Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

Component: Thermal Gene Switch; Description: 7H-YB: seven repeats of HSE motif (5'-nGAAnnTTCnnGAAn-3') upstream of synthetic YB core promoter

Component: Alternative Promoters Tested; Description: HSPB1, HSPA1A, HSPH1, HSPA6 (endogenous), YB (synthetic)

Component: Transgenes Controlled; Description: • Gluc (Gaussia luciferase) — reporter<br>• Fluc (firefly luciferase) — in vivo reporter<br>• IL-15 SA (IL-15 superagonist, sushi domain-IL-15 fusion) — cytokine<br>• NKG2DL BiTE (bispecific T cell engager targeting NKG2D ligands) — redirects T cells to antigen-negative cells

Component: CARs; Description: • Constitutive αCD19 CAR (under EF1α promoter)<br>• Constitutive αHER2 CAR

Component: Heat Source (In Vitro); Description: Thermal cycler (30 min at 42°C, pulsed or continuous)

Component: Heat Source (In Vivo); Description: Gold nanorods (AuNRs, PEGylated, 10 mg/kg i.v.) + 808 nm NIR laser; photothermal conversion; thermal camera guidance

Component: Nanoparticle Properties; Description: PEG-coated gold nanorods; passively accumulate in tumors via EPR effect

Component: T Cell Sources; Description: • Primary human CD3⁺ T cells (7 donors; AllCells)<br>• Pmel-1 TCR transgenic murine T cells (recognizes gp100 melanoma antigen)

Component: Mouse Models; Description: • NSG: CD19+ Raji/K562, K562, HER2+/- MDA-MB-468 (breast cancer)<br>• C57BL/6J: B16-F10 melanoma (syngeneic, Pmel-1 TCR)

Parameter: Thermal Switch Screening; Details: Jurkat T cells transduced with 2H-7H-B1 constructs; Gluc reporter measured after 30 min at 40-42°C; primary human T cells tested with 4 core promoters

Parameter: Thermal Specificity; Details: CoCl₂ (hypoxia mimic, 0-1000 µM) and CdCl₂ (heavy metal, 0-1000 µM) vs. HSP70 and HSPA6 promoters

Parameter: T Cell Function After Heat; Details: • Viability: PI/Annexin V staining (24 h post-heat)<br>• Proliferation: CellTrace Violet dilution (CD3/28 beads)<br>• Migration: Transwell chemotaxis (CXCL12)<br>• Cytotoxicity: αCD19 CAR T cells + CD19+/CD19− K562 (luciferase loss)

Parameter: Photothermal In Vivo (Fluc); Details: NSG mice with bilateral K562 (CD19−) and Raji (CD19+) tumours; TS-Fluc αCD19 CAR T cells; one tumour heated; luminescence quantified

Parameter: IL-15 SA Therapy; Details: NSG (CD19+ K562) and C57BL/6 (B16-F10, Pmel-1) models; TS-IL15 CAR or TCR T cells; heat treatments every 3-4 d (5 total); tumour volume and survival

Parameter: BiTE Therapy / Antigen Escape; Details: NSG mice with mixed MDA-MB-468 tumours (3:1 HER2+:HER2−); TS-BiTE or TS-Rluc αHER2 CAR T cells; heat treatments (days 45, 47, 52, 59, 66, 72); tumour volume and Fluc+ (HER2−) luminescence

Parameter: Sample Sizes; Details: In vitro: n=3 biologically independent wells; In vivo: n=4-7 mice per group

Parameter: Controls; Details: Untransduced T cells; TS-Fluc (no transgene); TS-Rluc (no BiTE); no heat; AuNRs alone; no T cells

Parameter: Statistical Tests; Details: Two-way ANOVA with Tukey/Sidak/Dunnett post-test; two-tailed t-test; log-rank (Mantel-Cox) for survival

Analysis Category: Gene Expression; Methods: Gluc/Fluc luminescence (IVIS Spectrum CT; Gen5 2.07); IL-15 SA ELISA (R&D Systems); Cytokine CBA kit (BD)

Analysis Category: Flow Cytometry; Methods: BD LSR Fortessa; FACSDiva v8; FlowJo X; Antibodies: CD69, CD4, CD8, CD3, CD45, CD19, PD-1, CD107a, HisTag, HER2, NKG2D-Fc chimera

Analysis Category: T Cell Function; Methods: • Proliferation: CFSE/CTV dilution<br>• Cytotoxicity: Luciferase loss (Firefly/Renilla); LDH release assay<br>• Migration: Transwell (CXCL12)

Analysis Category: Viability/Apoptosis; Methods: PI/Annexin V (BD apoptosis detection kit)

Analysis Category: In Vivo Imaging; Methods: IVIS Spectrum CT (Fluc: D-luciferin i.p./i.v.; Rluc: Rluc substrate); Living Image 4.4.5

Analysis Category: Thermal Imaging; Methods: FLIR thermal camera; Research IR Max software

Analysis Category: Histology; Methods: Not specified in text (not a major component)

Parameter: Best HSE repeat number; Result: 7 repeats (5H-7H best; 2H-4H lower response)

Parameter: Best core promoter; Result: YB (synthetic) — ~60-fold induction at 42°C

Parameter: Basal activity at 37°C; Result: Low (similar to untransduced)

Parameter: Activation at 38-40°C (24 h); Result: Negligible (fever range)

Parameter: Donor dependency; Result: None (tested 3 donors)

Parameter: Specificity (hypoxia, CdCl₂); Result: 7H-YB not activated; endogenous HSP70/HSPA6 showed dose-dependent activation

Function: Viability (continuous 30 min); Effect of Heat: No significant reduction

Function: Viability (pulsed 30-60 min); Effect of Heat: Significantly improved vs. continuous (>40 min)

Function: Proliferation (30 min); Effect of Heat: Unaffected (both continuous and pulsed)

Function: Proliferation (60 min); Effect of Heat: Reduced

Function: Migration (CXCL12 chemotaxis); Effect of Heat: Unaffected at 42°C (affected at 50°C)

Function: Cytotoxicity (αCD19 CAR, all E:T); Effect of Heat: >90% maintained (no significant difference vs. unheated)

Function: Repeated heating (4× over 8 d); Effect of Heat: No loss of cytotoxicity

Parameter: CD19+ Raji tumours + heat; Result: >30-fold increase in Fluc signal vs. unheated

Parameter: CD19− K562 tumours + heat; Result: No increase (fewer CAR T cells, no antigen)

Parameter: Heated vs. unheated (same mouse); Result: 40-fold increase in heated tumour only; spleen and distal tumour remained at baseline

Model: NSG (CD19+ K562); Condition: TS-IL15 αCD19 CAR + heat; Result: Marked tumour reduction; 0/7 reached euthanasia (within study)

Model: NSG (CD19+ K562); Condition: TS-IL15 CAR alone; Result: 6/7 reached euthanasia by day 39

Model: NSG (CD19+ K562); Condition: Heat only (no T cells); Result: No benefit

Model: C57BL/6 (B16-F10, Pmel-1); Condition: TS-IL15 Pmel-1 + heat (2 cycles); Result: Significant tumour control; survival extended to day 42

Model: C57BL/6 (B16-F10); Condition: Pmel-1 + IL-2 (no heat); Result: All reached euthanasia by day 33

Parameter: TS-BiTE Jurkat T cell activation (CD69); Result: Autocrine activation (BiTE+ cells activated; bystander cells not)

Parameter: Cytokine secretion (IFN-γ, TNF-α); Result: Temperature-dependent increase at 42°C (TS-BiTE αCD19 CAR)

Parameter: Cytotoxicity against CD19− K562; Result: Significantly enhanced by TS-BiTE vs. controls at 42°C (P < 0.0001)

Group: Untreated; Tumour Volume at Day 100: Largest; Complete Responders: 0/6; HER2− Cell Luminescence: High

Group: TS-Rluc αHER2 CAR (no BiTE); Tumour Volume at Day 100: ~12× larger than TS-BiTE; Complete Responders: 1/6; HER2− Cell Luminescence: Significantly increased (relapse)

Group: TS-BiTE αHER2 CAR + heat; Tumour Volume at Day 100: Significantly smaller; Complete Responders: 3/6 complete responders; HER2− Cell Luminescence: Significantly reduced (P < 0.001)

1. Light penetration depth: The authors acknowledge that "photothermal targeting by NIR light is limited by penetration depth to a few centimetres and by the need for a plasmonic transducer." Alternative heating modalities (e.g., focused ultrasound) are suggested.

2. Repeat heating required: The authors note that "another limitation of our study is the need for the repeat application of heat." However, they point to clinical precedents (LITT) for repeated thermal treatments.

3. No direct comparison to constitutive expression: The authors state that "a direct comparison with systemic administration of constitutively expressed transgenes is necessary before clinical application."

4. Not applicable to disseminated metastases: The authors acknowledge that "targeting individual metastases would preclude therapy" and that the approach "will probably not be applicable for the treatment of broadly disseminated tumours."

5. In vivo T cell trafficking not tracked: The study did not track whether thermally activated T cells migrated out of tumours over longer time periods (beyond the 24 h imaging window).

6. Autocrine BiTE mechanism context-dependent: The authors note that "our in vitro experiments showing that BiTE activation occurs primarily by an autocrine mechanism can be affected by secretion rates, diffusion and effector-to-bystander ratios, as these parameters are tunable."

7. NSG models lack intact immune system: Many experiments used NSG mice (immunodeficient); syngeneic B16-F10 experiments addressed this but only with TCR transgenic T cells, not CAR T cells.

8. Bystander cell activation not fully characterized: While TS-BiTE Jurkat cells were activated, the study did not extensively characterize whether endogenous immune cells (in immunocompetent models) were also activated by secreted BiTE.

9. No toxicity assessment in immunocompetent models: While heat and transgene expression were localized, potential off-target effects on normal tissues expressing NKG2DL were not systematically evaluated.

10. AuNRs remain in tissues: Gold nanorods accumulate in the liver and spleen; long-term clearance and potential toxicity were not assessed beyond the study duration.

11. Thermal gene switch activation kinetics: While transgene expression peaked at ~6 h, the duration of expression and whether repeated heating leads to promoter desensitization were not fully characterized.

12. IL-15 SA effects on Tregs: IL-15 can also expand regulatory T cells; this was not assessed in immunocompetent models.

Report prepared based on the published Nature Biomedical Engineering article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.