Methods and apparatus for applying tumor therapy electric fields in combination with personalized hyper-segmented stereotactic adaptive radiation therapy

Abstract

A method of treating a tumor in a subject, the method comprising applying a tumor treatment electric field to the tumor at a frequency of from about 50 kHz to about 1,000 kHz; and delivering a personalized hyper-segmented adaptive radiation therapy (PULSAR) protocol.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 362,369, filed on April 1, 2022, which is incorporated herein by reference in its entirety.

[0003] This application is further related to provisional application serial number 62 / 900,166 filed on September 13, 2019, PCT / US2020 / 050745 (WIPO WO2021051089A1) filed on September 14, 2020, U.S. application serial number 17 / 208,107 filed on March 22, 2021, U.S. application serial number 17 / 693,108 filed on March 11, 2022, and U.S. application serial number 15 / 938,088 filed on March 28, 2018, which are incorporated herein by reference in their entirety. Background of the Invention

[0005] Tumor treating fields (TTFields) are low intensity alternating electric fields in the mid-frequency range that can be used to treat tumors, as described in U.S. Pat. No. 7,565,205. TTFields are induced non-invasively into a target area by placing transducers directly on the patient's body and applying an AC voltage between the transducers. An AC voltage is applied between a first pair of transducers for a first time interval to generate an electric field having field lines that generally run in a front-to-back direction. Then, an AC voltage is applied between a second pair of transducers at the same frequency for a second time interval to generate an electric field having field lines that generally run in a right-to-left direction. The system then repeats this two-step sequence throughout the treatment.

[0006] In addition, conventional techniques for the treatment of cancer patients with radiotherapy are almost limited to fractionated radiation doses. In a short time frame, the fractionated dose, or the fractionated portion, is administered for several consecutive days, or even multiple times (over the course of several weeks) every day or every other day. This is referred to as conventional fractionated radiotherapy (CFRT). In this time frame, the patient will receive daily or near daily radiation doses. The radiation is typically administered with a linear accelerator and is used to control or kill the malignant cells that constitute the tumor.

[0007] Various attempts in the application of fractionated treatment of radiation therapy have been tried, but have never reached a point of success. Fractionated treatment is the deliberate separation or deployment of radiation doses. In the past, split or rest periods occurred between clusters of fractionated doses, for example, at the midpoint of a 6-week course of treatment, a 1-4 week break without therapy was inserted. Historically, the motivation for doing so was to reduce toxicity, thereby allowing the normal tissues surrounding the tumor to have more time to heal from what would otherwise be a difficult and toxic long radiation process. However, the tumor control penalty caused by tumor proliferation has troubled these attempts, meaning that with the extra time between doses, the tumor will begin to grow back.

[0008] Therefore, radiotherapy treatments usually tend to be very rigid and do not allow too much adjustment or personalization. It is not uncommon that the only customization in radiotherapy is based on the type and location of the tumor determined before the start of therapy. For example, a patient with primary lung cancer can be treated with a treatment sequence consisting of 2Gy (Gray) ionizing radiation in 30 fractions over 6 weeks, while a patient with metastatic renal cancer can receive 8Gy in 5 fractions every other day over 2 weeks. However, there is little customization or adjustment before this, such as narrowing the radiation field due to the shrinkage of tumor size. Since there are few real changes during the whole conventional therapy process, the whole process is planned and executed before all therapies start without modification or adjustment. Due to this rigidity, there may be a tendency for overtreatment or undertreatment for patients. Further, even if adjustments are made, with such a short amount of time between fractions and typically the completion of all radiotherapy in a total of 4-6 weeks, there is little time for the tumor or its environment to show significant or noteworthy changes that may affect adjustment or personalization. SUMMARY OF THE INVENTION

[0010] As will be discussed in more detail below, in some aspects, the following new methods and instruments involve a combination of TTField and a radiation delivery regimen called Personalized Ultra-fractionated Adaptive Radiotherapy (PULSAR). PULSAR is an ablative and adaptive method for radiotherapy. Ablative strategies such as stereotactic ablative radiotherapy (SAbR) are now common in radiation oncology, in more and more treatment scenarios. Conventional ablative treatments such as SAbR involve radiotherapy, in which many fractionated portions of radiation are delivered over weeks or days. However, PULSAR has nothing to do with conventional treatment, but introduces radiation pulses at intervals much longer than conventional radiotherapy or SAbR, and the radiation therapy is called pulses. As an improvement over conventional methods, it is highly effective to apply pulse therapy at customized time intervals in combination with the application of TTField or in combination with the application of TTField together with specific chemotherapeutic agents and immune checkpoint inhibitors. Indeed, PULSAR therapy can be adapted to the response of the tumor, the tumor microenvironment, and normal tissue to therapy, thereby allowing treatment to be tailored to the patient's clinical situation.

[0011] In addition to the effectiveness of PULSAR, TTFields are also effective because they create conditional vulnerability to agents that damage DNA (e.g., ionizing radiation). Clinical data created to facilitate this new combination therapy demonstrate striking radiation sensitization in tumors grown subcutaneously in immunocompetent subjects (e.g., mice). As will be discussed below, since PULSAR relies on extended time periods between radiation "pulses" (e.g., time intervals that have never been used or considered for conventional radiotherapy), in some cases, TTFields are applied during those time periods. Further, other agents that cause DNA damage, inhibit DNA replication fork travel or maintenance, or cause DNA replication fork collapse are also applied during the time interval between pulses. In addition, because TTField triggers an innate immune response via blocking mitochondrial autophagy, and high-dose radiation exposure (e.g., those used in PULSAR) will do the same thing, it is sufficient to combine PULSAR and TTField with immuno-oncology methods (e.g., immune checkpoint inhibition).

[0012] One aspect of the invention relates to a method of treating a tumor in a subject, the method comprising: applying a tumor treating electric field to the tumor at a frequency of about 80 kHz to about 300 kHz; and delivering an ablative, adaptive, and novel personalized hyperfractionated adaptive radiation therapy (PULSAR) protocol.

[0013] One aspect of the invention relates to a method for treating a tumor in a subject, comprising: applying a tumor treating electric field to the tumor at a frequency of about 80 kHz to about 300 kHz; delivering an ablative, adaptive, and novel personalized hyperfractionated adaptive radiation therapy (PULSAR) protocol; and delivering an agent that causes DNA damage, inhibits DNA replication fork progression or maintenance, or causes DNA replication fork collapse in the intervals between pulses.

[0014] In another aspect of the invention, a method may include applying a tumor treating electric field to the tumor at a frequency of about 80 kHz to about 300 kHz; administering a first pulse dose of radiation to the tumor in the subject; and measuring a biological characteristic of at least one of the subject and the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Depicted are exemplary methods for treating a tumor in a subject according to exemplary embodiments of the present disclosure.

[0017] Figure 2 Depicted are exemplary methods of treating a tumor in a subject according to exemplary embodiments of the present disclosure.

[0018] Figure 3 Depicted are exemplary effects of TTField on a subject, according to an exemplary embodiment of the present disclosure.

[0019] Figure 4A Depicted are exemplary effects of TTField on genes according to exemplary embodiments of the present disclosure.

[0020] Figure 4B Depicted are exemplary effects of TTField on the Fanconi anemia pathway according to exemplary embodiments of the present disclosure.

[0021] Figure 4C Depicted are exemplary effects of TTFields and irradiation on DNA repair kinetics, according to exemplary embodiments of the present disclosure.

[0022] Figure 4D Depicted are exemplary effects of TTFields on cells according to exemplary embodiments of the present disclosure.

[0023] Figure 5 Depicted are exemplary effects of a radioimmunotherapy pulse in a hot tumor microenvironment, according to an exemplary embodiment of the present disclosure.

[0024] Figure 6 Depicted are exemplary effects of a radioimmunotherapy pulse in a cold tumor microenvironment, according to an exemplary embodiment of the present disclosure.

[0025] Figure 7 Depicted are exemplary effects of PULSAR and immunotherapy on cancer according to exemplary embodiments of the present disclosure.

[0026] Figure 8 Depicted are exemplary survival results in a tumor microenvironment according to exemplary embodiments of the present disclosure.

[0027] Fig. 9 Depicted is an exemplary effect of a single round of TTField and irradiation combination according to an exemplary embodiment of the present disclosure.

[0028] Fig.10 Depicted is an exemplary effect of a combination of two rounds of TTFields and irradiation, according to an exemplary embodiment of the present disclosure.

[0029] Fig.11 Depicted is an exemplary effect of a combination of two rounds of TTFields and irradiation, according to an exemplary embodiment of the present disclosure.

[0030] Fig.12 Depicted are exemplary effects of TTFields and irradiation on tumor volume, according to exemplary embodiments of the present disclosure.

[0031] Fig.13 Depicted are tumor growth delay curves according to exemplary embodiments of the present disclosure.

[0032] Fig.14 Depicted are tumor growth delay curves according to exemplary embodiments of the present disclosure.

[0033] Fig.15 Depicted are multiple tumor growth delay curves by individual treatments according to exemplary embodiments of the present disclosure.

[0034] Fig.16 An example of an apparatus for applying a TTField having a modulated electric field to a subject's body is depicted.

[0035] Fig.17 An exemplary computer apparatus is depicted.

[0036] Description of the implementation plan

[0037] Disclosed are techniques for treating a tumor in a subject. The present disclosure relates to tumor treating fields (TTFields) delivered in combination with personalized hyperfractionated adaptive radiation therapy (PULSAR) that, when applied in a protocol, can induce synergistic cell killing via disruption of the DNA damage response, enhanced DNA replication stress, and DNA replication fork collapse.

[0038] The disclosed TTFields can reduce tumor cells by disrupting mitosis. Further, the disclosed TTFields can affect DNA damage repair and replication stress pathways of tumor cells. For example, the disclosed TTField treatment can reduce Fanconi anemia (FA) pathway signaling proteins by impairing the DNA damage repair process induced by irradiation (IR). The length of newly replicated DNA can slow down with TTField exposure time, and TTField can increase R-loop formation, indicating that TTField induces replication stress. The disclosed TTField can increase the sensitivity of chemotherapeutic agents that target and increase replication stress in new combination therapy options.

[0039] Ablative strategies such as stereotactic ablative radiotherapy (SAbR) are now common in radiation oncology, in more and more treatment scenarios. However, PULSAR has nothing to do with conventional radiotherapy, where many "fractions" of radiation are delivered over weeks rather than days (SAbR) or now with PULSAR over weeks or months, and because the intervals between radiation fractions are much longer than conventional radiotherapy or SAbR, the radiation therapy is called a pulse. This pulse interval is highly suitable for applying TTField or TTField plus specific chemotherapy agents and immune checkpoint inhibitors. Since PULSAR was developed for the purpose of integrating the response of tumors, tumor microenvironments, and normal tissues to therapy, it is ready to integrate additional innovations in response to the patient's clinical situation.

[0040] Figure 1Depicted are exemplary inventive methods for treating tumors in subjects according to exemplary embodiments of the present disclosure. Each of these inhibitors can target, for example, DNA repair, DNA replication, DNA crosslinking, DNA complexes, or cell cycle regulation in order to increase replication stress. For example, an ATR inhibitor 104 (a reagent that blocks key DNA repair proteins) can be introduced into tumor cells or tissues for inhibiting cell cycle checkpoints when DNA is damaged. Irradiation (IR) 102 (a process by which an object is exposed to radiation) can be applied to tumor cells or tissues for interrupting DNA chains. Platinum reagents 106 (a substance that binds DNA nucleotides together and blocks DNA synthesis) can be delivered to tumor cells or tissues for inducing intrachain crosslinks that can inhibit DNA replication. Crosslink repair can be downregulated by TTField. PARP inhibitors 110 (inhibitors prevent PARP from implementing cell repair processes) can be applied to tumor cells or tissues for inhibiting replication fork maintenance. Topoisomerase inhibitors 108 (a class of enzymes that change the supercoiling of double-stranded DNA) can be delivered to tumor cells or tissues for inhibiting DNA replication and chromosome condensation.

[0041] The disclosed combination therapy options using chemotherapeutic agents can synergistically increase replication stress in combination with TTFields. The disclosed chemotherapeutic agents can target replication stress, which can be a major cause of genomic instability. Cancer cells can maintain unlimited proliferation by maintaining low to mild levels of replication stress, with a defective DNA damage response (DDR) and loss of cell cycle checkpoints. Normal cells can maintain genomic stability through the coordinated action of the DDR and cell cycle checkpoints. Defects in the DDR and mild to low levels of replication stress are unique to cancer cells and can therefore be exploited therapeutically. In order to exploit replication stress, the disclosed TTFields can be combined with the disclosed chemotherapeutic agents (which can also cause replication stress at several key steps).

[0042] Figure 2An exemplary method 200 for treating a tumor in a subject is depicted. At 206, a tumor treatment field (TTField) may be applied to the tumor. In a non-limiting embodiment, the TTField may be applied with predetermined parameters. As an example, the TTField may include a frequency within a frequency range from about 50 kHz to about 1000 kHz. As an example, the frequency of the tumor treatment field may be about 200 kHz to about 500 kHz. As an example, the frequency of the tumor treatment field may be about 100 kHz, about 150 kHz, about 200 kHz, or about 250 kHz. As an example, the TTField may include an intensity within an intensity range from about 1 V / cm to about 20 V / cm. As an example, the TTField may include an intensity within an intensity range from about 1 V / cm to about 10 V / cm. As an example, the intensity of the tumor treatment field may be about 1 V / cm to about 4 V / cm. Other possible exemplary parameters regarding the TTField may include, among others, active time, dimming time, and duty cycle (all of which may be measured, for example, in ms). The parameters can be modified based on the size of the tumor, the type of tumor, the subject, or the purpose of the treatment. In one example, for treating glioblastoma cancer cells, the intensity of the tumor treating field is about 1 V / cm to about 4 V / cm, and the frequency of the tumor treating field is about 150 kHz to about 250 kHz.

[0043] The disclosed TTField treatments can induce DNA damage, impair the DNA damage response, and create conditional vulnerability. The application of the disclosed TTFields can increase in γ-H2AX foci over time in cells (e.g., which are not irradiated but exposed to TTFields). Because γ-H2AX is also an early sensor of stalled replication forks during replication stress, TTField exposure can induce replication stress because the reduced expression of BRCA1 and other members of the Fanconi anemia pathway adversely affects the repair of collapsed or stalled replication forks. Further, the MCM6 and MCM10 genes (constituent members of the DNA replication complex) can also be downregulated by the application of TTFields.

[0044] In certain embodiments, TTFields may be applied to a tumor before or after application of the disclosed inhibitors and / or PULSAR therapy. In certain embodiments, TTFields may be applied to a target tissue simultaneously with the disclosed inhibitors and / or PULSAR therapy. As an example, TTFields may be applied to a tumor prior to PULSAR therapy to establish conditional vulnerability, which causes damaged cells to become more vulnerable to PULSAR therapy. Alternatively, however, at least a portion of the applying step 202 may be performed simultaneously / concomitantly with at least a portion of the delivering step 202 and / or at least a portion of 204. In non-limiting embodiments, the disclosed agents / inhibitors may be delivered concomitantly with TTFields and / or PULSAR therapy. As an example, at least a portion of the applying step 204 may be performed simultaneously with at least a portion of the delivering step 202. In certain embodiments, fluctuations in the subject's weight may serve as a condition for switching from treatment with TTFields at 202 to starting a PULSAR procedure at 204. Additionally, in one non-limiting embodiment, TTFields may be applied for a predetermined period of time (e.g., approximately 7 days) followed by a rest period for a predetermined period of time (e.g., approximately 3 days). Alternatively, TTFields may be applied continuously throughout the procedure, along with the delivery of PULSAR at 204.

[0045] At 204, PULSAR can be delivered to the tumor. In one example, a first test dose of radiation, referred to as the first pulse, can be administered to a tumor in a subject. The tumor can be benign or malignant. In all embodiments, the dose can be substantial, sufficient to enable changes in the tumor, tumor environment, or within the patient to better promote adjustment and personalization. For this purpose, the pulse dose can be at least 6-8Gy, possibly more. In some embodiments, the dose can be ablative (defined as destroying both proliferation capacity and cell function) and is conventionally used as an independent therapy for both primary cancer and metastasis. The range of such doses is 15-50Gy / treatment. Often, ablative dose ranges cause widespread tumor death. For example, the dose can be a dose similar or identical to that to be applied in stereotactic ablative radiotherapy (SABR). The range of the dose currently used for the first or only segmented portion in existing SABR treatments for solid tumors is 8-50Gy. For example, the dose can be applied with high accuracy and precision and intentionally localized to the bulk tumor / cancer, which can mitigate the effects of radiation on normal tissue surrounding the tumor.

[0046] It is worth noting that PULSAR is an adaptive procedure, which means that it is modified based on the efficacy of the treatment and is tailored to a specific subject. Therefore, a second application of TTField can be applied to the subject, followed by a second pulse dose of radiation (or more) to the subject. In some embodiments, the time period between the first and second doses of radiation (i.e., the rest / observation period) can be at least 6-7 days or more. For example, a first 10Gy dose can be administered, and 10 days later, a second 10Gy dose can be administered. In some embodiments, the rest / observation period length can be 10 days, 20 days, 30 days or even months. In some embodiments, the second dose can also be ablative and similar or identical to the dose that will be applied in stereotactic ablative radiotherapy. The second dose can have the same or different efficacy as the first dose, thereby targeting a larger or smaller target field. In some embodiments, the second dose and subsequent pulse dose levels can be modified or adjusted based on information obtained during the rest / observation period.

[0047] For example, if a tumor shrinks dramatically after a first pulse, adjustments affecting the dose level of the second pulse may be as simple as reducing the dose. Conversely, if the tumor fails to respond after the first pulse or even grows, the second pulse dose may be greater than the first. When a tumor responds after the first or any previous pulse, the next planned pulse may treat a smaller volume as an adjustment. Another example may be that a previous pulse caused new hypoxia based on imaging that constitutes focal radiation resistance. In response, another dose may be "painted" to these hypoxic areas by using dosimetric modulation when planning the upcoming pulse. In response, a hypoxic cell sensitizing drug may be administered in addition to the next radiation pulse.

[0048] Another example could be that sampling of circulating tumor cells or repeated biopsies or other laboratory or imaging changes indicate that the targeted tumor may benefit from the addition of a particular class of drugs, targeted therapies, or immunotherapies. In this case, subsequent pulses could be delivered along with that drug or combination therapy in a manner known to optimize the combination. Another example could be that tumor response indicates that radiation alone will never eliminate a patient's particular tumor. In this case, the patient could be referred for surgery or drug therapy without radiation. The process of providing dose pulses in this manner could be repeated until the cancer is eliminated (cured), the treatment causes unacceptable toxicity, or tumor progression occurs despite exhaustion of all adaptation options / opportunities.

[0049] In some embodiments, before performing 204 and administering a second dose of radiation to a subject with a tumor, the level of radiation or efficacy, the duration of the rest / observation period, and the target range for the second dose can be determined. For example, treatment can be personalized and adjusted for a specific patient based on its response to the initial radiation dose. The patient's response to the initial dose can cover a variety of factors, including but not limited to symptoms, examinations, imaging, tumor response, blood tests, body fluid analysis, biopsy, histology, grade, stage, genomics, sequencing, gene expression, performance, patient tolerance, attitude, social environment, or any other personal test. Because the rest / observation period between doses is relatively long, which means not a few days immediately and ideally more than seven days, there is ample time for the patient's body and tumor to adapt. For example, if the tumor is determined to have features that suggest a high risk of rapid growth, the rest period between doses can be shorter than when tumor shrinkage has been detected. Further, the extra time between pulses can allow the immune cascade to run its designed process throughout the body, making subsequent pulses more effective.

[0050] In some embodiments, radiomic and biological signatures can be analyzed to further tailor treatment. Biological signatures can include target tissue vascularity, normal tissue vascularity, target tissue oxygenation status, normal tissue oxygenation status, target tissue cytokine profile, normal tissue cytokine profile, target tissue gene expression, normal tissue gene expression, target tissue receptor expression, normal tissue receptor expression, target tissue leukocyte infiltration, normal tissue leukocyte infiltration, tumor markers, tumor burden, systemic immune status, changes in subject health, and changes in patient weight.

[0051] In some embodiments, a DNA replication stress inducing agent is delivered to the tumor. The DNA replication stress inducing agent includes at least one of the following: a platinum compound, an alkylating agent, a wee1 inhibitor, a Chk1 inhibitor, a thymidylate synthase inhibitor, a ribonucleotide reductase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a maternal embryonic leucine zipper kinase (MELK) inhibitor, or a NEDD8-activating enzyme (NAE) inhibitor.

[0052] In some embodiments, radiomic features may include features extracted from or images from: anatomical imaging features (e.g., tumor response, tumor infiltration, margin features, density features, shape features, etc.), functional imaging features (e.g., blood flow, enhancement, etc.), and metabolic imaging features (e.g., glucose uptake, proliferation, hypoxia, etc.).

[0053] Using certain embodiments disclosed herein, TTFields in combination with PULSAR and various others are applied to tumors.

[0054] Experimental Results

[0055] For example, Figure 3 Depicted are exemplary methods for treating a tumor in a subject according to an exemplary embodiment of the present disclosure. Tumor growth delay (TGD) experiments were performed in three different mouse tumor models treated with TTFields. TTField exposure alone (7 days of continuous exposure) did not reduce the rate of tumor growth with any statistical significance in two of the three mouse tumor models, while MC38 tumors grew slower when exposed to TTFields. TTField treatment reduced mouse body weight by 7-16% after 7 days of treatment, but the mice quickly regained their weight within 2 days of being moved back to regular cages. Then, at the end of TTField exposure, radiation delivered as a single dose of 8Gy was added to the treatment regimen for MC38 tumors. Here, there was a significant delay in tumor growth after a round of TTFields and subsequent single dose of radiation.

[0056] In the next step, two rounds of TTField and radiation were applied. This is a PULSAR treatment strategy in which there is a significant gap in the treatment time. The tumor growth delay was much more significant after two rounds of treatment compared to one round of treatment. Then, at the end of the TTField exposure, radiation delivered as a single dose of 8Gy was added to the treatment protocol for MC38 tumors. Here, there was a significant delay in tumor growth after one round of TTField and subsequent single dose of radiation.

[0057] In the next step, two rounds of TTField and radiation were applied. This is a PULSAR treatment strategy, where there are significant gaps in the treatment time. Compared to one round of treatment, tumor growth delay was much more significant after two rounds of treatment.

[0058] Figures 4A-4D The exemplary effects of TTField on various subjects are depicted. In particular, Figure 4A Depicted is a genomic expression analysis performed using a panel of NSCLC cells using microarrays. Figure 4BDownregulation of the Fanconi anemia (FA) pathway upon TTField exposure is depicted. From the microarray results, it was found that BRCA1 DNA damage response pathway genes were downregulated upon TTField exposure, in addition to the already known cell cycle-related defects. In addition, downregulation of some FA pathway proteins was confirmed by Western blotting. Based on the established role of the FA pathway in DNA damage response, experiments on the kinetics of DNA damage repair induced by IR were performed, which produced results that indicate that TTField exposure impairs the DNA damage response induced by IR, as in Figure 4C In addition, some experiments demonstrated that TTField treatment alone induced some degree of DNA double-strand breaks, which were evident by increased g-H2AX foci. As discussed above, TTFields interfere with DNA damage repair kinetics, and it is hypothesized that in novel combination therapy options using TTFields with radiation, TTField treatment would increase cell death induced by IR. As described in Figure 4D As depicted in , results from some experiments indicate that TTFields increase cell killing induced by IR more than additively. In light of these findings, experiments were performed in which TTFields were applied to subjects prior to radiation, which produced results that further indicated that TTFields enhanced sensitivity to radiation, as evident by the increased CI values.

[0059] Figure 5 Depicted are exemplary effects of a radioimmunotherapy pulse in a hot tumor microenvironment, according to one or more embodiments. For example, Figure 5 The timing of radioimmunotherapy pulses is depicted to produce effects on tumor growth in a "hot" tumor microenvironment using MC38 cells.

[0060] Figure 6 Depicted are exemplary effects of a radioimmunotherapy pulse in a cold tumor microenvironment according to an exemplary embodiment of the present disclosure. For example, Figure 6 The timing of radioimmunotherapy pulses in a "cold" tumor microenvironment is depicted to produce effects on tumor growth.

[0061] Figure 7 Depicted are exemplary effects of PULSAR and immunotherapy on cancer according to exemplary embodiments of the present disclosure. For example, Figure 7 Depicted are the effects of PULSAR and immunotherapy on rectal cancer in mice.

[0062] Figure 8 Depicted are exemplary survival results in a tumor microenvironment according to an exemplary embodiment of the present disclosure. For example, Figure 8Survivability data across hot and cold microenvironments are depicted. Given the vulnerability of TTField-induced conditions, experiments were performed using TTField in combination therapy. In one experiment, tumor growth delay (TGD) was applied in combination with radiation. In this experiment, one round of TTField and IR treatment cycles were used. The results of this TGD approach suggested a significant tumor growth delay between the TTField group and the hot group in the MC38 mouse model after one round of TTField plus IR treatment: when the tumor volume reached 40mm 3 TTField and thermal treatment was started when the average volume of MC38 cells was reached. After 7 days of treatment (TTField or thermal), mice were exposed to 8Gy radiation. After radiation, mice were transferred to regular cages and tumor volume and body weight were measured every other day. TTField treatment slowed tumor growth, but significant tumor growth inhibition was observed in the TTField plus IR group of mice compared to their controls. In this figure, historical controls from other studies in which exact amounts of MC38 cells were injected in C57BL / 6 and TGD was monitored were used.

[0063] Fig. 9 Depicted is an exemplary effect of a single round of TTField and irradiation combination according to an exemplary embodiment of the present disclosure. For example, to facilitate when it involves Figure 8 The experiment discussed in Fig. 9 Depicted are the results of an experiment using a combination of TTField and radiation therapy in one cycle. For example, when the tumor volume reached 40 mm 3 TTField and thermal treatment was started when the average volume of the tumor reached about 100 μg / cm2. After 7 days of treatment (TTField or thermal), the mice were exposed to 8 Gy radiation and allowed to gain weight for 3 days in regular cages. After 3 days, the mice were given a second round of 7 days of treatment (TTField or thermal) and then exposed to 8 Gy radiation. The mice were transferred to regular cages immediately after the second round of irradiation, and tumor volume and body weight were measured every other day. One round of TTField and IR treatment slowed tumor growth, but further significant tumor growth inhibition was observed in the two-round TTField plus IR treatment group mice compared to their controls.

[0064] Fig.10 Depicted is an exemplary effect of a combination of two rounds of TTField and irradiation according to an exemplary embodiment of the present disclosure. Fig. 9 The experiment discussed in Fig.10 Depicted is a case where two cycles of TTField and radiation therapy combination were applied. When the tumor volume reached 40 mm 3TTField and thermal treatment was started when the average volume of the mice reached about 100 μg / cm2. After 7 days of treatment (TTField or thermal), the mice were exposed to 8 Gy radiation and allowed to gain weight in regular cages for 3 days. After 3 days, the mice were given a second round of 7 days of treatment (TTField or thermal) and then exposed to 8 Gy radiation. The mice were transferred to regular cages immediately after the second round of irradiation, and tumor volume and body weight were measured every other day. One round of TTField and IR treatment slowed tumor growth, but further significant tumor growth inhibition was observed in the two-round TTField plus IR treatment group of mice compared to their controls. It is noteworthy that although two rounds of TTField and IR treatment were administered, several rounds may be delivered to the subject, based on one or more factors, including but not limited to the subject's response to previous rounds of treatment.

[0065] In further embodiments, a third round of TTFields will further affect tumor growth delay. Notably, normal tissue responses advance the clinical use of radiation, and each tissue found in a radiation field has a tolerance dose that limits the individual doses of radiation as well as the cumulative dose. Therefore, one goal will be to minimize the number of radiation doses that an animal (e.g., mouse, patient, etc.) will receive as a way to limit the amount of normal tissue irradiated. Therefore, in this case, in view of the potential for enhanced immunogenic responses to a combination of radiation and immune checkpoint inhibitors or TTFields combined with immune checkpoint inhibitors, the anti-PD-L1 antibody atezolizumab will be added to the radiation / TTField protocol.

[0066] Fig.11 Depicted is an exemplary effect of a combination of two rounds of TTField and irradiation according to an exemplary embodiment of the present disclosure. Figure 9-10 The experiment discussed in Fig.11 Similar experiments are described using the LLC model in which mice were exposed to two cycles of TTField and radiation therapy combination. 3TTField and thermal treatment was started when the average volume of the tumor reached about 100 μg / cm2. After 7 days of treatment (TTField or thermal), the mice were exposed to 12 Gy radiation and allowed to gain weight in regular cages for 3 days. After 4 days, the mice were given a second round of 7 days of treatment (TTField or thermal) and then exposed to 8 Gy radiation. The mice were transferred to regular cages immediately after the second round of irradiation, and tumor volume and body weight were measured every other day. It was found that there were more ulcer formation and aggressive forms of tumors in this model. Therefore, the results of this experiment suggest that the trend of reduced TGD in the TTField plus IR treatment group mice compared to their controls is not as significant as in the MC38 model. It is worth noting that although two rounds of TTField and IR treatment were administered, several rounds can be delivered to the subject, based on one or more factors, including but not limited to the subject's response to previous rounds of treatment. In addition, various chemotherapeutic agents can be delivered between rounds of TTField and IR treatment. For example, after the second round of TTField and IR treatment, the first chemotherapeutic agent can be delivered to the subject. The subject can then be evaluated to determine the effectiveness of the first treatment with TTField, IR, and a chemotherapeutic agent. In one instance, if the subject (e.g., the subject's tumor) is found to be non-responsive (i.e., resistant) to a chemotherapeutic agent, then a second chemotherapeutic agent can be administered to the subject in a third round (or any number of subsequent rounds of treatment) of TTField and IR treatment. Alternatively, if the first chemotherapeutic agent is determined to be effective in treating the subject, then the first chemotherapeutic agent can be administered to the subject again in a subsequent round of treatment. However, the first chemotherapeutic agent can be delivered at a different dose or in combination with one or more other chemotherapeutic agents based on the determined effectiveness in an earlier round of treatment.

[0067] Fig.12 Depicted are exemplary effects of TTField and irradiation on tumor volume according to exemplary embodiments of the present disclosure. For example, Fig.12 Depicted are data predicting the outcome of treatment combinations of TTField, PULSAR, and one or more additional tumor damaging therapies.

[0068] Fig.13Tumor growth delay curves according to one or more examples are depicted. As depicted, an exemplary effect is shown, where after 7 days of treatment (TTField), mice were exposed to 8Gy of X-rays and allowed to gain weight for 3 days. The mice were then treated with TTField for an additional 7 days and subsequently exposed to an additional 8Gy of X-rays. At the time points shown, PD-L1 antibodies were injected subcutaneously into specific groups of mice, but not in other groups of mice. Significant tumor growth inhibition was observed with TTField combined with radiation and PD-L1, relative to the groups treated with TTField plus PD-L1 antibody or TTField plus radiation or radiation plus PD-L1 antibody. Further, in the triple combination group of TTField, radiation and PD-L1 antibody, tumor cure appeared to be observed in 3 of 6 mice (as in Fig.15 Here, the individual growth delays for each mouse in the triple combination group of TTField, radiation, and immune checkpoint inhibitors are described individually. For 3 of the 6 mice, the tumors were not easily measurable. This treatment protocol is an exemplary group in which tumor cures were seen.

[0069] Fig.14 Tumor growth delay curves according to one or more examples are depicted. To facilitate treatment, for example, Fig.13 The tumor size of each patient (e.g., mouse) was analyzed over a period of time after the treatment in question. As depicted, for some patients receiving TTField, radiation, and PD-L1 antibody, tumor volume significantly declined.

[0070] As in Fig.15As depicted in , multiple tumor growth delay curves by individual treatments are depicted. As depicted, individual growth delays are depicted for a group of patients (e.g., mice) that received a triple combination of TTField, radiation, and an immune checkpoint inhibitor. Statistical significance: The Bliss test for the significance of synergistic effects in combination therapy (as discussed in Demidenko, E., & Miller, TW (2019). Statistical Determination of Synergy based on Bliss Definition of Drugs Independence. PloS one, 14(11), e0224137. https: / / doi.org / 10.1371 / journal.pone.0224137, which is hereby incorporated by reference) is used to determine whether there is a synergistic combination of TTField and radiation (i.e., where the effect is greater than what would be obtained if the individual independent effects were combined). In this method, the TTField and radiation effects from the combination therapy are combined. Fig.13 and 14 The tumor growth delay curve is Fig.15 The data were replotted as a log-linear function and linear regression of the data was applied. The slopes of those regressions were considered independent effects and then analyzed to detect synergy with respect to combinations of independent effects. The solid line represents the linear regression, whose slope represents tumor growth in "percent / week". Each independent treatment was plotted as in the control group. Then, the combination therapies were plotted. The synergistic combination therapies included a combination of two rounds of TTField and radiation, a combination of radiation and anti-PD-L1, and a triple combination of TTField, radiation, and anti-PD-L1 antibody. The p-values ​​indicating statistical significance for these three combinations are depicted in Fig.15 1, and they are highly significant. As depicted, TTFields added to radiation resulted in a synergistic combination effect. Adding TTFields to the standard of care model (i.e., radiation plus a checkpoint inhibitor anti-PD-L1 antibody (which combination is also synergistic)) resulted in strong synergy, and most importantly, tumor cure, after only two rounds of radiation (including an immune checkpoint inhibitor).

[0071] Exemplary Instruments

[0072] Fig.16An example of an instrument for applying a TTField with a modulated electric field to a subject's body is depicted. A first transducer 1601 includes 16 electrode elements 1603 (which are located on a substrate 1604), and the electrode elements 1603 are electrically and mechanically connected to each other by wires 1609. A second transducer 1602 includes 20 electrode elements 1605 (which are located on a substrate 1606), and the electrode elements 1605 are electrically and mechanically connected to each other by wires 1610. The first transducer 1601 and the second transducer 1602 are connected to an AC voltage generator 1607 and a controller 1608. The controller 1608 may include one or more processors and a memory accessible by the one or more processors. The memory may store instructions that, when executed by the one or more processors, control the AC voltage generator 1607 to implement one or more embodiments of the present invention. In some embodiments, the AC voltage generator 1607 and the controller 1608 may be integrated into the first transducer 1601 and the second transducer 1602 and form a first electric field generator and a second electric field generator.

[0073] Fig.17 An exemplary computer instrument for use with embodiments described herein is depicted. As an example, the instrument 1700 may be a computer for implementing certain inventive techniques disclosed herein. As an example, the instrument 1700 may be a controller instrument for applying a TTField having a modulated electric field for embodiments described herein. The instrument 1700 may be used as Fig.13 The controller 1608 of the apparatus 1700 may include one or more processors 1702, one or more output devices 1705 and a memory 1703.

[0074] In one example, based on input 1701, the one or more processors generate control signals to control the voltage generator to implement embodiments of the present invention. In one example, input 1701 is user input. In another example, input 1701 can come from another computer in communication with instrument 1700. Output device 1705 can provide the status of the operation of the present invention, such as transducer selection, voltage being generated, and other operational information. Output device 1705 can provide visualization data according to certain embodiments of the present invention.

[0075] The memory 1703 is accessible by the one or more processors 1702 via a link 1704 so that the one or more processors 1702 can read information from and write information to the memory 1703. The memory 1703 may store instructions that, when executed by the one or more processors 1702, implement one or more embodiments of the present invention.

[0076] Many modifications, changes and variations to the described embodiments are possible without departing from the scope of the invention as defined in the claims.It is intended that the invention not be limited to the described embodiments, but has the full scope defined by the following claims and the language of equivalents thereof.

Claims

1. A method for treating a tumor in a subject, comprising: applying a tumor treating electric field to the tumor at a frequency of 50 kHz to about 1,000 kHz; and Delivering personalized hyperfractionated adaptive radiation therapy to the subject.

2. The method of claim 1, wherein the tumor treating electric field establishes a conditional vulnerability with respect to the tumor.

3. The method of claim 1, wherein delivering personalized hyperfractionated adaptive radiation therapy further comprises: administering a first pulse dose of radiation to a tumor in the subject, administering a second pulse dose of radiation to the tumor, wherein the second pulse dose is administered after an observation period having a duration of at least 7 days; and The subject is concurrently treated with immunotherapy.

4. The method of claim 3, wherein the radiation of the first and second pulse doses is ablative.

5. The method according to claim 4, further comprising: In response to administering the first pulse dose, at least one of: a radiation level for the second pulse dose, a duration of the observation period, and a target range for the second pulse dose is determined. The method according to claim 5 , wherein the first pulse dose of radiation is 8 Gy.

7. The method of claim 1, wherein the tumor treating electric field has a strength of about 1 V / cm to about 4 V / cm.

8. The method of claim 1, further comprising delivering a DNA replication stress inducing agent to the tumor.

9. The method of claim 1, wherein the frequency of the alternating electric field is 80 to 300 kHz.

10. The method of claim 1, wherein the tumor treating electric field is applied for 7 consecutive days.

11. The method of claim 5, further comprising analyzing radiomic signatures and biological signatures.

12. The method of claim 11, wherein the biological signature comprises at least one of: target tissue vascularity, normal tissue vascularity, target tissue oxygenation status, normal tissue oxygenation status, target tissue cytokine profile, normal tissue cytokine profile, target tissue gene expression, normal tissue gene expression, circulating tumor DNA indicative of tumor response to therapy, levels of circulating tumor cells, target tissue receptor expression, normal tissue receptor expression, target tissue leukocyte infiltration, normal tissue leukocyte infiltration, tumor markers, tumor burden, systemic immune status, changes in subject health, and changes in patient weight.

13. The method of claim 11, wherein the radiomics feature comprises at least one of: an anatomical imaging feature, a functional imaging feature, and a metabolic imaging feature.

14. The method of claim 5, wherein the second pulse dose is 15 Gy to 50 Gy.

15. The method of claim 1, further comprising delivering a DNA replication stress inducing agent to the tumor.

16. The method of claim 15, wherein the DNA replication stress inducing agent comprises at least one of the following: a platinum compound, an alkylating agent, a wee1 inhibitor, a Chk1 inhibitor, a thymidylate synthase inhibitor, a ribonucleotide reductase inhibitor, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a maternal embryonic leucine zipper kinase (MELK) inhibitor, or a NEDD8-activating enzyme (NAE) inhibitor.

17. The method of claim 16, wherein the DNA replication stress inducing agent comprising a platinum compound is delivered to the tumor, the platinum compound further comprising at least one of the following: cisplatin, carboplatin, oxaliplatin, dicycoplatin or lipoplatin, Wherein if the DNA replication stress inducing agent delivered to the tumor is the alkylating agent, then the alkylating agent comprises at least one of the following: cyclophosphamide or temozolomide, Wherein if the DNA replication stress inducing agent delivered to the tumor is the wee1 inhibitor, then the wee1 inhibitor comprises at least one of the following: Adavosertib-MK1775 or PD0166285, Wherein if the DNA replication stress inducing agent delivered to the tumor is the Chk1 inhibitor, then the Chk1 inhibitor comprises at least one of the following: UCN-01, LY2606368, SAR-020106, AZD7762 or PD0166285, Wherein if the DNA replication stress inducing agent delivered to the tumor is the thymidylate synthase inhibitor, then the thymidylate synthase inhibitor includes at least one of the following: 5-FU or pemetrexed, Wherein if the DNA replication stress inducing agent delivered to the tumor is the ribonucleotide reductase inhibitor, then the ribonucleotide reductase inhibitor comprises gemcitabine, Wherein if the DNA replication stress inducing agent delivered to the tumor is the topoisomerase I inhibitor, then the topoisomerase I inhibitor comprises at least one of the following: irinotecan or topotecan, Wherein if the DNA replication stress inducing agent delivered to the tumor is the topoisomerase II inhibitor, then the topoisomerase II inhibitor comprises at least one of the following: etoposide or doxorubicin, Wherein if the DNA replication stress inducing agent delivered to the tumor is the MELK inhibitor, then the MELK inhibitor comprises OTS167, and Wherein if the DNA replication stress inducing agent delivered to the tumor is the NAE inhibitor, then the NAE inhibitor comprises MLN4924.

18. The method of claim 15, further comprising delivering an additional DNA replication stress inducing agent to the tumor.

19. The method of claim 1, further comprising delivering at least one of the following: an E2F inhibitor, a CDK4 / 6 inhibitor, or a PARP inhibitor.

20. The method of claim 19, wherein the E2F inhibitor is delivered to the tumor, and wherein the E2F inhibitor is HLM006474.

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