Methods and compositions for remote control of T cell therapy by thermal targeting

Abstract

The present disclosure relates to a promoter construct comprising one or more heat shock elements, a core promoter, and a gene of interest, a vector comprising the promoter construct, and an immune cell modified to comprise the promoter construct. The promoter construct provides the ability to remotely control immune cell therapy by thermal targeting. The present disclosure also provides a method of use for the promoter construct.

Description

[Technical field]

[0001] This invention was made with Government support under Grant No. DP2HD091793 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 214,761, filed June 24, 2021, the entirety of which is incorporated herein by reference. [Background technology]

[0003] Engineered T cell therapies, such as chimeric antigen receptor (CAR) T cells, have transformed clinical care in hematological malignancies, driving many efforts to expand their use for different cancer types and applications. However, this success has not been reliably translated to solid tumors. The factors contributing to low response rates are multifaceted and include a paucity of tumor-specific antigens, inefficient persistence and expansion of adoptively transferred T cells, and immune suppression by the tumor microenvironment (TME). Promising approaches to improve the antitumor activity of engineered T cells include systemic administration of potent immune stimulants such as cytokines, checkpoint blockade inhibitor antibodies, and bispecific T cell engagers (BiTEs). However, these biologics lack specificity, activate both engineered and endogenous immune cells, and exhibit toxicity in healthy tissues, limiting the maximum tolerated dose and narrowing their therapeutic window. What is needed, therefore, are CAR T cells with enhanced function locally at disease sites, such as tumors and draining lymph nodes, thereby improving the safety and efficacy of cell-based therapies. Summary of the Invention

[0004] The present invention relates to thermoactivatable promoter constructs and methods for their production and use.

[0005] In one aspect, disclosed herein is a promoter construct comprising: a) one or more heat shock elements (such as the heat shock elements set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and / or 9); b) a core promoter; and c) a gene of interest.

[0006] Also disclosed is a promoter construct of any of the preceding aspects, wherein the promoter is capable of being incubated at 40° C. to 45° C. (e.g., 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.8, 4 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0 °C, or 40 °C to 42 °C, or 41 °C to 43 °C, or 42 °C to 45 °C, etc. In some embodiments, the promoter is at least 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48. 4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0°C).

[0007] In some embodiments, the promoter construct of any preceding embodiment disclosed herein, the heat shock element is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times. For example, in some embodiments, the heat shock element comprises seven repeats of SEQ ID NO:1.

[0008] Also disclosed herein is a promoter construct of any preceding aspect, wherein the core promoter comprises a heat shock protein core promoter (including, but not limited to, a heat shock protein core promoter of heat shock protein HSPA1A, HSPH1, HSPB1, HSPA6, or YB, such as a heat shock protein core promoter comprising any one of the nucleotide sequences of SEQ ID NOs: 10-13).

[0009] In some embodiments, the promoter construct of any preceding embodiment disclosed herein, wherein the gene of interest encodes any combination of the following: a) a reporter protein (e.g., luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma fluorescent protein (DMF), or a combination thereof). striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, Photoactivated GFP (PA-GFP), Venus, Kaede, Monomeric Kusabira Orange (mKO), Dronpa, Enhanced CFP (ECFP), Emerald, Energy-transfer Cyan Fluorescent Protein (CyPet), Super CFP (SCFP), Cerulean, Photoswitchable CFP (PS-CFP2), Photoactivated RFP1 (PA-RFP1), Photoactivated mCherry (PA-mCherry), Monomeric Teal Fluorescent Protein (mTFP1), Eos Fluorescent Protein (EosFP), Dendra, TagBFP, TagRFP, Enhanced YFP (EYFP), Topaz, Citrine, Energy-transfer Yellow Fluorescent Protein (YPet), Super YFP (SYFP), Enhanced GFP (EGFP), SuperfolderGFP, T-Sapphire, Fucci, mKO2, mOrange2, mApple, Sirius, Azurite, EBFP, and / or EBFP2, b) immunomodulatory agents (e.g., including but not limited to chemokines (CCL2, CCL1, CCL19, CCL22, CXCL12, CCL17, MIP-1α, MCP-1, GRO / KC, and / or CXCR3) cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IL-22, IFN- gamma, TNF-alpha, TGF-beta, LIF, and / or cytotoxins (including but not limited to perforin and / or granzymes); c) bispecific T cell engager antibodies, including but not limited to bispecific T cell engager antibodies comprising an anti-CD3 binding domain and an NKG2D receptor extracellular domain; d) chimeric antigen receptors (CARs) (CD19, B cell maturation antigen (BCMA), CD22, CD33, CD38, NCAM1, CD5, CD70, MET, Muc1, L1CAM, CD44 and / or e) a recombinant T cell receptor (TCR) (including but not limited to a TCR targeting WT1, HPV E6, HPV E7, NY-ESO-1, HA-1, MAGE, Gp100, MART-1, HBV, p53, CEA, SL9, TGFβ11, TRAIL, MCPyV, PRAME, EBV, CMV, or KRAS).

[0010] Also disclosed herein is a kit comprising the promoter construct of any preceding embodiment, further comprising a heating component that activates the promoter construct. In some embodiments, the heating component can be a light source (e.g., a laser (including but not limited to a near-infrared laser), a filament, an infrared emitting light source, or a light emitting diode (LED), a thermal pad, or a thermally regulated needle, probe, or scalpel, etc.).

[0011] In some aspects, disclosed herein is an immune cell comprising the promoter construct of any preceding aspect. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, or a dendritic cell. In some embodiments, the T cell comprises a recombinant TCR. In some embodiments, the immune cell is a chimeric antigen receptor (CAR) T cell and / or a CAR natural killer (NK) cell. For example, disclosed herein is a CAR T or CAR NK cell comprising a promoter construct comprising a) one or more heat shock elements (such as the heat shock element set forth in SEQ ID NO: 1), b) a core promoter, and c) a gene of interest. In some embodiments, the gene of interest encodes a chimeric antigen receptor, a recombinant TCR, an immunomodulator, or any combination thereof.

[0012] Also disclosed herein is a method of treating, lowering, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., a solid tumor, including, but not limited to, an epithelial carcinoma, a sarcoma, a lymphoma, a blastoma, or a melanoma), the method comprising administering to the subject a promoter, an immune cell, a T cell (e.g., a CAR T cell), an NK cell (e.g., a CAR NK cell), or a dendritic cell, or applying a kit of any preceding aspect. For example, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., solid tumors, including, but not limited to, epithelial carcinoma, sarcoma, lymphoma, blastoma, or melanoma), the methods comprising administering to the subject thermoregulated CAR immune cells (e.g., CAR T cells or CAR NK cells comprising a promoter construct comprising a) one or more heat shock elements (e.g., the heat shock element set forth in SEQ ID NO:1), b) a core promoter, and c) a gene of interest), and inducing activation of the CAR T cells and / or CAR NK cells at the tumor site (e.g., the CAR T cells and / or CAR NK cells were incubated at 40℃ to 45℃ (40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, and heating to 40-42°C or 41-43°C or 42-45°C, including but not limited to 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0.In some embodiments, the promoter is at least 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48. (The method may further comprise administering an additional anti-cancer agent or immunotherapy, including but not limited to, a checkpoint inhibitor such as those used in anti-PD-1 immunotherapy, anti-PD-L1 immunotherapy, anti-CTLA-4 immunotherapy, or a combination thereof.)

[0013] Additional aspects and advantages of the disclosure will be set forth in part in the detailed description and any claims that follow, and in part will be derived from the detailed description and may be learned by practice of various aspects of the disclosure. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure.

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and, together with the description, serve to explain, but not to limit, the principles of the present disclosure. Like numerals represent the same elements throughout the drawings. [Brief description of the drawings]

[0015] [Figure 1A]Figures 1A-1I show the construction and activity of heat-specific gene switches. (Figure 1A) Schematic diagram of a panel of six heat gene switch constructs (labeled 2H-B1-7H-B1) containing two to seven heat shock elements (HSEs) upstream of the HSPB1 core promoter. Base pairs in capital letters within the HSEs were conserved, and base pairs indicated as n were randomized. [Figure 1B] Figures 1A-1I show the construction and activity of the heat-specific gene switch. (Figure 1B) HSE number or (Figure 1C) Gluc reporter expression by Jurkat T cells after heating as a function of temperature (ns=not significant, *P<0.05, **P<0.01, two-way ANOVA with Tukey post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). Three independent experiments were performed with similar results. [Figure 1C] Figures 1A-1I show the construction and activity of the heat-specific gene switch. (Figure 1B) HSE number or (Figure 1C) Gluc reporter expression by Jurkat T cells after heating as a function of temperature (ns=not significant, *P<0.05, **P<0.01, two-way ANOVA with Tukey post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). Three independent experiments were performed with similar results. [Figure 1D] Figures 1A-1I show the construction and activity of the heat-specific gene switch. (Figure 1D) Kinetics of Gluc reporter expression by primary human T cells after heat treatment at the indicated temperatures (****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 1E] Figures 1A-1I show the construction and activity of heat-specific gene switches. (Figure 1E) Activity of heat gene switches containing different core promoter constructs after heat treatment in primary human T cells (ns = not significant, *P < 0.05, one-way ANOVA and Tukey post-hoc test and correction, mean ± SEM shown, n = 3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 1F]1A-1I show the construction and activity of the heat-specific gene switch. (FIG. 1F) Gluc expression in primary human T cells incubated at the indicated temperatures for 24 h, n=3. [Figure 1G] Figures 1A-1I show the construction and activity of a heat-specific gene switch. (Figure 1G) Activity of 7H-YB in primary human T cells from multiple human donors after 30 min of heat treatment at the indicated temperatures (****P<0.0001, Student's t-test, mean ± SEM shown, n=3 biologically independent wells). [Figure 1H] 1A-1I show the construction and activity of a heat-specific gene switch. Activity of 7H-YB relative to endogenous HSP70 and HSPA6 promoters in primary human T cells following exposure to (Fig. 1H) CoCl2 to mimic hypoxia, or (Fig. 1I) CdCl2 to model heavy metal toxicity (ns=not significant, *P<0.05, ****P<0.0001, two-way ANOVA with Tukey's post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). [Figure 1I] 1A-1I show the construction and activity of a heat-specific gene switch. Activity of 7H-YB relative to endogenous HSP70 and HSPA6 promoters in primary human T cells following exposure to (Fig. 1H) CoCl2 to mimic hypoxia, or (Fig. 1I) CdCl2 to model heavy metal toxicity (ns=not significant, *P<0.05, ****P<0.0001, two-way ANOVA with Tukey's post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). [Figure 2-1] Figure 2 shows qPCR screening of HSPs in primary murine T cells. Splenic CD8+ T cells were isolated using a CD8+ T cell isolation kit according to (Miltenyi 130-104-075). Six hours after the indicated heat treatments, mRNA was collected and quantified using a Mouse HSP Profiler Kit (Qiagen PAMM-076Z) according to the manufacturer's instructions. Data are presented relative to the non-heated control. [Figure 2-2]Figure 2 shows qPCR screening of HSPs in primary murine T cells. Splenic CD8+ T cells were isolated using a CD8+ T cell isolation kit according to (Miltenyi 130-104-075). Six hours after the indicated heat treatments, mRNA was collected and quantified using a Mouse HSP Profiler Kit (Qiagen PAMM-076Z) according to the manufacturer's instructions. Data are presented relative to the non-heated control. [Figure 2-3] Figure 2 shows qPCR screening of HSPs in primary murine T cells. Splenic CD8+ T cells were isolated using a CD8+ T cell isolation kit according to (Miltenyi 130-104-075). Six hours after the indicated heat treatments, mRNA was collected and quantified using a Mouse HSP Profiler Kit (Qiagen PAMM-076Z) according to the manufacturer's instructions. Data are presented relative to the non-heated control. [Figure 2-4] Figure 2 shows qPCR screening of HSPs in primary murine T cells. Splenic CD8+ T cells were isolated using a CD8+ T cell isolation kit according to (Miltenyi 130-104-075). Six hours after the indicated heat treatments, mRNA was collected and quantified using a Mouse HSP Profiler Kit (Qiagen PAMM-076Z) according to the manufacturer's instructions. Data are presented relative to the non-heated control. [Figure 2-5] Figure 2 shows qPCR screening of HSPs in primary murine T cells. Splenic CD8+ T cells were isolated using a CD8+ T cell isolation kit according to (Miltenyi 130-104-075). Six hours after the indicated heat treatments, mRNA was collected and quantified using a Mouse HSP Profiler Kit (Qiagen PAMM-076Z) according to the manufacturer's instructions. Data are presented relative to the non-heated control. [Diagram 3]Figure 3 shows the transduction efficiency of primary human T cells from three donors. Flow cytometry plots of primary human T cells derived from three donors and transduced with Gluc expressing a 7H-YB thermoswitch containing a constitutively expressed mCherry reporter. The inset shows the mean fluorescence intensity (MFI) of mCherry-transduced cells. [Figure 4A] 4A-4B show thermoswitch specificity in Jurkat T cells. Gluc activity by Jurkat T cells transduced with a synthetic thermoswitch construct (blue) or the endogenous HSPA6 promoter (red) after exposure to (FIG. 4A) CoCl2 to mimic hypoxia, or (FIG. 4B) CdCl2 to model heavy metal toxicity (ns=not significant, *P<0.05, ****P<.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=3). [Figure 4B] 4A-4B show thermoswitch specificity in Jurkat T cells. Gluc activity by Jurkat T cells transduced with a synthetic thermoswitch construct (blue) or the endogenous HSPA6 promoter (red) after exposure to (FIG. 4A) CoCl2 to mimic hypoxia, or (FIG. 4B) CdCl2 to model heavy metal toxicity (ns=not significant, *P<0.05, ****P<.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=3). [Figure 5A] 5A-5E show that heat treatment is well tolerated by primary human T cells. (FIG. 5A) Gluc activity of the 7H-YB thermoswitch in primary human T cells after continuous (light grey) and pulsed (dark grey) heat treatment with temperature, total duration, and heating profile as indicated (ns=not significant, *P<0.05, **P<0.01, ****P<0.0001, two-tailed t-test, mean±SEM shown, n=3 biologically independent wells). [Figure 5B]Figures 5A-5E show that heat treatment is well tolerated by primary human T cells. (Figure 5B) Propidium iodide (PI) and Annexin V flow staining of CD3+ T cells. Bars represent viable population (PI-Annexin V-) normalized to non-heated samples (ns=not significant, ****P<0.0001, one-way ANOVA with Dunnett's post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 5C] Figures 5A-5E show that heat treatment is well tolerated by primary human T cells. (Figure 5C) CellTrace Violet (CTV) flow histogram of T cells after heat treatment and incubation with CD3 / 28 beads at a bead-to-T cell ratio of 3:1. Two independent experiments were performed with similar results. [Figure 5D] Figures 5A-5E show that heat treatment is well tolerated by primary human T cells. (Figure 5D) Cell counts in the bottom wells of transwell plates containing CXCL12. T cells were heated and added to the top wells before sampling at the indicated time points (ns=not significant between 37°C and 42°C, two-way ANOVA and Tukey post-hoc test and correction, mean±SEM shown, n=3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 5E] Figures 5A-5E show that heat treatment is well tolerated by primary human T cells. (Figure 5E) Percentage of cytotoxicity observed in CD19- or CD19+ luciferase-labeled K562 cells after incubation with T cells constitutively expressing CAR after heating with effector-to-target ratios as indicated (ns=not significant, *P<0.05, two-way ANOVA with Sidak post-hoc test and correction, mean±SEM shown, n=3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 5F] Figure 5F. [Figure 6]Figure 6 shows the gating strategy for viability flow staining. Primary human T cells were heated at 42°C for 60 minutes as a positive control for heat injury. The shorter regimen was used in subsequent experiments. This conservative gating strategy was used to better represent the overall viability of the sample, as many of the Annexin V+ or PI+ events were not within the more stringent FSC / SSC gating. [Figure 7] Figure 7 shows long-term heating of primary human T cells. Primary human T cells transduced with the HSPA6-GFP switch were repeatedly heated once the GFP signal returned to baseline after the previous heat treatment (n=3 biologically independent wells, error bars indicate SEM). Two independent experiments were performed with similar results. [Figure 8A] Figures 8A-8B show that repeated heat treatment does not affect CAR T cytotoxicity. (Figure 8A) Primary human T cells were transduced to constitutively express the αCD19 CAR after CD3 / CD28 bead activation. Heat treatment was performed at the indicated time points and co-cultured with luciferase-activated CD19+K562 according to the timeline. [Figure 8B] Figures 8A-8B show that repeated heat treatment does not affect CAR T cytotoxicity (Figure 8B). Percent cytotoxicity was quantified by loss of luminescence in wells compared to control wells containing target cells only (b) (ns = not significant, two-way ANOVA with Sidak post-hoc test and correction, mean ± SEM shown, n = 3 biologically independent wells). [Figure 9A] Figures 9A-9F show in vivo photothermal activation of engineered T cells. (Figure 9A) Thermal and luminescence images of a well containing TS-Fluc T cells after irradiation with NIR laser light. The thermal image (left) was acquired using a FLIR thermal camera, and the luminescence image (right) was acquired using an IVIS Spectrum CT system after 6 hours of heating. [Figure 9B]Figures 9A-9F show in vivo photothermal activation of engineered T cells. (Figure 9B) Schematic of TS-Fluc αCD19 CAR construct transduced into primary human T cells before introduction into NSG mice bearing two flank (K562 or Raji) tumors, followed by photothermal heating of only one tumor. [Figure 9C] Figures 9A-9F show in vivo photothermal activation of engineered T cells. (Figure 9C) Thermal images of a mouse during laser irradiation of the tumor site at 0 and 3 minutes. [Figure 9D] 9A-9F show in vivo photothermal activation of engineered T cells. (FIG. 9D) Kinetic trace (colored line) showing the average skin temperature of a 3×3 pixel ROI centered on the laser site. Shaded areas indicate the standard deviation of three heating runs. [Figure 9E] Figures 9A-9F show in vivo photothermal activation of engineered T cells. (Figure 9E) Left: Luminescence images of heated mice bearing either K562 (CD19-) or Raji (CD19+) tumors. Signals indicate luciferase activity by transduced TS-Fluc T cells. Right: Luminescence of each tumor site relative to luminescence from non-heated tumors in the same animals. ROIs were drawn as indicated in the left panel. To confirm the reproducibility of the experimental results, another experiment was performed in which Raji tumors were repeatedly heated (Figure 11). [Figure 9F] Figures 9A-9F show in vivo photothermal activation of engineered T cells. (Figure 9F) Mice bearing two Raji (CD19+) tumors and one site heated. Left: Luminescence images of excised tumors (heated and unheated) and spleen. Right: Quantification of luminescence after heat treatment (0, 6, 12, 18, 24 hours). ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean ± SEM shown, n=4-5 biologically independent mice. [Figure 10] Figure 10 shows CD19 expression in K562 and Raji tumor cells. Representative flow cytometry plots of CD19 staining in K562 and Raji cell lines (Iso = isotype control). [Figure 11]Figure 11 shows the long-term control of intratumoral CAR T cells using photothermal pulses. Mice bearing Raji tumors (CD19+) were iv injected with TS-Fluc T cells. The tumor site was irradiated on days 2 and 4 using NIR laser light, as shown in Figure 18d. Luminescence was quantified daily via iv injection of D-luciferin (n=3, biologically independent wells, error bars show SEM). [Figure 12] Figure 12 shows TS-Fluc αCD19 CAR T cell infiltration into K562 and Raji flank tumors. When tumors reached approximately 250 mm3, tumor-bearing mice were injected iv with TS-Fluc αCD19 CAR T cells. After 7 days, tumors were excised, dissociated, stained, and cellular infiltration was quantified using bead counting flow cytometry (n=3 biologically independent wells, error bars show SEM). [Figure 13A] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13A) Schematic of co-culture assay of heated TS-IL15 αCD19 cells and CFSE-labeled wild-type cells. CD3 / 28 beads were added at a bead-to-T cell ratio of 1:10. [Figure 13B] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13B) Representative flow histograms (left), quantified proliferation (middle), and division index (right) calculated by the FlowJo proliferation tool of CFSE-labeled wild-type T cell populations after 30 min of heat treatment at the indicated temperatures (*P<0.05, two-tailed t-test, mean ± SEM shown, n=3 biologically independent wells). [Figure 13C]Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13C) IL-15 superagonist concentration in the supernatant of TS-IL15 αCD19 T cells after heat treatment. Temperature and duration of treatment are as indicated (ns=not significant, **P<0.01, ****P<0.0001, after two-way ANOVA and Tukey post-hoc test and correction, mean values ​​± SEM are shown, n=3 biologically independent wells, comparison is to non-heated control). Two independent experiments were performed with similar results. [Figure 13D] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13D) Schematic of the TS-IL15 αCD19 CAR vector used to transduce primary human T cells prior to transfer into tumor-bearing NSG mice. [Figure 13E] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13E) CD19+K562 tumor growth curves after transfer of TS-IL15αCD19 CAR T cells on day 0 and heat treatment on days 2, 6, 9, 13, and 16 (ns=not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean±SEM shown, n=7 biologically independent mice). Three independent experiments were performed with similar results. [Figure 13F] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13F) Survival curves of tumor-bearing mice after transfer and heat treatment of TS-15 αCD19 CAR T cells (Figure 13D) and (Figure 13E) (**p<0.01, log-rank (Mantel-Cox) test with correction for 6 multiple comparisons, n=7 biologically independent mice). [Figure 13G]Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13G) Schematic of TS-IL15 vector transduced into primary murine Pmel-1 T cells transferred into tumor-bearing C57BL / 6J mice. [Figure 13H] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13H) Tumor growth curves after transfer of TS-IL15 Pmel-1 T cells on day 0 and heat treatment on days 1 and 3 after inoculation with B16-F10 (ns=not significant, ***P<0.001, two-way ANOVA with Tukey post-hoc test and correction, mean±SEM shown, n=6-7 biologically independent mice). [Figure 13I] Figures 13A-13I show that photothermal control of IL-15 SA enhances adoptive T cell transfer and overall survival in mice. (Figure 13I) Survival curves of tumor-bearing mice after transfer and heat treatment of TS-15 Pmel-1 T cells in (Figure 13G) and (Figure 13H) (**p<0.01, log-rank (Mantel-Cox) test with correction for 6 multiple comparisons, n=7 biologically independent mice). [Figure 14] Figure 14 shows that cytokine maintenance improves proliferation of T cells receiving low levels of CD3 / 28 stimulation. T cells were labeled with CFSE and incubated with low levels of activation beads. For reference, routine expansion and culture of T cells uses 3 beads per T cell. Increasing amounts of IL-2 were added to each bead ratio. All samples were assayed after 4 days of incubation in the indicated conditions. Two independent experiments were performed with similar results. [Figure 15] Figure 15 shows the gating strategy for the mixed proliferation experiment. Transduced TS-15 αCD19 CAR T cells were identified by CAR expression. Proliferation of CFSE+ wild type cells was assessed by dye dilution and the FlowJo proliferation tool. [Figure 16A]Figures 16A-16B show characterization of engineered Pmel-1 T cells. 48 hours after isolation and peptide activation, Pmel-1 derived splenocytes were transduced with a TS-IL15 vector containing a constitutive GFP reporter. (Figure 16A) Pmel-1 T cells were characterized via flow cytometry prior to adoptive transfer to assess pure expansion of CD8+ cells (left) and transduction efficiency (right) (US = unstained Pmel-1 T cells, WT = wild type Pmel-1 T cells). [Figure 16B] Figures 16A-16B show characterization of engineered Pmel-1 T cells. 48 hours after isolation and peptide activation, Pmel-1 derived splenocytes were transduced with a TS-IL15 vector containing a constitutive GFP reporter. (Figure 16B) IL-15 production from transduced murine T cells measured via ELISA after heating for 20 minutes at the indicated temperatures (***P<0.001, unpaired t-test, mean±SEM shown, n=3 biologically independent wells). [Figure 17A] 17A-17B show that engineered Pmel-1 T cells enhance adoptive cell therapy in a high tumor burden setting. (FIG. 17A) Schematic of a large B16-F10 tumor-bearing C57BL / 6J mouse. [Figure 17B] Figures 17A-17B show that engineered Pmel-1 T cells enhance adoptive cell therapy in a high tumor burden setting. (Figure 17A) Schematic of a large tumor B16-F10-bearing C57BL / 6J mouse. (Figure 17B) Tumor growth curves following B16F10 inoculation, transfer engraftment of engineered murine T cells on day 0, and heat treatment on days 1, 3, and 5 (*P<0.05, **P<0.01, two-way ANOVA and Tukey post-hoc test and correction, mean ± SEM shown, n=6-7 biologically independent mice). [Figure 18A] Figures 18A-18I show enhanced CAR T cell targeting via heat-induced BiTEs. (Figure 18A) Schematic of heat-induced BiTEs or TS-BiTEs and TS-Fluc thermoswitches containing Fluc reporters. Both constructs contained the constitutive αCD19 CAR. [Figure 18B]Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18B) Histograms of HisTag flow staining in TS-BiTE and TS-Fluc primary T cells after heating. [Figure 18C] Figures 18A-18I show heat-induced BiTE-mediated enhanced CAR T cell targeting. (Figure 18C) NKG2DL flow staining on primary human T cells and K562 using NKG2D-Fc chimera and αFc-A488 secondary antibody. Stain=full staining, 2° Ctrl=secondary antibody only. [Figure 18D] Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18D) Schematic showing BiTE-mediated targeting of K562 target cells lacking the CAR target antigen via BiTE binding to NKG2DL and CD3. [Figure 18E] Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18E) Flow gating strategy to define bystander cells based on CD19 CAR expression in Jurkat co-culture assays with K562. UTD controls were gated on the lower (CAR-) population for graphing in (Figure 18G). [Figure 18F] Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18F) Flow staining of CD69 on Jurkat T cells after heating and incubation with K562. TS-BiTE CAR+ histograms (f) and summary data (Figure 18G) of the indicated populations are graphed (statistics indicate comparison to UTD, ns = not significant, **P<0.01, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean + SEM shown, n = 3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 18G]Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18F) Flow staining of CD69 on Jurkat T cells after heating and incubation with K562. TS-BiTE CAR+ histograms (f) and summary data (Figure 18G) of the indicated populations are graphed (statistics indicate comparison to UTD, ns = not significant, **P<0.01, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean + SEM shown, n = 3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 18H] Figures 18A-18I show heat-induced BiTE-mediated expanded CAR T cell targeting. (Figure 18H) Cytokine concentrations in the supernatants of primary human T cells after heat treatment and incubation with K562 cells. T cells were either untransduced or transduced with the indicated heat switch (ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean+SEM shown, n=3 biologically independent wells). [Figure 18I] Figures 18A-18I show thermally induced BiTE-mediated expanded CAR T cell targeting. (Figure 18I) Cytotoxicity against K562 quantified by luciferase assay after incubation with primary human T cells. T cells were either untransduced or transduced with the indicated thermal switches (ns=not significant, **P<0.01, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, comparisons are to UTD control, mean+SEM shown, n=3 biologically independent wells). [Figure 19A] Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19A) Schematic of TS-BiTE and TS-Rluc αHER2 CAR vectors. [Figure 19B]Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19B) Flow cytometry quantification of activation markers CD69, PD-1, and CD107a by TS-BiTE αHER2 CAR T cells after 30 min of heating and co-culture with HER2- MDA-MB-468 target cells. (ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=4 biologically independent wells, MFI=median fluorescence intensity). [Figure 19C] Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19C) Tumor growth curves of MDA-MB-468 tumors inoculated at a 3:1 HER2+ to HER2- ratio treated with TS-BiTE or TS-Rluc αHER2 CAR T cells. Heat treatment was performed on days 45, 47, 52, 59, 66, and 72. (*P<0.05, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, mean ± SEM shown, n=6-7 biologically independent mice). [Figure 19D] Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19D) Spider plots of individual tumors with vertical dashed lines indicating thermal treatment. [Figure 19E]Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19E) In vivo luminescence imaging time course and (Figure 19F) individual spider plots acquired by IVIS Spectrum CT system representing HER2- / Fluc+ cell populations in a mixed MDA-MB-468 tumor model. Transfer of engineered human T cells occurs on day 44 and heat treatment on days 45, 47, 52, 59, 66, and 72. Grey background represents the mean ± 2 standard deviations (n=6) above and below background measurements taken throughout the time course of the experiment. (ns=not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA and Tukey post-hoc test and correction, mean ± SEM shown, n=6-7 biologically independent mice). [Figure 19F] Figures 19A-19F show that photothermal control of TS-BiTE αHER2 CAR T cells mitigates the growth of antigen-negative tumors in vivo. (Figure 19E) In vivo luminescence imaging time course and (Figure 19F) individual spider plots acquired by IVIS Spectrum CT system representing HER2- / Fluc+ cell populations in a mixed MDA-MB-468 tumor model. Transfer of engineered human T cells occurs on day 44 and heat treatment on days 45, 47, 52, 59, 66, and 72. Grey background represents the mean ± 2 standard deviations (n=6) above and below background measurements taken throughout the time course of the experiment. (ns=not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA and Tukey post-hoc test and correction, mean ± SEM shown, n=6-7 biologically independent mice). [Figure 20A] Figures 20A-20C show validation of MDA-MB-468 transduced with HER2 or Renilla luciferase. (Figure 20A) Representative flow plots of NKG2DL and HER2 staining of HER2+ MDA-MB-468. MDA-MB-468 was transduced with lentivirus to achieve stable surface expression of HER2. [Figure 20B] Figures 20A-20C show validation of MDA-MB-468 transduced with HER2 or Renilla luciferase. (Figure 20B) Luminescence of Fluc-transduced HER2- MDA-MB-468 tumor cells (****P<0.0001, one-way ANOVA with Sidak post-hoc test and correction, mean±SEM shown, n=3 biologically independent wells). [Figure 20C] Figures 20A-20C show validation of MDA-MB-468 transduced with HER2 or Renilla luciferase. (Figure 20C) Percentage of cytotoxicity observed by LDH assay in HER2- or HER2+ MDA-MB-468 cells after incubation with T cells constitutively expressing CAR (****P<0.0001 between HER2- and HER2+ groups, two-way ANOVA and Sidak post-hoc test and correction, mean ± SEM shown, n=3 biologically independent wells). Two independent experiments were performed with similar results. [Figure 21-1] Figure 21 shows that TS-BiTE aHER2 CAR T cells are activated when incubated with HER2+ MDA-MB-468 cells. MFI of activation and degranulation markers CD69, PD-1, and CD107a in TS-BiTE aHER2 CAR T cells heated at the indicated temperatures prior to co-incubation with HER2+ MDA-MB-468 target cells. (ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=4 biologically independent wells). [Figure 21-2]Figure 21 shows that TS-BiTE aHER2 CAR T cells are activated when incubated with HER2+ MDA-MB-468 cells. MFI of activation and degranulation markers CD69, PD-1, and CD107a in TS-BiTE aHER2 CAR T cells heated at the indicated temperatures prior to co-incubation with HER2+ MDA-MB-468 target cells. (ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=4 biologically independent wells). [Figure 21-3] Figure 21 shows that TS-BiTE aHER2 CAR T cells are activated when incubated with HER2+ MDA-MB-468 cells. MFI of activation and degranulation markers CD69, PD-1, and CD107a in TS-BiTE aHER2 CAR T cells heated at the indicated temperatures prior to co-incubation with HER2+ MDA-MB-468 target cells. (ns=not significant, ****P<0.0001, two-way ANOVA with Tukey post-hoc test and correction, error bars indicate SEM, n=4 biologically independent wells). [Figure 22A] FIG. 22A shows a schematic diagram of IT injection of IL-2 in B16 tumors. [Figure 22B] Figure 22B shows tumor growth curves following ACT of Pmel CD8+ T cells. Six injections of iphIL-2 were administered at the doses indicated following ACT. [Figure 23] FIG. 23 shows that dendritic cells engineered with TS-IL15SA produce IL-15SA upon heat treatment at 42° C. for 30 minutes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The following description of the present disclosure is provided as an enabling teaching of the present disclosure in its best, currently known embodiment. To this end, those skilled in the art will recognize and understand that many changes can be made to the various embodiments of the present invention described herein and still obtain the beneficial results of the present disclosure. It will also be apparent that some of the desired advantages of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Thus, those skilled in the art will recognize that many modifications and adaptations to the present disclosure are possible and may even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as an illustration of the principles of the present disclosure, but is not limited thereto.

[0017] definition As used herein and in the claims that follow, reference will be made to a number of terms that are defined to have the following meanings.

[0018] As used in this 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 "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0019] As used herein, ranges can be expressed as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by use of the antecedent "about," the particular value forms another embodiment. It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein 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. As will be appreciated by those of skill in the art, when a value is disclosed, it is also understood that "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between the values ​​are also disclosed. For example, if the value "10" is disclosed, then "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and this data represents the end and beginning points, as well as ranges of any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than 10, less than 10, less than 10, and equal to 10, greater than 15, greater than 15, less than 15, less than 15, and equal to 15 are considered to be disclosed. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0020] As used herein and in the claims that follow, reference will be made to a number of terms that are defined to have the following meanings.

[0021] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur.

[0022] "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 statistically significant amount in a condition, symptom, activity, or composition. Thus, an 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.

[0023] "Reduction" can refer to any change that results in a lower amount of symptoms, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product containing the substance is less compared to the output of the gene product without the substance. Also, for example, a reduction can be a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual value, median, or average reduction in a statistically significant amount in a condition, symptom, activity, composition. Thus, a reduction 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% reduction, as long as the reduction is statistically significant.

[0024] "Inhibit", "inhibiting", and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0025] "Reduce" or other forms of this term, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means reducing the rate of growth of a tumor compared to a standard or control.

[0026] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction, and does not require comparison to a control. As used herein, some things can be reduced, but not prevented, and some things can be reduced and prevented. Similarly, some things can be prevented, but not reduced, and some things can be prevented and reduced. It is to be understood that when reduction or prevention is used, the use of the other words is expressly disclosed unless specifically specified otherwise.

[0027] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one embodiment, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0028] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to alleviate one or more causes or symptoms of a disease or disorder. Such alleviation requires only a reduction or alteration, not elimination.

[0029] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, condition or disorder, and also includes causal treatment, i.e., treatment directed to eliminating the cause of the associated disease, condition or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms but not cure the disease, condition or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed to ameliorating the associated disease, condition or disorder.

[0030] "Biocompatible" generally refers to a material and any metabolic or breakdown products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects in the subject.

[0031] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements but excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from the isolation and purification methods and pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0032] A "control" is a substitute control or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0033] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the age and general condition of the subject, the specific drug(s), and the like. Thus, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" in any subject's case can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug can refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as dictated by the exigencies of the therapeutic situation.

[0034] A "pharmaceutical acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., an ingredient that can be incorporated into a pharmaceutical formulation provided by the present disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is included. When used in relation to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing or that it is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0035] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally means a carrier or excipient useful in the preparation of a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0036] "Pharmacologically active" (or simply "active") can refer to a derivative or analog (e.g., a salt, ester, amide, complex, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound, and to approximately the same extent, in a "pharmacologically active" derivative or analog.

[0037] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0038] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a drug) refers to an amount effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is control of type I diabetes. In some embodiments, the desired therapeutic outcome is control of obesity. The therapeutically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The exact desired therapeutic effect will vary according to the condition being treated, the tolerability of the subject, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), as well as various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after administration of multiple doses of the composition over a period of days, weeks, or years.

[0039] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the sentence in which they are relied upon, and are individually and specifically incorporated by reference herein for the material contained therein.

[0040] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the sentence in which they are relied upon, and are individually and specifically incorporated by reference herein for the material contained therein.

[0041] composition Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a particular promoter construct or modified CAR T cell is disclosed and discussed, and several modifications that can be made to several molecules, including the promoter construct or modified CAR T cell, each and every combination and permutation of the promoter construct or modified CAR T cell, and modifications that are possible unless specifically indicated to the contrary, are specifically contemplated. Thus, when classes of molecules A, B, and C, as well as classes of molecules D, E, and F, and AD as an example of a combination molecule, are disclosed, each is individually and collectively contemplated, even if each is not individually listed, i.e., AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0042] It is understood that the compositions disclosed herein have a specific function. There are disclosed herein specific structural requirements for performing the disclosed function, and it is understood that there are various structures that can perform the same function related to the disclosed structure, and that these structures will ultimately achieve the same result.

[0043] Unless otherwise expressly stated, it is never intended that any method set forth herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed or specifically states in the claims or description that the steps are to be limited to a particular order, no order is ever intended to be inferred in any respect. This holds true for any possible implicit basis for interpretation, including the obvious meaning derived from the arrangement of steps or operational flow, grammatical construction or punctuation, and logical matters regarding the number or type of aspects described in the specification.

[0044] Emerging strategies to control engineered T cells and enhance their antitumor activity include the use of biomaterials to codeliy adjuvants to the TME, as well as genetic constructs for autonomous expression of immune-stimulating genes. For example, implantation of a biopolymer scaffold loaded with tumor-specific T cells and immune-stimulating adjuvants at the surgical site improved postoperative responses after primary tumor resection in a mouse model. To provide a localized source of adjuvant, T cells tethered to nanoparticle “backpacks” at their cell surface allowed infiltrating T cells to carry cargo and release a one-time dose of drug within the tumor. Increasingly sophisticated genetic circuits are also enabling T cells to locally produce biologics to overcome immune suppression or target antigens after tumor infiltration. For example, “armored CARs” leverage constitutive expression of biologics such as IL-12, αPD-1 scFv, and BiTEs to improve antitumor activity. T cells have also been engineered with sense-and-response biological circuits that conditionally activate in the presence of specific input signals. These strategies include NFAT-inducible cassettes that upregulate cytokine expression following T cell recognition of tumor-associated antigens. To further increase specificity, T cells have been engineered to target unique combinations of epitopes expressed in the TME, allowing differentiation from healthy cells expressing single epitopes. Such approaches, based on Boolean logic, require the presence of both target antigens for T cell activation and have demonstrated efficacy in multiple models of localized tumors. Taken together, these approaches demonstrate the need to develop strategies to control and improve intratumoral T cell activity.

[0045] Thus, in one aspect, disclosed herein is a promoter construct comprising: a) one or more heat shock elements (such as, for example, the heat shock element set forth in SEQ ID NO:1); b) a core promoter; and c) a gene of interest.

[0046] A heat shock element is a cis-acting regulatory motif that mediates the transcriptional response of a target gene when exposed to heat. An example of a heat shock element is nGAAnnTTCnnGAAn (SEQ ID NO: 1). In some embodiments, n=A, T, C, or G. A heat shock element can be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times. For example, in some embodiments, a heat shock element comprises 7 repeats of SEQ ID NO: 1. Example sequences for one or more heat shock elements are provided in Table 1. In some embodiments, one or more heat shock elements comprise or consist of the nucleotide sequence of any one of SEQ ID NOs: 2-9. [Table 1]

[0047] In some embodiments, the core promoter comprises a heat shock protein transcription start site. Such heat shock protein transcription start sites are known in the art and can include, but are not limited to, heat shock protein transcription start sites of HSPA1A, HSPH1, HSPB1, HSP6, HSP70, HSPA6, or YB. In some embodiments, the core promoter comprises a core promoter of heat shock proteins HSPA1A, HSPH1, HSPB1, HSP6, HSP70, HSPA6, or YB. Examples of core promoter sequences are provided in Table 2. In some embodiments, the core promoter sequence comprises or consists of any one of SEQ ID NOs: 10-13. [Table 2]

[0048] Examples of one or more heat shock element sequences and YB core promoter sequences together are provided in Table 3. In some embodiments, the one or more heat shock element sequences and the core promoter sequence together comprise a set of sequences set forth in any one of SEQ ID NOs: 14-21. [Table 3]

[0049] The use of heat shock elements allows for selective transcriptional control of a gene of interest such that the gene is only activated upon application of heat within a desired temperature range. Thus, for example, promoter constructs are disclosed herein that are capable of activating at temperatures between 40° C. and 45° C. (e.g., 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 45.5, 45.6, 45.8, 45.9, 46.1, 46.1, 46.2, 46.3 The invention may require heat activation at temperatures between 40°C and 42°C, or between 41°C and 43°C, or between 42°C and 45°C, including but not limited to 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0°C. In some embodiments, the promoter is at least 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48. 4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0°C).

[0050] The gene of interest used in the constructs of the present disclosure can be a reporter gene, an immunomodulator, a bispecific T cell engager (BiTE), a chimeric antigen receptor (CAR), a recombinant TCR, or any combination thereof.

[0051] BiTE refers to a bispecific fusion protein, which refers to a single chain protein consisting of two linked scFvs, one targeting T cells (CD3) and the other targeting a tumor cell antigen. Examples of BiTE molecules include those that contain an anti-CD3 binding domain and an NKG2D receptor extracellular domain, an anti-CD3 binding domain and an anti-EGFRvIII binding domain, and an anti-CD3 binding domain and an anti-CD19 binding domain.

[0052] Reporter genes are known in the art and can include any gene whose transcription and / or translation can be easily assayed following transfection. Examples of reporter genes for use in the disclosed promoter constructs include, for example, luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, Photoactivated GFP (PA-GFP), Venus, Kaede, Monomeric Kusabira Orange (mKO), Dronpa, Enhanced CFP (ECFP), Emerald, Energy-transfer Cyan Fluorescent Protein (CyPet), Super CFP (SCFP), Cerulean, Photoswitchable CFP (PS-CFP2), Photoactivated RFP1 (PA-RFP1), Photoactivated mCherry (PA-mCherry), Monomeric Teal Fluorescent Protein (mTFP1), Eos Fluorescent Protein (EosFP), Dendra, TagBFP, TagRFP, Enhanced YFP (EYFP), Topaz, Citrine, Energy-transfer Yellow Fluorescent Protein (YPet), Super YFP (SYFP), Enhanced GFP (EGFP), Superfolder These include GFP, T-Sapphire, Fucci, mKO2, mOrange2, mApple, Sirius, Azurite, EBFP, and / or EBFP2.

[0053] In some embodiments, the gene of interest can be an immunomodulatory agent, such as, for example, a chemokine, a cytokine, an interferon, a cytotoxin (including, but not limited to, perforin and / or granzyme), or any combination thereof. Examples of chemokines that can be used in the disclosed promoter constructs include, but are not limited to, CCL2, CCL1, CCL19, CCL22, CXCL12, CCL17, MIP-1α, MCP-1, GRO / KC, CXCL2, CXCR3, or any combination thereof. Cytokines that can be used in the disclosed promoter constructs include, but are not limited to, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-γ, TNF-α, TGF-β, LIF, or any combination thereof. In some embodiments, the cytokine is an IL-15 superagonist molecule. An example of an IL-15 superagonist molecule is ALT-803, which is a multimeric complex composed of the IL-15 N72D:IL-15Rα sushi domain fused to an IgG1 Fc domain.

[0054] Chimeric antigen receptors (CARs) are transgenic receptors expressed by T cells (CAR T cells) or NK cells (CAR NK cells) that target the T cells or NK cells to cells that express a ligand for the receptor. Such chimeric antigen receptors are typically membrane-bound single chain variable regions of immunoglobulins specific for a target. CAR targets include, but are not limited to, CD19, B cell maturation antigen (BCMA), CD22, CD33, CD38, NCAM1, CD5, CD70, MET, Muc1, L1CAM, CD44 SLAMF7, EGFR, EPHA2, HER2, mesothelin, GPC3, or PDCD1.

[0055] Recombinant T cell receptor (also called engineered TCR) refers to a TCR used to engineer T cells with a desired specificity, such as a tumor antigen. Examples of TCR targets include, but are not limited to, WT1, HPVE6, HPVE7, NY-ESO-1, HA-1, MAGE, Gp100, MART-1, HBV, p53, CEA, SL9, TGFβ11, TRAIL, MCPyV, PRAME, EBV, CMV, and KRAS.

[0056] The promoter constructs of the present disclosure may also include intervening nucleotides between the recited components. The linking nucleotides may be natural or non-natural (e.g., due to construct design). For example, the linking nucleotides may result from a restriction enzyme site used to link one domain to another or to clone a polynucleotide into a vector.

[0057] Examples of promoter constructs of the present disclosure are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0058] In some aspects, the disclosure provides a vector comprising a promoter construct according to any one of the embodiments disclosed herein. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, a cosmid, a virus, or a phage. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of a nucleic acid into a cell. An "expression vector" is a vector that, when present in the appropriate environment, can direct the expression of a protein encoded by one or more genes carried by the vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, gamma retroviral vectors, and lentiviral vectors. A "retrovirus" is a virus that has an RNA genome. A "gamma retrovirus" refers to a genus of the Retroviridae family. Examples of gamma retroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentivirus" refers to a genus of retroviruses that can infect dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types 1 and 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0059] It is understood and contemplated herein that the disclosed promoter constructs are particularly useful for creating adoptive immunotherapies (e.g., T cells) where the therapeutic effect is limited to the site where heat is applied, thereby preventing off-site expression and cytotoxicity. Accordingly, disclosed herein are immune cells comprising the promoter constructs or vectors disclosed herein. In some embodiments, the immune cells are T cells, natural killer (NK) cells, or dendritic cells. In some embodiments, the T cells are CD4+ T cells or CD8+ T cells. In some embodiments, the T cells comprise a recombinant TCR. In some embodiments, the immune cells comprise a CAR. In some embodiments, the immune cells are chimeric antigen receptor (CAR) T cells and / or CAR NK cells. For example, disclosed herein are CAR T, CAR NK cells, or recombinant TCR T cells comprising a promoter construct comprising a) one or more heat shock elements (such as the heat shock element set forth in SEQ ID NO:1), b) a core promoter, and c) a gene of interest. In some embodiments, the gene of interest encodes a chimeric antigen receptor, a recombinant TCR, an immunomodulatory agent, or any combination thereof.

[0060] It is understood and contemplated herein that the disclosed promoter constructs can be applied to cells to generate immunotherapy against a target. In one embodiment, disclosed herein is a kit comprising any of the promoter constructs disclosed herein, and further comprising a heating component that activates the promoter construct. In some embodiments, the heating component is coupled to a light source (e.g., a laser (e.g., a laser emitting light at 700 nm to about 1400 nm, e.g., 705, 730, 735, 760, 783, 785, 792, 793, 797, 808, 825, 830, 850, 852, 850, 860, 878, 880, 885, 888, 891, 900, 905, 915, 938, 940, 946, 960, The laser may be a near infrared laser, such as, but not limited to, a laser emitting at 975, 976, 980, 1030, 1040, 1053, 1064, 1123, 1177, 1210, 1280, 1300, 1317, 1319, and / or 1370 nm), a filament, an infrared emitting light source, or a light emitting diode (LED), a thermal pad, or a thermally regulated needle, probe, or scalpel.

[0061] How to Treat Cancer The disclosed promoter constructs, vectors, and immune cells (e.g., recombinant TCR T cells, CAR T cells, and / or CAR NK cells) comprising the promoter constructs can be used to treat any disease, such as cancer, in which uncontrolled cell proliferation occurs. A representative, but non-limiting list of cancers that can be treated using the compositions of the present disclosure are: sarcoma, blastoma, lymphomas, such as B-cell lymphoma and T-cell lymphoma; mycosis fungoides; Hodgkin's disease; myeloid leukemia (including, but not limited to, acute myeloid leukemia (AML) and / or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of the head and neck; kidney cancer; small cell lung cancer, non-small cell lung cancer, and other cancers. lung cancer, such as non-small cell lung cancer (NSCLC), lung squamous cell carcinoma (LUSC), and lung adenocarcinoma (LUAD); neuroblastoma / glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; liver cancer; melanoma; squamous cell carcinoma of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer, including but not limited to triple-negative breast cancer; genitourinary cancer; lung cancer; esophageal cancer; head and neck carcinoma; colorectal cancer; hematopoietic cancer; testicular cancer; and colon and rectal cancer.

[0062] Disclosed herein are methods of treating, lowering, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., solid tumors including, but not limited to, epithelial cancers, sarcomas, carcinomas, etc.), the methods comprising administering to the subject any of the promoter constructs, vectors, immune cells (e.g., recombinant TCR T cells, CAR T cells, or CAR NK cells), and / or utilizing any of the kits disclosed herein. For example, disclosed herein are methods of treating, lowering, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., solid tumors, including, but not limited to, epithelial carcinoma, sarcoma, lymphoma, blastoma, or melanoma), the methods including: i) administering to the subject thermo-regulated immune cells (e.g., recombinant TCR T cells, CAR T cells, or CAR T cells) comprising a promoter construct comprising a) one or more heat shock elements (e.g., the heat shock element set forth in SEQ ID NO:1); b) a core promoter (e.g., a core promoter comprising a heat shock protein transcription start site encoding HSPA1A, HSPH1, HSPB1, HSP6, HSP70, HSPA6, or YB); and c) a gene of interest (e.g., a reporter gene, an immunomodulator, a bispecific T cell engager (BiTE) (e.g., an anti-CD3 binding region and an NKG2D receptor extracellular domain), a recombinant TCR, a chimeric antigen receptor (CAR), or any combination thereof). NK cells, etc.) and ii) inducing activation of immune cells at the site of the tumor (e.g., incubating immune cells at 40°C to 45°C (40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.1, 46.2, 45.4, 45.5, 45.6, 45.8, 45.9, 4 40.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0, andIn some embodiments, the promoter is at least 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48. (The promoter construct may be activated by heating to a temperature of 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, or 45.0° C.) In some embodiments, heating can be achieved by applying a heating component to activate the promoter construct. In some embodiments, the heating component is a light source (e.g., a laser (e.g., a laser emitting light at 700 nm to about 1400 nm, e.g., 705, 730, 735, 760, 783, 785, 792, 793, 797, 808, 825, 830, 850, 852, 850, 860, 878, 880, 885, 888, 891, 900, 905, 915, 938, 940, 946, 960, The laser may be a near infrared laser, such as, but not limited to, a laser emitting at 975, 976, 980, 1030, 1040, 1053, 1064, 1123, 1177, 1210, 1280, 1300, 1317, 1319, and / or 1370 nm), a filament, an infrared emitting light source, or a light emitting diode (LED), a thermal pad, or a thermally regulated needle, probe, or scalpel.

[0063] In some embodiments, the method can further include administering an additional anti-cancer agent or immunotherapy, including but not limited to a checkpoint inhibitor such as anti-PD-1 immunotherapy, anti-PD-L1 immunotherapy, anti-CTLA-4 immunotherapy. Immunotherapies targeting immune checkpoint molecules can be antibodies or antigen-binding fragments thereof, or antibody fusion proteins.

[0064] It is understood and contemplated herein that the therapeutic regimens disclosed herein can be used alone or in combination with any anti-cancer therapy known in the art, including, but not limited to, abemaciclib, abiraterone acetate, avitrexate (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), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alumbrig (brigatinib), Ambochlorin (chlorambucil), Ambochlorin clorambucil), amifostine, aminolevulinic acid, anastrozole, aprepitant, Aredia (pamifostine), disodium dronate), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, bec Sar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Boslif (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, Carmbrys (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cervizin (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabi) (Clofarabine), CMF, cobimetinib, Cometrik (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyphos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin Shin, 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), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Eliedge (vismodegib), erlotinib hydrochloride,Erwinase (asparaginase erwinia chrysanthemi), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Everset (doxorubicin hydrochloride liposome), everolimus, Evista, (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fareston (toremifene), Faridak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole) ), Filgrastim, Fludara (Fludarabine phosphate), Fludarabine phosphate, Fluoroplex (Fluorouracil - topical), Fluorouracil injection, Fluorouracil - topical, Flutamide, Forex (methotrexate), ForexPFS (methotrexate), Forfiri, Forfiribevacizumab, Forfirisetuximab, Forfirinox, Forfox, Forothin (pralatrexate), FU-LV, Fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine) chin), Gadiva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Glivec (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 9-valent vaccine, recombinant, HPV 4-valent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper CVAD, Ibrance (palbociclib), ibritumomab tiucetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelarib, Idifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidam (ifosfamide), IL-2 (aldesleukin),Imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imligic (talimogene laherparepvec), Inrita (axitinib), inotuzumab ozogamicin, interferon alfa-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), iodine I131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixane Pura (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlucrucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Raltruvo (olatumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), Leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolidine (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Leupron (leuprolide acetate), Lupron Depo (leuprolide acetate), Lupron Depo-Ped (leuprolide acetate), Lynparza (olaparib), Marchibo (vincristine sulfate liposome), Matulan (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), Mustagen (Mechlorethamine hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulf , Myrosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupisant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, nilutamide , Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepecacide, Oncaspar (pegaspargas), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, 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 (per tuzumab), pertuzumab, Platinol (cisplatin), Platinol AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portolazza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Prinethol (mercaptopurine), Prixan (mercaptopurine), radium dichloride 2 23, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Liristor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hysera (rituximab) mab and hyaluronidase human), rituximab, rituximab and hyaluronidase human,, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, redapt (midostaurin), sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel T, somatilin depot (lanreotide acetate), sonidegib, sorafenib tosylate, sprycel (dasatinib), stanford V, sterile talc powder (talc),Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Silatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxine mepeccate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, Talimogene laherparepvec, tamoxifen citrate, TarabinPFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (ni Lotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenleucel, Trac (fluorouracil-topical), topotecan hydrochloride, toremifene, Tolicel (temsirolimus), tositumomab and iodine I131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Trianda (Benda mustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tycarb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), bi Darza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Bistogard (uridine triacetate), Boraxase (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Zygeva (denosumab),These include, but are not limited to, Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (dibuflibercept), Zalcio (filgrastim), Zejula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiusetan), Zinecard (dexrazoxane hydrochloride), dibu-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelaryb), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Therapeutic methods may include or further include checkpoint inhibitors, such as PD-1 (e.g., nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, MK-3475, etc.), PD-L1 (e.g., atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010 718C, etc.), PD-L2 (e.g., rHIgM12B7, etc.), CTLA-4 (e.g., ipilimumab (MDX-010), tremelimumab (CP-675,206), etc.), IDO, B7-H3 (e.g., MGA271, MGD009, omburtamab, etc.), B7-H4, B7-H3, Ig and ITIM domain-containing T cell immunoreceptor (TIGIT) (e.g., BM S-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO, etc.), CD96 and T lymphocyte attenuator (BTLA), V domain Ig suppressor of T cell activation (VISTA) (e.g., JNJ-61610588, CA-170, etc.), TIM3 (e.g., TSR-022, MBG4 53, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661, etc.), LAG-3 (e.g., BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep),These are not limited to: EXAMPLES

[0065] To further illustrate the principles of the present disclosure, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely illustrative of the present invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., amounts, temperatures, etc.), however, some errors and deviations should be accounted for. Unless otherwise specified, temperatures are in °C or are at ambient temperature, and pressures are at or near atmospheric pressure. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation is required to optimize such process conditions.

[0066] Example 1 result Manipulating a heat-specific gene switch The cellular response to elevated body temperature is mediated by the trimerization of the temperature-sensitive transcription factor heat shock factor 1 (HSF1) and its subsequent binding to HSEs. HSEs consist of multiple inverted repeats of the consensus sequence 5'-nGAAn-3', which are arranged upstream of the transcription start sites of heat shock proteins (HSPs), allowing their upregulation after heat stress. The response of endogenous HSP genes is selective to heat but not specific, as their promoters contain additional regulatory elements (e.g., hypoxia response elements, metal response elements) that mediate transcription after exposure to a diverse set of cues, including hypoxia, heavy metals, and mechanical forces. Furthermore, differences in the core promoters (e.g., initiator elements, TATA boxes) affect the composition of the preinitiation complex (PIC) and its interaction with transcriptional enhancers, including HSF1, leading to distinct heat responses across tissues and cell types. Due to this complexity and cross-activation with non-heat responsive pathways, the synthetic gene switch is activated by heat but not by other stress sources.

[0067] Six candidate constructs containing 2–7 repeats of the HSE motif 5'-nGAAnnTTCnnGAAn-3' upstream of the HSPB1 core promoter were cloned into Jurkat T cells (denoted 2H-B1–7H-B1, Fig. 1a). The HSPB1 core promoter was initially selected because its parent gene was one of two upregulated >20-fold at 42°C in primary murine T cells, relative to over 80 HSP and HSP-related genes that did not respond to heat treatment (Fig. 2). Selecting a core promoter from an endogenous gene with high thermoresponsiveness would promote transcriptional activity when integrated with HSE repeats. To quantify the response of the thermoswitch, transduced Jurkat T cells were transiently heated to 3–5°C above body temperature (i.e., 40–42°C), a mild temperature range in contrast to the temperature range used for ablation therapy (>50°C). 25Compared to the control samples at 37 °C, increased expression of the reporter Gaussia luciferase (Gluc) was observed upon increasing temperature and number of HSEs (Fig. 1b). Constructs containing 5 to 7 HSE repeats (5H-B1–7H-B1) yielded significantly higher thermal responses compared to those with 2 to 4 HSEs (2H-B1–4H-B1) (Fig. 1c).

[0068] To test the thermal response in primary human T cells, T cells were transduced with the 7H-B1 construct and peak thermal activity was observed approximately 6 h after heating at temperatures above 40 °C (Fig. 1d). The HSPB1 core promoter was initially selected from a screen of murine T cells, so the thermal response in primary human T cells is further dependent on the core promoter sequence. Therefore, based on previous studies, core promoters from the human HSPA6 gene (A1A, A6, B1) (Fig. 2), and a synthetic core promoter (YB), were identified and compared in a qPCR screen. Among the panel, the 7H-YB construct produced the highest increase in Gluc reporter levels after 30 min at 42 °C, corresponding to an approximately 60-fold increase in activity (Fig. 1e). Basal activity at 37 °C remained statistically identical to the non-transduced control, and negligible activation was observed at temperatures between 37 and 40 °C, corresponding to a 24-h fever range (Fig. 1f). Heat activation of 7H-YB was further validated in T cells from three separate donors, confirming the lack of donor dependency (Fig. 1g, Fig. 3). Based on this data, 7H-YB was selected for subsequent experiments.

[0069] We tested thermal specificity using hypoxia and heavy metal toxicity as two representative non-thermal stresses. As a benchmark, we compared with the endogenous HSPA6 or HSP70 promoters, which are highly stress-inducible and have been used previously for thermal control of gene expression. The gene switch was tested by incubating transduced primary human T cells and Jurkat T cells with the hypoxia mimetic CoCl2, a stabilizer of hypoxia-inducible factor-1α (HIF-1α), a master regulator of the hypoxic response, and the heavy metal complex cadmium chloride (CdCl2), which accumulates in the body through dietary or environmental exposure. While the HSP70 or HSPA6 promoters showed dose-dependent activation by hypoxia and cadmium toxicity in primary human T cells or Jurkat T cells, respectively (Fig. 1h, i; Fig. 4), 7H-YB was not activated and remained statistically identical to untransduced (UTD) up to concentrations (1000 mM CoCl2 and 1000 μM CdCl2) that are beyond the ranges commonly used to test cellular responses to hypoxia and cadmium. These results indicate that these constructs have increased thermal specificity compared to endogenous HSPs when exposed to non-thermal stress.

[0070] Primary T cells maintain primary functions following heat treatment Second, a thermal delivery profile was identified that was well tolerated by primary T cells without affecting their critical functions, including proliferation, migration, and cytotoxicity. Thermal medicine involves heating the target site to temperatures above 50°C to locally remove tissue by inducing tumor cell apoptosis and coagulation necrosis. In contrast, mild hyperthermia therapy (40-42°C) is used to promote the delivery of small molecules, such as in hyperthermic intraperitoneal chemotherapy (HIPEC), where abdominal infusion of heated chemotherapy serves as an adjuvant treatment after surgical debulking in patients with advanced ovarian cancer. At temperatures below 45°C, transient exposure to mild hyperthermia is well tolerated by cells and tissues due to induction of stress response pathways, including HSPs. In addition, T cell responses to continuous and fractionated heat treatments were also considered. Dose fractionation is commonly used in radiation therapy to reduce damage to normal tissues while maximizing the effect of radiation on cancer. Based on previous observations that trains of heat pulses increased Jurkat T cell resistance compared with continuous heat treatment with identical treatment areas under the curve (AUC), the effect of heat dose fractionation on primary T cells was further investigated.

[0071] Pulsed heat treatments at 67% duty cycle, consisting of three individual heat pulses (5 or 10 min each) separated by rest periods (2.5 or 5 min each) at 37 °C, were compared to their non-split counterparts (15 or 30 min continuous heating) (Figure 5a). In primary human T cells transduced with 7H-YB Gluc vector, pulsed heat treatments resulted in significantly higher reporter expression, up to approximately 87%, compared to continuous delivery at 30 min AUC (Figure 5b). To assess T cell viability, cell death (PI) and apoptosis (Annexin V) markers were quantified, and significant improvements were observed in primary T cells subjected to pulse treatments for 30–60 min at 67% duty cycle (Figure 5c). In contrast, a high, approximately 33% decrease in T cell viability was observed in samples subjected to continuous heat treatments for more than 40 min. A similar trend was observed in the dye (CTV) dilution T cell proliferation assay, where the percentage of proliferated T cells after incubation with CD3 / 28 beads was unaffected by both continuous and pulsed heating at 42 or 43°C for 30 min, whereas samples heated for 60 min resulted in reduced T cell proliferation (Figure 5d, Figure 6).

[0072] To probe chemotactic T cell migration, a transwell assay was used and it was observed that heat treatment (42°C for 30 min) had no significant effect on T cell migration to the bottom well containing the chemokine CXCL12, whereas T cells heated to 50°C were affected (Figure 5e). To test long-term activation, T cells were reheated over the course of 8 days and GFP mean fluorescence intensity (MFI) and GFP activation and decay half-life (t 1 / 2Similar increases in T cell response time were observed at 0.5 and 1 day, respectively, indicating that the magnitude and kinetics of the T cell response were unaffected by multiple heat treatments (Figure 7). To quantify the effect of heat on T cell cytotoxicity, primary human T cells expressing the αCD19 CAR under the constitutive EF1α promoter were incubated with either CD19+ or CD19- K562 containing a luciferase reporter, allowing quantification of cell death by loss of luminescence (Figure 5e). At all effector-to-target cell ratios tested (1:1, 5:1, 10:1), heated T cells maintained over 90% of the cytotoxicity observed in non-heated samples, whereas no significant differences in cytotoxicity were observed in samples containing CD19-K562 target cells (Figure 5f). Similarly, no statistical differences were observed in prolonged heat treatment, where αCD19 CAR T cells were heated four times over the course of 8 days prior to co-incubation with CD19+K562 cells (Figure 8). Taken together, these data demonstrate that primary human T cells maintain their ability to proliferate, migrate, and kill target cells after brief heat treatment delivered in continuous or pulsed waveforms of less than 30 min duration.

[0073] Photothermal activation of T cells in vivo Next, we sought to demonstrate spatially targeted activation of adoptively transferred T cells by photothermal heating. To locally heat tumors, plasmonic gold nanorods (AuNRs) were used as antennas to convert incident near-infrared (NIR) light (~650-900 nm) into heat. PEG-coated AuNRs are well-tested nanomaterials with long circulation periods that passively accumulate in tumors after intravenous administration. To confirm photothermal heating and thermal switch activation, primary T cells transduced with 7H-YB Fluc (TS-Fluc) αCD19 CAR were co-incubated with AuNRs in 96-well plates and irradiated with 808 nm laser light. In wells that reached 40–45 °C as monitored by a thermal camera, a significant increase in luminescence signal was observed after 6 h in the presence of TS-Fluc αCD19 CAR T cells, but not in wells containing non-transduced controls ( Figure 9 a), confirming plasmonic photothermal control of engineered T cells.

[0074] To perform photothermal targeting in vivo, NSG mice bearing bilateral flank tumors were inoculated with one cohort receiving CD19-K562 cells and another cohort receiving CD19+Raji cells to model CAR antigen negative and positive tumors, respectively (Figure 10). After intravenous injection of AuNRs and adoptive transfer of TS-Fluc αCD19 CAR T cells (Figure 9b), the tumors were irradiated with NIR laser light under the guidance of a thermal camera (Figure 9c) to maintain the target skin temperature (Figure 9d). After 20 minutes of heat treatment, luminescence increased more than 30-fold in Raji tumors receiving NIR light compared to unheated tumors in the same animals (Figure 9e), and similar to the in vitro experiments, thermal activation repeated twice over the course of 4 days did not result in loss of luminescence signal (Figure 11). In contrast, no increased luminescence was observed in K562 tumors treated with or without NIR light (Figure 9e). This lack of heat-induced activity was due to the absence of CD19 CAR antigen, and the density of intratumoral αCD19 CAR T cells was 20-fold lower in resected K562 tumors compared to CD19+Raji tumors (Figure 12). Although heat-induced expression of the transgene is spatially controlled by photothermal targeting, heat-activated T cells migrate from the tumor, thus resulting in off-target expression of the transgene. Therefore, in mice bearing bilateral CD19+Raji flank tumors, a single tumor site was heated and Fluc activity was quantified in the distal tumor and spleen. Luminescence in the heated tumor increased approximately 40-fold within 15 hours after heating, whereas non-heated tumor and spleen were statistically identical to baseline levels, indicating that transgene expression in TS-Fluc αCD19 CAR T cells was spatially restricted to the heated site (Figure 9f). Taken together, these data demonstrated photothermal control of intratumoral T cells engineered with a thermal gene switch.

[0075] Remote thermoregulation of IL-15 SA enhances adoptive T cell transfer We next investigated whether thermal control would enhance the efficacy of adoptive T cell therapy in vivo. To do this, we cloned a single-chain IL-15 superagonist (IL-15 SA), which contains a cytokine linked to the sushi domain of the IL-15Rα subunit under the control of our thermal vector (TS-IL15). IL-15 SA is a potent stimulator of CD8 T cells and NK cells, and the clinical candidate, ALT-803, is currently under investigation for a wide range of cancers. To test whether the thermally induced IL-15 SA was functionally active, we developed a T cell proliferation assay using CFSE-labeled wild-type T cells incubated with CD3 / 28 beads at a ratio of 10:1, without supplemental cytokines. This condition was found to be insufficient to induce T cell proliferation compared to conditions when cytokines such as IL-2 were present in the medium (Figure 14). Therefore, to test the heat regulation of IL-15 SA, heated or unheated TS-IL15 αCD19 CAR T cells were added to samples containing CFSE-labeled wild-type T cells with CD3 / 28 beads at a T cell to bead ratio of 10:1 (Figure 13a, Figure 15). Compared to the unheated control, CFSE-labeled T cells in the heated samples were found to expand with significantly higher proliferation and mitotic indices (Figure 13b), demonstrating that TS-IL15 αCD19 T cells are capable of producing physiologically active levels of IL-15 SA after a single heat treatment. To further characterize the heat effect of heat-induced secretion of IL-15 SA, conditioned medium was analyzed by ELISA and found that IL-15 SA levels increased with the duration and temperature of heat treatment (Figure 13c).

[0076] To explore the therapeutic effects of thermal targeting, tumors with an average size of 70 mm 3TS-IL15 αCD19 CAR T cells were adoptively transferred into NSG mice bearing CD19+K562 tumors when tumor volume was 100% (Fig. 13d). Photothermal heating of tumors was then performed every 3-4 days (days 2, 6, 9, 13, and 16) after ACT for a total of 5 treatments. Compared to control mice that did not receive CAR T cells or heat treatment (black), heat treatment of the tumor site alone did not result in a reduction in tumor burden or improved survival (grey) (Fig. 13e-f). Transfer of TS-IL15 αCD19 CAR T cells alone significantly reduced tumor burden (blue), but more than 85% (6 / 7) of animals reached euthanasia criteria within 39 days of ACT. In contrast, ACT of TS-IL15 αCD19 CAR T cells combined with NIR treatment significantly reduced tumor burden, and no animals reached euthanasia criteria within the study time frame.

[0077] Because NSG mice lack an intact immune system, this platform was further tested in immunocompetent C57BL / 6J mice bearing syngeneic B16-F10 melanoma tumors with transgenic TCR Pmel-1 T cells that recognize the melanoma autoantigen gp100 (Figure 13g). 25-33 Following peptide activation of Pmel-1 splenocytes with IL-15 SA, the CD8+ purity, transduction efficiency, and thermogenesis of IL-15 SA were verified (Figure 16). TS- IL15 Pmel-1 T cells were cultured at day 9 after lymphodepletion (approximately 52 mm 3 2 x 10 to 4 x 10 cells were adoptively transferred to a tumor volume of 1 x 10 (average tumor volume of 1 x 10 cells) and to expand the transferred cells. 5IU of IL-2 was given twice daily for three days. Under these conditions, two cycles of photothermal treatment were observed to result in significantly enhanced control of tumor growth (red) compared to cohorts of animals that received TS-IL15 Pmel-1 + IL-2 but no heat treatment (blue), heat treatment only (grey), or untreated (days 1 and 3 after ACT) (Figure 13h). All control mice reached euthanasia criteria within 33 days after ACT, whereas photothermal treatment of TS-IL15 Pmel-1 T cells resulted in significantly extended survival up to day 42 (Figure 13i). The experiments were performed on well-established, vascularized B16-F10 tumors (approximately 120 mm 3 The results were repeated with a mean tumor volume of 1000 ng / mL (mean tumor volume of 100 ng / mL) and similarly, significant improvement in tumor control in heat-treated mice was observed ( FIG. 17 ). These results are consistent with previous studies that showed that combined IL-2 and IL-15 improved antitumor activity compared to treatment with IL-2 alone. Taken together, these data demonstrated that photothermal control of IL-15 SA production by CAR or TCR engineered T cells significantly improved tumor control.

[0078] TS-BiTE αHER2 CAR T cells reduce antigen escape Heterogeneous expression of antigens can result in tumor escape from CAR T cells directed against a single antigen. Thus, NKG2K ligands (NKG2DLs)-a wide range of cancers as well as suppressor cells 70-73We sought to determine whether heat-induced expression of a bispecific T cell engager (BiTE) targeting NKG2DL-, which is upregulated in NKG2D, could alleviate antigen escape. We cloned a previously described NKG2DL-BiTE, which contains the CD3 recognition domain from the OKT3 antibody linked to the extracellular domain of the human NKG2D receptor. This vector (TS-BiTE) contained an Igκ leader sequence for BiTE secretion, a HisTag reporter, and a constitutive αCD19 CAR (Figure 18a). After heat treatment, TS-BiTE T cells were observed to stain positively with an anti-HisTag antibody compared to TS-Fluc control cells (Figure 18b). TS-BiTE T cells can undergo autocrine activation before the BiTE engages bystander T cells for paracrine activation. To test this, a mixture of TS-BiTE Jurkat T cells was heated with untransduced cells as bystanders prior to co-incubation with NKG2DL+CD19-K562 target cells (Fig. 18c-e) to isolate T cell activation by BiTE engagement without the confounding factor of CD19 CAR binding. Expression of the early activation marker CD69 in TS-BiTE Jurkat T cells was found to be significantly upregulated compared to bystander cells as heating times were extended (red vs. black) (Fig. 18f, g). In contrast, CD69 was minimally upregulated in bystander cells compared to untransduced (UTD) Jurkat T cells incubated with K562 cells and heated in a separate well as a control (black vs. grey). These data provided support that TS-BiTE T cells are activated primarily by an autocrine pathway. To quantify cytotoxicity from heat-induced expression of BiTEs, primary human TS-BiTE αCD19 CAR T cells were co-incubated with NKG2DL+CD19-K562 cells. In contrast to untransduced or TS-Fluc αCD19 CAR controls, TS-BiTE αCD19 CAR T cells expressed increasing levels of T h1 cytokines IFN-γ and TNF-α (Fig. 18h). A temperature-dependent increase in K562 cytotoxicity was observed at 37°C, but not at UTD controls, indicating a lack of BiTE-induced killing at basal temperature (Fig. 18i). These data demonstrated that TS-BiTE αCD19 CAR T cells can be redirected to target antigen-negative tumor cells expressing NKG2DL by thermal control.

[0079] To test the mitigation of antigen escape in vivo, we developed a heterogeneous model of breast cancer containing a mixture of HER2+ and HER2- MDA-MB-468 tumor cells. Endogenous expression of NKG2DL was verified in wild-type cells, which were transduced with either HER2 (Fig. 20a) or Fluc (Fig. 20b) to allow luminescent quantification of antigen-negative cells in vivo. We confirmed that both TS-BiTE or TS-Rluc αHER2 CAR T cells selectively targeted and killed HER2+ MDA-MB-468 cells (Fig. 19a, Fig. 20c). In contrast, targeting of HER2- cells required heat treatment of TS-BiTE αHER2 CAR T cells, as confirmed by the temperature-dependent increase in activation markers CD69, PD-1, and CD107a (Fig. 19b, Fig. 21). To test whether thermoregulation of NKG2DL BiTEs could treat tumors with heterologous antigen expression, NSG mice were inoculated with HER2+ and HER2- MDA-MB-468 cells at a 3:1 ratio and incubated at 44 days when tumors were well established and vascularized (mean volume ∼110 mm). 3) were transferred with TS-BiTE or TS-Rluc αHER2 CAR T cells (Fig. 19c). Approximately 40 days after ACT with prolonged heating (days 45, 47, 52, 59, 66, and 72), significant tumor regression was observed in mice treated with either TS-BiTE or TS-Rluc αHER2 CAR T cells, the latter due to killing of the HER2+ fraction of the tumor. However, by day 74, tumors in mice treated with TS-Rluc αHER2 CAR T cells began to recur compared to the TS-BiTE-treated cohort, resulting in tumors of an average of 12-fold larger volume by day 100. Tumors from 4 of 6 TS-BiTE mice and 1 of 6 TS-Rluc mice were undetectable by caliper measurement and palpation (Fig. 19d). To further support these results and determine whether recurrence could be attributed to proliferation of antigen-negative cells, tumor luminescence was quantified from HER2- cells, and a significant signal reduction was observed in the TS-BiTE compared to the TS-Rluc group by day 60, before tumor volume began to diverge (Fig. 19e). One mouse with residual disease was observed from both the TS-BiTE and TS-Rluc groups and was initially considered a complete response by caliper measurement, but had a luminescence signal above background defined as 2 standard deviations above the mean. Three of six TS-BiTE mice with nonpalpable tumors and luminescence within background levels for approximately 45 days were considered to be complete responses (Fig. 19f). These data demonstrate that thermal control of NKG2DL BiTEs has the potential to mitigate antigen escape in tumors with heterogeneous antigen expression.

[0080] Consideration The ability to better control engineered T cell activity within the tumor site has the potential to improve therapy against solid tumors. Thus, a platform for remote thermal control of T cell activity was developed. To provide T cells with the ability to respond to heat, a synthetic thermal gene switch was designed containing an array of heat shock elements upstream of the core promoter. This architecture eliminated sensitivity to non-thermal stresses such as hypoxia, and the thermal response was tunable based on the number of HSEs or different core promoters. Importantly, negligible activation of the thermal gene switch was observed at temperatures ≤40°C when T cells were incubated for >24 h, providing support that the temperature threshold for activation is higher than the typical range of fever (approximately 38-40°C) in patients with cytokine release syndrome (CRS), which would prevent activation of T cells without targeted thermal input.

[0081] Thermal control of T cell activity with IL-15 superagonist and NKG2DL BiTEs was demonstrated to enhance antitumor responses. Engineered T cells constitutively expressing similar classes of molecules have shown potent antitumor efficacy, but their therapeutic application is limited by off-tumor effects and toxicity in healthy tissues. Targeted expression of these genes within tumors would therefore contain potent T cell activity and improve therapeutic outcomes. Thermal induction of the transgene was shown to be transient and reversible, and it was found that thermally activated T cells remained localized at the heating site when transgene expression was on, reducing the possibility of off-target expression of the transgene. In K562 and syngeneic B16-F10 tumors, photothermal control of IL-15 SA expression by either αCD19 CAR or TCR transgenic Pmel-1 T cells resulted in enhanced antitumor activity compared to adoptive transfer of T cells alone. Remote thermal control of NKG2DL BiTEs was further demonstrated by targeting CAR T cells to antigen-negative tumor cells expressing NKG2D ligands, which mitigates antigen escape. In a mixed model of HER2+ and HER2- breast cancer, treatment with TS-BiTE αHER2 CAR T cells led to the elimination of well-established tumors without detectable residual disease in three of six mice, or significantly delayed recurrence compared to treatment with αHER2 CAR T cells targeting a single antigen. In light of these results, a broad range of biologics can be subjected to thermal control without potential loss of function due to protein misfolding or aggregation in T cells due to heat stress.

[0082] Finally, some of the conclusions from these studies are context-specific. For example, in vitro experiments showing that BiTE activation occurs primarily by an autocrine mechanism may be influenced by secretion rate, diffusion, and effector-to-bystander ratio, parameters that are tunable. Collectively, these results support remote thermal targeting of engineered T cell therapies to improve responses to solid tumors.

[0083] method Plasmid construction. Synthetic thermal switches were generated as gene blocks by IDT and cloned into Lego-C (Addgene Plasmid No. 27348) or pMKO.1 (Imgenex, San Diego, CA) backbones. Core promoters were truncated at their 5' end just upstream of their previously reported TATA box and at their 3' end at their translation start site. Genomic HSPA6 promoters were amplified from genomic DNA using PCR primers listed in a previous publication. The NKG2DL BiTE sequence (US20120294857A1) was previously reported and modified to include an Igκ leader sequence to facilitate secretion from T cells, as well as a HisTag for construct detection. This combined sequence was synthesized (ATUM) and cloned downstream of the synthetic thermal gene switch. The IL-15 superagonist sequence was previously reported and synthesized by ATUM without modification. The constitutive αCD19 CAR (US9499629B2) was kindly provided by Dr. Krishnendu Roy (Georgia Institute of Technology). The αHER2 CAR (US20180326032A1) was previously reported. 93 All proprietary material may be made available by the corresponding author upon reasonable request.

[0084] Culture of primary human T cells and cell lines. CD19+K562 (obtained from Dr. Yvonne Chen) and wild type K562 (obtained from Dr. Krishnendu Roy) were cultured in Isocove's modified Dulbecco's medium (ThermoFisher No. 12440053) supplemented with 10% FBS (Fisher No. 16140071) and 10 U / mL penicillin-streptomycin (Life Technologies No. 15140-122). Raji cells were obtained from Dr. Krishnendu Roy and cultured in RPMI-1640 medium supplemented with 10% FBS. MDA-MB-468 (ATCC, HTB-132) and B16-F10 (ATCC, CRL-6475) cells were cultured in Dulbecco's modified Eagle's medium (Gibco No. 11995073) supplemented with 10% FBS (Fisher No. 16140071) and 10 U / mL penicillin-streptomycin (Life Technologies No. 15140-122). Primary human CD3+ cells were obtained from anonymous donor blood after apheresis (AllCells) and cryopreserved in 90% FBS and 10% DMSO until further use. After thawing, cells were cultured in human T cell medium composed of X-VIVO10 (Lonza No. 04-380Q), 5% human AB serum (Valley Biomedical No. HP1022), 10 mM N-acetyl-L-cysteine ​​(Sigma No. A9165), and 55 μM 2-mercaptoethanol (Sigma No. M3148-100ML) supplemented with 50 units / mL human IL-2 (Sigma No. 11147528001). A total of seven donors were utilized for the experiments. Figure 1 used donors 1, 2, 6, and 7, Figure 5 used donors 2 and 3, Figure 9 used donor 2, Figure 13 used donor 4, and Figures 18 and 19 used donor 2.

[0085] Isolation and expansion of Pmel-1 T cells. Splenocytes from Pmel-1 transgenic mice were depleted of red blood cells using RBC lysis buffer (Biolegend No. 420302) and incubated with 1 μg / mL hgp100 25-33Splenocytes were cultured for 2 days in complete medium supplemented with 100 units / mL recombinant human IL-2 (Sigma No. 11147528001) in the presence of peptide (Tufts University Core Facility). Splenocytes were cultured at 10 × 10 cells in medium spiked with retrovirus at a multiplicity of infection of 10. 6 The cells were resuspended at 1 × 10 cells / mL and centrifuged at 2000 × g for 90 minutes. 6 Cells were cultured at a concentration of 100 cells / mL in complete medium supplemented with 100 units / mL of IL-2 and then transferred intravenously into C57BL / 6J mice 6 days after isolation.

[0086] Virus production and primary human T cell transduction. VSV-G pseudotyped lentivirus was produced via transfection of HEK293T cells (ATCC, CRL-3216) using psPAX2 (Addgene No. 12260) and pMD2.G (Addgene No. 12259), and viral supernatants were concentrated using PEG-it virus precipitation solution (System Biosciences LV825A-1) according to the manufacturer's instructions. Retrovirus was produced via transfection of HEK293T cells (ATCC, CRL-3216) using pCL-Eco and the pMKO.1 vector encoding the thermal switch circuit (TS-IL15) (Imgenex, San Diego, CA), and after 48 hours, viral supernatants were concentrated using Retro-Concentin retrovirus concentration reagent (System Biosciences RV100A-1) according to the manufacturer's instructions and frozen at -80°C. For viral transduction of primary human T cells, cells were thawed, incubated for 24 hours, and activated with human T-activator Dynabeads (Life Technologies No. 11131D) at a 3:1 bead:cell ratio for 24 hours. To transduce activated T cells, concentrated lentivirus was added to non-TC treated 6-well plates coated with Retronectin (Takara No. T100B) according to the manufacturer's instructions and spun at 1200 x g for 90 minutes at room temperature. After centrifugation, the virus solution was aspirated and 2 mL of human T cells (250,000 cells / mL) in human T cell medium containing 100 units / mL hIL-2 was added, spun at 1200 x g for 60 minutes at 37°C, and moved to an incubator. Cells were incubated in the virus-coated plates for 24 hours before expansion, and the Dynabeads were removed 7 days after T cell activation. For cells that were flow sorted prior to adoptive cell transfer, Dynabead was added at a 3:1 ratio for 48 hours immediately after sorting.

[0087] Staining and flow cytometry. To detect CAR expression, biotinylated CD19 (10 μg / mL, Acro Biosystems No. CD9-H8259) and streptavidin-APC (ThermoFisher No. S868) were used according to the manufacturer's instructions. NKG2DL expression was assessed by staining with NKG2D-Fc chimera (10 μg / mL, Fisher 1299NK050) followed by αFc secondary staining (Invitrogen No. A-10631). NIR Live / Dead (ThermoFisher No. L34976), CFSE (LifeTech No. C34554), and CellTrace Violet (CTV, LifeTech No. C34557) were used according to the manufacturer's instructions. Human Fc block (BD No. 564220) was used prior to staining with any antibody. For intracellular staining of granzyme B, intracellular fixation and permeabilization buffer (eBioscience No. 88-8823-88) was used according to the manufacturer's instructions, and brefeldin A was added approximately 4 hours before staining. Antibodies against GranzymeB (GB12, ThermoFisher), CD69 (FN50, BD), CD4 (RPA-T4, BioLegend), hCD8 (RPA-T8, BioLegend), CD3 (UCHT1, BD), CD45 (HI30, BD), CD19 (HIB19, BioLegend), PD-1 (EH12.2H7, Biolegend), CD107a (H4A3, Biolegend), mCD8 (53-6.7, BioLegend), HER2 (24D2, Biolegend), as well as HisTags (4E3D10H2 / E3, ThermoFisher) and human Fc (Invitrogen No. A-10631) were all used at a dilution of 1:100.

[0088] In vitro luciferase and heat tolerance assays. Primary human T cells were heated in a thermal cycler and transferred to culture plates for incubation at 37°C. Cell supernatants were sampled for luciferase activity 24 hours after the end of heat treatment unless otherwise stated. Non-heat treatments were performed by incubating engineered cells with the indicated concentrations of CoCl2 (Sigma No. 232696-5G) or CdCl2 (Sigma No. 202908). Where indicated, luminescence was compared to a ladder of recombinant Gaussia luciferase (NanoLight No. 321-500) quantified using a Gaussia luciferase luminescence assay kit (ThermoFisher No. 16161) according to the manufacturer's instructions. For viability and proliferation studies, primary human T cells were heated in a thermal cycler and then assayed with an Apoptosis Detection Kit (BD No. 556547) or CellTrace Violet (Fisher No. C34571). Viability was assessed 24 hours after heating, and the gating strategy is shown in Figure 6. For migration studies, wild-type cells were added to the upper insert of a transwell plate (Sigma No. CLS3421) and CXCL12 (50 ng / mL, Peprotech No. 300-28A) was added to the lower chamber. Cells in the lower chamber were counted by hemocytometer at the indicated times.

[0089] Cytotoxicity and T cell activation assays. For cytometric analysis, TS-CAR T cells were heated in a thermal cycler and co-incubated with K562 target cells at an effector cell to target cell ratio of 10:1 for 24 h before staining as described above. For luciferase-based assays, K562 were luciferized with either firefly luciferase (CD19+) or Renilla luciferase (CD19-) and incubated with effector cells after heating. An effector to target ratio of 10:1 was used unless otherwise stated. After incubation, either D-luciferin (Fisher No. LUCK-2G, 150 μg / mL lead concentration) or Rluc substrate (VWR No. PAP1232, 17 μM lead concentration) was added to the samples. Maximum cytotoxicity was defined as the luminescence signal from wells containing only medium, whereas no cytotoxicity was defined by wells containing only target cells. Supernatants were collected after incubation and assayed for cytokines using a human Th1 / Th2 / Th17 CBA kit (BD No. 560484). IL-15 superagonists were quantified using a human IL-15 / IL-15R alpha complex DuoSet ELISA (R&D Systems DY6924). For BiTE experiments with primary human T cells, T cells were subjected to two heat treatments (42°C, 30 min), 6 h apart, before incubation with target cells. The ability of engineered T cells to kill tumor target cells was also measured by lactate dehydrogenase (LDH) release assay. Briefly, engineered T cells were co-cultured with target cells at an effector cell to target ratio of 2:1 in 96-well plates for 24 h. LDH release was then measured using an LDH Cytotoxicity Assay Kit (fluorimetric) (Abcam No. 197004) according to the manufacturer's instructions.

[0090] IL-15 superagonist dynabead experiments: Wild type primary human T cells were labeled with CFSE and incubated with either heated or unheated TS-IL15 cells. Beads were added at a T cell to bead ratio of 10:1, which was determined not to induce strong proliferation in untransduced T cells without cytokine support (Figure 15). CFSE labeling allowed for discrimination from TS-IL15 cells (Figure 16) and proliferation and mitotic index were calculated in FlowJo using the proliferation tool.

[0091] Animals: NSG mice were bred and housed at Georgia Tech Physiological Research Laboratory (GT PRL) prior to use at 8-16 weeks of age. C57BL / 6 and transgenic Pmel-1 mice (B6.Cg-Thy1a / Cy Tg(TcraTcrb)8Rest / J) were purchased from Jackson Laboratories. C57BL / 6 and Pmel-1 mice aged 6-8 weeks were used at the beginning of the experiments. All animal protocols were approved by Georgia Tech IACUC (protocols No. A100190 and A100191). All authors adhered to the relevant ethical regulations when conducting this study.

[0092] Photothermal heating and in vivo bioluminescence imaging: AuNRs were purchased from Nanopartz (No. A12-10-808-CTAB-500) and PEGylated (Laysam Bio No. MPEG-SH-5000-5g) to replace the CTAB coating. These AuNRs were intravenously injected (10mg / kg) into tumor-bearing mice 24-48 hours prior to adoptive transfer of T cells. Mice were anesthetized with isoflurane gas and the target site was illuminated using an 808nm laser (Coherent) under the guidance of a thermal camera (FLIR model 450sc). Fluc activity was measured using an IVIS Spectrum CT (Perkin Elmer) approximately 5 minutes after intravenous injection or 20 minutes after intraperitoneal injection of D-luciferin (Fisher No. LUCK-2G). The detection limit was identified by calculating the mean ± 2 standard deviations of the background measurements.

[0093] Adoptive cell transfer (ACT) experiments: After shaving the site and sterilizing it using isopropyl wipes (GT PRL), NSG mice were inoculated with 5 × 10 6 For heterogeneous expression of HER2, 5 × 10 5 MDA-MB-468 cells were inoculated at a 1:3 HER2- to HER2+ ratio 44 days prior to ACT. Engineered primary human T cells were injected via the tail vein in 200 μL of sterile saline. For the B16 tumor model, 5 × 10 5 B16F10 melanoma cells were inoculated into the flank of C57BL / 6 mice. Eight days after tumor cell inoculation, mice were sublethally lymphodepleted by total body irradiation (100 cGy / min, 5 min). Engineered Pmel-1 T cells (6 × 10 cells) were 6 Mice were administered 2 × 10 5 Units of recombinant human IL-2 (Peprotech No. 200-02) were administered intraperitoneally twice daily at least 10 hours apart for a total of six doses. All mice received PEGylated AuNRs intravenously via the tail vein approximately 24 hours prior to adoptive transfer of human T cells. 25 hours after ACT, photothermal heat treatment was administered and monitored as described above.

[0094] Software and statistical analysis. All results are expressed as the mean and error bars indicate SEM. Statistical analysis was performed using GraphPad Prism statistical software. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. Flow cytometry data were analyzed using FlowJo X (FlowJo, LLC). In vitro luminescence data were collected using Gen5 2.07 (Biotek). In vivo luminescence data were collected and analyzed using Living Image 4.4.5 (PerkinElmer). Flow cytometry data were collected using BD FACSDIVA v8 (BD Biosciences). Thermal imaging data were acquired and analyzed using Research IR Max (FLIR). Figures were designed in Adobe Illustrator.

[0095] Example 2 Immunotherapy has enormous therapeutic potential and is used to treat a myriad of diseases, such as asthma, transplant rejection, and cancer. Notably, chimeric antigen receptor (CAR) T-cell therapy has led to sustained long-term survival in certain types of cancer patients with B-cell malignancies. However, their effectiveness in treating solid tumors is limited by factors such as tumor heterogeneity and severe immunosuppression. Potent immunomodulators, such as cytokines (IL-2, -12, -15), growth factors (Flt3L, IGF-IR), or chemokines (CXCL12, CCL19), can potently modulate immune function, making immune cell therapy more effective. However, traditional delivery mechanisms that rely on systemic administration are associated with poor pharmacokinetics and numerous immune-related side effects (irAEs), including on-target, extratumoral toxicity, cytokine release syndrome, neurotoxicity, and in some cases, life-threatening autoimmunity. As immunotherapies are developed for more disease indications, creating strategies to safely deliver immune modulating agents with spatial precision is critical to enhance systemic responses while mitigating irAEs. Here, we introduce a non-invasive method to spatially control T cell-mediated drug delivery at different anatomical sites, including but not limited to the brain, spleen, lymph nodes, tumors, kidneys, stomach, intraperitoneal space, lungs, heart, liver, pancreas, and bladder. In contrast to drug delivery by passive diffusion, immune cells can infiltrate deep within tissues, target diseased sites, and home to lymphoid organs, thus providing an opportunity to engineer immune cells both as therapeutics and as delivery vehicles to improve therapeutic efficacy and mitigate irAEs. We describe a platform in which cells are engineered to controllably deliver therapeutic molecules, including but not limited to CARs, cytokines, chemokines, transcription factors, and nucleases, under conditional control by thermal cues that can be spatially positioned by various mechanisms (e.g., focused ultrasound, light, radiation, etc.). We demonstrate spatial control of engineered cells and demonstrate enhanced therapeutic efficacy resulting from localized delivery of immunomodulatory molecules.Using multiple preclinical models, we demonstrate: 1) spatial delivery of reporter molecules using engineered T cells as drug delivery vehicles to various anatomical sites, 2) enhanced antitumor therapy due to localized delivery of cytokines, and 3) mitigation of tumor growth resulting from tumor heterogeneity. Collectively, this platform represents a modular approach to locally deliver immune-modulating molecules and augment cell-based therapy to overcome challenges associated with systemic treatments while mitigating adverse off-target events.

[0096] Spatial control of engineered immune cells In one embodiment, we engineered a synthetic thermal gene switch (TS) consisting of a DNA nucleotide sequence encoding a series of heat shock elements (from various species including, but not limited to, H. sapiens, M. musculus, C. dromedarius, or D. rerio) upstream of a natural or synthetically derived core promoter for transgene activation upon mild temperature elevation (40-44°C). In one embodiment, we combined TS with focused ultrasound (FUS) as a trigger to deliver proteins upon mild temperature elevation in various anatomical organs. T cells were virally transduced with a transgene (TS-Fluc, SEQ ID NO: 22 or TS-Gluc, SEQ ID NO: 26) encoding a thermal switch driving firefly luciferase or Gaussia luciferase and adoptively transferred into mice. FUS was used to locally apply heat and deliver molecules in the brain, tumor, or lymph node. For additional data, including local delivery mediated by near-infrared (NIR) light, please see the attached manuscript.

[0097] Localized delivery of cytokines by engineered immune cells enhances immunotherapy. Spatial control of immune-modulating genes, such as those encoding stimulatory (e.g., IL-2, -12, -15, TNFα, IFNγ, etc.), inhibitory (e.g., IL-6, -10, TGFβ, etc.), and chemotactic (e.g., CXCL12, CCL2, CCL19, etc.) molecules, can enhance therapeutic outcomes beyond systemically administered molecules (Figure 22). In one embodiment, dendritic cells were transduced with a transgene encoding heat-driven production of IL-15SA (TS-IL15SA SEQ ID NO: 23) and heated for 30 minutes (66% duty cycle) using a thermocycler with a pulsed profile. After 12 hours of heating, IL-15SA production by dendritic cells was quantified via ELISA (Figure 23).

[0098] Localized delivery of recombinant proteins enhances antitumor activity Spatial control can also be implemented to regulate the production of recombinant proteins, including but not limited to antibodies and nanobodies (e.g., αPDL1, αCTLA-4, αIL-6r), transcription factors (e.g., NFAT, NFκB, T-bet), caspases (e.g., caspase 3, caspase 8), or bispecific T cell engagers (BiTEs) (e.g., NKG2DL, EGFRvIII, CD19 BiTEs).

Claims

**Claim 1** A promoter construct comprising the following regions: a) one or more heat shock elements, b) a core promoter, and c) a promoter construct comprising a gene of interest. **Claim 2** The promoter construct according to claim 1, wherein the promoter requires heat activation at 40 °C or higher and 45 °C or lower. **Claim 3** The promoter construct according to claim 1, wherein the promoter construct is activated by a light source, and optionally the light source is a laser or a near-infrared laser. **Claim 4** The promoter construct according to claim 1, wherein the heat shock element is repeated 2, 3, 4, 5, 6, 7 times or more. **Claim 5** The promoter construct according to claim 1, wherein the heat shock element comprises SEQ ID NO:

1. **Claim 6** The promoter construct according to claim 1, wherein the one or more heat shock elements comprise any one nucleotide sequence of SEQ ID NOs: 2-9. **Claim 7** The promoter construct according to claim 1, wherein the core promoter comprises the nucleotide sequence of SEQ ID NO:

13. **Claim 8** The promoter construct according to claim 1, wherein the one or more heat shock elements and the core promoter together comprise any one nucleotide sequence of SEQ ID NOs: 14-21, and optionally, the one or more heat shock elements and the core promoter together comprise the nucleotide sequence of SEQ ID NO:

20. **Claim 9** The gene of interest is a) a reporter protein, b) an immunomodulator, c) a bispecific T cell engager antibody, d) a chimeric antigen receptor, e) a recombinant T cell receptor, encode these or any combination thereof, and optionally, the reporter protein is luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mStrawberry, mPlum, photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric Kusabira Orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, energy transfer cyan fluorescent protein (Cypet), Super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP1), photoactivatable mCherry (PA-mCherry), monomeric Teal fluorescent protein (mTFP1), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, energy transfer yellow fluorescent protein (YPet), Super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T-Sapphire, Fucci, mKO2, mOrange2, mApple, Sirius, Azurite, EBFP, and / or EBFP2, the promoter construct according to claim 1.

10. The promoter construct according to claim 9, wherein the immunomodulator is a cytokine, chemokine, interferon protein, or cytotoxin.

11. The promoter construct according to claim 10, wherein the cytokine is a cytokine selected from the group consisting of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-γ, TNF-α, TGF-β, and LIF, or the cytokine is an IL-15 superagonist molecule.

12. The promoter construct according to claim 9 or 10, wherein the immunomodulator is a chemokine selected from the group consisting of CCL2, CCL1, CCL19, CCL22, CXCL12, CCL17, MIP-1α, MCP-1, GRO / KC, CSCL12, and / or CXCR3.

13. The promoter construct according to claim 9, wherein the bispecific T cell engager antibody comprises an anti-CD-3 binding domain and an NKG2D receptor extracellular domain.

14. The promoter construct according to claim 1, comprising any one nucleotide sequence of SEQ ID NO: 23, 24, and 29.

15. A vector comprising the promoter construct according to claim 1, Optionally, the vector is a viral vector, and optionally, the viral vector is a lentiviral vector.

16. An immune cell comprising the promoter construct according to claim 1 or the vector according to claim 15.

17. The immune cell according to claim 16, wherein the immune cell is a T cell or an NK cell, optionally, the T cell is a recombinant TCR T cell or a CAR T cell, and optionally, the NK cell is a CAR NK cell.

18. A kit comprising the promoter construct according to claim 1 or the vector according to claim 15, and further comprising a heating component for activating the promoter construct.

19. A pharmaceutical composition for use in the treatment of cancer in a subject, comprising the promoter construct according to claim 1 or the vector according to claim 15.

20. A pharmaceutical composition for use in the treatment of cancer in a subject, wherein the treatment comprises: i) administering to the subject the immune cell according to claim 16 or 17; and ii) activating the immune cell with a heating component, optionally, the immune cell is activated at a temperature in the range of 40°C or higher and 45°C or lower.

21. The pharmaceutical composition according to claim 20, wherein the subject is administered an anti-cancer agent or immunotherapy, optionally, the immunotherapy is anti-PD1 immunotherapy, or the immunotherapy is anti-PD-L1 immunotherapy.

22. The pharmaceutical composition according to claim 20, wherein the cancer causes the formation of a solid tumor, and optionally, the solid tumor is an epithelial cancer, sarcoma, lymphoma, blastoma, or melanoma.

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