TWI869971B - Methods for reducing viability of cancer cells by activation of the sting pathway with ttfields - Google Patents

Abstract

Viability of cancer cells (e.g., glioblastoma cells) can be reduced by applying an alternating electric field with a frequency between 100 and 500 kHz to the cancer cells for about 3-10 days and administering a checkpoint inhibitor to the cancer cells.

Description

Methods for reducing cancer cell viability by activating the STING pathway using TTFields

The present invention relates to reducing the viability of cancer cells by applying an alternating electric field having a frequency between 100 and 500 kHz to the cancer cells and administering a checkpoint inhibitor to the cancer cells. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/898,290, filed on September 10, 2019, which is incorporated herein by reference in its entirety.

All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

Tumor Treating Fields (TTFields) are an effective anti-proliferative therapeutic modality delivered via non-invasive application of low-intensity, medium-frequency (e.g., 100-500 kHz) alternating electric fields. TTFields exert directed forces on polar microtubules and interfere with the normal assembly of mitotic spindle bodies. Such interference with microtubule dynamics causes abnormal spindle body formation and subsequent mitotic arrest or delay. Cells may die while in mitotic arrest or proceed to cell division, resulting in the formation of normal or abnormal aneuploid progeny. The formation of tetraploid cells may occur due to exit from mitosis via slippage or may occur during inappropriate cell division. Abnormal daughter cells may die during subsequent interphases, may undergo permanent arrest, or may proliferate via additional mitosis, where they will be subjected to further TTFields attack. Giladi M et al. Sci Rep. 2015;5:18046.

In an in vivo setting, TTFields therapy can be delivered using a wearable and portable device (Optune®). The delivery system includes an electric field generator, 4 adhesive patches (non-invasive, insulated transducer arrays), a rechargeable battery, and a carrying case. The transducer array is applied to the skin and connected to the device and battery. The therapy is designed to be worn for as long as possible throughout the day and night.

In a preclinical setting, TTFields can be applied in vitro using, for example, the Inovitro™ TTFields benchtop system. The Inovitro™ consists of a TTFields generator and a base plate containing 8 ceramic disks per plate. Cells are plated on a cover slip placed inside each disk. TTFields are applied using two perpendicular pairs of transducer arrays insulated by a high dielectric constant ceramic in each disk. The orientation of the TTFields in each disk is switched 90° every 1 second, thereby covering the different orientation axes of cell division.

Recently, the immune sensing molecule cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING, encoded by TMEM 173 ) pathway has been identified as an important component of cytoplasmic DNA sensing and plays an important role in regulating the immune response of cells. Ghaffari et al., British Journal of Cancer, Vol. 119, pp. 440-449 (2018); see, e.g., FIG. 3. Activation of the STING pathway mediates immune responses by responding to abnormalities in cells, such as the presence of cytoplasmic double-stranded DNA (dsDNA).

Checkpoint proteins act as inhibitors of the immune system they can direct (e.g., T cell proliferation and IL-2 production). Azoury et al., Curr Cancer Drug Targets. 2015;15(6):452-62. Checkpoint proteins may have adverse effects on cancer by shutting down immune responses. Blocking the function of checkpoint proteins could be used to activate dormant T cells to attack cancer cells. Checkpoint inhibitors are cancer drugs that inhibit checkpoint proteins in order to recruit the immune system to attack cancer cells.

Therefore, there is interest in using checkpoint inhibitors as cancer treatments to block the activity of checkpoint proteins, thereby enabling the production of interleukins and the recruitment of T cells to attack cancer cells and is an active area of immunotherapy drug development.

What is needed are methods for activating the immune response and increasing and stimulating the response to cancer therapies, such as checkpoint inhibitors.

The methods described herein reduce cancer cell viability by applying an AC electric field to the cancer at a frequency between 100 and 500 kHz for 3 days and administering a checkpoint inhibitor to the cancer cells. The AC electric field may be applied to the cancer cells continuously or intermittently over the 3 days. In another aspect, the AC electric field may be applied to the cancer cells for at least 4 hours per day on each of the 3 days or for at least 6 hours per day on each of the 3 days.

As described herein, exposure of cancer cells (e.g., glioblastoma cells) to TTFields induces the STING pathway, leading to production of proinflammatory cytokines (e.g., type I interferons) and pyroptosis. In one aspect, activation of the STING pathway with TTFields is analogous to "vaccinating" cancer cells, rendering them particularly susceptible to treatment with anticancer drugs such as checkpoint inhibitors. Therefore, continuous, intermittent, or intermittent exposure of cancer cells to TTFields may render the cancer cells susceptible to further treatment by inducing the STING pathway, followed by treatment with one or more checkpoint inhibitors and/or other oncology drugs.

Despite aggressive chemoradiation, glioblastoma (GBM) is the most common and lethal malignant brain cancer in adults. Tumor treating fields (TTFields) in combination with adjuvant temozolomide chemotherapy have recently been approved for use in newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that interfere with mitotic macromolecular assembly, resulting in disruption of chromosome segregation, integrity, and stability. In many patients, a transient phase of increased peritumoral edema is often observed early in the course of TTFields treatment, followed by objective radiographic responses, suggesting that a major component of the therapeutic efficacy of TTFields may be an immune-mediated process. However, the mechanisms underlying these observations remain unclear.

As described herein, TTFields-activated micronucleus-dsDNA sensor complexes lead to: i) induction of pyroptotic cell death, as measured by specific LDH release assays, and cleavage of apoptotic caspase 1 and pyroptosis-specific gasdermin D via recruitment of AIM2; and ii) activation of STING pathway components, including type I interferons (IFNs), and proinflammatory cytokines downstream of the NFkB pathway. See, e.g., FIG3 . GBM cell-specific shRNA depletion of AIM2 or STING or both in co-culture experiments of bone marrow cells or spleen cells with supernatants from GBM cells expressing self-blocked genes was able to reverse the induction of immune cells.

GBM cell lines were treated with TTFields using the in vitro TTFields system at a clinically approved frequency of 200 kHz. In one aspect, 24-hour TTFields-treated GBM cells had a significantly higher rate of micronuclei structures released into the cytoplasm (19.9% vs. 4.3%, p=0.0032) due to TTFields-induced chromosomal instability. Almost 40% of these micronuclei colocalized with two upstream dsDNA sensors, absent melanoma 2 (AIM2) and interferon (IFN)-inducing protein cyclic GMP-AMP synthase (cGAS), compared to the absence of colocalization in untreated cells. These results demonstrate that TTFields activates the immune system in GBM cells.

The aspects described herein provide a method of reducing the viability of cancer cells by applying an alternating electric field to cancer cells at a frequency between 100 and 500 kHz for 3 to 10 days and administering a checkpoint inhibitor to the cancer cells. The alternating electric field may be applied to the cancer cells continuously or intermittently over the 3 to 10 days. In another aspect, the alternating electric field may be applied to the cancer cells for at least 4 hours per day or for at least 6 hours per day on each day of the 3 to 10 days. The alternating electric field may be applied to the cancer cells for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days as needed. In another aspect, the alternating electric field can be applied to the cancer cells for 3-5, 3-6, 3-7, 3-8, 3-9 or 3-15 days.

The term "reducing cancer cell viability" refers to shortening, limiting, or having a negative impact on the ability of cancer cells to remain viable. For example, reducing the growth or reproduction rate of cancer cells will reduce their viability.

The term "administering a checkpoint inhibitor" means providing a checkpoint inhibitor to a patient by a healthcare professional or under the care of a healthcare professional or as part of an approved clinical trial, by any appropriate and accepted route of administration (e.g., oral, intravenous, parenteral, topical, etc.) as approved by a regulatory agency on the product label. Prescribing a checkpoint inhibitor may also mean "administering" a checkpoint inhibitor.

The term "continuously" means that the alternating electric field is applied for a substantially constant period of time. The alternating electric field may be applied continuously even if it is interrupted for shorter periods of time (e.g., seconds) to properly position the equipment or to briefly disconnect the power supply.

The term "intermittently" refers to applying an AC field for a period of time with periodic interruptions or interruptions for seconds, minutes, an hour, or longer. In this aspect, the patient may have an AC field applied for a period of time (e.g., 1, 2, 3, or 4 hours), with periods of 15 minutes, 30 minutes, 45 minutes, or 1 hour without the application of the AC field. In another aspect, the patient may have the AC field applied continuously during sleep and intermittently during wakefulness. In another aspect, the patient may have the AC field applied continuously except during mealtimes or during social events.

In another aspect, the alternating electric field is applied to the cancer cells for at least 4 or 6 hours per day, each day for 3 to 10 days.

In another embodiment, an AC electric field is applied to the cancer cells for 3 days, followed by a 3-day period in which the AC electric field is not applied to the cancer cells, and followed by a 3-day period in which the AC electric field is applied to the cancer cells.

In another aspect, the alternating electric field is applied to the cancer cells at least 3 days per week.

In another aspect, an AC electric field is applied to the cancer cells for a first period of 3 to 10 days, followed by no AC electric field being applied for a second period of time. In another aspect, the second period of time is at least the same as the first period of time.

This aspect can significantly improve patient comfort and convenience because the device used to apply TTFields can be worn by the patient at home or during periods when it is more appropriate to wear the device continuously while sleeping. The patient does not have to continue wearing the device during periods when the patient does not want to be hindered by the medical device (e.g., working, exercising, participating in social activities).

Thus, the patient would receive the desired TTFields treatment, then take a pill of, for example, a checkpoint inhibitor, without having to continue wearing the device in a public or social environment. Compliance with treatment would be improved along with patient comfort. It has not been previously disclosed or suggested to interrupt the use of TTFields during a treatment cycle as described herein.

In another aspect, the AC electric field is applied to the cancer cells in short pulses. The term "short pulses" refers to a non-continuous AC electric field applied to the cancer cells, wherein each pulse has a duration of, for example, less than 5 seconds.

The cancer cell can be selected from the group consisting of neuroglioblastoma cells, pancreatic cancer cells, ovarian cancer cells, non-small cell lung cancer (NSCLC) cells and mesothelioma cells. In another aspect, the cancer cell is a neuroglioblastoma cell.

The checkpoint inhibitor may be selected, for example, from the group consisting of ipilimumab, pembrolizumab and nivolumab.

The frequency of the AC field can be between 180 and 220 kHz.

In another aspect, at least a portion of administering the checkpoint inhibitor to the cancer cells is performed after cessation of applying an alternating electric field at a frequency between 100 and 500 kHz to the cancer cells for 3 to 10 days.

Another aspect provides a method of treating neuroglioblastoma by applying an alternating electric field at a frequency between 100 and 500 kHz to the head of an individual with neuroglioblastoma for 3 days and administering a checkpoint inhibitor to the individual. The alternating electric field is applied to the individual continuously or intermittently over the 3 days. In another aspect, the alternating electric field is applied to the individual for at least 4 hours per day on each of the 3 days. The checkpoint inhibitor can be selected from the group consisting of: ipalimumab, pembrolizumab, and nivolumab. The frequency of the alternating electric field can be between 180 and 220 kHz.

In another aspect, at least a portion of administering the checkpoint inhibitor to the individual is performed after cessation of application of an alternating electric field at a frequency between 100 and 500 kHz to the head of the individual with neuroglioblastoma for 3 to 10 days.

Another aspect provides a method of reducing the viability of cancer cells, comprising applying an alternating electric field at a frequency between 100 and 500 kHz to the cancer cells for a period of time sufficient to kill about 1-2% of the cancer cells; and administering a checkpoint inhibitor to the cancer cells. In one aspect, the period of time sufficient to kill about 12% of the cancer cells is 3, 4, 5, 6, 7, 8, 9 or 10 days.

TTFields can induce the formation of cytoplasmic micronuclei in GBM cells exposed to TTFields. Figure 1 shows the results of an exemplary experiment in which LN827 cells were treated with TTFields for 24 hours and then fixed with 4% PFA for 20 min. DAPI (4',6-dicarboxamidino-2-phenylindole) (1:5000) stain was incubated for 5 minutes at room temperature to stain nuclei and micronuclei. Figure 1 (right panel) shows the percentage of control cells with micronuclei (approximately 4%) compared to TTFields exposed cells (approximately 20%). Therefore, TTFields induce the formation of cytoplasmic micronuclei (dsDNA) that can induce the STING pathway.

While small molecule STING activators (e.g., STING agonists) are known and in clinical development (Ryan Cross, STING fever is sweeping through the cancer immunotherapy world. Vol. 96, No. 91, pp. 24-26, Chemical & Engineering News (Feb. 26, 2018)), these drugs can have significant side effects on patients. In contrast, TTFields have virtually no side effects and thus present a safer and more comfortable alternative to small molecule STING activators.

Nuclear lamella B1 structure is disrupted upon exposure to TTFields, resulting in the release of dsDNA into the cytoplasm of LN827 cells. Figure 2 shows additional nuclear damage in LN827 cells treated with TTFields for 24 hours, fixed with 4% PFA for 20 min, and blocked with 0.2% Triton/0.04% BSA for 1 hour. DAPI-stained cells are shown on the left panel (not treated with TTFields and treated as indicated). The middle panel shows the results when cells were incubated with nuclear lamella B1 antibody overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 hour (not treated with TTFields and treated as indicated). The right panel shows the merged image (DAPI/nuclear lamella B1). These results indicate that dsDNA is released into the cytoplasm following application of TTFields that induce the STING pathway.

FIG3 depicts induction of the proinflammatory STING and pyroptotic pathways by dsDNA. dsDNA can be generated by aberrant mitosis-induced micronuclei. Aberrant mitosis can be induced, for example, by TTFields. As shown, TTFields can also reduce nuclear envelope integrity by disrupting the nuclear lamin B1 structure, thereby causing dsDNA to enter the cytoplasm and induce the STING pathway as shown.

cGAS (cyclic GMP-AMP synthase) and AIM2 independently colocalize with micronuclei in response to exposure to TTFields. cGAS and AIM2 are immunosensors that detect the presence of cytoplasmic dsDNA. In Figure 4, LN827 cells were treated with TTFields for 24 hours, fixed with 4% PFA for 20 min, and blocked with 0.2% Triton/0.04% BSA (bovine serum albumin) for 1 hour. Flag and cGAS antibodies were incubated overnight at 4°C, followed by incubation with secondary antibodies for 1 hour, and DAPI staining for 5 minutes at room temperature.

Thus, cGAS and AIM2 each independently colocalize with micronuclei in response to TTFields, indicating that TTFields induce the presence of cytoplasmic dsDNA, activating the STING pathway. Figure 5 quantifies the percentage of cGAS, AIM2, and micronuclei from the results of Figure 4 in the presence or absence of exposure to TTFields.

In U87 and LN827 cells, IRF3 and p65 are phosphorylated after one day of exposure to TTFields. In Figure 6, total protein was collected after U87 and LN827 cells were treated with TTFields for 24 hours. The presence of IRF3 and p65 downstream of the STING pathway and their activated phosphorylated forms were measured by western blot. B-actin was used as an internal control. The STING-induced pathway (IRF3 and p65) is activated after TTFields, as shown by the presence of phosphorylated forms of IRF3 and p65. Therefore, IRF3 or interferon regulatory factor 3 and p65 phosphorylation is triggered by STING activation.

TTFields induce type I IFN responses and proinflammatory interleukins downstream of STING. The term "downstream of STING" refers to interleukins that are induced after activation of the STING pathway. In this aspect, TTFields induce a STING response as described herein.

LN428 cells were treated with/without TTFields for 24 hours (Figure 7). Total RNA was extracted and converted to cDNA. Quantitative PCR was used to detect the transcript levels of IL1α, IL1β, IL6, IL8 and ISG15, IFNα, IFNβ. Total LN428 protein was collected in protein lysis buffer and cell number was determined. Endogenous protein content of IFNβ was determined by ELISA. Final protein content was normalized by cell number.

As shown in Figure 7, TTFields induces the expression of interleukins, such as interferon b (IFNB) in LN428. Specifically, exposure of LN428 cells to TTFields for 3 days increased IFNB levels 300-fold compared to the control group and 100-fold compared to exposure to TTFields for 1 day. In this aspect, application of TTFields for about 3 days significantly increased the levels of pro-inflammatory interleukins.

STING is degraded after activation by TTFields in GBM cells. In the experiments summarized in Figure 8, LN428 (human) and KR158 (mouse) proteins were collected at the indicated time points. STING, p65 and phospho-p65 protein levels were determined by Western blotting. B-actin/GAPDH was used as an internal control. As shown in Figure 8, STING protein levels and phospho-p65 levels decreased during TTFields treatment over a 24-hour period.

STING is required for inflammatory response induced by dsDNA and TTFields treatment in human GBM cells (LN428 human cells). In the experiment summarized in Figure 9, the human GBM cell line LN428 was stably infected with lentivirus-shScramble or shSTING. The cells were treated with dsDNA or TTFields alone for 24 hours. Polyethylenimine (PEI) was used as a transfection buffer to induce dsDNA migration into the cytoplasm. Total RNA was extracted and converted into cDNA. Quantitative PCR was used to detect the transcript levels of IL1α, IL1β, IL8, ISG15 and STING.

As shown in FIG. 9 , induction of the STING pathway by both dsDNA and TTFields in LN428 cells reduced the levels of various cytokine RNA transcripts in the absence of STING (shSTING).

In KR158 and F98 GBM cells, autophagy and dsDNA or TTFields synergistically induce STING-dependent proinflammatory responses. In the experiments summarized in Figure 10, the mouse GBM cell line KR158 and the rat GBM cell line F98 were stably infected with lentivirus-shScramble or shSTING. The cells were isolated and treated with dsDNA or TTFields for 24 hours. PEI was used as a transfection buffer to induce dsDNA into the cytoplasm. Total RNA was extracted and converted into cDNA. Quantitative PCR was used to detect the transcription levels of IL1α, IL6, ISG15, IFNβ and STING.

In related experiments, cells were isolated as described above and treated with dsDNA or TTFields for 24 hours in the presence/absence of chloroquine (autophagy inhibitor). PEI was used as transfection buffer to induce dsDNA into the cytoplasm. Total RNA was extracted and converted to cDNA. Quantitative PCR was used to detect the transcript levels of IL6, ISG15, IFNβ.

As shown in Figure 10, induction of the STING pathway by both dsDNA and TTFields in KR158 and F98 cells reduced the levels of various cytokine RNA transcripts in the absence of STING (shSTING). The levels of cytokine transcripts were further reduced by the autophagy inducer coenzyme Q (CQ).

In the F98 rat neuroglioma model, TTFields-induced inflammatory interleukin production is dependent on STING and AIM2. In the experiment summarized in Figure 11, the rat GBM cell line F98 was stably infected with a lentivirus-scramble control group (WT) or a double knock down (DKD) of STING and AIM2. The cells were injected into the brain of male Fischer rats using a stereotaxic system. Seven days after the injection of cells, the rats were applied to heat or TTFields for an additional 7 days. At the end of treatment, the rats were killed, and tissues were collected and further analyzed. Quantitative PCR was used to detect the transcript levels of IL1α, IL1β, IL6, ISG15 and IFNβ.

As shown in FIG. 11 , double knockdown (DKD) of STING and AIM2 significantly reduced the levels of the indicated interleukins.

Tumor size correlates with fold changes in inflammatory interleukin expression in response to TTFields. Figure 12 shows images of rat brains used in the experiments summarized in Figure 11. Quantitative PCR results from Figure 11 (i.e., relative mRNA levels for each individual rat's MRI image) are shown below each image at day 15 post-injection.

Figure 13 provides an exemplary heat map showing that in the F98 rat neuroglioma model, recruitment of CD45 cells into GBM is lower in GBM lacking STING and AIM2. In the experiments summarized in Figure 13, the rat GBM cell line F98 was stably edited by lentiviral-scramble control (WT) or double knockout gene expression (DKD) of STING and AIM2. Cells were injected into the brain of male Fischer rats using a stereotaxic system. Seven days after cell injection, heat or TTFields were applied to the rats for an additional 7 days.

At the end of treatment, the rats were sacrificed, tissues were collected and split for further analysis. Here, bulk tumors were dissociated into single cell suspensions. Multiple flow antibodies were used to stain for CD45. The single cell suspensions were then fixed and analyzed on a flow cytometry machine the next day.

Figure 14 provides an exemplary heat map showing that CD3 (T cell) recruitment is lower in GBM lacking STING and AIM2. Figure 14 summarizes the same experiment as Figure 13 but using antibodies against CD3.

Figure 15 provides an exemplary heat map showing that DC/macrophage recruitment is low and MDSC recruitment is high in GBM lacking STING and AIM2. Figure 15 summarizes the same experiment as Figure 13, involving antibodies detecting CD11b/c and MHC II (macrophages).

FIG. 16 provides quantitative data from the flow cytometry results of FIG. 15 .

Figure 17 shows "ghosting" induced by three days of exposure to TTFields in human GBM cell lines LN308 and LN827 human GBM cell lines. The term "ghosting" refers to the presence of cell remnants that remain after immunogenic cell death. In these experiments, LN308 and LN827 cells were treated with/without TTFields for 3 days. Images were acquired under bright field microscopy. Images show increased immunogenic cell death after 3 days of TTFields exposure.

Figure 18 shows that TTFields induces membrane damage and reduces GSDMD in U87 GBM cells exposed to TTFields. In the experiment summarized in Figure 18, the human GBM cell line U87 was treated for 24 hours under the indicated conditions. Lactate dehydrogenase (LDH) released into the cell culture medium was detected by cytotoxicity analysis. U87 cells were forced to express dermatolin D (GSDMD) by lentivirus-GSDMD-Flag-N and treated with/without TTFields. Total protein was collected at the indicated time points. The level of over-expressed protein GSDMD was determined by Western blotting using flag antibody. B-actin was used as an internal control group. As shown in Figure 18, exposure to TTFields killed 12% of the cells.

Figure 19 shows that TTFields induce membrane damage and cleavage of GSDMD in human leukemia monocytic cell line THP-1 macrophages. In the experiment summarized in Figure 19, the human leukemia monocytic cell line THP-1 was treated with 150nM PMA treatment agent for 24 hours to stimulate differentiation into macrophages. LDH release was tested on the third day of TTFields treatment to examine the electric field frequency range. THP-1 cells were forced to express GSDMD and treated with/without TTFields by lentivirus-GSDMD-Flag-N. Total protein was collected at the indicated time points. The over-expressed protein GSDMD content and its cleaved N-fragment were determined by Western blotting using flag antibodies. B-actin was used as an internal control group. The positive control group showed LPS treatment for 6 h, followed by ATP for 1 h.

Figure 20 shows THP-1 cells labeled with GFP lentivirus and pretreated with 150 nM PMA for 24 hours. After the 24 hour period, cells were exposed to TTFields for 24 hours and time course images were captured every 20 minutes.

Figure 21 shows THP-1 cells labeled with GFP lentivirus and pretreated with 150 nM PMA for 24 hours. After the 24 hour period, cells were grown in normal culture conditions for 24 hours and time course images were captured every 20 minutes.

As shown in Figures 20 and 21, treatment with TTFields caused greater immunogenic cell death (Figure 20) compared to control cells (Figure 21).

Figure 22 shows that TTFields induce pyroptosis-dependent caspase-1 activation after 1 day and 3 days of exposure to TTFields. In the experiment summarized in Figure 22, THP-1 cells were pretreated with 150nM PMA for 24 hours. After the 24 hour period, cells were treated with and without TTFields for the indicated time points. The caspase-1 activation detection kit was used to mark cells with the cleaved caspase-1 form. Samples were analyzed on a flow cytometry machine.

Figure 23 shows that TTFields-induced apoptosis proteinase-1 activation and cell pyroptosis are consistent with lower levels of full-length IL-1 β and higher levels of LDH release after 1 day and 3 days of exposure to TTFields. In the experiment summarized in Figure 23, THP-1 cells were pretreated with 150nM PMA for 24 hours. After the 24-hour period, the cells were treated for 3 days with and without TTFields. Nigericin was used for 12 hours as a positive control group. The apoptosis proteinase-1 activation detection kit was used to mark cells with a cleaved apoptosis proteinase-1 form. Samples were analyzed on a flow cytometry machine. Cell culture media were collected at the same time points on day 3. The levels of IL1β and LDH in the culture medium were determined by ELISA and cytotoxicity assay.

Figure 24 shows that TTFields-induced STING/AIM2 activation and inflammatory cytokine production are maintained for at least 3 days after TTFields treatment has ended. As shown in Figure 24, inflammatory cytokine production induced by TTF is dependent on STING and AIM2 and remains above baseline for several days after a short pulse of TTF treatment. In the experiment summarized in Figure 24, K-LUC cells were transduced with empty virus or virus carrying double-stranded shRNA that targets and inhibits STING and AIM2. 30k of these cells per plate were then treated with TTFields for 3 days, then cultured for an additional 3 days after TTF cessation and collected for inflammatory cytokine assays (IL-6 and ISG15). As shown in Figure 24, elevated IL6 and ISG15 production continued for at least 3 days after cessation of TTFields (blue bars, EV).

Figure 25 shows that short pulses of TTF-induced STING/AIM2 activity are associated with reduced tumor growth and increased recruitment of DCs (dendritic cells) to deep cervical draining lymph nodes. This persistent inflammatory activation via STING/AIM2 by TTFields activates the immune system after cessation of TTFields treatment.

In the experiments summarized in Figure 25, K-LUC cells were transduced with empty virus or virus expressing dual shRNA targeting STING and AIM2 and then left untreated or treated with TTF for 3 days. 3×10 ⁵ of these K-LUC cells were orthotopically implanted into B6 mice. Tumor growth was measured by luc BLI. As shown in the middle panel, tumor size two weeks after implantation was greatly reduced in empty virus (EV) TTFields mice compared to double knockout (DKD) mice. These mice also displayed the highest levels of DC cells (e.g., T cells) (right panel). The deep cervical lymph nodes are considered to be where DC recruitment and priming of naive T cells by antigens from the brain occurs.

Thus, TTFields stimulate the immune system to generate an anti-tumor immune response, similar to an in situ "vaccination" in which cells are primed for additional cancer therapies (e.g., TTFields treatment for at least three days followed by treatment with a checkpoint inhibitor).

Figure 26 shows that caspase 1 was not detected in TTFields-treated cells without AIM2. Caspase 1 is immediately downstream of the AIM2-double-stranded DNA complex and is a molecular marker of cell pyroptosis. Caspase 1 activation was detected using a commercially available kit that detects the cleavage products (activation) of caspase 1. Activated caspase 1 was detected when the second FITC peak of the blue curve shifted to the right side that was only present in TTFields-treated cells with normal AIM2 content (EV (empty virus) vs. EV + TTFields). However, no such peak was observed in TTFields-treated cells without AIM2 (AIM2 KD vs. AIM2 KD + TTFields). Therefore, the effect of TTFields on cell pyroptosis is at least partially mediated by AIM2.

Although the present invention has been disclosed with reference to certain specific examples, numerous modifications, changes and variations of the described specific examples are possible without departing from the field and scope of the present invention as defined in the appended claims. It is therefore intended that the present invention not be limited to the specific examples described, but may have the full scope defined by the language of the following claims and their equivalents.

without

[Figure 1] shows that TTFields can induce the formation of cytoplasmic micronuclei (double-stranded DNA or dsDNA) in glioblastoma (GBM) cells exposed to TTFields (TTFields) compared with control GBM cells (control group);

[Figure 2] shows that the nuclear laminin B1 structure is disrupted after exposure to TTFields, thereby releasing dsDNA into the cytoplasm in LN827 cells;

[Figure 3] shows an example of biochemical pathways induced by cytoplasmic dsDNA (pro-inflammatory (STING) and pyroptotic pathways);

[Figure 4] shows that cGAS and AIM2 independently colocalize with micronuclei in response to exposure to TTFields.

[FIG. 5] provides a graph showing the percentage of AIM2/cGAS co-localized with micronuclei from the results of FIG. 4;

[Figure 6] shows the phosphorylation of IRF3 and p65 in U87 and LN827 cells after one day of exposure to TTFields;

[Figure 7] shows that TTFields induce type I IFN response and pro-inflammatory cytokines downstream of STING;

[Figure 8] shows that STING is degraded after being activated by TTFields in GBM cells (LN428 human cells and KR158 mouse cells);

[ FIG. 9 ] shows that STING is required for the inflammatory response induced by dsDNA and TTFields treatment in GBM cells (LN428 human cells, KR158 mouse cells, and F98 rat cells);

[ FIG. 10 ] shows that autophagy and dsDNA or TTFields synergistically induce STING-dependent proinflammatory responses in KR158 and F98 GBM cells;

[Figure 11] shows that TTFields-induced inflammatory interleukin production in the F98 rat neurofibroma model is dependent on STING and AIM2;

[Figure 12] shows that tumor size is correlated with the fold change in inflammatory interleukin expression in response to TTFields;

[ FIG. 13 ] provides an exemplary heat map showing that in the F98 rat neurofibroma model, the recruitment of CD45 cells into GBM is lower in GBM lacking STING and AIM2;

[ FIG. 14 ] provides an exemplary heat map showing that CD3 (T cell) recruitment is lower in GBM lacking STING and AIM2;

[ FIG. 15 ] provides an exemplary heat map showing that in GBM lacking STING and AIM2, DC/macrophage recruitment is low and MDSC recruitment is high;

[Figure 16] provides quantitative results of the data in Figure 17;

[Figure 17] shows "ghosting" induced by three-day exposure to TTFields in human GBM cell lines LN308 and LN827;

[Figure 18] shows that TTFields induces membrane damage and reduces GSDMD in U87 GBM cells exposed to TTFields;

[ FIG. 19 ] shows that TTFields induce membrane damage and cleave GSDMD in human leukemia monocytic cell line THP-1 macrophages;

[FIG. 20] shows THP1-GFP PMA-pretreated cells exposed to TTFields for 24 hours;

[FIG. 21] shows THP1-GFP PMA pre-treated control cells not exposed to TTFields;

[FIG. 22] shows that TTFields induces cell pyroptosis-dependent caspase-1 activation after 1 day and 3 days of exposure to TTFields;

[Figure 23] shows that TTFields-induced caspase-1 activation and pyroptosis are consistent with lower levels of full-length IL-1β and higher LDH release after 1 and 3 days of exposure to TTFields;

[FIG. 24] shows that TTFields-induced STING/AIM2 activation and inflammatory interleukin production are maintained for at least 3 days after TTFields treatment has ended;

[FIG. 25] shows that short pulse TTF-induced STING/AIM2 activity is associated with reduced tumor growth and increased DC (dendritic cell) recruitment to deep cervical draining lymph nodes; and

[Figure 26] shows that caspase 1 is detected in TTFields-treated cells without AIM2.

Claims (18)

A use of a checkpoint inhibitor for the manufacture of a pharmaceutical for reducing the viability of cancer cells, wherein the pharmaceutical is administered in a method comprising: applying an alternating electric field at a frequency between 100 and 500 kHz to the cancer cells; interrupting the application of the alternating electric field to the cancer cells; and administering the pharmaceutical to the cancer cells. The use as claimed in claim 1, wherein the AC electric field is applied for a period sufficient to kill approximately 1-2% of the cancer cells. The use as claimed in claim 1, wherein the AC electric field is applied for a period sufficient to increase the interferon B content in the cancer cells by 300 times. The use of any one of claims 1 to 3, wherein the AC electric field is applied to the cancer cells for a first period of time, and the AC electric field is interrupted for a second period of time, wherein the second period of time is at least the same as the first period of time. The use as claimed in any one of claims 1 to 3, wherein the AC electric field is applied to the cancer cells in short pulses, wherein the duration of each short pulse is less than 5 seconds. The use of any one of claims 1 to 3, wherein the cancer cells are selected from the group consisting of neuroglioblastoma cells, pancreatic cancer cells, ovarian cancer cells, non-small cell lung cancer (NSCLC) cells and mesothelioma. The use as claimed in any one of claims 1 to 3, wherein the cancer cells are glioblastoma cells. The use of any one of claims 1 to 3, wherein the checkpoint inhibitor is selected from the group consisting of: ipilimumab, pembrolizumab and nivolumab. The use as claimed in any one of claims 1 to 3, wherein the frequency of the AC electric field is between 180 and 220 kHz. The use of any one of claims 1 to 3, wherein at least a portion of the administration of the drug to the cancer cells is performed after the application of an alternating electric field at a frequency between 100 and 500 kHz to the cancer cells is interrupted. A use of a checkpoint inhibitor for the manufacture of a medicament for treating glioblastoma, wherein the medicament is administered in a method comprising: applying an alternating electric field at a frequency between 100 and 500 kHz to the head of an individual suffering from glioblastoma; interrupting the application of the alternating electric field to the head of the individual; and administering the medicament to the individual. The use of claim 11, wherein the neuroglioblastoma comprises neuroglioblastoma cells, and wherein the alternating electric field is applied for a period sufficient to kill approximately 1-2% of the neuroglioblastoma cells. The use of claim 11, wherein the neuroglioblastoma comprises neuroglioblastoma cells, and wherein the alternating electric field is applied for a period sufficient to increase the interferon B content in the neuroglioblastoma cells by 300 times. The use as claimed in any one of claims 11 to 13, wherein the AC electric field is applied to the head of the individual for a first period of time, and the AC electric field is interrupted for a second period of time, wherein the second period of time is at least the same duration as the first period of time. The use as claimed in any one of claims 11 to 13, wherein the AC electric field is applied to the head of the individual in short pulses, wherein the duration of each short pulse is less than 5 seconds. The use of any one of claims 11 to 13, wherein the checkpoint inhibitor is selected from the group consisting of: ipalizumab, pembrolizumab and nivolumab. The use as claimed in any one of claims 11 to 13, wherein the frequency of the AC electric field is between 180 and 220 kHz. The use of any one of claims 11 to 13, wherein at least a portion of the administration of the drug to the individual is performed after the application of an alternating electric field at a frequency between 100 and 500 kHz to the individual suffering from neuroglioblastoma is interrupted.