead cell material and cross-present it to cytotoxic T cells through MHC-I [Wculek SK, Nat Rev Immunol 2020, Volovitz I, 2016 Int Rev Immunol]. Regarding the activation of cDC1 (Table 3), except for the 200kHz group, the proportion of fully activated (double-positive) DCs in all treatment groups was significantly increased. In the 150kHz treatment group without LPS, an increase in double-positive DCs was also detected (P=0.003); compared with the control group, the proportion of double-positive DCs in the 200kHz treatment group did not increase. The 150kHz+LPS group and the 200kHz+LPS group showed a similar double-positive ratio to the control group (both 63%-70%), indicating that cDC1 can be effectively activated under TTField. Although the double positive ratio of the 200kHz+LPS group was significantly higher than that of the 200kHz group, the double positive ratio of the 150kHz+LPS group was only slightly higher than that of the 150kHz group (NS). Of the total increase in double positive cDC1 (total increase = control group on day 2 minus 150kHz+LPS group), 93% of the increase was attributable to exposure of cDC1 to 150kHz TTField.

[0130] Table 3 Individual and summary results of the proportion of double-positive fully mature cDC1 (CD80+, CD83+)

[0131]

[0132] Proportion of double positive DCs at day 0 and after 48 hours of culture. Shown are individual proportions for each treatment group or averages of 2-3 technical replicates. The averages of 8 experiments are given, with two rows of statistical comparisons below: the top row compares all groups with the control group on day 2 (not treated with TTFields); the bottom row compares each treatment group with the same group treated with LPS, for example, the 150kHz group is compared with the 150kHz+LPS group. In the proportion of responding cells, NA indicates an experiment in which a specific DC subset could not be clearly identified using the gating strategy.

[0133] To illustrate the effect of 150kHz on cDC1, Figure 4 Shown is a comparison of cDC1 double positive cells (CD80+ and CD83+) in the control group versus the 150 kHz group in all 8 experiments.

[0134] Effects of TTF on cDC2 Dendritic Cells

[0135] cDC2 is the most common conventional DC and the only DC subtype found in non-negligible numbers in glioblastoma. cDC2 is able to effectively activate helper T cells. Compared with cDC1, cDC2 secretes more inflammatory cytokines such as IL1β, IL6, TNFα, and IL8. Depending on their activation and maturation status, they may play a pro-tumor or anti-tumor role within the tumor [Wculek SK, Nat Rev Immunol 2020, Volovitz I, Int Rev Immunol, 2016].

[0136] Table 4 shows that the activation of cDC2 is similar to that of cDC1 in many aspects. Therefore, except for the 200kHz group, the proportion of double-positive, fully activated DCs in all treatment groups was significantly higher than that in the control group on day 2. Exposing cDC2 to a 150kHz but LPS-free environment significantly increased the proportion of double-positive cDC2 (P=0.006), while 200kHz had no effect on the proportion of double-positive cDC2. The 150kHz+LPS group and the 200kHz+LPS group showed a double-positive ratio similar to that of the control group (both 48%-50%), indicating that cDC2 can be effectively activated under TTField. Although the double-positive ratio of the 200kHz+LPS group was significantly higher than that of the 200kHz group, the double-positive ratio of the 150kHz+LPS group was only slightly higher than that of the 150kHz group (NS). The double-positive cDC2 induced by the 150kHz frequency accounted for 72% of the total increase in double-positive cDC2 that could be achieved (subtracting the 150kHz+LPS group from the control group on day 2).

[0137] Table 4 Individual and summary results of the proportion of double-positive fully mature cDC2 (CD80+, CD83+)

[0138]

[0139]

[0140] Proportion of double positive cDC2s at day 0 and after 48 hours of culture. Shown are individual proportions for each treatment group or averages of 2-3 technical replicates. The averages of 8 experiments are given, with two rows of statistical comparisons below: the top row compares all groups with the control group on day 2 (not treated with TTFields); the bottom row compares each treatment group with the same group treated with LPS, for example, the 150kHz group is compared with the 150kHz+LPS group. In the proportion of responding cells, NA indicates an experiment in which a specific DC subset could not be clearly identified using the gating strategy.

[0141] To illustrate the effect of 150 kHz TTF on cDC2 activation, Figure 5Comparison results between double positive cells in the control group (day 2) and the 150 kHz group are shown.

[0142] Effects of TTF on pDCs

[0143] pDCs are the main producers of type I IFN (IFN-α / β) in the early response to viruses, bacteria, or autologous 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 TTField has a relatively small effect on the proportion of double-positive pDCs compared with cDCs. In the 150kHz group, the average proportion of double-positive cells was significantly higher than that in the control group (P=0.018), but only 11% of the cells in this group were double-positive, compared with 2% in the control group (a difference of 9%). Similarly, the 200kHz group had no effect on DC maturation. The activation rate in the presence of LPS but without TTField was always higher (mean 35%) than 150kHz (mean 18%) or 200kHz (mean 12%), but these differences did not reach statistical significance. Similarly, the 200kHz+LPS group was significantly higher than the 200kHz group, while the 150kHz+LPS group was not significantly higher than the 150kHz group. Therefore, the double-positive pDCs induced by 150kHz treatment only accounted for 56% of the double-positive pDC increase.

[0144] Table 5 Individual and summary results of the proportion of pDC double positive fully mature DC (CD80+, CD83+)

[0145]

[0146] The proportion of double positive pDCs on day 0 and after 48 hours of culture. Shown are individual proportions for each treatment group or the average of 2-3 technical replicates. The average of 8 experiments is given, and there are two rows of statistical comparisons below: the top row compares all groups with the control group on day 2 (not treated with TTField). The bottom row compares each treatment group with the same group treated with LPS, for example, the 150kHz group is compared with the 150kHz+LPS group. In the proportion of responding cells, NA indicates an experiment in which a specific DC subset could not be clearly identified using the gating strategy.

[0147] To illustrate the effect of 150kHz TTF on pDC double-positive cells, Figure 6 Mean values ​​of cell frequency for control and matched 150 kHz samples are shown. Although the activation scale of pDCs was lower than that of cDCs, consistent activation of pDCs driven by 150 kHz was observed in all experiments.

[0148] in conclusion

[0149] Only slight differences in activity were found for TTField-treated DC cells. In addition, no significant differences were found in the proportion of double-positive (CD80+CD83+) DCs between the control group + LPS and the TTField-treated samples + LPS, indicating that TTField did not inhibit DC maturation achieved by activation agents such as LPS and R848.

[0150] Data from eight experiments showed that physiological DCs, namely cDC1, cDC2, and pDC, were activated upon 150kHz TTField treatment. In fact, for all DC subsets, much of the DC maturation achieved by exposure to strong activating factors such as LPS could also be achieved by exposing PBMCs to 150kHz TTField alone. These results suggest that TTFields can act as a "physical adjuvant" to enhance the maturation state of DCs in a non-antigen-specific manner.

[0151] DCs play a key role in tumors, achieving their effective anti-tumor response by attracting T cells to the tumor area, entering the tumor area and then activating these T cells [Wculek SK, Nat Rev Immunol 2020]. The immunostimulatory effect of 150kHz can drive DCs in tumors to fully mature after only 48 hours of exposure. DC maturation is a key parameter for its ability to drive a potent anti-tumor response. The difference between 150kHz and 200kHz TTField treatment may not only affect the killing potential of specific tumor cells. As we have shown before, it may also affect the activity and function of tumor-infiltrating T cells [Diamant G, 2021, J Immunol], or affect the maturation state or function of DCs within the treatment range.

[0152] Example 2

[0153] The main goal of cancer immunotherapy is to activate pre-existing endogenous immune responses in cancer patients. Although significant progress has been made in this field, the effectiveness of treatment still needs to be further improved. Personalized cancer vaccines are a promising strategy to enhance anti-tumor immune responses through immunogenic forms of apoptosis, also known as immunogenic cell death (ICD). ICD is characterized by the release of danger-associated molecular patterns that are used to recruit immune cells to tumor sites. Previous studies have shown that TTField treatment can enhance immunogenic cell death of cancer cells, ultimately stimulating immune responses by phagocytosis of cancer cells. In turn, it can also present new antigens to further initiate adaptive immunity.

[0154] To justify the use of TTField as an immunomodulator, INOVITRO TM The mice were treated with TTFields for 72 hours using the Novocure system (Novocure GmbH, Root, Switzerland). Cancer cells were then isolated from the mice. PBMCs were also isolated from mice before or after TTField exposure, or from HLA-matched donors. PBMCs were co-cultured with cancer cells to activate and load neoantigens into dendritic cells. The activated and antigen-loaded dendritic cells were separated from the co-culture and cancer cells and then administered to mice as a vaccine to trigger an immune response against cancer development.

[0155] In this way, a method to enhance cancer cell immunity is combined with TTField therapy. Combining TTField therapy with the administration of a personalized composition containing activated dendritic cells has a more synergistic effect than either treatment alone and triggers an immune response in the patient, enabling the immune system to eliminate cancer cells.

[0156] Example 3

[0157] Dendritic cells are activated by exposure to TTFields as described in Example 1. Dendritic cells exposed to TTFields are then antigen loaded by pulse with an antigen of interest or co-cultured with an antigen source (e.g., cancer cells, bacterial cells, virally infected cells, fungal infected cells, etc.). The activated and antigen-loaded dendritic cells are separated from the culture and then administered as an immunomodulator or vaccine to the same subject or an allogeneic subject to trigger an immune response. In this way, TTField therapy is equivalent to a physical adjuvant. Non-limiting illustrative embodiments of the present inventive concept

[0158] Illustrative Embodiment 1 A method for activating dendritic cells, the method comprising the steps of: applying an alternating electric field to a composition comprising immature dendritic cells or precursors thereof in vivo for a sufficient period of time to produce activated dendritic cells.

[0159] Illustrative embodiment 2 According to the method of illustrative embodiment 1, the method further comprises the step of contacting the activated dendritic cells with an antigen source to produce antigen-loaded dendritic cells.

[0160] Illustrative embodiment 3 A method for preparing an immunogenic composition, the method comprising the steps of: applying an alternating electric field to a composition comprising immature dendritic cells or precursors thereof in vivo for a sufficient period of time to produce activated dendritic cells; contacting the activated dendritic cells with an antigen source to produce antigen-loaded dendritic cells; and isolating the antigen-loaded dendritic cells to form the immunogenic composition.

[0161] Illustrative Embodiment 4 The method according to Illustrative Embodiment 2 or 3, wherein the dendritic cells are pulsed with an antigen.

[0162] Illustrative Embodiment 5 The method according to Illustrative Embodiment 2 or 3, wherein the dendritic cells are co-cultured with an antigen source.

[0163] Illustrative Embodiment 6 The method according to Illustrative Embodiment 5, wherein the contacting step is further defined as co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells.

[0164] Illustrative embodiment 7 The method according to any one of illustrative 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.

[0165] Illustrative Embodiment 8 A method for preparing an immunogenic composition, the method comprising the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells; and (3) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell in (2) to form the immunogenic composition.

[0166] Illustrative Embodiment 9 A method for treating cancer in a subject, the method comprising the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to produce mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell in (2); and (4) administering the antigen-loaded dendritic cells to the subject.

[0167] Illustrative Embodiment 10 The method according to Illustrative Embodiment 8 or 9, wherein at least a portion of steps (1) and (2) are performed simultaneously, so that during the co-cultivation process, the alternating electric field is also applied to the at least one cancer cell.

[0168] Illustrative Embodiment 11 The method according to any one of Illustrative Embodiments 8-10, wherein the at least one separated cancer cell is exposed to an alternating electric field before step (2).

[0169] Illustrative Embodiment 12 The method according to Illustrative Embodiment 11, wherein an alternating electric field is applied to a target area of ​​the subject before separating the at least one cancer cell from the subject.

[0170] Illustrative Embodiment 13 The method according to Illustrative Embodiment 11 or 12, wherein the at least one cancer cell is exposed to an alternating electric field in vitro prior to co-culturing.

[0171] Illustrative Embodiment 14 The method according to any one of Illustrative Embodiments 8-13, wherein the method comprises isolating the composition from the subject before step (1).

[0172] Illustrative Embodiment 15 The method according to Illustrative Embodiment 14, wherein the composition comprises peripheral blood mononuclear cells (PBMC).

[0173] Illustrative Embodiment 16 The method according to Illustrative Embodiment 14 or 15, wherein the separation of the composition is further defined as comprising the steps of: isolating immature monocytes (dendritic cell precursors) from the bloodstream of the subject; and generating immature dendritic cells from the immature monocyte / dendritic cell precursors.

[0174] Illustrative Embodiment 17 The method according to any one of Illustrative Embodiments 8 to 16, wherein the composition described in (1) comprises PBMCs isolated from an HLA-matched donor.

[0175] Illustrative Embodiment 18 The method according to any one of Illustrative Embodiments 8 to 17, wherein the at least one cancer cell is further defined as at least a portion of a solid tumor.

[0176] Illustrative embodiment 19 The method according to any one of illustrative embodiments 8 to 18, wherein step (2) is 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, and combinations thereof.

[0177] Illustrative Embodiment 20 According to the method described in any one of Illustrative Embodiments 9 to 19, the method further comprises the following steps: (5) applying the alternating electric field to the target area of ​​the subject.

[0178] Illustrative embodiment 21 According to the method described in any one of illustrative embodiments 9-20, the method is further defined as a method for reducing the volume of a tumor and / or preventing the volume of the tumor from increasing, wherein the tumor is present in the body of a living subject and comprises a plurality of cancer cells, and wherein at least one cancer cell is separated from the tumor before step (2).

[0179] Illustrative embodiment 22 According to the method of illustrative embodiment 21, the method further comprises the following steps: before separating the at least one cancer cell, applying an alternating electric field to a target area of ​​the subject, and wherein the target area includes the tumor.

[0180] Illustrative Embodiment 23 An immunogenic composition comprising a population of isolated and antigen-loaded dendritic cells, wherein the antigen-loaded dendritic cells are produced by co-culturing dendritic cells with at least one cancer cell isolated from a subject, and wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro prior to co-culturing with the dendritic cells.

[0181] Illustrative Embodiment 24 A method for preparing an immunogenic composition, the method comprising the steps of: co-culturing dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells, wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro prior to co-culturing with the dendritic cells; and isolating a population of antigen-loaded dendritic cells to form the immunogenic composition.

[0182] Illustrative Embodiment 25 A method for preparing an immunogenic composition, the method comprising the following steps: (1) applying an alternating electric field to a target area of ​​a subject; (2) isolating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to produce antigen-loaded dendritic cells; and (4) isolating the antigen-loaded dendritic cells from the co-culture and the cancer cells in (3) to form the immunogenic composition.

[0183] Illustrative Embodiment 26 A method for treating cancer in a subject, the method comprising the following steps: (1) applying an alternating electric field to a target area of ​​the subject; (2) isolating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to produce antigen-loaded dendritic cells; (4) isolating antigen-loaded dendritic cells from the co-culture and the cancer cells in (3); and (5) administering the antigen-loaded dendritic cells to the subject.

[0184] Illustrative Embodiment 27 According to the method of Illustrative Embodiment 25 or 26, the method further comprises the following step: isolating the dendritic cells or precursors thereof used in step (3) from the subject before step (1).

[0185] Illustrative Embodiment 28 The method according to Illustrative Embodiment 27, wherein the isolation of the dendritic cells is further defined as comprising the following steps: isolating immature monocytes (dendritic cell precursors) from the bloodstream of the subject; generating immature dendritic cells from the immature monocytes / dendritic cell precursors; and culturing the dendritic cell precursors to induce differentiation into mature dendritic cells.

[0186] Illustrative Embodiment 29 According to the method of any one of Illustrative Embodiments 25 to 28, the method further comprises the following step: isolating dendritic cells or precursors thereof from the subject after step (1).

[0187] Illustrative Embodiment 30 The method according to any one of Illustrative Embodiments 25-29, wherein the dendritic cells are isolated from an HLA-matched donor.

[0188] Illustrative Embodiment 31 The method according to any one of Illustrative Embodiments 25 to 30, wherein step (3) is 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, and combinations thereof.

[0189] Illustrative Embodiment 32 According to the method described in any one of Illustrative Embodiments 26 to 31, the method further comprises the following steps: (6) applying the alternating electric field to the target area of ​​the subject.

[0190] Illustrative Embodiment 33 The method according to any one of Illustrative Embodiments 26-32 is further defined as a method for reducing tumor volume and / or preventing the tumor volume from increasing, wherein the tumor is present in the body of a living subject and contains a plurality of cancer cells, and step (1) is further defined as applying an alternating electric field to a target area of ​​the subject, wherein the target area includes the tumor.

[0191] Illustrative embodiment 34 A method for preparing an immunogenic composition, the method comprising the following steps: (1) isolating at least one cancer cell from a subject; (2) applying an alternating electric field to the isolated at least one cancer cell in vitro; (3) co-culturing the isolated at least one cancer cell to which the alternating electric field has been applied with dendritic cells to produce antigen-loaded dendritic cells; and (4) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell in (3) to form the immunogenic composition.

[0192] Illustrative Embodiment 35 A method for treating cancer in a subject, the method comprising the following steps: (1) isolating at least one cancer cell from the subject; (2) applying an alternating electric field to the at least one isolated cancer cell in vitro; (3) co-culturing the at least one isolated cancer cell to which the alternating electric field has been applied with dendritic cells to produce antigen-loaded dendritic cells; (4) isolating antigen-loaded dendritic cells from the co-culture and the at least one cancer cell in (3); and (5) administering the antigen-loaded dendritic cells to the subject.

[0193] Illustrative Embodiment 36 The method according to Illustrative Embodiment 34 or 35, wherein the at least one cancer cell is further defined as at least a portion of a tumor.

[0194] Illustrative Embodiment 37 The method according to any one of Illustrative Embodiments 34-36, wherein the dendritic cells are isolated from the subject and / or an HLA-matched donor.

[0195] Illustrative embodiment 38 The method according to any one of illustrative embodiments 34 to 37, wherein step (3) is 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, and combinations thereof.

[0196] Illustrative Embodiment 39 According to the method described in any one of Illustrative Embodiments 35 to 38, the method further comprises the following steps: (6) applying the alternating electric field to the target area of ​​the subject.

[0197] Illustrative Embodiment 40 According to the method described in any one of Illustrative Embodiments 35-39, the method is further defined as a method for reducing tumor volume and / or preventing the tumor volume from increasing, wherein the tumor is present in the body of a living subject and contains a plurality of cancer cells, and wherein steps (1) to (3) are further defined as: (1) removing at least a portion of the tumor from the subject; (2) applying an alternating electric field to at least a portion of the removed tumor in vitro; and (3) co-culturing the at least a portion of the removed tumor to which the alternating electric field has been applied with dendritic cells to produce antigen-loaded dendritic cells.

[0198] Illustrative Embodiment 41 The method according to any one of Illustrative Embodiments 6-40, wherein the at least one cancer cell is selected from the group consisting of the following items: Cancer / cancer cell is selected from the group consisting of the following items: 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.

[0199] Illustrative Embodiment 42 The method according to any one of Illustrative Embodiments 9-22, 26-33 and 35-41, wherein the antigen-loaded dendritic cells are administered intradermally, subcutaneously, intravenously and / or intranodally.

[0200] Illustrative Embodiment 43 The method according to any one of illustrative embodiments 9-22, 26-33 and 35-42, wherein the antigen-loaded dendritic cells are administered to the subject in the form of at least one immunogenic composition, and wherein the at least one immunogenic composition further comprises at least one 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-LAG-3 agent (such as but not limited to OPDUALAG), TM and / or relalizumab (Bristol-Myers Squibb, New York, NY)) and combinations thereof.

[0201] Illustrative embodiment 44 An immunogenic composition comprising: a population of isolated and antigen-loaded dendritic cells produced by the method of any one of illustrative embodiments 3-8, 10-19, 24-25, 27-31, 34, 36-38 and 41.

[0202] Illustrative Embodiment 45 The method or immunogenic composition according to any one of Illustrative Embodiments 1-44, wherein at least one of the following is achieved: the alternating electric field is applied at a frequency in the range of about 50 kHz to about 1 MHz; in at least a portion of the cancer cells, the field strength of the alternating electric field is at least about 1 V / cm; and the time period for which the alternating electric field is applied is at least about 24 hours.

[0203] Illustrative Embodiment 46 The method or immunogenic composition according to Illustrative Embodiment 45, wherein the alternating electric field is applied at a frequency in the range of about 50 kHz to about 500 kHz, or about 50 kHz to about 190 kHz, or about 50 kHz to about 180 kHz, or about 50 kHz to about 175 kHz, or about 50 kHz to about 160 kHz, or about 50 kHz to about 150 kHz.

[0204] Illustrative Embodiment 47 The method or immunogenic composition according to Illustrative Embodiment 46, wherein the alternating electric field is applied at a frequency of about 150 kHz, a field strength of about 2.7 V / cm, and a duration of about 48 hours.

[0205] Illustrative Embodiment 48 The immunogenic composition according to any one of Illustrative Embodiments 23 and 44-47, further comprising a pharmaceutically acceptable carrier.

[0206] Illustrative Embodiment 49 The immunogenic composition according to any one of Illustrative Embodiments 23 and 44-48, wherein the immunogenic composition is formulated for intradermal, subcutaneous, intravenous and / or intranodal administration.

[0207] Illustrative embodiment 50 The immunogenic composition according to any one of illustrative 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-LAG-3 agent, and a combination thereof.

[0208] Illustrative Embodiment 51 The method or immunogenic composition according to any one of Illustrative Embodiments 1-50, wherein the dendritic cells include at least one of conventional DC1 (cDC1), cDC2, and plasmacytoid DC (pDC).

[0209] Illustrative embodiment 52 A use of the immunogenic composition according to any one of illustrative embodiments 23 and 44-51 in a method for treating cancer.

[0210] Illustrative Embodiment 53 A use of an immunogenic composition in a method for treating cancer, wherein the use comprises a method according to any one of Illustrative Embodiments 9-22, 26-33, 35-43, 45-47 and 51.

[0211] Although the above disclosure describes the inventive concept in conjunction with the specific experiments, results and language of elaboration, it is obvious that many substitutions, modifications and variations are obvious to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A method for activating dendritic cells, the method comprising the following steps: An alternating electric field is applied to a composition comprising immature dendritic cells or precursors thereof in vivo for a sufficient period of time to produce activated dendritic cells.

2. The method according to claim 1, further comprising the following steps: The activated dendritic cells are contacted with an antigen source to generate antigen-loaded dendritic cells.

3. A method for preparing an immunogenic composition, the method comprising the following steps: applying an alternating electric field to a composition comprising immature dendritic cells or precursors thereof in vivo for a sufficient period of time to produce activated dendritic cells; contacting the activated dendritic cells with an antigen source to generate antigen-loaded dendritic cells; as well as The antigen-loaded dendritic cells are isolated to form the immunogenic composition.

4. The method of claim 2 or 3, wherein the dendritic cells are pulsed with an antigen. The method according to claim 2 or 3, wherein the dendritic cells are co-cultured with an antigen source.

6. The method according to any one of claims 2-5, 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. A method for preparing an immunogenic composition, the method comprising the steps of: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell of (2) to form the immunogenic composition.

8. A method for treating cancer in a subject, the method comprising the following steps: (1) applying an alternating electric field to a composition comprising immature dendritic cells and / or dendritic cell precursors in vitro to generate mature dendritic cells; (2) co-culturing the mature dendritic cells with at least one cancer cell isolated from the subject to produce antigen-loaded dendritic cells; (3) isolating the antigen-loaded dendritic cells from the co-culture and the at least one cancer cell of (2); and (4) administering the antigen-loaded dendritic cells to the subject.

9. The method according to claim 7 or 8, wherein at least part of steps (1) and (2) are performed simultaneously, so that during the co-cultivation process, the alternating electric field is also applied to the at least one cancer cell.

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

11. The method of claim 10, wherein an alternating electric field is applied to a target area of ​​the subject before separating the at least one cancer cell from the subject.

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

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

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

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

16. The method according to any one of claims 7 to 15, wherein step (2) is 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, and combinations thereof.

17. The method according to any one of claims 8 to 16, further comprising the following steps: (5) Applying the alternating electric field to the target area of ​​the subject.

18. An immunogenic composition, comprising: A population of isolated and antigen-loaded dendritic cells, wherein the antigen-loaded dendritic cells are produced by co-culturing dendritic cells with at least one cancer cell isolated from a subject, and wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro prior to co-culturing with the dendritic cells.

19. A method for preparing an immunogenic composition, the method comprising the steps of: Co-culturing dendritic cells with at least one cancer cell isolated from a subject to produce antigen-loaded dendritic cells, wherein the at least one cancer cell has been exposed to an alternating electric field in vivo or in vitro prior to co-culturing with the dendritic cells; as well as A population of antigen-loaded dendritic cells is isolated to form the immunogenic composition.

20. A method for preparing an immunogenic composition, the method comprising the steps of: (1) applying an alternating electric field to a target area of ​​the subject; (2) separating cancer cells from the target area to which the alternating electric field has been applied; (3) co-culturing the isolated cancer cells with dendritic cells to produce antigen-loaded dendritic cells; as well as (4) Isolating antigen-loaded dendritic cells from the co-culture and the cancer cells described in (3) to form the immunogenic composition.

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

22. The method according to claim 20 or 21, further comprising the steps of: The dendritic cells or precursors thereof used in step (3) are isolated from the subject before step (1).

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

24. The method according to any one of claims 20-23, wherein step (3) is 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, and combinations thereof.

25. The method according to any one of claims 21 to 24, further comprising the steps of: (6) Applying the alternating electric field to the target area of ​​the subject.

26. A method for preparing an immunogenic composition, the method comprising the steps of: (1) isolating at least one cancer cell from a subject; (2) applying an alternating electric field to the at least one isolated cancer cell in vitro; (3) co-culturing the separated at least one cancer cell to which the alternating electric field has been applied with dendritic cells to generate antigen-loaded dendritic cells; as well as (4) isolating antigen-loaded dendritic cells from the co-culture described in (3) and the at least one cancer cell to form the immunogenic composition.

27. A method for treating cancer in a subject, the method comprising the steps of: (1) isolating at least one cancer cell from the subject; (2) applying an alternating electric field to the at least one isolated cancer cell in vitro; (3) co-culturing the separated at least one cancer cell to which the alternating electric field has been applied with dendritic cells to generate antigen-loaded dendritic cells; (4) isolating antigen-loaded dendritic cells from the co-culture of (3) and the at least one cancer cell; and (5) administering the antigen-loaded dendritic cells to the subject.

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

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

30. The method according to any one of claims 26-29, wherein step (3) is 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, and combinations thereof.

31. The method according to any one of claims 27 to 30, further comprising the steps of: (6) Applying the alternating electric field to the target area of ​​the subject.

32. The method of any one of claims 7-31, 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 8-17, 21-25 and 27-32, wherein the antigen-loaded dendritic cells are administered intradermally, subcutaneously, intravenously and / or intranodally.

34. according to the method described in any one of claims 8-17, 21-25 and 27-33, wherein the dendritic cells of the antigen load are administered to the study subject in the form of at least one immunogenic composition, and wherein the at least one immunogenic composition further comprises at least one compound selected from the group consisting of the following: adjuvant, cytokine, interferon, TLR agonist, STING (stimulator of interferon genes) agonist, GM-CSF, CD40L, FMS-related tyrosine kinase 3 ligand (FLT3L), C-type lectin receptor (CLR), anti-LAG-3 agent (such as but not limited to OPDUALAG), TM and / or relalizumab (Bristol-Myers Squibb, New York, NY)) and combinations thereof.

35. An immunogenic composition, comprising: A population of isolated and antigen-loaded 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. The method or immunogenic composition of any one of claims 1-35, wherein at least one of the following is achieved: the alternating electric field is applied at a frequency in the range of about 50 kHz to about 1 MHz; the field strength of the alternating electric field is at least about 1 V / cm in at least a portion of the cancer cells; and the alternating electric field is applied for a period of at least about 24 hours.

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 about 50 kHz to about 190 kHz, or about 50 kHz to about 180 kHz, or about 50 kHz to about 175 kHz, or about 50 kHz to about 160 kHz, or about 50 kHz to about 150 kHz.

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

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

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

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-LAG-3 agent, and combinations thereof.

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

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

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

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