CN117425515A

CN117425515A - Compositions and methods for treatment with a combination of alternating electric field and trastuzumab

Info

Publication number: CN117425515A
Application number: CN202280039734.3A
Authority: CN
Inventors: E·H·金
Original assignee: Novokule Co ltd
Current assignee: Novokule Co ltd
Priority date: 2021-06-01
Filing date: 2022-05-31
Publication date: 2024-01-19
Also published as: EP4346998A1; JP2024520616A; WO2022254340A1

Abstract

Disclosed are methods of treating a subject in need thereof, comprising applying an alternating electric field to a target site of a subject in need thereof at a frequency for a period of time; and administering trastuzumab to a target site in a subject in need thereof. Disclosed are methods of increasing the accumulation of trastuzumab at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof at a frequency for a period of time; and administering trastuzumab to a target site of a subject in need thereof, wherein the accumulation of trastuzumab at the target site is increased as compared to administering trastuzumab at the target site without applying an alternating electric field. Disclosed are methods of inhibiting proliferation or increasing apoptosis of cancer cells at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof for a period of time and at a frequency; and administering trastuzumab to a target site in a subject in need thereof, wherein proliferation of the cancer cells is inhibited or wherein apoptosis of the cancer cells is increased.

Description

Compositions and methods for treatment with a combination of alternating electric field and trastuzumab

Background

One of the reasons for the increased mortality rate in women in the united states is the increasing number of breast cancer cases. According to the american cancer society 2015 census, it is estimated that about 60,290 breast cancer cases (in situ) are to be identified. In addition, in all malignant tumors, breast cancer accounts for 7-10%, increasing about 3-4% per year in China. Increased activation of the human epidermal growth factor receptor 2 (HER 2) tyrosine kinase receptor gene has also been found to be a negative prognostic factor for early stage nodule positive breast cancer. Thus, an enhanced interpretation of the mechanism and effect of the HER2 gene may lead to classification, prognosis and remodeling of disease treatment. In addition, it was found that 15-20% of the highly expressed HER2 gene in various breast cancers, which tends to regulate various cellular processes including cell survival, proliferation, angiogenesis, invasion and metastasis. Recently, humanized monoclonal anti-ERBB 2 antibodies, known as Trastuzumab (Herceptin), have been discovered which significantly improve the clinical outcome of early and late HER2 positive breast cancers. Trastuzumab affects HER2 by inhibiting dimerization of HER2 and thus affecting the growth signal that ultimately shuts down HER2 receptor gene expression. The drug also targets the phosphatidylinositol 3-kinase (PI 3K)/AKT/mTOR pathway and the RAS/RAF/MEK/MAP kinase (MAPK) pathway. In addition, the Fc portion of trastuzumab is involved in antibody-dependent cellular cytotoxicity (ADCC) function. Although the antibody shows high initial efficacy, patients with metastatic breast cancer stages develop primary resistance to the antibody. Thus, in this case, such treatment cannot prove to be effective. Thus, this disadvantage poses a greater challenge in the treatment of HER2 positive breast cancer patients.

Tumor treatment fields (better abbreviated as ttfields or TTFs) are therapies that contain an alternating electromagnetic field used with a low-intensity electric field to inhibit cancer cell proliferation in vivo. The therapy targets induced electric fields in humans to inhibit proliferation and invasion of cancer cells. This treatment induces apoptosis and thus shows the ability to destroy cancer cells. The FDA has approved the use of TTField to treat patients with recurrent glioblastoma multiforme or GBM. This technique was found to increase the life expectancy of the patient. In the case of a newly diagnosed GBM, this technique can be combined with temozolomide prior to surgery (www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpma/pma.cfmid=p 100034) (www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpma.cfmid=p 100034S 013). The american clinical oncology institute (ASCO) regards this treatment as an advancement in cancer treatment due to its novel methods, efficacy and low toxicity properties. The national integrated cancer network (NCCN) recommends ttfieldas a class 2A treatment for patients with newly diagnosed GBM and results in a good performance status (www.nccn.org/pro-files/physiologias/f guide. In contrast to commonly used methods (chemotherapy and radiotherapy), TTF does not cause side effects such as pain, nausea, fatigue or diarrhea. However, this technique is associated with some of the drawbacks noted in the test, including localized rash caused by prolonged electrode use. However, the efficacy of TTF is considered to be similar to that of chemotherapy or radiotherapy. The combined anti-tumor effect of TTF and trastuzumab on HER2 positive breast cancer representative cell lines was subsequently studied along with tumor xenograft models. The results show that TTF therapy can significantly enhance trastuzumab-induced growth inhibition.

Disclosure of Invention

Disclosed are methods of treating a subject in need thereof, comprising applying an alternating electric field to a target site of a subject in need thereof at a frequency for a period of time; and administering trastuzumab to a target site in a subject in need thereof.

Disclosed are methods of increasing the accumulation of trastuzumab at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof at a frequency for a period of time; and administering trastuzumab to a target site of a subject in need thereof, wherein the accumulation of trastuzumab at the target site is increased as compared to administering trastuzumab at the target site without applying an alternating electric field.

Disclosed is a method of inhibiting proliferation of cancer cells at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof for a period of time and at a frequency; and administering trastuzumab to a target site in a subject in need thereof, wherein proliferation of the cancer cells is inhibited.

Disclosed are methods of increasing apoptosis of cancer cells at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof for a period of time and at a frequency; and administering trastuzumab to a target site in a subject in need thereof, wherein apoptosis of the cancer cells is increased.

Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and together with the description, serve to explain the principles of the disclosed methods and compositions.

Figures 1A-1B depict the effect of trastuzumab treatment on HER2 positive cell viability. Fig. 1A: TRZ inhibits HER2 positive cell viability in a dose-dependent manner. Fig. 1B: cell viability of Jimt1 and BT474 cells treated with specific amounts TRZ was assessed using the MTT assay; * p <0.05.

Figures 2A-2C depict trastuzumab treatment and the effect of TTF on HER2 positive cell viability. Fig. 2A: jimt1 cells were treated in dimethylsulfoxide (DMSO, vehicle), TRZ (5 μg/ml), TTF (1.5V/cm), combination for 48 hours and monitored for morphological changes under a phase contrast microscope (magnification X400). Fig. 2B: cell viability was determined by trypan blue exclusion assay. Fig. 2C: colony formation assays were performed using Jimt1 cells treated with the indicated treatments for 7-9 days (n=3); * p <0.05, < p <0.01, < p <0.001.

Figures 3A-3C depict TTF anti-trastuzumab resistant tumor effects in a BT474 cell line human breast cancer xenograft model. Fig. 3A: control (saline; "CTL"), trastuzumab ("TRZ"), TTF, or a combination of trastuzumab and TTF ("ttf+ TRZ") was used to treat nude mice bearing BT474R cells as xenografts. Fig. 3B: therapeutic effect on tumor volume. Fig. 3C: representative hematoxylin and eosin (H & E) staining of Ki-67. Data are expressed as mean ± standard error of mean (n=5), P <0.05.

Figures 4A-4C depict the combined treatment induced apoptosis in trastuzumab-resistant HER2 positive cell lines. Fig. 4A: jimt1 cells were treated with DMSO, TRZ (5 μg/ml), TTF, or a combination for 48 hours. Cell lysates (30 μg) were immunoblotted (or western blotted) with antibodies to pHER2, HER2, cleaved PARP, bcl-2 and β -actin. Fig. 4B: representative TUNEL. Data are expressed as mean ± standard error of mean (n=10), P <0.05. Fig. 4C: TUNEL positive cells were stained and imaged with a 10X objective using an In cell analyzer. Apoptotic cells are shown in light grey.

Figure 5 depicts the effect of TTF on growth factor receptor 2 of the human epidermis and downstream signaling pathways. The xenografts were examined for pAKT, pERK and pHER2 expression using immunohistochemistry. * p <0.05, p <0.01.

Fig. 6 depicts TTF increasing permeation of trastuzumab. Fluorescence images were obtained with a 10X objective using an In cell analyzer. The channels are as follows: DAPI was used for nuclei (shown in blue), alexa 488-TRZ (shown in green) and rhodamine lectin to detect blood vessels (shown in red). TRZ infiltration from tumor vessels is plotted as a linear distribution measured by the intensities of the vessels in the various ROIs from the peripheral and central regions of each tumor. In addition, the area under the curve was calculated from three tumors with 10 sections in each tumor in both groups. * p <0.05.

Detailed Description

The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and the examples included therein and the figures and their previous and following description.

It is to be understood that the disclosed methods and compositions are not limited to particular synthetic methods, particular analytical techniques, or particular reagents, and thus may vary, unless otherwise indicated. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Materials, compositions, and components are disclosed that can be used in, in conjunction with, or as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed, and an example of a combination molecule a-D is disclosed, each is contemplated individually and collectively, even if each is not individually recited. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F is specifically contemplated, and should be considered as being from A, B and C; D. e and F and example combinations A-D. Also, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, a subgroup of A-E, B-F and C-E is specifically contemplated, and should be considered as being from A, B and C; D. e and F and example combinations A-D. This concept applies to all aspects of the present application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

A. Definition of the definition

It is to be understood that the disclosed methods and compositions are not limited to the particular methods, protocols, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, reference to "the cell" refers to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

As used herein, a "target site" is a particular site or location present in or on a subject or patient. For example, a "target site" may refer to, but is not limited to, a cell (e.g., a cancer cell), a cell population, an organ, a tissue, or a tumor. Thus, the phrase "target cell" may be used to refer to a target site, wherein the target site is a cell. In some aspects, a "target cell" may be a cancer cell. In some aspects, the organ that may be the target site includes, but is not limited to, an abdominal organ (e.g., stomach, intestine) or breast. In some aspects, the cell or population of cells that can be the target site or target cell includes, but is not limited to, breast tissue cells or abdominal cells. In some aspects, the "target site" may be a tumor target site.

A "tumor target site" is a site or location present in or on a subject or patient that comprises or is adjacent to one or more cancer cells, previously comprising one or more tumor cells, or suspected of comprising one or more tumor cells. For example, a tumor target site may refer to a site or location that exists in or on a subject or patient susceptible to metastasis. In addition, a target site or tumor target site may refer to a excision site or location of a primary tumor present in or on a subject or patient. In addition, a target site or tumor target site may refer to a site or location adjacent to a primary tumor resection that is present in or on a subject or patient.

As used herein, "alternatingAn electric field (alternating electric field) "or" alternating electric field (alternating electric fields) "refers to a very low intensity, directional, medium frequency alternating electric field delivered to, obtained from, or delivered to a particular location (e.g., a target site, such as a cell) within a subject or patient. In some aspects, the alternating electric field may be unidirectional or multidirectional. In some aspects, the alternating electric field may be delivered by two pairs of transducer arrays that generate a vertical field within the target site. For example, for Optune ^TM System (alternating electric field delivery system), one pair of electrodes is located to the Left and Right (LR) of the target site, and the other pair of electrodes is located to the front and rear (AP) of the target site. Cycling the field between these two directions (i.e., LR and AP) ensures that the maximum range of cell orientations is targeted.

As used herein, an "alternating electric field" applied to a tumor target site may be referred to as a "tumor treatment field" or "TTField". "TTFields" have been identified as forms of anti-mitotic cancer treatment because they interfere with proper microtubule assembly during metaphase and ultimately destroy cells during the terminal, cytokinesis, or subsequent intervals. TTField targets solid tumors and is described in U.S. patent No. 7,565,205, the teachings of which are incorporated herein by reference in their entirety.

In vivo and in vitro studies have shown that the efficacy of TTField therapy increases with increasing electric field strength. Thus, optimizing array placement on a subject to increase the target location or intensity in target cells is a standard practice of the Optune system. Array placement optimization may be performed by "rules of thumb" (e.g., placing the array on the subject as close as possible to the target site or target cell), measurements describing the geometry of the patient's body, target site size, and/or target site or cell location. The measurement used as input may be derived from the imaging data. Imaging data is intended to include any type of visual data, such as Single Photon Emission Computed Tomography (SPECT) image data, x-ray computed tomography (x-ray CT) data, magnetic Resonance Imaging (MRI) data, positron Emission Tomography (PET) data, data that may be captured by an optical instrument (e.g., a photographic camera, a Charge Coupled Device (CCD) camera, an infrared camera, etc.), and so forth. In some implementations, the image data may include 3D data (e.g., point cloud data) obtained from or generated by a 3D scanner. Optimization may rely on an understanding of how the electric field is distributed within the target sites or target cells as a function of array position, and in some aspects, account for variations in the distribution of electrical properties within the head of different patients.

The term "subject" refers to a target, such as an animal, to which it is administered. Thus, the subject of the disclosed methods can be a vertebrate, e.g., a mammal. For example, the subject may be a human. The term does not denote a particular age or gender. A subject may be used interchangeably with "individual" or "patient. For example, the subject to be administered may refer to the recipient of the alternating electric field. For example, the subject administered may be a subject with early and late HER2 positive breast cancer.

"treatment" refers to the administration or application of therapeutic agents, such as alternating electric fields and vectors, to a subject (e.g., a human or other mammal (e.g., an animal model)) having or having an increased susceptibility to developing breast or gastric cancer, to prevent or delay the progression of the effect of a disease or infection, or to partially or fully reverse the effect of breast or gastric cancer. For example, treating a subject having breast cancer or gastric cancer may include delivering a therapeutic agent to cells in the subject.

"preventing" refers to minimizing or reducing the chance of a subject developing breast or gastric cancer.

As used herein, the terms "administering" and "administering" refer to any method of providing trastuzumab to a target site or subject. Such methods are well known to those skilled in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ocular administration, intra-aural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable, e.g., intravenous administration, intra-arterial administration, intramuscular administration and subcutaneous administration. Administration may be continuous or intermittent. In various aspects, the formulation may be administered therapeutically; i.e., to treat breast or gastric cancer. In further aspects, the formulation may be administered prophylactically; that is, administration is for the prevention of breast cancer or gastric cancer. In one aspect, the skilled artisan can determine an effective dose, an effective schedule, or an effective route of administration in order to treat the subject. In some aspects, the applying comprises exposing or applying. Thus, in some aspects, exposing or applying an alternating electric field to a target site or subject refers to applying an alternating electric field to the target site or subject.

As used herein, a "therapeutically effective amount" is an amount of trastuzumab or a composition that provides a therapeutic benefit to an individual or subject. For example, a therapeutically effective amount of trastuzumab is an amount that treats, reduces, improves, alleviates, reduces symptoms of, delays onset of, inhibits progression of, reduces the severity of, and/or reduces the incidence of breast or gastric cancer. In one embodiment, a therapeutically effective amount of trastuzumab will result in an improvement in, or prevent or slow the progression of, one or more indicators or symptoms (such as those described herein) of breast or gastric cancer. As used herein, "treating" a subject having breast cancer or gastric cancer includes administering a therapeutically effective amount of trastuzumab.

"optional" or "optionally" means that the subsequently described event, circumstance or material may or may not occur or be present, and that the description includes instances where the event, circumstance or material occurs or is present and instances where it does not.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, it is specifically contemplated and considered that a range from one particular value and/or to another particular value is disclosed unless the context clearly dictates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another particularly contemplated embodiment, which should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint, unless the context clearly indicates otherwise. Finally, it is to be understood that all individual values and subranges of values included within the explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies whether or not some or all of these embodiments are explicitly disclosed in particular instances.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the materials to which they refer are specifically incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprises" and "comprising", will be used in the sense of "including but not limited to", and are not intended to exclude, for example, other additives, components, integers or steps. In particular, in a method that is stated to include one or more steps or operations, it is specifically contemplated that each step includes what is listed (unless the step includes a limiting term, such as "consisting of … …"), meaning that each step is not intended to exclude other additives, components, integers or steps, such as those not listed in the step.

B. Alternating electric field

The methods disclosed herein include an alternating electric field. In some aspects, the alternating electric field used in the methods disclosed herein is a tumor treatment field. In some aspects, the alternating electric field may vary depending on the type of cell or the conditions under which the alternating electric field is applied. In some aspects, the alternating electric field may be applied by one or more electrodes placed on the subject's body. In some aspects, there may be two or more pairs of electrodes. For example, the arrays may be placed on the anterior/posterior and lateral sides of a patient and may be used with the systems and methods disclosed herein. In some aspects, when two pairs of electrodes are used, the alternating electric field may alternate between the two pairs of electrodes. For example, a first pair of electrodes may be placed in front of and behind a subject, while a second pair of electrodes may be placed on either side of the subject, then an alternating electric field may be applied, and may alternate between front and rear electrodes, then to side electrodes.

In some aspects, the frequency of the alternating electric field is between 100kHz and 1 MHz. In some aspects, the frequency of the alternating electric field is between 100-500 kHz. The frequency of the alternating electric field may also be, but is not limited to, between 50-500kHz, between 100-500kHz, between 25kHz to 1MHz, between 50-190kHz, between 25-190kHz, between 180-220kHz, or between 210-400 kHz. In some aspects, the frequency of the alternating electric field may be an electric field at 50kHz, 100kHz, 150kHz, 200kHz, 250kHz, 300kHz, 350kHz, 400kHz, 450kHz, 500kHz, or any frequency in between. In some aspects, the frequency of the alternating electric field is about 200kHz to about 400kHz, about 250kHz to about 350kHz, and may be about 300kHz.

In some aspects, the field strength of the alternating electric field may be between 0.5 and 4V/cm RMS. In some aspects, the field strength of the alternating electric field may be between 1-4V/cm RMS. In some aspects, different field strengths (e.g., between 0.1-10V/cm) may be used. In some aspects, the field strength may be 1.75V/cm RMS. In some embodiments, the field strength is at least 1V/cm RMS. In some aspects, the field strength may be at least 0.9V/cm RMS. In other embodiments, a combination of field strengths is applied, e.g., two or more frequencies are combined simultaneously, and/or two or more frequencies are applied at different times.

In some aspects, various different intervals of alternating electric field in the range of 0.5 hours to 72 hours may be applied. In some aspects, different durations (e.g., between 0.5 hours and 14 days) may be used. In some aspects, the application of the alternating electric field may be repeated periodically. For example, the alternating electric field may be applied daily for a duration of two hours.

In some aspects, the exposure may last for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more.

The disclosed methods include applying one or more alternating electric fields to a cell or subject. In some aspects, an alternating electric field will be applied to the target site or tumor target site. When an alternating electric field is applied to the cells, this may generally apply an alternating electric field to the subject comprising the cells. Thus, applying an alternating electric field to the target site of the subject results in applying an alternating electric field to the cells.

C. Trastuzumab

Trastuzumab antibodies to be administered to a target site of a subject in need thereof are disclosed. In some aspects, trastuzumab is administered as a combination therapy or treatment with an alternating electric field.

Compositions comprising one or more trastuzumab antibodies are disclosed.

In some aspects, the composition may be a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising trastuzumab as described herein as an active ingredient, and, or consisting essentially of, or consisting of, a pharmaceutically acceptable carrier, diluent, or excipient.

Compositions and formulations of trastuzumab with a pharmaceutically acceptable carrier or diluent are disclosed. For example, pharmaceutical compositions comprising trastuzumab and a pharmaceutically acceptable carrier are disclosed.

For example, the compositions described herein may comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant that the material or carrier selected will minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is well known to those skilled in the art. Examples of carriers include dimyristoyl phosphatidyl (DMPC), phosphate buffered saline, or polycystic liposomes. For example, PG: PC: cholesterol: peptide or PC: peptide may be used as a carrier in the present invention. Other suitable pharmaceutically acceptable carriers and formulations thereof are described in Remington: the Science and Practice of Pharmacy (19 th edition) editors A.R. Gennaro, mack Publishing Company, easton, pa.1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, ringer's solution, and dextrose solution. The pH of the solution may be about 5 to about 8, or about 7 to about 7.5. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in blood vessels during the angioplasty procedure), liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition to be administered. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

The pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives and the like, provided that the intended activity of the polypeptides, peptides, nucleic acids, carriers of the invention is not compromised. The pharmaceutical composition may also include one or more active ingredients (in addition to the compositions of the present invention), such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. In the methods described herein, delivery of the disclosed compositions to cells can be via a variety of mechanisms. The pharmaceutical composition may be administered in a variety of ways depending on whether local or systemic treatment is desired, and depending on the area to be treated.

1. Delivery of compositions

Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g. olive oil) and injectable organic esters (e.g. ethyl oleate). Aqueous carriers include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, ringer's lactate solution or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases.

Formulations for topical application may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily matrices, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Some compositions may potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids (e.g., hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric and phosphoric) and organic acids (e.g., formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic and fumaric) or by reaction with inorganic bases (e.g., sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (e.g., mono-, di-, trialkylamines and aryl amines and substituted ethanolamines).

D. Therapeutic method

In some aspects, the target site comprises a cell. For example, the cell may be, but is not limited to, a cancer cell. In some aspects, the cancer cells may be, but are not limited to, breast cancer cells or gastric cancer cells.

In some aspects, the alternating electric field is applied before, after, or simultaneously with the administration of trastuzumab. In some aspects, trastuzumab is administered before, simultaneously with, or after application of the alternating electric field. Applying the alternating electric field prior to administration of trastuzumab may include seconds, minutes or hours prior to administration of trastuzumab. Applying the alternating electric field after administration of trastuzumab may include seconds, minutes, hours or days after administration of trastuzumab. Applying the alternating electric field simultaneously with the administration of trastuzumab may comprise seconds or minutes before or after the administration of trastuzumab. In some aspects, simultaneously applying an alternating electric field and trastuzumab may include applying trastuzumab while applying an alternating electric field. In some aspects, the step of applying the alternating electric field begins at least one hour prior to the given time.

In some aspects, trastuzumab comprises a detectable agent. As used herein, a detectable agent or label is any molecule that can bind directly or indirectly to trastuzumab and directly or indirectly result in a measurable detectable signal. Many such labels for conjugation or coupling to antibodies are known to those skilled in the art. Examples of detection agents may be, but are not limited to, radioisotopes, fluorescent molecules (fluorophores), phosphorescent molecules, enzymes, antibodies, and ligands.

In some cases, the label may be, but is not limited to, an isotopic label, a colorimetric biosensor, or a fluorescent label. For example, the fluorescent label may be, but is not limited to, green Fluorescent Protein (GFP) or Rhodamine Fluorescent Protein (RFP). Other labels may include biotin, streptavidin, horseradish peroxidase or luciferase. Other examples of suitable fluorescent labels include, but are not limited to, fluorescein (FITC), 5, 6-carboxymethylfluorescein, texas red, nitrobenzene-2-oxa-1, 3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4' -6-diamidino-2-phenylindole (DAPI), and cyanine dyes Cy3, cy3.5, cy5, cy5.5, and Cy7. Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine (5, 6-tetramethyl rhodamine). Preferred fluorescent labels for combination multicolor encoding are FITC and cyanine dyes Cy3, cy3.5, cy5, cy5.5, and Cy7. The absorption and emission maxima of these fluorescent agents are respectively: FITC (490 nm;520 nm), cy3 (554 nm; 218 nm), cy3.5 (581 nm; 5888 nm), cy5 (652 nm:67 nm), cy5.5 (682 nm;703 nm) and Cy7 (755 nm;778 nm) thus allowing their simultaneous detection. Fluorescent labels are available from a variety of commercial sources, including Molecular Probes, eugene, OR and Research Organics, cleveland, ohio.

In some aspects, the frequency of the alternating electric field is any of those described herein. In some aspects, the frequency of the alternating electric field is between 100-500 kHz. For example, in some aspects, the frequency of the alternating electric field is 150kHz.

In some aspects, the field strength of the alternating electric field is any of those described herein. In some aspects, the field strength of the alternating electric field is between 0.5 and 4V/cm RMS. For example, in some aspects, the field strength of the alternating electric field is 0.9V/cm RMS.

In some aspects, trastuzumab is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intradurally, intravascular, intravenous (targeted or non-targeted), intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, intranasal, via intratumoral injection (e.g., computer tomography guided, during surgery or biopsy), or via inhalation. In some aspects, trastuzumab is administered in a pharmaceutical composition. For example, the pharmaceutical composition may be any of those described herein.

In some aspects, a subject in need thereof suffers from cancer. In some aspects, the cancer may be breast cancer or gastric cancer. For example, the cancer may be a HER2 positive cancer.

In some aspects, one or more of pAKT, pERK, and pHER2 expression is reduced as a result of treatment with an alternating electric field and trastuzumab.

Disclosed are methods of treating a subject in need thereof, comprising applying an alternating electric field to a target site of a subject in need thereof at a frequency for a period of time; administering trastuzumab to a target site in a subject in need thereof, and further comprising administering a second therapeutic agent to the subject. For example, the second therapeutic agent may be a chemotherapeutic agent. The chemotherapeutic agent may be, but is not limited to, an alkylating agent, an antimetabolite, an anthracycline, an antitumor antibiotic, a monoclonal antibody, platinum, or a plant alkaloid.

In some aspects, trastuzumab is administered in a therapeutically effective amount.

E. Methods for increasing antibody accumulation

In some aspects, the increase in trastuzumab accumulation is any amount that is greater than the amount of trastuzumab at the target site without application of an alternating electric field. In some aspects, the increase in trastuzumab accumulation is one, two, three, or more times the amount of trastuzumab at the target site without application of an alternating electric field.

In some aspects, the target site comprises a cell. For example, the cell may be a cancer cell. In some aspects, the cancer cell may be, but is not limited to, a breast cancer cell or a gastric cancer cell or any Her2 positive cell.

In some aspects, the alternating electric field is applied before, after, or simultaneously with the administration of trastuzumab. In some aspects, trastuzumab is administered before, simultaneously with, or after application of the alternating electric field. Applying the alternating electric field prior to administration of trastuzumab may include seconds, minutes or hours prior to administration of trastuzumab. Applying the alternating electric field after administration of trastuzumab may include seconds, minutes, hours or days after administration of the antibody. Applying the alternating electric field simultaneously with the administration of trastuzumab may comprise seconds or minutes before or after the administration of trastuzumab. In some aspects, simultaneously applying an alternating electric field and trastuzumab may include applying trastuzumab while applying an alternating electric field. In some aspects, the step of applying the alternating electric field begins at least one hour prior to the given time.

In some aspects, one or more of pAKT, pERK, and pHER2 expression is reduced as a result of increased accumulation of trastuzumab at the target site.

Disclosed are methods of increasing the accumulation of trastuzumab at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof at a frequency for a period of time; administering trastuzumab to a target site of a subject in need thereof, wherein the accumulation of trastuzumab at the target site is increased as compared to administering trastuzumab at the target site without applying an alternating electric field; and further comprising administering a second therapeutic agent to the subject. For example, the second therapeutic agent may be a chemotherapeutic agent. The chemotherapeutic agent may be, but is not limited to, an alkylating agent, an antimetabolite, an anthracycline, an antitumor antibiotic, a monoclonal antibody, platinum, or a plant alkaloid.

F. Methods of inhibiting proliferation of cancer cells

In some aspects, the increase in inhibition of cancer cell proliferation is compared to cancer cell proliferation at the target site when the alternating electric field is administered alone, wherein trastuzumab is administered alone, or wherein no treatment is administered to the subject. In some aspects, cancer cell proliferation is inhibited compared to administration of trastuzumab in the absence of an applied alternating electric field. In some aspects, there is an increase in inhibition of proliferation of cancer cells. For example, the increase in inhibition of cancer cell proliferation is compared to administration of trastuzumab at the target site without application of an alternating electric field. In some aspects, the increase in inhibition of cancer cell proliferation is any amount that is greater than the amount of inhibition of cancer cell proliferation at the target site without application of an alternating electric field. In some aspects, the inhibition of cancer cell proliferation is an amount that is one, two, three, or more times the amount of inhibition of cancer cell proliferation in the absence of an applied alternating electric field.

In some aspects, the target site comprises a cell. For example, the cell may be a cancer cell. In some aspects, the cancer cell may be a breast cancer cell or a gastric cancer cell. For example, in some aspects, the target site is a cancer cell.

In some aspects, the alternating electric field is applied before, after, or simultaneously with the administration of trastuzumab. In some aspects, trastuzumab is administered before, simultaneously with, or after application of the alternating electric field. Applying the alternating electric field prior to administration of trastuzumab may include seconds, minutes or hours prior to administration of trastuzumab. Applying the alternating electric field after administration of trastuzumab may include seconds, minutes, hours or days after administration of trastuzumab. Applying the alternating electric field simultaneously with the administration of trastuzumab may comprise seconds or minutes before or after the administration of trastuzumab. In some aspects, simultaneously applying an alternating electric field and an antibody may include applying trastuzumab simultaneously with applying the alternating electric field. In some aspects, the step of applying the alternating electric field begins at least one hour prior to the given time.

Disclosed is a method of inhibiting proliferation of cancer cells at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof for a period of time and at a frequency; administering trastuzumab to a target site in a subject in need thereof, wherein proliferation of cancer cells is inhibited; and further comprising administering a second therapeutic agent to the subject. For example, the second therapeutic agent may be a chemotherapeutic agent. The chemotherapeutic agent may be, but is not limited to, an alkylating agent, an antimetabolite, an anthracycline, an antitumor antibiotic, a monoclonal antibody, platinum, or a plant alkaloid.

G. Methods for increasing apoptosis in cancer cells

In some aspects, the increase in apoptosis of the cancer cell is compared to apoptosis of the target site when the alternating electric field is administered alone, wherein trastuzumab is administered alone, or wherein no treatment is administered to the subject.

In some aspects, the increase in apoptosis of the cancer cells is inhibited as compared to administration of trastuzumab in the absence of the application of the alternating electric field. In some aspects, there is an increase in apoptosis of the cancer cells. For example, the increase in apoptosis of cancer cells is compared to administration of trastuzumab at the target site without application of an alternating electric field. In some aspects, the increase in cancer cell apoptosis is any amount that is greater than the amount of increase in cancer cell apoptosis at the target site without the application of an alternating electric field. In some aspects, the increase in apoptosis of the cancer cell is one, two, three, or more times the amount of increase in apoptosis of the cancer cell without application of an alternating electric field.

In some aspects, a subject in need thereof suffers from a cancer selected from breast cancer or gastric cancer. For example, the cancer may be a HER2 positive cancer.

Disclosed are methods of increasing apoptosis of cancer cells at a target site in a subject in need thereof, comprising applying an alternating electric field to the target site of the subject in need thereof for a period of time and at a frequency; administering trastuzumab to a target site in a subject in need thereof, wherein apoptosis of the cancer cells is increased; and further comprising administering a second therapeutic agent to the subject. For example, the second therapeutic agent may be a chemotherapeutic agent. The chemotherapeutic agent may be, but is not limited to, an alkylating agent, an antimetabolite, an anthracycline, an antitumor antibiotic, a monoclonal antibody, platinum, or a plant alkaloid.

H. Kit for detecting a substance in a sample

The above materials, as well as other materials, may be packaged together in any suitable combination as a kit for or to aid in the practice of the disclosed methods. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed methods. For example, kits comprising antibodies and one or more materials for delivering an alternating electric field, such as an Optune system, are disclosed.

Examples

A. Example 1 tumor treatment field is effective in overcoming trastuzumab-resistant breast cancer proliferation

Human epidermal growth receptor 2 or HER2 is one of the most expressed negative receptors, which constitutes about 15-20% of malignant breast cancer tumors in women. HER2 epidemic has untimely and adverse consequences for breast cancer and its underlying cancer cell processes, structure and growth. Trastuzumab (derived from a relatively recently based humanized antibody) has been found to be operable in its construction in the treatment of anti-HER 2 positive breast cancer. The agent is combined with radiation therapy or chemotherapy to relieve control of HER2 gene in vivo. However, patients with advanced cancer evolutionary tumors show good tolerability to trastuzumab treatment alone. In contrast, factorization of tumor test fields (ttfields or TTFs) into cancer therapies restores the function of trastuzumab treatment plans by sensitizing the HER2 gene to drugs. In turn, this promotes continued limitation of cancer cell proliferation and toxicity levels by trastuzumab during treatment. The study evaluated the aspects and effects of this pairing both in vivo and in vitro by BT474 cells. TTField conducts electromagnetic boundaries that generate sine wave radiation to manipulate HER2 gene structure. The method followed by this example also checks the genetic cell culture and its viability by solutions such as tryptophan blue or crystal violet (which may or may not deliver some testers to the experiment). Western blot testing and IHC confirm the presence of antibodies and negative receptors in BT474 cells. These procedures help to formulate a treatment plan that overcomes the trastuzumab-resistant properties of tumors, which is basically the purpose of the study. This example shows that a healthy combination of TTF and trastuzumab can inhibit dimerization and expression of dangerous gene structures.

1. Materials and methods

i. Experimental setup of electric field

TTF is generated by a pair of insulated wires attached to a functional generator and a high voltage amplifier [17] that generate a sine wave signal in the range of 0-800V. Which results in an applied electric field strength of 0.9V/cm and a frequency of 150kHz. The field strength was maintained at 0.9V/cm due to its use in a clinical setting. Cells were then plated in 100mm dishes and incubated at 37 ℃ under a humidity and 5% CO2 atmosphere for irradiation treatment purposes until 70-80% confluence was achieved.

Cell culture

BT-474 and JIMT-1 (ACC-589) ductal carcinoma cells were obtained from the American type culture Collection. BT-474 and JIMT-1 cells were maintained in DMEM-F12 supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2mmol/L glutamine and 1% penicillin G-streptomycin. Cells were maintained at 37℃with 5% CO 2.

Cell viability assay

Cell viability was determined by trypan blue exclusion assay. An equal volume of trypan blue reagent was added to the cell suspension and the percentage of living cells was assessed by microscopy. Assays were performed in triplicate.

Colony Formation Assay (CFA)

After 6 hours of trastuzumab exposure at a final concentration of 5 μmol/L, cells were subjected to TTField, after which the cells were incubated for 48 hours. After 14-20 days, colonies were stained with 0.4% crystal violet (Sigma, st.Louis, MO, USA). Plating Efficiency (PE) indicates the percentage of seeded cells of a particular cell line that formed colonies under a particular culture condition. The survival score, expressed as a function of radiation, was calculated as follows: survival fraction = counted colonies/(seeded cells)

×PE/100)。

Western blot analysis

Total protein was extracted from OS cells using RIPA buffer (50 mM Tris-Cl, pH7.4;1% NP-40;150mM NaCl and 1mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 1. Mu.g/ml aprotinin, 1. Mu.g/ml leupeptin and 1mM Na3VO 4) and quantified using the Bradford method. SDS/polyacrylamide gel electrophoresis was used to separate protein samples (30. Mu.g) which were then transferred to nitrocellulose membranes. The membranes were then incubated with mouse monoclonal antibodies overnight at 4 ℃ by blocking the non-specific antibody binding sites. After 1 hour incubation with peroxidase-conjugated secondary antibodies at 37 ℃, the protein strips were visualized by enhanced chemiluminescent reagents (GE Healthcare Biosciences, pittsburgh, PA, USA) and detected using Amersham Imager 680 (GE Healthcare Biosciences).

Immunohistochemistry

For immunohistochemical evaluation purposes, breast sections embedded with paraffin at a thickness of 4 μm were fixed on coated slides to study the protein under study, followed by blocking endogenous peroxidase, recovery antigen and non-specific protein binding. Slide sections were then first incubated with primary antibody (from Cell Signaling Technology [ Danvers, MA, USA ]) followed by conjugation with horseradish peroxidase on the secondary antibody. All slides were developed with 3,3' -diaminobenzidine and counterstained with hematoxylin followed by blind analysis.

Conjugation of Alexa Fluor 488 with trastuzumab

A solution of Alexa 488-NHS Ester (Invitrogen, waltham, mass., USA) was prepared using DMSO. It contained 1% acetic acid, dissolved in 500. Mu.L TRZ (10 mg/mL) in 1M sodium bicarbonate solution at pH 8.4. The resulting reaction was incubated for 1 hour at room temperature, then purified by size exclusion PD-10 column (GE Healthcare Bio-Sciences AB, uppsala, sweden) and connected to an ultraviolet/visible light detector set at a maximum wavelength of 517 nm. Unconjugated aliquots (100. Mu.g/. Mu.L, PBS, pH 7.2) and conjugated Alexa 488-TRZ were measured using Nano Drop Spectrophotometer (ThermoFisher Scientific, waltham, MA, USA). The Alexa 488 molecules around TRZ were estimated by peak intensity comparison with conjugated Alexa 488-TRZ and the eluting solution without Alexa 488 using high performance liquid chromatography profiles.

In vivo antibody penetration studies

BT-474 cells (5X 10) ⁶ ) Is administered to male BALB/nude mice (n=6 per group). Tumor size was measured using a digital caliper and volumes were calculated using the following formula: width 2 x length 0.5. For all experiments related to mice (5-6 weeks old; weighing 18-20 g), the Institutional Animal Care and Use Committee (IACUC) protocol of the korean institute of radiology and medicine (KIRAMS) (no KIRAMS2018-0016; approval date: day 5, 15 of 2018) was followed.

When the tumor size reaches 200mm ³ At this time, 150 μg Alexa 488-TRZ was injected intravenously and TTFied (0.9V/cm) was exposed for a period of 5 days, after which the mice were exsanguinated and dissected by cardiac puncture. Isolated tumors were immediately fixed with paraformaldehyde (4%) and kept overnight at 4 ℃. Tumor tissue was then embedded in an Optimal Cutting Temperature (OCT) compound and frozen until further use at-70 ℃. Frozen tissue samples were separated into 8 μm thick sections using a Leica CM 1850 cryostat (Leica Microsystems, USA), rehydrated with PBS, stained with DAPI, and observed under a fluorescence microscope (In cell analyzer 2200,GE Healthcare,USA). By Click-TUNEL Alexa/>647 imaging assay kit (Invitrogen, carlsbad, CA, USA) stained TUNEL positive cells. Furthermore, apoptotic cells were calculated by TUNEL assay.

Histological image acquisition

Since the In cell analyzer was custom-built with a fluorescence microscope and a mosaic stitching software (In cell developer kit, GE Healthcare, USA), three independent channels were used to obtain fluorescence images with a 10x objective. In addition, DAPI was used for nuclei (shown in blue), alexa 488-TRZ (green) and rhodamine lectin to detect blood vessel (red) images. The offset is determined by autofocus.

Image analysis of Alexa 488 trastuzumab accumulation in tumors

Image analysis was performed using internal programs written in MATLAB (Math Works, natick, mass., USA), ZEN (blue) and MIPAV (NIH, USA). The individual channels are output in the TIFF file from ZEN (blue). The resulting graph, which depicts line intensity versus line distance, was read using an internal MATLAB program and MIPAV application (National Institutes of Health, bethesda, MD, USA). TRZ signal intensity was recorded as 60 μm from tumor vessels. TRZ penetration from tumor vessels was plotted using line graphs by measuring each tumor intensity in various ROIs from the vessels from peripheral and central regions. In addition, the area under the curve was calculated from 10 sections in three tumors of each of the two groups.

Statistical analysis of

Statistical significance was determined using Student's t test. If the P value is less than 0.05, 0.001 or 0.001, the difference is considered significant. P <0.05, P <0.01, P < 0.001).

2. Results

i. Sensitization of trastuzumab-resistant BT474 cell lines in vitro with TTF treatment

To determine the ability of TTF to control trastuzumab resistance, two HER2 positive (Jimt 1 and BT 474) cell lines were treated with 1-100 μg (4-fold dilution) for 48 hours.

The data obtained indicated that the cells showed dose-dependent sensitivity to trastuzumab (fig. 1A). Furthermore, treatment with trastuzumab significantly inhibited cell growth in trastuzumab-resistant cells (fig. 1B), and both cancer cell lines had reduced cell viability, depending on the various doses, with about 10% viability inhibition observed at 100 μg.

inhibition of cell proliferation in trastuzumab-resistant HER 2-positive human breast cancer cells in vitro following TTF treatment

Jimt1 cells were then treated with a fixed dose of trastuzumab to evaluate their effect on breast cancer cells in cell morphology and 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation assay (fig. 2A-2C). Jimt1 cells were treated with TTF (0.9V/cm), TRZ or a combination of both, and morphological related changes were observed with a phase contrast microscope in the combined treatment group. In addition, it was noted that cell growth was significantly inhibited after 48 hours of treatment with 100 μg trastuzumab. These data show that TTF increases trastuzumab sensitivity in trastuzumab-resistant HER 2-positive human breast cancer cells.

Trastuzumab promotes TTField sensitivity in vivo

To study the combined effect of TTF and trastuzumab on breast cancer growth in vivo, a subcutaneous HER2 positive breast cancer model was utilized, which was achieved by injecting mice with human BT474 cells. As shown in fig. 3A, xenografts treated with the TTF and trastuzumab combination showed reduced growth compared to the control group and the group receiving either treatment. Thus, the tumors of the single treatment group were significantly larger than those of the combined treatment group. At the same time, the tumor volume in the combination treated mice was reduced compared to the volume in mice receiving either treatment (fig. 3A).

Xenograft depictions of mice receiving either treatment were stained with the proliferation marker Ki-67 stronger than the combination treated mice (fig. 3B-3C). Taken together, the data show that TTF in combination with trastuzumab slowed breast cancer growth in vivo.

Treatment with TTF+ TRZ inhibits P-HER2 expression and increases apoptosis marker levels in vitro

The level of p-HER2 expression and the cleaved PARP of the apoptosis marker as well as the anti-apoptosis marker Bcl-2 were studied by protein (or western blot) blotting techniques. After 24 hours of treatment with ttf+ TRZ, the expression levels of p-HER2 and Bcl-2 were down-regulated, and cleaved PARP was up-regulated in Jimt1 cell line (fig. 4A). To calculate the ability of combination therapies to induce apoptosis in vivo, the rate of apoptosis was assessed by a terminal deoxynucleotidyl transferase mediated dUTP nick end-marker (TUNEL) assay. Notably, cell death in apoptotic cells increased after the combination treatment (fig. 4B). TTF increased the apoptotic area, as calculated by TUNEL assay (fig. 4C).

Inhibition of P-HER2 and its downstream mediators AKT and MAPK expression levels in vivo by TTF treatment

Histopathological studies showed that tumors treated with TTF expressed low HER2 levels compared to control. In addition, tumors treated with TTF in combination with trastuzumab expressed lower levels of pAKT, pERK and pHER2 compared to the control group (fig. 5).

Increased penetration of Alexa 488-trastuzumab from tumor vessels in vivo by TTF treatment

Antibody accumulation was determined by analysis of fluorescence intensity in tumor sections. The results show that Alexa 488-TRZ (green) accumulation was improved with TTF. In TTF-combination treated tumors, TRZ penetration from tumor vessels in whole tumor sections was about 33% higher

(p=0.0211) (fig. 6). TTF significantly improved extravasation of TRZ from tumor vessels within 60 μm.

3. Discussion of the invention

The present study confirms the hypothesis that supporting a broad sense of trastuzumab drug for controlling the life threatening HER2 positive breast cancer gene, inhibition of such trastuzumab resistant cells can be amplified using tumor electric field therapy. The aim of this study was also to identify and elucidate the work of the TTField system on the trastuzumab-resistant HER 2-positive breast cancer cell lines involved. By doing so, it produces TTF value for future research. At 9 1998, the FDA approved the use of trastuzumab and its inclusion in designing a therapeutic regimen for GBM or glioblastoma multiforme (rare malignancy). The need for trastuzumab for use in therapy arises from the excessive activity of the HER2 tyrosine kinase receptor gene. In response to proliferation, the rate at which breast cancer progresses to its secondary stage increases surprisingly. Thus, trastuzumab was predicted to function as a humanized anti-HER 2 monoclonal IgG1 antibody [18]. However, this study, as well as other examples in the past, clearly demonstrated that the effect of HER2 gene on trastuzumab remains unchanged through its primary stage. Thus, therapeutic therapies for breast cancer are a major disadvantage due to their faced trastuzumab resistance. The mechanism contributing to failure of trastuzumab to HER2 operation was studied in depth and included the following general observations: i) Trastuzumab faces a hurdle in binding HER2; ii) upregulation of the signaling pathway causes downstream HER2; iii) Trastuzumab has a tendency to signal through alternative pathways, and iv) is unable to trigger immune-mediated mechanisms to destroy tumor cells [6].

This study showed that trastuzumab-resistant properties of the oncogene can be compensated by TTF. The study was first of all self, destroying cancer cells by using trastuzumab antibodies and making them sensitive in constructing a good electromagnetic TTF. This finding demonstrates enhanced permeation and uptake of Alexa 488-TRZ trastuzumab after TTF treatment. Fig. 4 shows that ttTield increases apoptotic areas as measured by TUNEL assay. Increased apoptotic areas may induce a decrease in interstitial pressure. The enhanced penetration and uptake of Alexa 488-TRZ may be explained by reduced interstitial pressure following TTF treatment.

The result of these applications is a prohibitive result of inhibiting HER2 receptor cells. This study is in contrast to the environment where oncology currently considers chemotherapy in combination with trastuzumab as the most accurate way to treat tumor cells. Despite this release, many patients with breast cancer that overexpresses HER2 either re-develop or acquire resistance to such methods. However, the results of this study show that a regimen of TTF integration with trastuzumab can enhance HER2 therapy.

This objective was achieved by analyzing the background-dependent effects of TTF on trastuzumab-resistant breast cancer cells, a general consensus being a positive outcome.

This objective was supported by previous studies in which TTF was responsible for GBM arrest, deregulation of cyclin-dependent kinase expression, induction of caspase-3 activation and spindle formation, and cell death in vitro [20, 21]. TTF is also responsible for inhibiting GBM proliferation and invasion [3, 21]. In addition, in vivo and in vitro experiments [16] are consistent with the desirability of TTField, as it specifically targets cancer cells and is an attractive alternative to conventional cancer therapies. Many in vivo experiments have pointed to promising results of TTF in performing anti-tumor activity. In addition, TTF can also block human lung tumors as demonstrated in xenograft models and in GBM or GBM patient-derived stem cell tumor carrying models [22, 23].

The data disclosed show a clear positive summary of TTF display, which must be studied and examined to discuss the therapeutic potential of TTF in combination with trastuzumab in HER2 positive breast cancer. In this study, the BT474 cell line was used for in vivo and in vitro experiments and the changes they produced were recorded and analyzed. From the observations, it was shown that the use of TTField resulted in the sensitivity of the primary trastuzumab-resistant cell line. Thus, this promotes apoptosis by increasing the expression of caspase 3/7 assay. In addition, TTF can inhibit phosphorylation of HER2, p-AKT, and p-MAPK expression levels in HER 2-positive BT474 cells. In turn, this inhibits migration and invasion of cells and successfully inhibits growth of tumor cells in vivo models. By evaluation route, the study also identified TTF therapy as an alternative solution to patients resistant to chemotherapy from antibodies (e.g., trastuzumab).

In summary, the data presented herein show that TTF can overcome trastuzumab resistance in vitro and in vivo.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating a subject in need thereof, comprising:

a) Applying an alternating electric field to a target site of a subject in need thereof at a frequency for a period of time; and

b) Trastuzumab is administered to a target site in a subject in need thereof.

2. A method of increasing accumulation of trastuzumab at a target site in a subject in need thereof, comprising:

b) Trastuzumab is administered to a target site in a subject in need thereof,

wherein the accumulation of trastuzumab at the target site is increased as compared to administration of trastuzumab at the target site without application of an alternating electric field.

3. A method of inhibiting proliferation of a breast or gastric cancer cell at a target site in a subject in need thereof, comprising:

b) Trastuzumab is administered to a target site in a subject in need thereof,

wherein proliferation of the mammary gland or gastric cancer cells is inhibited.

4. The method of claim 3, wherein the increase in inhibition of breast or gastric cancer cell proliferation is compared to breast or gastric cancer cell proliferation at the target site when the alternating electric field is administered alone, wherein the antibody is administered alone, or wherein no treatment is administered to the subject.

5. A method of increasing apoptosis of a breast or gastric cancer cell at a target site in a subject in need thereof, comprising:

b) Trastuzumab is administered to a target site in a subject in need thereof,

wherein apoptosis of the breast or gastric cancer cells is increased.

6. The method of claim 5, wherein the increased apoptosis of the breast or gastric cancer cells is compared to apoptosis at the target site when the alternating electric field is administered alone, wherein trastuzumab is administered alone, or wherein no treatment is administered to the subject.

7. The method of any one of the preceding claims, wherein the target site comprises one or more breast or gastric cancer cells.

8. The method of any one of the preceding claims, wherein the alternating electric field is applied before, after, or simultaneously with the administration of the antibody.

9. The method of any of the preceding claims, wherein the step of applying the alternating electric field begins at least one hour before a given time.

10. The method of any one of the preceding claims, wherein the antibody comprises a detectable agent.

11. The method of claim 10, wherein the detectable agent is a fluorophore.

12. The method of any of the preceding claims, wherein the frequency of the alternating electric field is between 100-500 kHz.

13. The method of any of the preceding claims, wherein the frequency of the alternating electric field is 150kHz.

14. The method of any of the preceding claims, wherein the field strength of the alternating electric field is between 0.5-4V/cm RMS.

15. The method of any of the preceding claims, wherein the field strength of the alternating electric field is 0.9V/cm RMS.

16. The method of any one of the preceding claims, wherein trastuzumab is administered intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intravascularly, intravenously (targeted or non-targeted), intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, via intratumoral injection (e.g., computer tomography guided, during surgery or biopsy), or via inhalation.

17. The method of any one of the preceding claims, wherein trastuzumab is administered in a pharmaceutical composition.

18. The method of any one of the preceding claims, wherein the subject in need thereof has a cancer selected from breast cancer or gastric cancer.

19. The method of claim 18, wherein the cancer is breast cancer and the subject has trastuzumab resistance.

20. The method of claims 18-19, wherein the cancer is HER2 positive cancer.

21. The method of any one of the preceding claims, wherein pAKT, pERK, and pHER2 expression are reduced.

22. The method of any one of the preceding claims, wherein trastuzumab is administered before, simultaneously with, or after the alternating electric field is applied.

23. The method of any one of the preceding claims, further comprising administering a chemotherapeutic agent to the subject.

24. The method of any one of the preceding claims, wherein trastuzumab is administered in a therapeutically effective amount.