Methods and apparatus for applying tumor treating electric fields in conjunction with individualized hyperfractionated stereotactic adaptive radiation therapy - Patents.com

Abstract

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 50 kHz to about 1,000 kHz and delivering a personalized hyperfractionated adaptive radiation therapy (PULSAR) regimen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 362,369, filed April 1, 2022, which is incorporated by reference in its entirety.

[0002] This application is further related to Provisional Application No. 62 / 900,166, filed September 13, 2019, PCT / US2020 / 050745, filed September 14, 2020 (WIPO WO2021051089A1), U.S. Application No. 17 / 208,107, filed March 22, 2021, U.S. Application No. 17 / 693,108, filed March 11, 2022, and U.S. Application No. 15 / 938,088, filed March 28, 2018, which are incorporated by reference in their entireties herein. [Background technology]

[0003] Tumor treating fields (TTFields) are low intensity alternating electric fields in the mid-frequency range that may be used to treat tumors as described in U.S. Patent No. 7,565,205. TTFields are non-invasively induced in a region of interest by applying an AC voltage between transducers, where the transducers are placed directly on the patient's body. An AC voltage is applied between a first pair of transducers for a first time interval to generate an electric field whose field lines run generally in a front-to-back direction. An AC voltage is then applied at the same frequency between a second pair of transducers for a second time interval to generate an electric field whose field lines run generally in a side-to-side direction. The system then repeats this two-step sequence throughout the treatment.

[0004] Furthermore, conventional techniques for radiation therapy of cancer patients have been almost exclusively limited to the delivery of fractionated doses. The fractionated doses, or fractions, are administered within a short time frame on consecutive days, or even multiple times daily or every other day over a period of several weeks. This is known as conventional fractionated radiotherapy (CFRT). During this time frame, patients receive daily or nearly daily doses of radiation. The radiation is typically delivered with a linear accelerator and is used to control or kill the malignant cells that create tumors.

[0005] Various attempts at applying split-course treatments of radiotherapy have been attempted without success. Split-course treatment involves intentionally dividing or spreading out the radiation dose. In the past, there has been a split or rest period between clusters of fractionated doses, for example at the midpoint of a 6-week course, with a 1-4 week break without treatment. Traditionally, the motivation for this was to reduce toxicity and allow more time for the normal tissue surrounding the tumor to heal from what is sometimes a painful and harmful prolonged irradiation. However, the disadvantage of tumor control caused by tumor growth has plagued these attempts, and with the extra time between doses, the tumor begins to grow back.

[0006] As a result, radiation therapy in general tends to be extremely inflexible and does not allow much for adaptation or individualization. It is not uncommon that the only customization in radiation therapy is based on the type and location of the tumor, which is determined before the start of therapy. For example, a patient with primary lung cancer may be prescribed a treatment sequence consisting of 30 fractions of 2 Gy (gray) ionizing radiation over 6 weeks, while a patient with metastatic kidney cancer may receive 5 fractions of 8 Gy every other day over 2 weeks. Beyond that, however, there is little customization or adaptation, such as narrowing the radiation field due to a decrease in tumor size. Since few actual changes occur throughout the traditional course of therapy, the entire course is planned before the start of all therapy and performed without any changes or adaptations. This inflexibility may tend to result in patients being either overtreated or undertreated. Furthermore, even when adaptations are implemented, the amount of time between fractions is very short, generally with all radiation therapy completed within 4-6 weeks, leaving little time for the tumor or its environment to reveal any noticeable or significant changes that may affect adaptation or individualization. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 7,565,205 [Non-patent literature]

[0008] [Non-Patent Document 1] Demidenko, E., and 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 Summary of the Invention [Means for solving the problem]

[0009] As described in more detail below, the following novel methods and devices involve, in some respects, the combination of TTFields with a radiation delivery regimen called Personalized Ultra-fractionated Adaptive Radiotherapy (PULSAR). PULSAR is an ablative, adaptive approach to radiation therapy. Ablative strategies such as Stereotactic Ablative Radiotherapy (SAbR) are now common in radiation oncology and in a growing number of treatment settings. Conventional ablative treatments such as SAbR include radiation therapy in which multiple fractions of radiation are delivered over a period of weeks or days. However, PULSAR is unrelated to conventional treatments and instead introduces pulses of radiation at intervals much longer than those of conventional radiation therapy or SAbR, where the radiation treatment is referred to as a pulse. As an improvement over conventional approaches, the application of treatment in pulses at customized time intervals has been highly effective in combination with the application of TTFields or in combination with the application of TTFields in conjunction with certain chemotherapy agents and immune checkpoint inhibitors. Indeed, PULSAR therapy is adaptable to the response of the tumor, the tumor microenvironment, and normal tissues to the therapy, allowing treatment to be modified depending on the patient's clinical situation.

[0010] In addition to the efficacy of PULSAR, TTFields are also effective in that they create a conditional vulnerability to agents that damage DNA (e.g., ionizing radiation). Clinical data generated to advance this novel combination therapy demonstrates its dramatic radiosensitization of tumors growing subcutaneously in immunocompetent subjects (e.g., mice). As explained below, PULSAR relies on extended time periods between radiation "pulses" (e.g., time intervals not used or considered in traditional radiation therapy), so in some cases TTFields are applied within those time periods. Furthermore, other agents that cause DNA damage or inhibit the progression or maintenance of DNA replication forks or cause the collapse of additional DNA replication forks during the time intervals between pulses. Furthermore, because TTFields trigger an innate immune response by disrupting mitophagy, and high-dose radiation exposure, such as that used in PULSAR, does the same, there is a strong need to combine PULSAR and TTFields with immuno-oncology approaches such as immune checkpoint inhibition.

[0011] One aspect of the present invention is directed 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 a novel ablative, adaptive, personalized hyperfractionated adaptive radiation therapy (PULSAR) regimen.

[0012] One aspect of the invention is directed to a method of 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, novel personalized hyperfractionated adaptive radiation therapy (PULSAR) regimen, and delivering an agent between pulses that causes DNA damage or inhibits progression or maintenance of DNA replication forks, or causes collapse of DNA replication forks.

[0013] In yet another aspect of the invention, a method may include applying a tumor treating electric field to a tumor at a frequency of about 80 kHz to about 300 kHz, administering a first pulsed dose of radiation to the tumor in a subject, and measuring biological characteristics of at least one of the subject and the tumor. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 illustrates an exemplary approach for treating a tumor in a subject, according to an exemplary embodiment of the present disclosure. [Diagram 2] FIG. 1 shows an exemplary method of treating a tumor in a subject, according to an exemplary embodiment of the present disclosure. [Diagram 3] FIG. 1 illustrates an exemplary effect of TTFields on a subject, according to an exemplary embodiment of the present disclosure. [Figure 4A] FIG. 1 shows an exemplary effect of TTFields on genes according to an exemplary embodiment of the present disclosure. [Figure 4B] FIG. 1 illustrates an exemplary effect of TTFields on the Fanconi Anemia pathway, according to an exemplary embodiment of the present disclosure. [Figure 4C] FIG. 1 shows an exemplary effect of TTFields and radiation on DNA repair rates, according to an exemplary embodiment of the present disclosure. [Figure 4D] FIG. 1 illustrates an exemplary effect of TTFields on a cell, according to an exemplary embodiment of the present disclosure. [Diagram 5] FIG. 1 illustrates an exemplary effect of a pulse of radioimmunotherapy in a hot tumor microenvironment, according to an exemplary embodiment of the present disclosure. [Figure 6] FIG. 1 shows an exemplary effect of a pulse of radioimmunotherapy in a cold tumor microenvironment, according to an exemplary embodiment of the present disclosure. [Figure 7] FIG. 1 shows an exemplary effect of PULSAR and immunotherapy on cancer, according to an exemplary embodiment of the present disclosure. [Figure 8] FIG. 1 shows exemplary survival results in a tumor microenvironment, according to an exemplary embodiment of the present disclosure. [Figure 9] FIG. 1 shows an exemplary effect of combined TTFields and radiation in a single round, according to an exemplary embodiment of the present disclosure. [Figure 10] FIG. 1 shows an exemplary effect of the combination of TTFields and radiation in two rounds, according to an exemplary embodiment of the present disclosure. [Figure 11] FIG. 1 shows an exemplary effect of the combination of TTFields and radiation in two rounds, according to an exemplary embodiment of the present disclosure. [Figure 12] FIG. 1 illustrates an exemplary effect of TTFields and radiation on tumor volume, according to an exemplary embodiment of the present disclosure. [Figure 13] FIG. 1 illustrates a tumor growth delay curve, according to an exemplary embodiment of the present disclosure. [Figure 14] FIG. 1 illustrates a tumor growth delay curve, according to an exemplary embodiment of the present disclosure. [Figure 15A] FIG. 1 shows multiple tumor growth delay curves with individual treatments, according to an exemplary embodiment of the present disclosure. [Figure 15B] FIG. 1 shows multiple tumor growth delay curves with individual treatments, according to an exemplary embodiment of the present disclosure. [Figure 16] FIG. 1 shows an example of a device for applying TTFields to a subject's body using modulated electric fields. [Figure 17] FIG. 1 illustrates an exemplary computing device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Techniques are disclosed for treating tumors in subjects. The disclosure relates to tumor treating electric fields (TTFields) delivered in combination with personalized hyperfractionated adaptive radiation therapy (PULSAR), which, when applied in a regimen, can induce synergistic cell killing by disrupting DNA damage response, enhancing DNA replication stress, and DNA replication fork collapse.

[0016] The disclosed TTFields can reduce tumor cells through mitotic collapse. Additionally, the disclosed TTFields can affect DNA damage repair and replication stress pathways in tumor cells. For example, the disclosed TTFields treatment can reduce Fanconi Anemia (FA) pathway signaling proteins by attenuating radiation (IR)-induced DNA damage repair processes. The length of newly replicated DNA can be slowed depending on the TTFields exposure time, and TTFields can increase R-loop formation, indicating that TTFields induced replication stress. The disclosed TTFields can enhance the sensitivity of chemotherapy agents that target and increase replication stress in novel combination therapy options.

[0017] Ablative strategies such as stereotactic ablative radiation therapy (SAbR) are now common in radiation oncology and in an increasing number of treatment settings. However, PULSAR is unrelated to traditional radiation therapy, where numerous "fractions" of radiation are delivered over weeks (SAbR) rather than days, or now over weeks or months in PULSAR, and the interval between radiation fractions is much longer than that in traditional radiation therapy or SAbR, so the radiation therapy is called a pulse. This interval between pulses is well suited to adding TTFields or TTFields plus certain chemotherapy drugs and immune checkpoint inhibitors. Because PULSAR was developed with the intention of integrating the tumor, the tumor microenvironment, and the normal tissue response to therapy with each other, PULSAR is open to integrating further innovations depending on the patient's clinical situation.

[0018] FIG. 1 illustrates an exemplary inventive approach for treating a tumor in a subject, according to an exemplary embodiment of the present disclosure. Each of these inhibitors may target, for example, DNA repair, DNA replication, DNA cross-linking, DNA complexing, or cell cycle control to increase replication stress. For example, ATR inhibitors 104 (drugs that block important DNA repair proteins) may be introduced into tumor cells or tissues to inhibit cell cycle checkpoints upon DNA damage. Radiation (IR) 102 (a process in which an object is exposed to radiation) may be applied to tumor cells or tissues to break DNA strands. Platinum agents 106 (substances that bind DNA nucleotides to each other and block DNA synthesis) may be delivered to tumor cells or tissues to induce intra-strand crosslinks that can inhibit DNA replication. Repair of crosslinks may be downregulated by TTFields. PARP inhibitors 110 (inhibitors that stop PARP from performing cellular repair processes) may be added to tumor cells or tissues for inhibition of replication fork maintenance. Topoisomerase inhibitors 108, a type of enzyme that alters the supercoiling of double-stranded DNA, can be delivered to tumor cells or tissues for the inhibition of DNA replication and chromosome condensation.

[0019] The disclosed combination therapy options using chemotherapeutic agents can be combined with TTFields to synergistically increase replication stress. The disclosed chemotherapeutic agents can target replication stress, which may be a major cause of genomic instability. Cancer cells can continue to proliferate unchecked by being at low to moderate levels of replication stress with impaired DNA damage response (DDR) and loss of cell cycle checkpoints. Normal cells can maintain genomic stability through the coordinated action of DDR and cell cycle checkpoints. Impaired DDR and moderate to low levels of replication stress are unique to cancer cells and can therefore be exploited therapeutically. To exploit replication stress, the disclosed TTFields may be combined with the disclosed chemotherapeutic agents, which can also cause replication stress at several critical steps.

[0020] FIG. 2 illustrates an exemplary method 200 of treating a tumor in a subject. At 206, tumor treating electric fields (TTFields) may be applied to the tumor. In non-limiting embodiments, the TTFields may be applied with predetermined parameters. By way of example, the TTFields may include a frequency within a frequency range of about 50 kHz to about 1000 kHz. By way of example, the frequency of the tumor treating electric field may be about 200 kHz to about 500 kHz. By way of example, the frequency of the tumor treating electric field may be about 100 kHz, about 150 kHz, about 200 kHz, or about 250 kHz. By way of example, the TTFields may include an intensity within an intensity range of about 1 V / cm to about 20 V / cm. By way of example, the TTFields may include an intensity within an intensity range of about 1 V / cm to about 10 V / cm. By way of example, the intensity of the tumor treating electric field may be about 1 V / cm to about 4 V / cm. Other possible exemplary parameters of TTFields may include active time, dimming time, and duty cycle (all of which may be measured in ms, for example), among other parameters. The parameters may be varied based on tumor size, tumor type, target, and goal of treatment. In one example, the strength of the tumor treatment electric field is about 1 V / cm to about 4 V / cm, and the frequency of the tumor treatment electric field is about 150 kHz to about 250 kHz for treating glioblastoma cancer cells.

[0021] The disclosed TTFields treatments can induce DNA damage, attenuate the DNA damage response, and create conditional vulnerability. Application of the disclosed TTFields can increase γ-H2AX foci over time in cells (e.g., not irradiated but exposed to TTFields). Since γ-H2AX is also an early sensor of stalled replication forks during replication stress, TTFields exposure can induce replication stress as a decrease in the expression of BRCA1 and other members of the Fanconi Anemia pathway, negatively impacting the repair of collapsed or stalled replication forks. Additionally, MCM6 and MCM10 genes, integral members of the DNA replication complex, can also be downregulated by application of TTFields.

[0022] In some embodiments, TTFields may be applied to the tumor before or after the disclosed inhibitors and / or PULSAR therapy are administered. In some embodiments, TTFields may be applied to the target tissue simultaneously with the disclosed inhibitors and / or PULSAR therapy. As an example, TTFields may be applied to the tumor prior to PULSAR therapy to create a conditional vulnerability that renders the injured cells 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 a non-limiting embodiment, the disclosed agent / inhibitor 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 some embodiments, a change in the subject's weight may serve as a condition to transition from treatment with TTFields at 202 to initiating a PULSAR regimen at 204. Additionally, in one non-limiting embodiment, TTFields may be applied for a predetermined period of time (e.g., about 7 days) followed by a rest period for a predetermined period of time (e.g., about 3 days). Alternatively, TTFields may be applied continuously throughout the regimen in parallel with the delivery of PULSAR at 204.

[0023] At 204, PULSAR may be delivered to the tumor. In one example, a first test dose of radiation, called a first pulse, may be administered to a tumor in a subject. The tumor may be either benign or malignant. In all embodiments, the dose may be sufficient to cause changes in the tumor, the tumor environment, or in the patient, making it more adaptive and individualized. To this end, the pulse dose may be at least 6-8 Gy, and possibly more. In some embodiments, the dose may be ablative, defined as destroying both proliferation and cellular function, and traditionally used as a monotherapy for both primary cancers and metastases. Such doses range from 15-50 Gy per treatment. Often, the ablative dose range will kill tumors that have spread. For example, the dose may be similar or the same dose delivered in stereotactic ablative radiotherapy (SABR). Doses currently used for the first or only fraction in existing SABR treatments for solid tumors range from 8-50 Gy. For example, the dose may be applied with high accuracy and precision and purposefully limited to large tumors / cancers, which may mitigate the effects of radiation on normal tissue surrounding the tumor.

[0024] In particular, PULSAR is an adaptive regimen intended to be modified based on the efficacy of the treatment and adapted to a particular subject. Thus, a second application of TTFields may be administered to the subject, followed by administration of a second pulse dose (or more) of radiation to the subject. In some embodiments, the time period between the administration of the first and second doses (i.e., rest / observation period) may be at least 6-7 days or more. For example, a first dose of 10 Gy may be administered, followed 10 days later by a second dose of 10 Gy. In some embodiments, the rest / observation period may be 10 days, 20 days, 30 days, or even months long. In some embodiments, the second dose is also ablative and may be a similar or the same dose as the dose applied in stereotactic ablative radiation therapy. The second dose may target a larger or smaller target field and have the same or different efficacy as the first dose. In some embodiments, the second, subsequent pulse dose level may be altered or adapted based on information obtained during one or more rest / observation periods.

[0025] For example, an adaptation affecting the second pulse dose level may be as simple as reducing the dose if the tumor shrinks dramatically after the first pulse. Conversely, if the tumor does not respond or even grows after the first pulse, the second pulse dose may be larger than the first. If the tumor responds after the first or any previous pulse, the next planned pulse may treat the smaller dose as an adaptation. Another example may be that the previous pulse caused a new hypoxic state based on imaging that constitutes the radioresistance of the lesion. In response, an additional dose may be "painted" using dosimetric modulation to these hypoxic regions when planning the next pulse. In response, a hypoxic cell sensitizer may be given in addition to the next radiation pulse.

[0026] Another example may be that circulating tumor cell sampling or re-biopsy or other experimental or imaging opportunities indicate that the targeted tumor may benefit from a particular class of drug, targeted therapy, or immunotherapy. In this situation, subsequent pulses may be delivered with this drug or combination treatment in a manner known to optimize this combination. Another example may be that tumor response indicates that radiation alone will not eliminate a particular tumor in a patient. In this situation, the patient may be referred for surgery or drug therapy without radiation. This process of providing pulses of dose may be repeated until the cancer is eliminated (cured), the treatment causes unacceptable toxicity, or tumor progression occurs despite all adaptive options / opportunities being exhausted.

[0027] 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 length of the rest / observation period, and the target field of the second dose may be determined. For example, treatment may be individualized and adapted for a particular patient based on the patient's response to the first dose of radiation. The patient's response to the first dose may encompass a wide variety of factors, including, but not limited to, symptoms, tests, imaging, tumor response, blood tests, body fluid analysis, biopsy, histology, grade, stage, genomics, sequencing, gene expression, performance, patient tolerance, attitude, social situation, or other personal tests. The rest / observation period between doses is relatively long, not consecutive days, ideally 8 days or more, so that there is enough time for the patient's body and the tumor to adapt. For example, if the tumor is determined to have characteristics indicative of a high risk of rapid growth, the rest period between doses may be shorter than when tumor shrinkage is detected. Furthermore, the extra time between pulses allows immune cascades to run their designed course throughout the body, making subsequent pulses more effective.

[0028] In some embodiments, radiomics and biological features may be analyzed to further adapt treatment. Biological features may include vascularity of the target tissue, vascularity of the normal tissue, oxygen status of the target tissue, oxygen status of the normal tissue, cytokine profile of the target tissue, cytokine profile of the normal tissue, gene expression of the target tissue, gene expression of the normal tissue, receptor expression of the target tissue, receptor expression of the normal tissue, leukocyte infiltration of the target tissue, leukocyte infiltration of the normal tissue, tumor markers, tumor burden, systemic immune status, changes in the subject's health, and changes in the patient's weight.

[0029] In some embodiments, a DNA replication stress inducing agent is delivered to the tumor. The DNA replication stress inducing agent comprises at least one of 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.

[0030] In some embodiments, radiomics features may include features extracted from, or images derived from, anatomical imaging features (e.g., tumor response, tumor infiltration, edge features, density features, shape features, etc.), functional imaging features (e.g., blood flow, dilation, etc.), and metabolic imaging features (e.g., glucose uptake, proliferation, hypoxia, etc.).

[0031] Using several embodiments disclosed herein, TTFields in combination with PULSAR, and a variety of others, have been administered to tumors.

[0032] Experimental Results For example, FIG. 3 illustrates an exemplary method of 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. TTFields exposure alone (7 days of continuous exposure) did not reduce tumor growth rates with statistical significance in two of the three mouse tumor models, but MC38 tumors slowed down more when exposed to TTFields. TTFields treatment reduced mouse body weight by 7-16% after 7 days of treatment, but the mice rapidly regained weight within 2 days after being returned to their normal cages. Radiation delivered as a single dose of 8 Gy was therefore added to the treatment regimen for MC38 tumors at the end of TTFields exposure. Here, there was a significant delay in tumor growth after one round of TTFields followed by a single dose of radiation.

[0033] In the next step, two rounds of TTFields and radiation were added. This is a PULSAR treatment strategy with a significant gap in the number of treatments. Tumor growth delay was much more pronounced after two rounds of treatment compared to one round. Radiation delivered as a single dose of 8 Gy was therefore added to the treatment regimen for MC38 tumors at the end of TTFields exposure. Here, there was a significant delay in tumor growth after one round of TTFields followed by a single dose of radiation.

[0034] In the next step, two rounds of TTFields and radiation were added, a PULSAR treatment strategy with a significant gap in the number of treatments. Tumor growth delay was much more pronounced after two rounds of treatment compared to one round.

[0035] Figures 4A-4D show exemplary effects of TTFields on various subjects. In detail, Figure 4A shows a genomic expression analysis performed using a microarray using a panel of NSCLC cells. Figure 4B shows the downregulation of the Fanconi Anemia (FA) pathway under TTFields exposure. The microarray results showed that, in addition to the already known cell cycle-related impairments, BRCA1 DNA damage response pathway genes were downregulated under TTFields exposure. Furthermore, the downregulation of several FA pathway proteins was confirmed by Western blot. Based on the established role of the FA pathway in DNA damage response, IR-induced DNA damage repair kinetics experiments were performed, which yielded results showing that TTFields exposure attenuated the IR-induced DNA damage response, as shown in Figure 4C. Furthermore, some experiments showed that TTFields treatment alone induced some degree of DNA double-strand breaks, evident by an increase in g-H2AX foci. As explained above, TTFields is hypothesized to increase IR-induced cell death, impeding DNA damage repair kinetics in a novel combination therapy option using TTFields with radiation. Results from several experiments showed that TTFields increased IR-induced cell killing beyond what was added, as shown in Figure 4D. In light of these findings, experiments were performed in which TTFields were applied to subjects prior to radiation, providing results that further demonstrated that TTFields increased radiation sensitivity, as evidenced by increased CI values.

[0036] Figure 5 shows an exemplary effect of a pulse of radioimmunotherapy in a hot tumor microenvironment, according to one or more embodiments. For example, Figure 5 uses MC38 cells to show that timing of a pulse of radioimmunotherapy affects tumor growth in a "hot" tumor microenvironment.

[0037] 6 shows an exemplary effect of a pulse of radioimmunotherapy in a cold tumor microenvironment, according to an exemplary embodiment of the present disclosure. For example, FIG. 6 shows that timing of a pulse of radioimmunotherapy affects tumor growth in a "cold" tumor microenvironment.

[0038] 7 shows an exemplary effect of PULSAR and immunotherapy on cancer according to an exemplary embodiment of the present disclosure. For example, FIG. 7 shows the effect of PULSAR and immunotherapy on mouse rectal cancer.

[0039] FIG. 8 shows exemplary survival results in a tumor microenvironment according to an exemplary embodiment of the present disclosure. For example, FIG. 8 shows survival data across hot and cold microenvironments. Given that TTFields induce conditional vulnerability, experiments were performed using TTFields in a combination therapy. In one experiment, the application of tumor growth delay (TGD) was added in combination with radiation. In this experiment, one round of TTFields and IR treatment cycles were used. The results of this TGD approach suggest significant tumor growth delay between the TTFields and hyperthermia groups in the MC38 mouse model after one round of TTFields plus IR treatment. TTFields and hyperthermia treatments were associated with a tumor volume of 40 mm 3 The treatment was started when the tumor reached an average volume of 1000 mg / kg / day. Seven days after treatment (TTFields and hyperthermia), the mice were exposed to 8 Gy of radiation. After radiation, the mice were transferred to regular cages and tumor volumes and body weights were measured every other day. TTFields treatment slowed tumor growth, but a significant tumor growth inhibition was observed in the mice in the TTFields plus IR group compared to their controls. For this graph, historical controls from other studies were used, in which precise amounts of MC38 cells were injected into C57BL / 6 mice and TGD was monitored.

[0040] 9 shows an exemplary effect of combining TTFields and radiation in a single round, according to an exemplary embodiment of the present disclosure. For example, to further the experiments described with respect to FIG. 8, FIG. 9 shows the results of an experiment using a combination of TTFields and radiation therapy for one cycle. For example, TTFields and hyperthermia treatment reduced tumor volume by an average volume of approximately 40 mm. 3 The study began when tumor growth reached 100%. Seven days after treatment (TTFields and hyperthermia), the mice were exposed to 8 Gy of radiation and the mice were allowed to gain weight in regular cages for three days. Three days later, a second round of 7 days of treatment (TTFields and hyperthermia) was given to the mice and then exposed to 8 Gy of radiation. Immediately after the second round of radiation, the mice were transferred to regular cages and tumor volumes and body weights were measured every other day. Although one round of TTFields and IR treatment slowed tumor growth, a more significant tumor growth inhibition was observed in mice in the group that received two rounds of TTFields plus IR treatment compared to their controls.

[0041] FIG. 10 shows an exemplary effect of combining TTFields and radiation in two rounds, according to an exemplary embodiment of the present disclosure. To further the experiment described with respect to FIG. 9, FIG. 10 shows a case where two cycles of combined TTFields and radiation therapy were added. TTFields or hyperthermia treatment reduced tumor volume to an average volume of approximately 40 mm. 3The tumor growth rate was started when the tumor reached 100%. Seven days after the treatment (TTFields and hyperthermia), the mice were exposed to 8 Gy of radiation and the mice were allowed to gain weight in a normal cage for three days. Three days later, the mice were given a second round of treatment (TTFields and hyperthermia) for seven days and then exposed to 8 Gy of radiation. Immediately after the second round of radiation, the mice were transferred to normal cages and tumor volume and body weight were measured every other day. Although one round of TTFields and IR treatment slowed tumor growth, a more significant tumor growth inhibition was observed in mice in the group that received two rounds of TTFields plus IR treatment compared to their controls. Notably, two rounds of TTFields and IR treatment were administered, although subjects may receive several rounds based on one or more factors, including but not limited to, the subject's response to the previous round of treatment.

[0042] In a further embodiment, a third round of TTFields further impacts tumor growth delay. In particular, normal tissue reaction is a determining factor in the clinical use of radiation, with all tissues found in the radiation field having a tolerable dose that limits the individual dose of radiation as well as the cumulative dose. Thus, one objective is to minimize the number of radiation doses received by an animal (e.g., mouse, patient, etc.) as a means of limiting the amount of normal tissue irradiated. Thus, in this case, the anti-PD-L1 antibody atezolizumab is added to the radiation / TTFields regimen, considering the possibility of enhanced immunogenic responses to the combination of radiation and immune checkpoint inhibitors, or TTFields combined with immune checkpoint inhibitors.

[0043] FIG. 11 shows an exemplary effect of combining TTFields and radiation in two rounds, according to an exemplary embodiment of the present disclosure. To further the experiments described with respect to FIGS. 9-10, FIG. 11 shows a similar experiment using an LLC model in which mice were exposed to two cycles of combined TTFields and radiation therapy. TTFields and heat treatment reduced tumor volume to an average volume of approximately 40 mm. 3The treatment was started when the tumor reached a tumor size of 1000 mg / kg / day. Seven days after the treatment (TTFields and hyperthermia), the mice were exposed to 12 Gy of radiation, and the mice were allowed to gain weight in a normal cage for three days. Four days later, the mice were given a second round of 7 days of treatment (TTFields and hyperthermia) and then exposed to 8 Gy of radiation. Immediately after the second round of radiation, the mice were transferred to normal cages and tumor volumes and body weights were measured every other day. Tumor ulceration and invasive morphology were found to be more prevalent in this model. Thus, the results of the experiment suggest that the trend of decreased TGD in mice in the group that received TTFields plus IR treatment was not as marked as in the MC38 model, compared to the control group. Notably, two rounds of TTFields and IR treatment were administered, although subjects may receive several rounds based on one or more factors, including but not limited to, the subject's response to the previous round of treatment. In addition, various chemotherapy agents may be delivered between rounds of TTFields and IR treatment. For example, after a second round of TTFields and IR treatment, the subject may be delivered a first chemotherapy agent. The subject may then be evaluated to determine the effectiveness of the first treatment of TTFields, IR, and chemotherapy agent. In one instance, if the subject (e.g., the subject's tumor) is found to be unresponsive (i.e., resistant) to the chemotherapy agent, the subject may be administered a second chemotherapy agent in a third round of TTFields and IR treatment (or any number of subsequent rounds of treatment). Alternatively, if the first chemotherapy agent is determined to be effective in treating the subject, the subject may again be administered the first chemotherapy agent in a subsequent round of treatment. However, the first chemotherapy agent may be delivered at a different dosage based on the determined effectiveness, or in conjunction with one or more other chemotherapy agents.

[0044] 12 illustrates an exemplary effect of TTFields and radiation on tumor volume, according to an exemplary embodiment of the present disclosure. For example, FIG. 12 shows data predicting the outcome of a therapeutic combination of TTFields, PULSAR, and one or more additional tumor impairing therapies.

[0045] FIG. 13 shows tumor growth delay curves according to one or more examples. As shown, an exemplary effect is shown in which mice were exposed to 8 Gy of X-rays after 7 days of treatment (TTFields) and allowed to gain weight for 3 days. The mice were then treated with TTFields for an additional 7 days, followed by an additional 8 Gy of X-rays. At the indicated time points, PD-L1 antibodies were subcutaneously injected into certain cohorts of mice and not the other mice. When TTFields was combined with radiation and PD-L1, substantial tumor growth inhibition was observed compared to groups treated with either TTFields plus PD-L1 antibodies, or TTFields plus radiation, or radiation plus PD-L1 antibodies. Furthermore, tumor cure appears to be observed in three out of six mice in the triple combination group of TTFields, radiation, and PD-L1 antibodies (as further shown in FIG. 15). The individual growth delays of each mouse in the triple combination group of TTFields, radiation, and immune checkpoint inhibitors will now be discussed individually. In 3 of 6 mice, tumors were not readily measurable. This treatment regimen represents an exemplary cohort in which tumor cure was observed.

[0046] Figure 14 shows tumor growth delay curves, according to one or more examples. To drive a treatment, such as that described with respect to Figure 13, the tumor size of each patient (e.g., mouse) was analyzed over a period of time following treatment. As shown, the tumor volume of some patients who received the combination of TTFields, radiation, and PD-L1 antibody was significantly reduced.

[0047] In FIG. 15, multiple tumor growth delay curves from individual treatments are shown. As shown, individual growth delays for patients (e.g., mice) receiving the triple combination of TTFields, radiation, and immune checkpoint inhibitors are shown. Statistical significance: The Bliss test of significance of synergy in combination therapy (described in Demidenko, E., and 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 and incorporated herein by reference) was used to determine whether there were combinations of TTFields and radiation that were synergistic (i.e., the effect was greater than the effect of the individual independent effects when combined). In this approach, the tumor growth delay curves from FIG. 13 and FIG. 14 were replotted in FIG. 15 as log-linear functions and a linear regression of the data was applied. The slopes of those regressions are treated as independent effects and then analyzed to detect synergistic effects of the combination of independent effects. The solid line represents the linear regression, whose slope represents the tumor growth as a percentage per week. Each independent treatment is plotted as if it were a control group. The combination therapies are then plotted. The combination therapies that are synergistic include the combination of two rounds of TTFields and radiation, the combination of anti-PD-L1 and radiation, and the triple combination of TTFields, radiation, and anti-PD-L1 antibody. The p-values ​​representing the statistical significance for these three combinations are shown in Table 1 of FIG. 15 and are highly significant. As shown, TTFields added to radiation produces a combination effect that is synergistic. When TTFields are added to the standard of care model, i.e., radiation plus checkpoint inhibitor anti-PD-L1 antibody, the combination is also synergistic, resulting in a strong synergistic effect and, most importantly, tumor cure after only two rounds of radiation including immune checkpoint inhibitors.

[0048] Exemplary Apparatus FIG. 16 illustrates an example of a device for applying TTFields with modulated electric fields to a subject's body. A first transducer 1601 includes 16 electrode elements 1603, the electrode elements 1603 located on a substrate 1604, the electrode elements 1603 electrically and mechanically connected to each other through conductive traces 1609. A second transducer 1602 includes 20 electrode elements 1605, the electrode elements 1605 located on a substrate 1606, the electrode elements 1605 electrically and mechanically connected to each other through conductive traces 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 with the first transducer 1601 and the second transducer 1602 to form a first electric field generator and a second electric field generator.

[0049] 17 illustrates an exemplary computing device for use with embodiments herein. As an example, device 1700 may be a computer implementing some of the inventive techniques disclosed herein. As an example, device 1700 may be a controller device that applies TTFields using modulated electric fields in embodiments herein. Device 1700 may be used as controller 1608 of FIG. 13. Device 1700 may include one or more processors 1702, one or more output devices 1705, and memory 1703.

[0050] In one example, based on the input 1701, one or more processors generate control signals to control a voltage generator to implement an embodiment of the present invention. In one example, the input 1701 is a user input. In another example, the input 1701 may be from another computer in communication with the device 1700. The output device 1705 may provide a status of the operation of the present invention, such as transducer selection, voltage generated, and other operating information. The output device 1705 may provide visualization data according to some embodiments of the present invention.

[0051] Memory 1703 is accessible by one or more processors 1702 via link 1704 such that the one or more processors 1702 can read information from and write information to memory 1703. Memory 1703 may store instructions that, when executed by the one or more processors 1702, implement one or more embodiments of the present invention.

[0052] Numerous modifications, variations, and variations to the described embodiments are possible without departing from the scope of the invention as defined in the claims. The invention is not limited to the described embodiments, but is intended to have full scope defined by the language of the appended claims and their equivalents. [Explanation of symbols]

[0053] 102 Radiation (IR) 104 ATR Inhibitors 106 Platinum Preparations 108 Topoisomerase Inhibitors 110 PARP Inhibitors 1601 First Transducer 1602 Second Transducer 1603 Electrode Element 1604 Board 1605 Electrode Element 1606 Board 1607 AC Voltage Generator 1608 Controller 1609 Conductive wiring 1610 Conductive wiring

Claims

1. 1. A method of 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; delivering individualized hyperfractionated adaptive radiation therapy to the subject; A method comprising:

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

3. 4. The method of claim 3, wherein the step of delivering the individualized hyperfractionated adaptive radiation therapy comprises: administering a first pulse dose of radiation to the tumor in the subject; administering a second pulse dose of radiation to the tumor, said second pulse dose being administered after an observation period, said observation period having a length of at least seven days; and concurrently treating said subject with immunotherapy. The method of claim 1, further comprising:

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

5. 5. The method of claim 4, further comprising determining at least one of a radiation level of the second pulse dose, the length of the observation period, and a target field of the second pulse dose in response to administration of the first pulse dose.

6. 6. The method of claim 5, wherein the first pulse dose of radiation is 8 Gy.

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

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

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

10. 13. 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 features and biological features.

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

13. The method of claim 11 , wherein the radiomics features include at least one of anatomical imaging features, functional imaging features, and metabolic imaging features.

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

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

16. The method of claim 15, wherein the DNA replication stress inducer comprises at least one of 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 DNA replication stress inducer is delivered to the tumor comprising a platinum compound, the platinum compound further comprising at least one of cisplatin, carboplatin, oxaliplatin, dicycloplatin, or lipoplatin; when the DNA replication stress-inducing agent delivered to the tumor is an alkylating agent, the alkylating agent comprises at least one of cyclophosphamide or temozolomide; When the DNA replication stress inducer delivered to the tumor is the wee1 inhibitor, the wee1 inhibitor comprises at least one of Adavosertib-MK1775 or PD0166285; when the DNA replication stress inducer delivered to the tumor is the Chk1 inhibitor, the Chk1 inhibitor comprises at least one of UCN-01, LY2606368, SAR-020106, AZD7762, or PD0166285; when the DNA replication stress-inducing agent delivered to the tumor is the thymidylate synthase inhibitor, the thymidylate synthase inhibitor comprises at least one of 5-FU or pemetrexed; when the DNA replication stress-inducing agent delivered to the tumor is the ribonucleotide reductase inhibitor, the ribonucleotide reductase inhibitor comprises gemcitabine; when the DNA replication stress-inducing agent delivered to the tumor is the topoisomerase I inhibitor, the topoisomerase I inhibitor comprises at least one of irinotecan or topotecan; when the DNA replication stress-inducing agent delivered to the tumor is the topoisomerase II inhibitor, the topoisomerase II inhibitor comprises at least one of etoposide or doxorubicin; when the DNA replication stress-inducing agent delivered to the tumor is the MELK inhibitor, the MELK inhibitor comprises OTS167; 17. The method of claim 16, wherein when the DNA replication stress-inducing agent delivered to the tumor is an NAE inhibitor, the NAE inhibitor comprises MLN4924.

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

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

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

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