Ultrasound-Inducible Hypoxia-Gated Solid Orthogonal Antigen for CAR-T Therapy in Solid Tumors

Introduction: : Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of certain hematologic malignancies but still faces critical obstacles in solid tumors, one of the main problems is tumor selectivity to avoid off-tumor toxicities that may lead to severe complications, further complicated by tumor heterogeneity and antigen escape mechanisms, which limit CAR-T cell therapy efficacy. To address this challenge, we designed SHIFTERS (Solid-tumor Hypoxia Inducible, Focused-ultrasound Triggered Enhanced Reprogramming System), a dual-input system that rewires the hypoxic tumor environment into a localized “kill-me” signal CD19 under spatiotemporal control of focused ultrasound (FUS). Central to SHIFTERS is an AND-gate circuit that integrates a split Gal4-VP64 transcription factor, with one module driven by a hypoxia-responsive promoter and the other by a heat shock promoter activated via focused ultrasound (FUS). Integrating with CD19 SynNotch EGFR CAR-T cells, which EGFR is a uniform tumor cell target, SHIFTERS triggered significant and sustained treatment efficacy upon FUS stimulation in hypoxic environment. In both spheroids and vivo models, SHIFTERS combined with SynNotch CAR-T demonstrated potent tumor clearance, highlighting its potential to reprogram the tumor microenvironment and improve CAR-T therapy for solid malignancies. Beyond hypoxia-driven regulation, SHIFTERS serves as a flexible platform that enables precise control of antigen expression through modular promoter exchange. By integrating tumor-specific promoters, such as the enhanced alpha-fetoprotein (enAFP) promoter, SHIFTERS reprograms the tumor specific cues to enhance the precision and efficacy of CAR-T therapy in solid malignancies.

Materials and

Methods: : Lentiviruses were used to deliver transgenes into the cells, with engineered cells selected via Fluorescence-activated cell sorting. The results were evaluated by flow cytometry. Gating is based on non-engineered cells. Luciferase assay is used for cytotoxicity reading compared with no T cell control. All animal procedures were approved under Protocol 21479 by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California. Experiments were conducted in compliance with institutional guidelines and ethical regulations for animal use. Male NOD.CG-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, aged 6–8 weeks, were obtained from The Jackson Laboratory. Our lab developed a focused ultrasound (FUS) system with real-time temperature control feedback for hyperthermia experiments. The system includes a custom-fabricated, 1.1-MHz single-element transducer constructed from a pre-focused, modified PZT (diameter: 70 mm, radius of curvature: 65 mm; DL-47, Del Piezo Specialties) with a 20 mm central aperture. It also contains a coupling cone (length: 65 mm) with a 4 mm diameter opening at the tip which was attached to the transducer. The cone held degassed deionized water as the acoustic medium and guided the ultrasound focus. The tip opening was sealed with an acoustically transparent thin film (Chemplex, 100). Degassing of deionized water was performed using a vacuum pump (Vevor). The transducer was driven by a function generator (Stanford Research Systems, SG386) and a 50 dB power amplifier (E&I, 325LA) to generate pulsed sine waves.

Results, Conclusions, and Discussions:: To address the challenges of antigen escape and off-tumor toxicity, in the meanwhile keeping the therapy safe from on-target off-tumor toxicity in solid tumor CAR-T therapy, we developed SHIFTERS—a dual-input gene regulation system integrating hypoxia and focused ultrasound (FUS) to drive tumor-localized expression of an orthogonal antigen, CD19. SHIFTERS employs an AND-gated split Gal4–VP64 circuit, with Gal4 driven by a heat shock promoter and VP64 by a hypoxia-responsive element. CD19 expression is thus restricted to regions experiencing both hypoxia and FUS-induced hyperthermia. More from hypoxia, we showed SHIFTERS can serve as a platform for other cancer-specific synthetic cues to enhance CAR-T therapeutics.

In vitro studies in U251 glioma cells confirmed that only the combinatorial condition (hypoxia + heat shock) robustly induced CD19, while background expression from individual stimuli was minimized by optimizing SunTag copy number and UAS response elements. SHIFTERS outperformed a 7HE-only system in sustaining CD19 production, enhancing the activation of CD19 SynNotch EGFR CAR-T cells. Co-culture assays demonstrated that even a small fraction (10%) of CD19+ "trainer" cells could prime SynNotch T cells to eliminate a mixed tumor population, confirming SHIFTERS’ capacity to form an effective in situ “training center.”

In 3D spheroid models, SHIFTERS induced complete tumor clearance under heat shock, whereas non-heat-shocked controls continued to grow, confirming spatial control and robust antitumor activity. We further showed SHIFTERS’ modularity by replacing the hypoxia promoter with a hepatocellular carcinoma-specific enAFP promoter. This configuration selectively activated CD19 expression and tumor killing in Hep3B cells under FUS, validating the system’s adaptability to tumor-specific cues. In vivo, subcutaneously implanted SHIFTERS tumors showed enhanced CD19 expression and tumor suppression under FUS, with systemic SynNotch CAR-T administration leading to selective clearance of FUS-treated flanks in a bilateral tumor model. This underscores SHIFTERS’ spatial precision and translational potential.

In conclusion, SHIFTERS enables spatiotemporally controlled antigen expression in solid tumors, enhancing CAR-T selectivity and overcoming antigen escape. Its modular design allows adaptation to various tumor-specific inputs, offering a broadly applicable strategy for safe and effective solid tumor immunotherapy. Future work will focus on clinical gene delivery approaches to fully realize SHIFTERS' translational promise.

Acknowledgements and/or References (Optional):: This work was supported in part by grants from National Institutes of Health (NIH) EB029122, GM140929, HL121365, HD107206, and CA262815 to Y. Wang and NIH K01EB035649 to L. Liu. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Jeong Min Oh and Hao Zhou from the University of Southern California for providing the U251-MG wild type cell lines. The authors also thank Yushun Zeng from the University of Southern California for the manufacture of the ultrasound transducer used in this study. Cartoon illustrations in this study were created with Biorender.com.