WO2025081190A1 - Image guided car t cell therapy to treat brain tumors - Google Patents

Abstract

Disclosed herein are thermal switch (TS)-engineered T cells and methods of their use in the treatment of cancers in the brain.

Description

IMAGE GUIDED CAR T CELL THERAPY TO TREAT BRAIN TUMORS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/589,791 filed October 12, 2023, which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. GR00022545 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cell therapy has made significant strides in the treatment of hematological malignancies, and emerging data from brain cancer trials suggest its potential in combating aggressive tumors like glioblastoma. Despite encouraging responses CAR T trials involving brain cancer patients, the unique challenges presented by brain tumors continue to limit the widespread success of this approach. A critical issue in glioblastoma is the heterogeneous expression of tumor-associated antigens (TAAs), which prevents uniform targeting by CAR T cells and allows antigen-negative tumor cells to escape immune surveillance. This antigenic diversity within the brain tumor microenvironment results in incomplete tumor eradication, leaving behind antigen-negative cells that drive relapse and disease progressions.

[0004] Moreover, the brain tumor microenvironment (TME) presents immunosuppressive barriers that hinder CAR T cell efficacy. Immune-modulating cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs) are prevalent in the glioblastoma TME, secreting inhibitory cytokines and expressing immune checkpoint molecules that impair CAR T cell activity. In glioblastoma, high infiltration of MDSCs is a hallmark of poor prognosis and resistance to immunotherapies, further complicating the use of CAR T cells in the brain. These immunosuppressive forces, combined with the blood-brain barrier and the immune-privileged status of the brain, create a particularly hostile environment for CAR T cells, making glioblastoma a challenging target for immunotherapy. Thus, there is a need for improved CAR T cell applications in the brain. This need and others are at least partially satisfied by the present disclosure. SUMMARY

[0005] Disclosed herein are thermal switch (TS)-engineered T cells comprising a promoter construct comprising one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements comprising 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs). In some aspects, thermal activation of the promoter construct to express the BTE occurs from about 40°C to about 45°C. In some aspects, the heat shock element is repeated 2, 3, 4, 5, 6, 7, or more times.

[0006] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis (such as for example, brain tumors including, but not limited to glioblastoma, astrocytic tumor, oligodendroglia tumor, ependymoma, craniopharyngioma, medulloblastoma, ganglioglioma, pituitary tumor, primary lymphoma of the brain, pineal gland tumor, primary germ cell tumor of the brain and metastasis in the brain such as breast cancer brain metastasis) in a subject with a brain tumor, the method comprising: a) intracranially providing a plurality of thermal switch (TS)-engineered T cells of any preceding aspect; and b) heating the brain tumor (including, but not limited heating the brain to 40°C to about 45°C), thereby inducing hyperthermia in the brain tumor; wherein the hyperthermia causes the TS-engineered T cells to produce bispecific T cell engagers (BTEs). For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis (such as for example, brain tumors including, but not limited to glioblastoma, astrocytic tumor, oligodendroglial tumor, ependymoma, craniopharyngioma, medulloblastoma, ganglioglioma, pituitary tumor, primary lymphoma of the brain, pineal gland tumor, primary germ cell tumor of the brain and metastasis in the brain such as breast cancer brain metastasis) in a subject with a brain tumor, the method comprising: a) intracranially providing a plurality of thermal switch (TS)-engineered T cells (including, but not limited to TS T cells comprising a promoter construct comprising one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements comprising 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs)) to the brain tumor; and b) heating the brain tumor (including, but not limited heating the brain to 40°C to about 45°C), thereby inducing hyperthermia in the brain tumor; wherein the hyperthermia causes the TS-engineered T cells to produce bispecific T cell engagers (BTEs). In some instances, the BTE targets both the brain tumor and the TS-engineered T cells. In some aspects, the TS-engineered T cells are administered intratum orally. In one aspect, it is understood and herein contemplated that the TS-engineered T cells are retained in the tumor microenvironment (TME), as opposed to migrating out of the TME and brain.

[0007] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein the promoter construct is activated by the heating in step b).

[0008] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein the core promoter comprises a heat shock protein transcription start site (such as, for example, the heat shock protein transcription start site of HSPA1A, HSPH 1. HSPA6. or YB). In some aspects, the core promoter comprises 80% similarity or more to any one of SEQ ID NOs: 10-13. In some aspects the one or more heat shock elements and core promoter together comprise 80% similarity or more to any one of SEQ ID NOs: 14-21.

[0009] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein the heating in step b) occurs via ultrasound (such as, for example, focused ultrasound including, but not limited to MR-guided focused ultrasound (MRgFUS)). In some aspects, the ultrasound is administered in a cycle comprising from about 5 minutes to about 15 minutes (including, but not limited to 5 to 10 minutes) such as, for example, administering ultrasound in a cycle for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes followed by from about 1 minute to about 10 minutes of rest (such, as for example, 1, 2, 3, 4,

5, 6, 7, 8, 9, or 10 minutes rest). In some aspects, the ultrasound is administered in 1, 2, 3, 4, 5,

6, or more cycles. In some aspects the ultrasound has a frequency of from about 1 MHz to about 3 MHz.

[0010] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein step b) uses a focused ultrasound system comprising a focused ultrasound transducer; a device for measuring temperature (such as, for example, a magnetic resonance (MR) thermometer); and a proportional-integral-derivative (PID) controller configured to modulate power output of the focused ultrasound transducer based on temperature data received from the device for measuring temperature. In some aspects, the PID controller is a closed-loop controller. In some aspects, the PID controller yields a temperature deviation of less than about 0.5°C from a target temperature. [0011] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein the method does not induce neuronal degeneration.

[0012] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, wherein, by from about 7 days to about 30 days after step b), expression of Iba-1 and/or glial fibrillary acidic protein (GFAP) in the subject has changed by less than about 5% and/or the cytotoxicity of the TS-engineered T cells is reduced by less than about 5%.

[0013] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis of any preceding aspect, further comprising administering to the subject an anti-cancer agent or immunotherapy ((including, but not limited to immune checkpoint inhibitors such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example chimeric antigen receptor (CAR) T cells, CAR Natural Killer (NK) (CAR NK cells), CAR Macrophage (CARMAC) CAR NK T cells, Tumor Infiltrating Lymphocytes (TILs), and/or Marrow Infiltrating Lymphocytes (MILs)).

[0014] In one aspect, disclosed herein is a system comprising a plurality of thermal switch (TS)-engineered T cells of any preceding aspect; a heat source; a device for measuring temperature; and a proportional-integral-derivative (PID) controller configured to modulate power output of the heat source based on temperature data received from the device for measuring temperature. For example, disclosed herein is a system comprising a plurality of thermal switch (TS)-engineered T cells (including, but not limited to TS T cells comprising a promoter construct comprising one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements comprising 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs)); a heat source; a device for measuring temperature (such as, for example, magnetic resonance (MR) thermometer); and a proportional-integral-derivative (PID) controller configured to modulate power output of the heat source based on temperature data received from the device for measuring temperature. In some instances, the BTE targets both the brain tumor and the TS-engineered T cells. In some aspects, thermal activation of the promoter construct to express the BTE occurs from about 40°C to about 45°C. In some aspects, the heat shock element is repeated 2, 3, 4, 5, 6, 7, or more times.

[0015] Also disclosed herein are systems of any preceding aspect, wherein the core promoter comprises a heat shock protein transcription start site (such as, for example, the heat shock protein transcription start site of HSPA1A, HSPHL HSPA6._j or YB). In some aspects, the core promoter comprises 80% similarity or more to any one of SEQ ID NOs: 10-13. In some aspects the one or more heat shock elements and core promoter together comprise 80% similarity or more to any one of SEQ ID NOs: 14-21.

[0016] In one aspect, disclosed herein are systems of any preceding aspect, wherein the device for measuring temperature can measure temperature in vivo and/or can measure temperature intracranially.

[0017] Also disclosed herein are systems of any preceding aspect, wherein the PID controller is a closed-loop controller.

[0018] Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF DRAWINGS

[0019] FIGURES 1A-1G depict that FUS-mediated hyperthermia redirects CAR T cells towards antigen-negative tumor cells. FIG. 1A is a schematic representation of TS.BTE aHER2 CAR construct redirecting T cell activity towards HER2- tumor cells by binding NKG2DL FIG. IB is a schematic representing in vitro FUS setup to evaluate effects of US sonication on transgene production of CAR T cells. FIGS. 1C-1D show representative flow staining of BTE expression (FIG. 1C) and quantification (FIG. ID) following 20-minute heating at either 41 °C or 42°C with continuous or pulsed heating strategy. FIG. IE shows quantification of cytotoxicity against HER2- MDA-MB-468 following coculture of WT or engineered T cells following heating. FIG. IF shows viability (left) and luminescence (right) of TS.Fluc aHER2 CAR T cells following FUS mediated hyperthermia at indicated parameters quantified by flow cytometry and IVIS, respectively. FIG. 1G shows BTE expression (left) and cytotoxicity (right) against HER2-MDA-MB-468 tumor cells following FUS mediated hyperthermia for 10 minutes at indicated temperatures. Mean ± s.e.m. depicted, n= 3-4 independent wells. [0020] FIGURES 2A-2C show that FUS mediated hyperthermia at 41 °C does not compromise key T cell functions. FIG. 2A shows that TS.Fluc aHER2 CAR T cells were heated at indicated temperatures for 20 minutes by FUS and then cocultured on HER2+ MDA- MB-468. FIGS. 2B-2C show that cytotoxicity (FIG. 2B) and fFNy production (FIG. 2C) were quantified 24 hours following coculture. One-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01, ns = nonsignificant, mean ± s.e.m. depicted, n= 3-4 independent wells, T= tumor only condition.

[0021] FIGURES 3A-3E depict that PID-based closed-loop MRgFUS can effectively induce transcranial hyperthermia and control the temperature. FIG. 3A is a schematic of MRgFUS system describing two different closed-loop controllers. FIG. 3B shows representative images of T2-weighted MRI and fused MRTI. FIG. 3C shows representative images of MRTI from bi-state and PID controllers. FIG. 3D shows temperature profiles from bi-state and PID controllers (n=4). FIG. 3E shows histogram variance of temperature measurements from bi-state and PID controllers.

[0022] FIGURES 4A-4E depict assessment of safety of PID closed-loop MRgFUS transcranial hyperthermia in healthy mice. FIG. 4A is a schematic showing the brain region safety was used for assessing safety. FIG. 4B shows T2-weighted MRI showing edema that dissolves after one week. FIGS. 4C-4D show quantification of microglia and astrocyte activation, demonstrating that they return to baseline 1 - 2 weeks after sonication. FIG. 4E shows representative images of GFAP, IBA-1, Fluoro-Jade and H&E staining at different time points after sonications. For all data n=4.

[0023] FIGURES 5A-5E depict that MRgFUS locally activates thermal sensitive CAR T cells in models of BCBM. FIG. 5A is a schematic representation of MRgFUS setup with braintumor bearing mice and representative in vivo heating strategy. FIG. 5B shows a representative image and quantification of luciferase signal from intratumoral TS.Fluc aHER2 CAR T cells 10 hours post MRgFUS-mediated heating. FIG. 5C shows transient and repeatable activation of thermal sensitive CAR T cells following MRgFUS mediated heating for 10 minutes at 41.5°C (red triangle) as measured by IVIS expression of luciferase. FIGS. 5D-5 show representative images (FIG. 5D) and quantification (FIG. 5E) of liver, spleen, and brain 10 hours post heating, organs were isolated and luciferase signal quantified. Student’s two-tailed T test, *p < 0.05, **p < 0.01, ****p < 0.0001, ns = nonsignificant, mean ± s.e.m. depicted.

[0024] FIGURES 6A-6D depict that MRgFUS mediated production of BTEs by CAR T cells mitigates antigen escape in heterogenous BCBM. FIG. 6A shows that NSG mice were inoculated with heterogenous tumors including a 75% HER2+ tumor mixture of MDA-MB- 468 tumor cells. Schematic representation of BTE production following 2 MRgFUS-mediated thermal treatments to redirect TS.BTE aHER2 CAR T cells towards HER2- tumors. FIG. 6B shows representative MR images and quantification following treatment strategy depicted in FIG. 6A. FIG. 6C shows representative IVIS images and quantification of HER2- Tumor burden following treatment strategy depicted in FIG. 6A. FIG. 6D shows survival curves of tumor-bearing mice following treatment, log-rank (Mantel-Cox) test with P-values depicted. [0025] FIGURE 7 depicts the experimental setup to evaluate effect of mBTE on T cell ability to overcome MDSC mediated suppression

[0026] FIGURES 8A-8I depict that thermal mediated BTE production overcomes immune suppression in glioblastoma. FIG. 8A shows a representative flow plot of T cell activation and proliferation following coincubation with MDSCs at a 0.5: 1 E:T ratio in either WT or mBTE conditioned media. FIG. 8B shows quantification of T cell activation by CD25 staining at varied MDSC:T cell ratios in mBTE conditioned media. FIG. 8C shows expansion index quantified by CTV staining of T cells following coincubation with MDSCs at various ratios. Two-way ANOVA with multiple comparisons, *p < 0.05, ****p < 0.0001, ns = nonsignificant, mean ± s.e.m. depicted. FIG. 8D shows a representative timeline for in vivo depletion of MDSCs by MRgFUS-mediated BTE production by TS.mBTE transduced T cells. FIGS. 8E- 8F show quantification of percent CAR+ cells (FIG. 8E) and percent and count of M-MDSCs cells (FIG. 8F) following MRgFUS treatment of tumor bearing mice. One-way ANOVA with multiple comparisons, **p < 0.01, ***p < 0.001, ns = nonsignificant, mean ± s.e.m. depicted. FIGS. 8G-8I show representative images of tumor burden (FIG. 8G), spider plots (FIG. 8H), and quantification (FIG. 81) of SB28 tumor burden following treatment. Two-way ANOVA with multiple comparisons, **p < 0.01, ***p < 0.001, ns = nonsignificant, mean ± s.e.m. depicted.

DETAILED DESCRIPTION

[0027] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS

[0028] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0029] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0030] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0031] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0032] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0033] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0034] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0035] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0036] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0037] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0038] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0039] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0040] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0041] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0042] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0043] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0044] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0045] Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0046] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. [0047] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. [0048] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0049] As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0050] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0051] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and/or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).

[0052] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability. In this case, the functional fragment would retain the ability to perform as a telomerase.

[0053] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0054] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0055] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine). [0056] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0057] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and/or the C-terminal region of a polypeptide or the 5 '-terminal region and/or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0058] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0059] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0060] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0061] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0062] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0063] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0064] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0065] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. [0066] Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0067] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

TS-ENGINEERED T CELLS AND METHODS OF USING THE SAME

[0068] Disclosed herein are thermal switch (TS)-engineered T cells including a promoter construct including one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements including 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs).

[0069] In some aspects, each of the one or more heat shock elements can include SEQ ID NO: 1 or a variant thereof. In some aspects, each of the one or more heat shock elements can consist of SEQ ID NO: 1 or a variant thereof. In some aspects, each of the one or more heat shock elements can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to any one of SEQ ID NOs: 2-9. In some aspects, each of the one or more heat shock elements can include any one of SEQ ID NOs: 2-9. In some aspects, each of the one or more heat shock elements can consist of any one of SEQ ID NOs: 2-9. In some aspects, each heat shock element can be repeated 2, 3, 4, 5, 6, 7, or more times.

[0070] In some aspects, thermal activation of the promoter construct to express the BTE can occur at a temperature of at least about 40°C (e.g., at least about 40.1°C, at least about 40.2°C, at least about 40.3°C, at least about 40.4°C, at least about 40.5°C, at least about 40.6°C, at least about 40.7°C, at least about 40.8°C, at least about 40.9°C, at least about 41°C, at least about 41.1 °C, at least about 41.2°C, at least about 41.3 °C, at least about 41.4°C, at least about 41.5°C, at least about 41.6°C, at least about 41 ,7°C, at least about 41.8°C, at least about 41.9°C, at least about 42°C, at least about 42.1°C, at least about 42.2°C, at least about 42.3°C, at least about 42.4°C, at least about 42.5°C, at least about 42.6°C, at least about 42.7°C, at least about 42.8°C, at least about 42.9°C, at least about 43°C, at least about 43.1°C, at least about 43.2°C, at least about 43.3°C, at least about 43.4°C, at least about 43.5°C, at least about 43.6°C, at least about 43.7°C, at least about 43.8°C, at least about 43.9°C, at least about 44°C, at least about 44.1°C, at least about 44.2°C, at least about 44.3°C, at least about 44.4°C, at least about 44.5°C, at least about 44.6°C, at least about 44.7°C, at least about 44.8°C, at least about 44.9°C, at least about 45°C). In some aspects, thermal activation of the promoter construct to express the BTE can occur at a temperature of up to about 45°C (e.g., up to about 44.9°C, up to about 44.8°C, up to about 44.7°C, up to about 44.6°C, up to about 44.5°C, up to about 44.4°C, up to about 44.3°C, up to about 44.2°C, up to about 44.1°C, up to about 44°C, up to about 43.9°C, up to about 43.8°C, up to about 43.7°C, up to about 43.6°C, up to about 43.5°C, up to about 43.4°C, up to about 43.3°C, up to about 43.2°C, up to about 43.1°C, up to about 43°C, up to about 42.9°C, up to about 42.8°C, up to about 42.7°C, up to about 42.6°C, up to about 42.5°C, up to about 42.4°C, up to about 42.3°C, up to about 42.2°C, up to about 42.1°C, up to about 42°C, up to about 41.9°C, up to about 41.8°C, up to about 41.7°C, up to about 41.6°C, up to about 41.5°C, up to about 41.4°C, up to about 41.3°C, up to about 41.2°C, up to about 41.1°C, up to about 41°C, up to about 40.9°C, up to about 40.8°C, up to about 40.7°C, up to about 40.6°C, up to about 40.5°C, up to about 40.4°C, up to about 40.3°C, up to about 40.2°C, up to about 40.1 °C, up to about 40°C).

[0071] It is considered that thermal activation of the promoter construct to express the BTE can occur at a temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, thermal activation of the promoter construct to express the BTE can occur at a temperature of from about 40°C to about 45°C (e.g., from about 40.1°C to about 44.9°C, from about 40.2°C to about 44.8°C, from about 40.3°C to about 44.7°C, from about 40.4°C to about 44.6°C, from about 40.5°C to about 44.5°C, from about 40.6°C to about 44.4°C, from about 40.7°C to about 44.3°C, from about 40.8°C to about 44.2°C, from about 40.9°C to about 44.1°C, from about 41°C to about 44°C, from about 41.1°C to about 43.9°C, from about 41.2°C to about 43.8°C, from about 41.3°C to about 43.7°C, from about 41.4°C to about 43.6°C, from about 41.5°C to about 43.5°C, from about 41.6°C to about 43.4°C, from about 41.7°C to about 43.3°C, from about 41.8°C to about 43.2°C, from about 41 ,9°C to about 43.1°C, from about 42°C to about 43 °C, from about 42.1°C to about 42.9°C, from about 42.2°C to about 42.8°C, from about 42.3°C to about 42.7°C, from about 42.4°C to about 42.6°C, from about 40°C to about 42.5°C, from about 40.1°C to about 42.4°C, from about 40.2°C to about 42.3°C, from about 40.3°C to about 42.2°C, from about 40.4°C to about 42.1°C, from about 40.5°C to about 42°C, from, about 40.6°C to about 41.9°C, from about 40.7°C to about 41.8°C, from about 40.8°C to about 41.7°C, from about 40.9°C to about 41.6°C, from about 41°C to about 41.5°C, from about 41.1°C to about 41.4°C, from about 41.2°C to about 41.3°C, from about 42.5°C to about 45°C, from about 42.6°C to about 44.9°C, from about 42.7°C to about 44.8°C, from about 42.8°C to about 44.7°C, from about 42.9°C to about 44.6°C, from about 43°C to about 44.5°C, from about 43.1°C to about 44.4°C, from about 43.2°C to about 44.3°C, from about 43.3°C to about 44.2°C, from about 43.4°C to about 44.1°C, from about 43.5°C to about 44°C, from about 43.6°C to about 43.9°C, from about 43.7°C to about 43.8°C).

[0072] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis (such as for example, brain tumors including, but not limited to glioblastoma, astrocytic tumor, oligodendroglia tumor, ependymoma, craniopharyngioma, medulloblastoma, ganglioglioma, pituitary tumor, primary lymphoma of the brain, pineal gland tumor, primary germ cell tumor of the brain and metastasis in the brain such as breast cancer brain metastasis) in a subject with a brain tumor, the method including: a) intracranially providing a plurality of thermal switch (TS)-engineered T cells; and b) heating the brain tumor (including, but not limited heating the brain to 40°C to about 45°C), thereby inducing hyperthermia in the brain tumor; wherein the hyperthermia causes the TS-engineered T cells to produce bispecific T cell engagers (BTEs). For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis (such as for example, brain tumors including, but not limited to glioblastoma, astrocytic tumor, oligodendroglial tumor, ependymoma, craniopharyngioma, medulloblastoma, ganglioglioma, pituitary tumor, primary lymphoma of the brain, pineal gland tumor, primary germ cell tumor of the brain and metastasis in the brain such as breast cancer brain metastasis) in a subject with a brain tumor, the method including: a) intracranially providing a plurality of thermal switch (TS)-engineered T cells (including, but not limited to TS T cells including a promoter construct including one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements including 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof as described above), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs)) to the brain tumor; and b) heating the brain tumor (including, but not limited heating the brain to 40°C to about 45°C), thereby inducing hyperthermia in the brain tumor; wherein the hyperthermia causes the TS-engineered T cells to produce bispecific T cell engagers (BTEs). In some instances, the BTE can target both the brain tumor and the TS-engineered T cells. In some aspects, the TS-engineered T cells can be administered intratum orally.

[0073] In some aspects, heating the brain tumor in step b) can include heating to a temperature of at least about 40°C (e.g., at least about 40.1°C, at least about 40.2°C, at least about 40.3°C, at least about 40.4°C, at least about 40.5°C, at least about 40.6°C, at least about 40.7°C, at least about 40.8°C, at least about 40.9°C, at least about 41°C, at least about 41.1°C, at least about 41.2°C, at least about 41.3 °C, at least about 41.4°C, at least about 41.5°C, at least about 41.6°C, at least about 41.7°C, at least about 41.8°C, at least about 41.9°C, at least about 42°C, at least about 42.1°C, at least about 42.2°C, at least about 42.3°C, at least about 42.4°C, at least about 42.5°C, at least about 42.6°C, at least about 42.7°C, at least about 42.8°C, at least about 42.9°C, at least about 43°C, at least about 43.1°C, at least about 43.2°C, at least about 43.3°C, at least about 43.4°C, at least about 43.5°C, at least about 43.6°C, at least about 43.7°C, at least about 43.8°C, at least about 43.9°C, at least about 44°C, at least about 44.1°C, at least about 44.2°C, at least about 44.3°C, at least about 44.4°C, at least about 44.5°C, at least about 44.6°C, at least about 44.7°C, at least about 44.8°C, at least about 44.9°C, at least about 45°C). In some aspects, heating the brain tumor in step b) can include heating to a temperature of up to about 45°C (e.g., up to about 44.9°C, up to about 44.8°C, up to about 44.7°C, up to about 44.6°C, up to about 44.5°C, up to about 44.4°C, up to about 44.3°C, up to about 44.2°C, up to about 44.1°C, up to about 44°C, up to about 43.9°C, up to about 43.8°C, up to about 43.7°C, up to about 43.6°C, up to about 43.5°C, up to about 43.4°C, up to about 43.3°C, up to about 43.2°C, up to about 43.1°C, up to about 43°C, up to about 42.9°C, up to about 42.8°C, up to about 42.7°C, up to about 42.6°C, up to about 42.5°C, up to about 42.4°C, up to about 42.3°C, up to about 42.2°C, up to about 42.1°C, up to about 42°C, up to about 41.9°C, up to about 41.8°C, up to about 41.7°C, up to about 41.6°C, up to about 41.5°C, up to about 41.4°C, up to about 41.3°C, up to about 41.2°C, up to about 41.1°C, up to about 41°C, up to about 40.9°C, up to about 40.8°C, up to about 40.7°C, up to about 40.6°C, up to about 40.5°C, up to about 40.4°C, up to about 40.3°C, up to about 40.2°C, up to about 40.1°C, up to about 40°C).

[0074] It is considered that heating the brain tumor in step b) can include heating to a temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, heating the brain tumor in step b) can include heating to a temperature of from about 40°C to about 45°C (e.g., from about 40.1°C to about 44.9°C, from about 40.2°C to about 44.8°C, from about 40.3°C to about 44.7°C, from about 40.4°C to about 44.6°C, from about 40.5°C to about 44.5°C, from about 40.6°C to about 44.4°C, from about 40.7°C to about 44.3°C, from about 40.8°C to about 44.2°C, from about 40.9°C to about 44.1°C, from about 41°C to about 44°C, from about 41.1°C to about 43.9°C, from about 41.2°C to about 43.8°C, from about 41.3°C to about 43.7°C, from about 41.4°C to about 43.6°C, from about 41.5°C to about 43.5°C, from about 41.6°C to about 43.4°C, from about 41.7°C to about 43.3°C, from about 41.8°C to about 43.2°C, from about 41.9°C to about 43.1°C, from about 42°C to about 43°C, from about 42.1°C to about 42.9°C, from about 42.2°C to about 42.8°C, from about 42.3°C to about 42.7°C, from about 42.4°C to about 42.6°C, from about 40°C to about 42.5°C, from about 40.1°C to about 42.4°C, from about 40.2°C to about 42.3°C, from about 40.3°C to about 42.2°C, from about 40.4°C to about 42.1°C, from about 40.5°C to about 42°C, from, about 40.6°C to about 41.9°C, from about 40.7°C to about 41.8°C, from about 40.8°C to about 41.7°C, from about 40.9°C to about 41.6°C, from about 41°C to about 41.5°C, from about 41.1°C to about 41.4°C, from about 41.2°C to about 41.3°C, from about 42.5°C to about 45°C, from about 42.6°C to about 44.9°C, from about 42.7°C to about 44.8°C, from about 42.8°C to about 44.7°C, from about 42.9°C to about 44.6°C, from about 43°C to about 44.5°C, from about 43.1°C to about 44.4°C, from about 43.2°C to about 44.3°C, from about 43.3°C to about 44.2°C, from about 43.4°C to about 44.1°C, from about 43.5°C to about 44°C, from about 43.6°C to about 43.9°C, from about 43.7°C to about 43.8°C). [0075] In one aspect, it is understood and herein contemplated that the TS-engineered T cells can be retained in the tumor microenvironment (TME), as opposed to migrating out of the TME and brain. For example, in some aspects, the TS-engineered T cells can be retained in the TME for at least about 1 hour, or at least about 2 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or at least about 7 hours, or at least about 8 hours, or at least about 9 hours, or at least about 10 hours, or at least about 11 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 36 hours, or at least about 48 hours, or at least about 60 hours, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days.

[0076] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein the promoter construct is activated by the heating in step b).

[0077] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein the core promoter can include a heat shock protein transcription start site (such as, for example, the heat shock protein transcription start site of HSPA1A, HSPHL HSPA6._j or YB).

[0078] In some aspects, the core promoter can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to any one of SEQ ID NOs: 10-13. In some aspects, the core promoter can include any one of SEQ ID NOs: 10-13. In some aspects, the core promoter can consist of any one of SEQ ID NOs: 10-13.

[0079] In some aspects, the one or more heat shock elements and core promoter together can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to any one of SEQ ID NOs: 14-21. In some aspects, the one or more heat shock elements and core promoter together can include any one of SEQ ID NOs: 14-21. In some aspects, the one or more heat shock elements and core promoter together can consist of any one of SEQ ID NOs: 14-21.

[0080] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein the heating in step b) occurs via ultrasound (such as, for example, focused ultrasound, including, but not limited to MR-guided focused ultrasound (MRgFUS)). In some aspects, the ultrasound can be administered in a cycle including from about 5 minutes to about 15 minutes (including, but not limited to 5 to 10 minutes) such as, for example, administering ultrasound in a cycle for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes followed by from about 1 minute to about 10 minutes of rest (such, as for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes rest). In some aspects, the ultrasound can be administered in 1, 2, 3, 4, 5, 6, or more cycles. In some such aspects, each cycle can have the same administration time and/or the same rest time. In other such aspects, each cycle can have a different administration time and/or a different rest time.

[0081] In some aspects, the ultrasound can have a frequency of at least about 1 MHz (e.g., at least about 1.1 MHz, at least about 1.2 MHz, at least about 1.3 MHz, at least about 1.4 MHz, at least about 1.5 MHz, at least about 1.6 MHz, at least about 1.7 MHz, at least about 1.8 MHz, at least about 1.9 MHz, at least about 2 MHz, at least about 2.1 MHz, at least about 2.2 MHz, at least about 2.3 MHz, at least about 2.4 MHz, at least about 2.5 MHz, at least about 2.6 MHz, at least about 2.7 MHz, at least about 2.8 MHz, at least about 2.9 MHz, at least about 3 MHz). In some aspects, the ultrasound can have a frequency of up to about 3 MHz (e.g., up to about 2.9 MHz, up to about 2.8 MHz, up to about 2.7 MHz, up to about 2.6 MHz, up to about 2.5 MHz, up to about 2.4 MHz, up to about 2.3 MHz, up to about 2.2 MHz, up to about 2.1 MHz, up to about 2 MHz, up to about 1.9 MHz, up to about 1.8 MHz, up to about 1.7 MHz, up to about 1.6 MHz, up to about 1.5 MHz, up to about 1.4 MHz, up to about 1.3 MHz, up to about 1.2 MHz, up to about 1.1 MHz, up to about 1 MHz).

[0082] It is considered that the ultrasound can have a frequency ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ultrasound can have a frequency of from about 1 MHz to about 3 MHz (e.g., from about 1.1 MHz to about 2.9 MHz, from about 1.2 MHz to about 2.8 MHz, from about 1.3 MHz to about 2.7 MHz, from about 1.4 MHz to about 2.6 MHz, from about 1.5 MHz to about 2.5 MHz, from about 1.6 MHz to about 2.4 MHz, from about 1.7 MHz to about 2.3 MHz, from about 1.8 MHz to about 2.2 MHz, from about 1.9 MHz to about 2.1 MHz, from about 1 MHz to about 2 MHz, from about 1.1 MHz to about 1.9 MHz, from about 1.2 MHz to about 1.8 MHz, from about 1.3 MHz to about 1.7 MHz, from about 1.4 MHz to about 1.6 MHz, from about 2 MHz to about 3 MHz, from about 2.1 MHz to about 2.9 MHz, from about 2.2 MHz to about 2.8 MHz, from about 2.3 MHz to about 2.7 MHz, from about 2.4 MHz to about 2.6 MHz).

[0083] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein the heating in step b) occurs via a light source (such as for example, a laser (including, but not limited a near infrared laser), filament, infrared emitting light source, or light emitting diode (LED)), thermal pad, or thermally regulated needle, probe, or scalpel.

[0084] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein step b) uses a focused ultrasound system including a focused ultrasound transducer; a device for measuring temperature (such as, for example, a magnetic resonance (MR) thermometer); and a proportional-integral-derivative (PID) controller configured to modulate power output of the focused ultrasound transducer based on temperature data received from the device for measuring temperature. In some aspects, the PID controller can be a closed-loop controller. In some aspects, the PID controller can yield a temperature deviation of less than about 0.5°C (e.g., less than about 0.45°C, less than about 0.4°C, less than about 0.35°C, less than about 0.3°C, less than about 0.25°C, less than about 0.2°C, less than about 0.15°C, less than about 0.1°C, less than about 0.05°C) from a target temperature. In some aspects, the PID controller can be an Al-based controller or a rule-based controller.

[0085] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein the method does not induce neuronal degeneration.

[0086] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, wherein, by from about 7 days to about 30 days (e.g., about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days) after step b), expression of Iba-1 and/or glial fibrillary acidic protein (GFAP) in the subject has changed by less than about 5% (e.g., less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.5%) and/or the cytotoxicity of the TS-engineered T cells is reduced by less than about 5% (e.g., less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.5%).

[0087] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing ameliorating, and/or preventing a cancer, noncancerous tumor, and/or metastasis, further including administering to the subject an anti-cancer agent or immunotherapy ((including, but not limited to immune checkpoint inhibitors such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example, chimeric antigen receptor (CAR) T cells, CAR Natural Killer (NK) (CAR NK cells), CAR Macrophage (CARMAC) CAR NK T cells, Tumor Infiltrating Lymphocytes (TILs), and/or Marrow Infiltrating Lymphocytes (MILs)).

[0088] In some aspects, the immunotherapy can include an immune checkpoint inhibitor. In some such aspects, the immune checkpoint can include an antibody that blocks PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT- 011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX- 1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7- H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS- 986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR- 022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0089] In some aspects, the immunotherapy can include an adoptive cell therapy. In some such aspects, the adoptive cell therapy can include the administration of tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen. [0090] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL- 2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado- Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPP A, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonaval ent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R- ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, T ARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil— Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and/or ZYTIGA® (Abiraterone Acetate).

[0091] In one aspect, disclosed herein is a system including a plurality of thermal switch (TS)-engineered T cells; a heat source; a device for measuring temperature; and a proportional- integral-derivative (PID) controller configured to modulate power output of the heat source based on temperature data received from the device for measuring temperature. For example, disclosed herein is a system including a plurality of thermal switch (TS)-engineered T cells (including, but not limited to TS T cells including a promoter construct including one or more heat shock elements (such as, for example, SEQ ID NO: 1 or heat shock elements including 80% similarity or more to any one of SEQ ID NOs: 2-9 or any variants thereof as described above), a core promoter, and a gene encoding a bispecific T cell engagers (BTEs)); a heat source; a device for measuring temperature (such as, for example, magnetic resonance (MR) thermometer); and a proportional-integral-derivative (PID) controller configured to modulate power output of the heat source based on temperature data received from the device for measuring temperature. In some aspects, the BTE can target both the brain tumor and the TS- engineered T cells. In some aspects, thermal activation of the promoter construct to express the BTE can occur at from about 40°C to about 45°C as described above. In some aspects, the heat shock element can be repeated 2, 3, 4, 5, 6, 7, or more times.

[0092] Also disclosed herein are systems, wherein the core promoter can include a heat shock protein transcription start site (such as, for example, the heat shock protein transcription start site of HSPA1A, HSPHL HSPA6._j or YB). In some aspects, the core promoter can include 80% similarity or more to any one of SEQ ID NOs: 10-13 as described above. In some aspects the one or more heat shock elements and core promoter together can include 80% similarity or more to any one of SEQ ID NOs: 14-21 as described above.

[0093] In one aspect, disclosed herein are systems, wherein the device for measuring temperature can measure temperature in vivo and/or can measure temperature intracranially. For example, in one aspect, the device for measuring temperature can be a magnetic resonance (MR) thermometer.

[0094] Also disclosed herein are systems, wherein the PID controller is a closed-loop controller. In some aspects, the PID controller can be an Al-based controller or a rule-based controller.

EXAMPLES

Example 1: MRgFUS Activation of T Cells in the Brain

[0095] To overcome these hurdles, innovative strategies that enhance the specificity and adaptability of CAR T cells within brain tumors are critically needed. A study was conducted which sought to engineer CAR T cells with inducible gene expression systems that can be externally regulated to modulate their activity in a spatially and temporally controlled manner. Thermal-sensitive (TS) gene switches offer such a mechanism, where transgene expression is induced upon exposure to mild hyperthermia. Focused ultrasound (FUS) mediated hyperthermia is particularly attractive due to its non-invasive nature, precise spatial targeting capabilities, and ability to penetrate deep tissues. By delivering controlled hyperthermia to specific tumor sites, FUS can activate TS-engineered CAR T cells locally within the tumor, minimizing off-target effects and systemic toxicity.

[0096] Here, the study explored the use of FUS-mediated hyperthermia to activate thermalsensitive CAR T cells engineered to produce bispecific T cell engagers (BTEs also referred to as BiTEs) upon thermal activation. BTEs are fusion proteins that can redirect T cells toward tumor cells by simultaneously binding to a tumor-associated antigen and the CD3 complex on T cells, effectively bridging the two and promoting targeted cytotoxicity. By inducing BTE production in CAR T cells upon hyperthermia, the study aims to redirect their cytotoxic activity toward antigen-negative tumor cells within heterogeneous tumors, thereby mitigating antigen escape. Additionally, the study investigated whether thermal activation of CAR T cells could modulate the immunosuppressive TME, particularly by overcoming MDSC-mediated suppression in glioblastoma models.

[0097] The study engineered primary human and murine T cells with a thermal-sensitive promoter driving the expression of BTEs and a CAR and evaluated the optimal FUS exposure settings for transgene production. The study assessed the effects of hyperthermia on T cell viability, activation, and cytotoxic function both in vitro and in vivo. In vivo studies utilized MR-guided focused ultrasound (MRgFUS) to deliver localized hyperthermia in models of breast cancer brain metastasis (BCBM) and glioblastoma. These findings demonstrate that controlled hyperthermia can effectively trigger TS-engineered CAR T cells to enhance antitumor activity against heterogeneous tumors and within immunosuppressive environments, offering a promising strategy to improve the efficacy of CAR T cell therapies for solid tumors.

Methods

[0098] Primary human T cell production'. Peripheral blood was drawn from healthy human donors, as approved by the Georgia Tech and Emory University Institutional Review Boards (IRB #H20288). Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep density gradient medium (STEMCELL Technologies, 07801) and SepMate- 15mL tube (STEMCELL Technologies, 85415), according to manufacturer’s instructions. CD3+ cells were then isolated using the EasySep Human CD3 Positive Selection Kit II (STEMCELL Technologies, 17851) and activated using Dynabeads (ThermoFisher, 1113 ID) at a 3: 1 bead-to-cell ratio. Activated cells were cultured in complete human T cell media (hTCM; X- vivo 10 [Lonza #04-380Q], 5% Human AB serum [Valley Biomedical, HP 1022], 10 mM N-acetyl L-Cysteine [Sigma A9165] , 55 uM 2-mercaptoethanol [Sigma, M3148- 100ML] supplemented with 50 U/mL recombinant human IL-2 (TECINTM Teceleukin, Bulk Ro 23-6019, National Cancer Institute, Frederick, MD) for 24 hours at 37°C in 5% CO2. To transduce the activated human T cells, concentrated lentivirus (MOI=30) was added to a 24- well suspension culture plate coated with Retronectin (Takara, T100B) according to the manufacturer’s instructions and subsequently centrifuged at l,200xg for 90 minutes at 37°C. Following centrifugation, activated human T cells in human T cell media supplemented with 100 units/mL of hIL-2 were added to each well and the plate was spun at l,200xg for 60 minutes at 37°C. Cells were incubated on the virus-coated plate for 24 hours before expansion. 7 days after activation, Dynabeads were removed. [0099] Primary murine CAR T cell production'. Cells from C57BL/6 mouse spleens were harvested by gently dissociating the tissues using frosted glass slides, then centrifuged at lOOOxg for 5 min and resuspended in lx RBC lysis buffer for 5 min at 4°C. lx PBS was added to quench the lysis reaction, and cells were centrifuged and resuspended in murine T cell media (mTCM; RPMI + 10% FBS + 1% Pen/Strep + lx NEAA + lx Sodium Pyruvate + 50 pM Betamercaptoethanol + 100 lU/mL rhIL-2) and passed through a 40 pm cell strainer. CD3+ cells from C57BL/6 mice isolated using the StemCell EasySep Mouse T Cell Isolation kit following the manufacturer’s protocol. After isolation, C57BL/6 CD3+ T cells were resuspended at a concentration of 1 * 10⁶ cells/mL in mTCM supplemented with murine Dynabeads at a 1 : 1 Bead to T cell ratio. Two days after activation, cells were collected, washed, and resuspended at 1 x 10⁶ cells/mL in concentrated retroviral supernatant supplemented with 100 lU/mL hIL2 and 8 pg/mL polybrene. Spinfection was performed in a U-bottom 96-well plate at 2,000xg for 90 min at 32°C. Transduced cells were maintained at l * 10⁶ cells/mL in complete murine T cell media supplemented with 100 lU/mL hIL-2 and passaged daily until use on Day 5.

[0100] Brain tumor inoculations'. SB28 glioma or MDA-MB-468 cells (0.5 - 2 * 10⁴ cells) were stereotactically implanted into the brain at 1 mm anterior, 1 mm to the right, and 3 mm deep of the bregma of 6- to 10-week-old female albino C57BL/6 J or NSG mice respectively (The Jackson Laboratory). After cell implantation, tumor growth was monitored using T2- weighted MRI and treatment occurred when bioluminescent signal reached sufficient signal. To minimize differences related to tumor size, before each experiment, the tumors in all animals were measured with MRI and IVIS and distributed equally among cohorts. The animals were considered as their endpoint if they exhibited severely impaired activity, significant weight loss, tumor dimensions exceeding 20 mm, or if treatment-related severe adverse events occurred that caused pain or distress and that could not be ameliorated.

[0101] Adoptive cell transfer of CAR T cells'. Transduced human CAR T cells were purified using the EasySep Human CD19 Positive Selection Kit II (STEMCELL Technologies, 17854) according to the manufacturer’s protocol after 9 days of activation. All Human T cells were maintained at a concentration of 0.7-2* 10⁶ cells/mL until Day 10-14 for use in downstream assays. In contrast, murine T cell transduction was evaluated by GFP reporter expression 5 days post isolation. For ACT, 5 x 10⁵ CAR T cells were resuspended in 3 pL of sterile PBS and were transferred by stereotactic injection into brain tumors 1 mm anterior, 1 mm to the right, and 3 mm deep of the bregma of mice upon tumor engraftment assessed by IVIS or MRI. [0102] Quantification of tumor-infiltrating immune cells and flow cytometry. To assess tumor composition, the study performed flow cytometry on single cell suspension of tumor milieu, mice were euthanized and then immediately received intracardiac perfusion of 10 mL sterile PBS. The whole brain was dissected, and the entire tumor mass was separated from the brain. Tumors were immediately minced with razor blades and washed with RPMI. Tumors were dissociated using the Mouse or Human Tumor Dissociation Kits and gentleMACS Dissociator. Homogenized cells were passed through a 40 pm cell strainer and depleted of red blood cells using lx RBC lysis buffer. TILs were isolated from the single cell suspension using a density gradient with Percoll Centrifugation Media and DMEM Media (10% FBS, 1% Pen- strep) at a 44:56 volume ratio. Cell viability was assessed by staining with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit) following manufacturer’s instructions.

Results

[0103] FUS-mediated hyperthermia redirects CAR T cells towards antigen-negative tumors'. The study first sought to evaluate the effects of FUS mediated heating on T cell function and thermal gene activation. The study engineered primary human T cells with a TS.BTE aHER2 CAR by lentiviral transduction as previously described278 to redirect cytotoxicity towards HER2- tumor cells expressing NKG2D-ligands (FIG. 1A). To evaluate optimal FUS exposure settings for reporter gene induction by TS-engineered CAR T cells, the study designed an in vitro system composed of i) a mylar window, ii) a layer of collagen, where the cells will be suspended, iii) followed by another layer of collagen that allows to further insulate the cells from fluid flow related to FUS exposure (shear stress mediated by acoustic streaming). The temperature in this setup is mapped using a thermochromic film and controlled using a thermocouple-based closed loop FUS controller (FIG. IB). Such control systems enable tight control over temperature profiles with minimal user-input to enable programmable and safe in vivo heating strategies. The study first evaluated BTE expression by HIS-tag staining 16 hours following thermocycler mediated heating at either 41 °C or 42°C with either a pulsed (66% duty cycle) or continuous profile. A temperature dependent increase in BTE production was observed as temperature increased from 41 °C to 42°C (FIGS. 1C-1D). Notably, a pulsed thermal profile did not reduce gene production suggesting that safer heating protocols to deposit mild hyperthermia intracranially can be implemented without decreasing switch activity. A correlation with BTE production and antitumoral cytotoxicity was further observed following coculture of TS.BTE aHER2 CAR T cells against HER2- MDA-MB-468s. Cytotoxicity was observed only in conditions in which thermal sensitive CAR T cells are heated to produce BTEs to redirect cytotoxicity against antigen-negative tumor cells (FIG. IE). [0104] Unlike thermocycler mediated heating, FUS mediated hyperthermia applies a shear stress onto the cells. Therefore, the study first sought to evaluate the effect of FUS mediated heating on primary human T cell viability and thermal switch activation. The study transduced primary human T cells with TS.Fluc aHER2 CAR and evaluated cell viability and luciferase expression following FUS treatments. A temperature and time dependent reduction in T cell viability was observed when cells were exposed to FUS-mediated hyperthermia, particularly as the temperature increased to 42°C with a mean normalized viability of -73% (FIG. IF, left). Despite the observed reduction in T cell viability at higher temperatures, thermal switch activation remained substantial. TS.Fluc aHER2 CAR T cells exhibited an approximately 80- fold increase in luciferase expression when heated to 42°C compared to baseline levels at 37°C (FIG. IF, right). This indicates that FUS-mediated hyperthermia can induce therapeutic gene expression without severely compromising cell viability.

[0105] The study next evaluated the impact of FUS-mediated heating on the cytotoxic function of TS.BTE CAR T cells when cocultured with HER2+ MDA-MB-468 tumor cells (FIG. 2A). Following FUS-mediated heating at 37°C, 41°C, or 42°C for 20 minutes, no significant differences were observed in tumor cell killing across all temperature conditions, indicating that FUS mediated mild hyperthermia does not impair the cytolytic activity of CAR T cells (FIG. 2B). However, a significant reduction in IFNy secretion was observed at 42°C compared to 37°C and 41 °C, while no significant difference was noted between 37°C and 41 °C (FIG. 2C). These findings suggest that elevated temperatures may modulate cytokine production without affecting the cytotoxic function of the T cells. To further evaluate the effect of FUS-mediated hyperthermia on BTE expression and the cytotoxic activity of TS.BTE aHER2 CAR T cells against HER2- MDA-MB-468 tumor cells, the study measured surface BTE expression and cytotoxicity following coculture following FUS-mediated heating. A significant increase in BTE expression was observed on the surface of T cells heated to 42°C compared to those maintained at 37°C, indicating functional activation of the thermal switch (FIGS. 1F-1G, left). Correspondingly, only the T cells heated to 42°C demonstrated enhanced cytotoxicity against HER2- tumor cells, consistent with the increased BTE expression (FIGS. 1F-1G, right). Taken together, these results suggest that FUS-mediated heating effectively triggers thermal switch activation in engineered CAR T cells, leading to enhanced cytotoxicity against antigen-negative tumor cells. However, careful modulation of hyperthermia is necessary to avoid compromising T cell viability, underscoring the need for controlled heating protocols in vivo. [0106] Development and Validation of a closed-loop MRgFUS system for in vivo murine applications'. Based on frequency and design parameters optimized through mathematical modeling, a custom-built closed-loop Magnetic Resonance-guided Focused Ultrasound (MRgFUS) system was developed for in silico validation. This system includes three critical components: Magnetic Resonance (MR) Thermometry, Transcranial Focused Ultrasound (FUS), and a Feedback Controller. The integration of transcranial FUS with MR thermometry facilitates precise monitoring and control of temperature elevation within the focal region. Additionally, the system incorporates a robust feedback control mechanism that utilizes rapid MR thermometry to regulate thermal exposure during heating, thereby allowing fine-tuning to achieve and sustain an optimal temperature.

[0107] To refine the MRgFUS system for use with clinically relevant tumor models in rodents, the study focused on developing and evaluating the most effective algorithm to achieve and maintain precise temperature targets. Two different closed-loop controller systems — bistate and Proportional-Integral-Derivative (PID) — were experimentally tested in a HER2+ Breast Cancer Brain Metastasis (BCBM) mouse model (HER2+ MDA-MB-468 in NSG mice) (FIG. 3A).

[0108] Experimental results, derived from temperature measurements using MR thermometry imaging (MRTI) (FIG. 3B), indicated that the PID closed-loop algorithm achieved a focal temperature that closely approximated the target, with significantly reduced variation compared to the bistate controller (FIG. 3C). Detailed analysis of temperature profiles over a 20-minute FUS hyperthermia session (67% duty cycles) at 41.5°C (FIG. 3D) demonstrated that while both controllers reached similar average target temperatures (bistate: 41.4°C vs. PID: 41.5°C), the PID controller exhibited substantially lower deviation values (AT < 0.1 °C vs. 0.5°C) (FIG. 3E). These findings highlight the ability of the PID-based closed- loop system to effectively induce localized hyperthermia and control the temperature within a very narrow range.

[0109] Safety assessment indicates FUS hyperthermia is well tolerated with no significant neuroinflammation'. Following the establishment of an optimized MRgFUS system with a precise controller algorithm to induce transcranial FUS hyperthermia in rodent brains, further validation was conducted to assess potential neuroinflammatory responses. Immunofluorescence staining was employed to analyze brain sections harvested at different time points (2, 7, and 14 days post 10-minute hyperthermia treatment; 67% duty cycle) (FIG. 4A), analyzing on markers indicative of astrocyte activation (GFAP), microglial activation (Iba-1), and neuronal degeneration (Fluoro- Jade). [0110] The results demonstrated a significant increase in GFAP and Iba-1 staining at 2 days post-treatment, persisting over several days, which indicates transient astrocyte and microglial activation (FIG. 4E). MRI imaging corroborated these findings, revealing mild edema at day 2, which resolved by day 5 (FIG. 4B). Importantly, Fluoro-Jade staining showed no evidence of neuronal degeneration, and Hematoxylin and Eosin (H&E) staining confirmed the absence of tissue damage in the treated regions (FIG. 4E).

[0111] Quantitative analysis of fluorescence intensity further validated that the levels of GFAP and Iba-1 in the treated regions were significantly elevated compared to the untreated contralateral regions at day 2. However, these levels returned to baseline or non-significant values over time. By day 7, the Iba-1 expression was no longer significantly different from the untreated regions, and by day 14, GFAP levels were comparable to baseline (FIGS. 4C-4D). This temporal analysis supports the conclusion that localized hyperthermia, as applied by the MRgFUS system, does not lead to long-term neuroinflammation or neuronal damage when administered through the skull in rodents.

[0112] Overall, these experimental findings confirm that the PID-based closed-loop MRgFUS system, operating at an optimal frequency of 1.7 MHz, can safely and effectively induce localized thermal stress in the brains of mice without triggering sustained neuroinflammatory or neurodegenerative responses.

[0113] MRgFUS locally activates thermal sensitive CAR T cells in models ofBCBM'. To evaluate the efficacy of FUS-mediated hyperthermia in activating thermal sensitive CAR T cells in vivo, the study utilized an MR-guided focused ultrasound (MRgFUS) system in brain tumor-bearing mice. Primary human T cells engineered with TS.Fluc aHER2 CARs were administered intratum orally. Following optimization of MR thermographic imagery based closed-loop control systems, the MRgFUS setup allowed precise heating of the tumor region to 41.5°C using two cycles of 10 minutes of heating followed by 5 minutes of rest, totaling 20 minutes (FIG. 5A). Ten hours after MRgFUS-mediated heating, a significant increase in luciferase expression was observed in the heated tumors compared to unheated controls, in which ~10-fold increase in luminescence was observed following heating, indicating successful activation of the thermal switch and subsequent reporter gene expression in intratumoral CAR T cells (FIG. 5B). Monitoring luciferase expression over time following adoptive cell transfer revealed transient and repeatable activation of the thermal sensitive CAR T cells in response to MRgFUS-mediated heating. Peaks in luminescence corresponded with the timing of the thermal treatments, demonstrating that thermal switch mediated protein production is transient and can be repeatedly activated in vivo through mild increases in temperature (FIG. 5C).

[0114] To assess the localization of activated TS.Fluc aHER2 CAR T cells following MRgFUS-mediated heating, the study conducted a biodistribution study using luminescence imaging ten hours post-treatment. Heated cohorts exhibited a significant increase in luminescence within the brain tumor site compared to unheated controls (**p < 0.01, FIG. 5D). Non-significant luminescent was observed in the liver and spleen, indicating that the activation of CAR T cells was confined to the targeted tumor region (FIG. 5E). This localized activation is crucial for immunomodulatory molecules that need to remain within the tumor microenvironment to minimize systemic exposure and potential off-target effects. These findings demonstrate that FUS-mediated hyperthermia can successfully activate thermal sensitive CAR T cells in vivo, leading to localized expression of therapeutic genes within the tumor microenvironment. The ability to control the timing and location of CAR T cell activation through MRgFUS offers a promising strategy for enhancing the efficacy and safety of CAR T cell therapies in solid tumors.

[0115] MRgFUS mediated production ofBTEs by CAR T cells mitigates antigen escape in heterogenous BCBM'. Breast cancer frequently metastasizes to the brain, resulting in tumors with heterogeneous antigen expression that challenge the efficacy of targeted therapies. To overcome the limitation of CAR T cells in addressing antigen-negative tumor cells within such heterogeneous tumors, the study evaluated the therapeutic potential of FUS-mediated hyperthermia to redirect CAR T cells toward CAR antigen-negative cells in a BCBM model. The study implemented the closed-loop MRgFUS system to deposit hyperthermia locally in mice bearing heterogeneous tumors composed of 70% HER2+ and 30% HER2- MDA-MB- 468 cells (FIG. 6A). Mice received adoptive transfer of TS.BTE aHER2 CAR T cells on day 8 post-tumor inoculation, followed by MRgFUS treatments on days 9 and 14 to activate the thermal switch and locally induce BTE production. Mice treated with TS.BTE CAR T cells and MRgFUS heating exhibited significant tumor regression compared to control groups (FIG. 6B) tumors in TS.BTE cohorts were undetectable by MRI throughought the duration of the study. In contrast, tumors in the WT Unheated and TS.Rluc Heated groups continued to grow, indicating that BTE production in response to thermal activation redirected CAR T cell activity towards antigen-negative tumor cells. Luminescent imaging further confirmed these findings by specifically tracking the HER2- tumor cells expressing Flue. Mice in the TS.BTE Heated group displayed a marked decrease in luminescent signal following MRgFUS treatments, reflecting the clearance of HER2- tumor cells (FIG. 6C). In contrast, signal remained high in the WT Unheated group and showed only a moderate reduction in the TS.Rluc Heated group. These results demonstrate that thermal activation of the TS.BTE CAR T cells effectively redirect cytotoxicity towards the antigen-negative tumor population. Survival analysis corroborated the therapeutic efficacy of this approach. Mice receiving TS.BTE CAR T cells with MRgFUS-mediated heating showed significantly improved survival compared to both control groups (FIG. 6D). The majority of mice in the TS.BTE Heated group survived beyond 100 days post-adoptive cell transfer, whereas mice in the WT Unheated group succumbed to tumor burden by around day 30 (WT Unheated vs. TS.BTE Heated: p = 0.0003; TS.Rluc Heated vs. TS.BTE Heated: p = 0.0191).

[0116] Collectively, these results indicate that MRgFUS-mediated hyperthermia can effectively activate thermal-sensitive CAR T cells in vivo, leading to the production of BTEs that redirect cytotoxicity towards antigen-negative tumor cells. This strategy not only suppresses tumor growth but also significantly extends survival, highlighting the potential of combining focused ultrasound with thermal sensitive CAR T cells for the treatment of heterogeneous tumors.

[0117] Thermal mediated BTE production overcomes immune suppression in glioblastoma'. Myeloid-derived suppressor cells (MDSCs) pose a significant challenge to effective CAR T cell therapy in glioblastoma due to their immunosuppressive effects within the tumor microenvironment279. To address this issue, the study investigated whether mBTE- engineered CAR T cells exhibit resistance to MDSC-mediated suppression and assessed the impact of heat-mediated production of mBTEs on T cell function and tumor burden. The study first sought to evaluate CAR T cell activation and proliferation in the presence of MDSCs in vitro. Briefly, WT T cells were cocultured in mBTE conditioned media in the presence of MDSCs at various cell ratios following CD3 and CD28 dynabead stimulation and measured CD25 expression as a marker of activation and CTV staining as a measure of proliferation (FIG. 7). Cells cultured in mBTE conditioned media CAR T cells demonstrated enhanced activation compared to WT CAR T cells across all ratios, as indicated by higher CD25 expression (FIG. 8A, left). Additionally, mBTE CAR T cells exhibited increased proliferation, as shown by greater CTV dilution at an effector-to-target ratio of 0.5: 1, suggesting reduced susceptibility to MDSC-mediated suppression (FIG. 8A, right). Quantitative analysis revealed that mBTE CAR T cells maintained significantly higher CD25 mean fluorescence intensity at all MDSC cell ratios tested compared to coculture in unconditioned media (FIG. 8B). The expansion index of mBTE CAR T cells was also significantly higher than that of unoconditioned media in the presence of MDSCs, indicating sustained proliferation despite the suppressive conditions (FIG. 8C).

[0118] To evaluate these findings in vivo, the study implemented a syngeneic murine glioblastoma model characterized by high MDSC infiltration280. Mice bearing EGFRvIIH SB28 tumors received adoptive transfer of TS.mBTE CAR T cells (non-specific to the tumor antigen) or WT T cells, followed by MRgFUS heating of the tumors at 41°C for 10 minutes on day 9 post- ACT (FIG. 8D). Flow analysis on day 11 post-inoculation showed that CAR T cells were still detectable in the tumors and remain unchanged following thermal treatment (FIG. 8E). Notably, the study also assessed the effect of MRgFUS heating on tumor-infiltrating monocytic MDSCs (M-MDSCs). Both the percentage and absolute count of M-MDSCs were significantly reduced in the TS.mBTE Heated group compared to unheated or WT controls, suggesting that MRgFUS-mediated hyperthermia may modulate the immunosuppressive tumor microenvironment by reducing MDSC populations (FIG. 8F).

[0119] The study next evaluated the therapeutic benefit of TS.mBTE aEGFRvIII CAR T cells in EGFRvIII+ SB28 brain tumor-bearing mice. Tumors were luciferized and Bioluminescent imaging implemented to quantify burden. Mice were divided into four groups: (1) WT CAR T cells without MRgFUS heating (WT Unheated), (2) TS.mBTE CAR T cells without MRgFUS heating (TS.mBTE Unheated), (3) WT CAR T cells with MRgFUS heating (WT Heated), and (4) TS.mBTE CAR T cells with MRgFUS heating (TS.mBTE Heated). In the WT Unheated and WT Heated groups, tumor burden increased steadily over time, indicating that heat alone does not alter tumor burden (FIGS. 8G-8I). Similarly, mice treatead with TS.mBTE CAR T cells had an initial reduction in tumor burden but was insufficient at controlling tumor growth. In contrast, the TS.mBTE Heated group demonstrated a significant reduction in tumor burden over time. Luminescence imaging revealed markedly lower signals in this group compared to all other groups, indicating effective tumor suppression (FIGS. 8G- 81). This reduction in tumor burden suggests that MRgFUS-mediated hyperthermia successfully activated the thermal switch in TS.mBTE CAR T cells, leading to the production of mBTEs to help overcome the immunosuppressive tumor microenvironment mediated by MDSCs contributed to a more favorable environment for CAR T cell activity and tumor cell killing. Taken together, these findings highlight the therapeutic potential of combining MRgFUS-mediated hyperthermia with TS.mBTE-engineered EGFRvIII CAR T cells to overcome immunosuppression in glioblastoma. By activating the thermal switch to locally induce mBTE production, this approach enhances CAR T cell infiltration and function within the tumor, leading to significant tumor burden reduction. Discussion

[0120] This study demonstrates that FUS-mediated hyperthermia effectively activates thermal-sensitive CAR T cells, leading to enhanced cytotoxicity against antigen-negative tumor cells without severely compromising T cell viability. In vitro optimization showed a substantial increase in transgene expression upon heating, with TS.Fluc aHER2 CAR T cells exhibiting an approximately 80-fold increase in luciferase expression at 42°C compared to baseline levels at 37°C. Importantly, this activation did not impair the cytolytic function of the CAR T cells against antigen-positive targets and only minimally affected cytokine production at elevated temperatures, indicating that key T cell functions are preserved under mild hyperthermia conditions.

[0121] In vivo, the use of MRgFUS enables precise and localized heating of tumor sites, resulting in the activation of thermal-sensitive CAR T cells specifically within the tumor microenvironment. In BCBM models with heterogeneous antigen expression, MRgFUS- mediated hyperthermia induced BTE production by the CAR T cells, effectively redirecting their cytotoxicity toward HER2-negative tumor cells. This strategy significantly suppressed tumor growth and extended survival compared to control groups, highlighting its potential to mitigate antigen escape — a major challenge in solid tumor immunotherapy.

[0122] Furthermore, in the glioblastoma model characterized by high MDSC infiltration, thermal activation of CAR T cells led to reduced MDSC populations within the tumor and enhanced CAR T cell infiltration. The production of murine BTEs upon heating appeared to overcome the immunosuppressive effects of MDSCs, resulting in significant tumor burden reduction. These findings suggest that FUS-mediated hyperthermia not only enhances CAR T cell function but also modulates the tumor microenvironment to favor antitumor immunity. By reducing immunosuppressive cell populations and enhancing CAR T cell function, this approach addresses two critical barriers in the treatment of solid tumors.

[0123] The ability to control the timing and location of CAR T cell activation through MRgFUS offers a significant advantage in terms of safety and efficacy. Localized activation minimizes systemic exposure to therapeutic agents, reducing the risk of off-target effects and cytokine release syndrome. Moreover, the transient and repeatable nature of thermal switch activation allows for precise modulation of CAR T cell activity, which could be adjusted based on the therapeutic response and tolerability in patients.

[0124] Collectively, this study highlights the potential of integrating FUS-mediated hyperthermia with thermal-sensitive CAR T cell therapy to address key limitations in solid tumor treatment. By enabling controlled, localized activation of CAR T cells and overcoming immunosuppressive barriers, this approach offers a promising avenue for enhancing the efficacy of CAR T cell therapies against solid tumors. Evaluating the long-term efficacy and safety of this strategy in clinical settings will be crucial for its potential translation into a viable treatment option for patients with solid tumors.

[0125] Any patents, applications and publications as listed throughout this document are hereby incorporated by reference in their entirety herein.

SEQUENCES

TABLE 1. HSEs

TABLE 2. Core Promoters

TABLE 3. HSE + Core Promoter

Claims

What is claimed is:

1. A method of treating a subject with a brain tumor, the method comprising: a) intracranially providing a plurality of thermal switch (TS)-engineered T cells to the brain tumor; and b) heating the brain tumor, thereby inducing hyperthermia in the brain tumor; wherein the hyperthermia causes the TS-engineered T cells to produce bispecific T cell engagers (BTEs).

2. The method of claim 1, wherein the BTE targets both the brain tumor and the TS- engineered T cells

3. The method of any one of claims 1-2, wherein the TS-engineered T cells are administered intratum orally.

4. The method of any one of claims 1-3, wherein the TS-engineered T cells are retained in the tumor microenvironment (TME).

5. The method of any one of claims 1-4, wherein the TS-engineered T cells comprise a promoter construct comprising: one or more heat shock elements; a core promoter; and a gene encoding the BTE.

6. The method of claim 5, wherein the promoter construct requires thermal activation from about 40°C to about 45°C.

7. The method of any one of claims 5-6, wherein the promoter construct is activated by the heating in step b).

8. The method of any one of claims 5-7, wherein the heat shock element is repeated 2, 3, 4, 5, 6, 7, or more times.

9. The method of any one of claims 5-8, wherein the heat shock element comprises SEQ ID NO: 1 or a variant thereof.

10. The method of claim 9, wherein the one or more heat shock elements comprises 80% similarity or more to any one of SEQ ID NOs: 2-9.

11. The method of any one of claims 5-10, wherein the core promoter comprises a heat shock protein transcription start site.

12. The method of claim 11, wherein the core promoter comprises the heat shock protein transcription start site of HSPA1 A, HSPH1, HSPA6, or YB.

13. The method of any one of claims 11-12, wherein the core promoter comprises 80% similarity or more to any one of SEQ ID NOs: 10-13.

14. The method of any one of claims 5-13, wherein the one or more heat shock elements and core promoter together comprise 80% similarity or more to any one of SEQ ID NOs: 14- 21.

15. The method of any one of claims 1-14, wherein the heating in step b) is focused ultrasound.

16. The method of claim 15, wherein the focused ultrasound is administered in a cycle comprising from about 5 minutes to about 15 minutes followed by from about 1 minute to about 10 minutes of rest.

17. The method of claim 16, wherein the focused ultrasound is administered in 1, 2, 3, 4, 5, 6, or more cycles.

18. The method of any one of claims 15-17, wherein the focused ultrasound has a frequency of from about 1 MHz to about 3 MHz.

19. The method of any one of claims 15-18, wherein step b) uses a focused ultrasound system comprising: a focused ultrasound transducer; a device for measuring temperature; and a proportional-integral-derivative (PID) controller configured to modulate power output of the focused ultrasound transducer based on temperature data received from the device for measuring temperature.

20. The method of claim 19, wherein the device for measuring temperature is a magnetic resonance (MR) thermometer.

21. The method of any one of claims 19-20, wherein the PID controller is a closed-loop controller.

22. The method of any one of claims 19-21, wherein the PID controller yields a temperature deviation of less than about 0.5°C from a target temperature.

23. The method of any one of claims 1-22, wherein the brain tumor is heated to from about 40°C to about 45°C.

24. The method of any one of claims 1-23, wherein the method does not induce neuronal degeneration.

25. The method of any one of claims 1-24, wherein, by from about 7 days to about 30 days after step b), expression of Iba-1 and/or glial fibrillary acidic protein (GFAP) in the subject has changed by less than about 5%.

26. The method of any one of claims 1-25, wherein cytotoxicity of the TS-engineered T cells is reduced by less than about 5%.

27. The method of any one of claims 1-26, wherein the brain tumor is glioblastoma, astrocytic tumor, oligodendroglial tumor, ependymoma, craniopharyngioma, pituitary tumor, primary lymphoma of the brain, pineal gland tumor, primary germ cell tumor of the brain, Medulloblastomas, Gangliogliomas, or any combination thereof.

28. The method of any one of claims 1-27, wherein the brain tumor is a primary tumor.

29. The method of any one of claims 1-27, wherein the brain tumor is a metastatic brain tumor (e.g., Lung cancer brain metastasis, breast cancer brain metastasis).

30. The method of any one of claims 1-29, further comprising administering to the subject an anti-cancer agent or immunotherapy.

31. The method of claim 30, wherein the immunotherapy comprises an immune checkpoint inhibitor.

32. The method of claim 31, wherein the immune checkpoint inhibitor comprises an antibody that blocks PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3.

33. A system comprising: a plurality of thermal switch (TS)-engineered T cells; a heat source; a device for measuring temperature; and a proportional-integral-derivative (PID) controller configured to modulate power output of the heat source based on temperature data received from the device for measuring temperature.

34. The system of claim 33, wherein the TS-engineered T cells comprise a promoter construct comprising: one or more heat shock elements; a core promoter; and a gene encoding the BTE.

35. The system of claim 34, wherein the promoter construct requires thermal activation from about 40°C to about 45°C.

36. The system of any one of claims 34-35, wherein the promoter construct is activated by the heating in step b).

37. The system of any one of claims 34-36, wherein the heat shock element is repeated 2, 3, 4, 5, 6, 7, or more times.

38. The system of any one of claims 34-37, wherein the heat shock element comprises SEQ ID NO: 1 or a variant thereof.

39. The system of claim 38, wherein the one or more heat shock elements comprises 80% similarity or more to any one of SEQ ID NOs: 2-9.

40. The system of any one of claims 34-39, wherein the core promoter comprises a heat shock protein transcription start site.

41. The system of claim 40, wherein the core promoter comprises the heat shock protein transcription start site of HSPA1 A, HSPH1, HSPA6, or YB.

42. The method of any one of claims 40-41, wherein the core promoter comprises 80% similarity or more to any one of SEQ ID NOs: 10-13.

43. The system of any one of claims 34-42, wherein the one or more heat shock elements and core promoter together comprise 80% similarity or more to any one of SEQ ID NOs: 14- 21.

44. The system of any one of claims 34-43, wherein the device for measuring temperature can measure temperature in vivo.

45. The system of claim 44, wherein the device for measuring temperature can measure temperature intracranially.

46. The system of any one of claims 44-45, wherein the device for measuring temperature is a magnetic resonance (MR) thermometer.

47. The system of any one of claims 33-36, wherein the PID controller is a closed-loop controller.