Abstract A021: Spatiotemporal Regulation of Chimeric Antigen Receptor T-cells Using Focused Ultrasound Therapy to Enable Use of High-Affinity Binders

Abstract Pediatric High Grade Glioma (HGG) is an aggressive cancer with a 5-year survival rate of just 10-20% (Fangusaro, 2012). While previous studies using chimeric antigen receptor (CAR) T-cells for brain malignancies demonstrated tolerability (Majzner et al., 2022; Vitanza et al., 2021; Vitanza et al., 2023), the low survival rates demand more efficacious therapies (Majzner et al., 2022). In this study, we perform in vitro modeling of spatiotemporally regulated high-affinity CARs targeting the GD2 antigen to enable use of highly potent CAR variants while mitigating on-target, off-tumor toxicity. By placing CAR affinity variants under control of a heat shock promoter, thermal energy delivered to the tumor tissue by focused ultrasound (FUS) induces CAR expression (Wu et al., 2021) at an optimal temperature of 43oC. We first determine FUS-controlled CAR induction and decay kinetics in vitro for selected signaling domain architectures as well as affinity variants. Following codon optimization, differences in basal expression at 37oC from the heat shock promoter were noted for the three signaling domain architectures, with 28z and BBz displaying the highest and lowest levels of aberrant expression, respectively. Next, we compare expression of CAR constructs with and without 3 cycles of heat shock at 43oC in CAR-T cells. Then, using live cell imaging, we analyze the kinetics of expression and decay of the CAR following heat shock. Finally, we perform effector function assays of the CAR-T cells to compare the cytotoxicity and persistence for each construct. We identify the CAR architecture harboring the CD28 transmembrane domain and the 4-1BB cytoplasmic domain as the optimal balance between basal and induced expression. We plan to move this architecture forward to in vivo studies to assess the ability to maintain robust tumor lysis and minimal on-target, off-tumor toxicity. Citation Format: Edward Trope, Yun-Yun Su, Rie Nakata, Magda Golyska, Sarah Richman. Spatiotemporal Regulation of Chimeric Antigen Receptor T-cells Using Focused Ultrasound Therapy to Enable Use of High-Affinity Binders [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A021.