ABSTRACT Chimeric antigen receptor (CAR) T cells are transforming clinical care for hematological malignancies, spurring numerous efforts to expand their use for different cancer types and applications. However, this success has not reliably translated to solid tumors, including breast cancer. Following adoptive transfer, a small fraction of CAR T cells manage to infiltrate tumor sites and the tumor microenvironment (TME) is highly immunosuppressive. Co-administration of biologics to enhance trafficking or to overcome the TME (e.g., cytokines or immune check- point inhibitors) have the potential to enhance CAR T cell activity. However, they affect both CAR and endoge- nous T cells populations, which can lead to off-target killing, systemic toxicities, and limited therapeutic windows. Moreover, noninvasive biomarkers of CAR T cell infiltration and trafficking are needed to assess early on treat- ment response. This proposal seeks to improve overall safety and efficacy of CAR T cell therapy against solid tumors by utilizing CAR T cells, simultaneously tagged with gold nanorods (AuNRs) and engineered with thermal gene switches (TGSs), by 1) confirming AuNR-TGS-CAR T cell infiltration at the tumor site using a combined ultrasound and photoacoustic (US/PA) imaging system, and 2) achieving precisely controlled local immune cell activation and therapy by mild heating of TGS-CAR T cells using focused ultrasound (FUS) guided by US/PA and thermal (US/PA/TH) imaging platform. TGS are genetic constructs that are transcriptionally inactive at body temperature but undergo a sharp thermal transition at 40–42°C to trigger transgene expression to levels greater than 200-fold above basal levels. TGS allows thermal targeting of tumors to activate infiltrated CAR T cells to locally produce potent therapeutic genes that would otherwise be toxic when administered systemically. The US/PA/TH imaging platform will confirm cell infiltration, guide FUS delivery of heat by noninvasive mapping of local temperatures within the tumor microenvironment, and quantify key biomarkers of therapy response. These synergistic advances in CAR T cell engineering and imaging will be tested in the context of HER2-CAR T cells for breast cancer where approximately 30 percent of patients carry an amplification of the HER2 gene and/or HER2 over-expression. Preclinical evaluation of the image-guided CAR T cell therapy approach will be per- formed in a syngeneic model of mammary adenocarcinoma by orthotopic injection of E0771 tumor cells express- ing human HER2 into B6-HER2 transgenic mice. HER2-CAR T cells will be engineered with TGS that control stimulatory cytokine IL-15SA. The successful completion of our studies will result in a new class of image-guided CAR T cell therapy to improve response against breast tumors while limiting systemic toxicity. The advances developed through these studies can be extended to other CAR T cell receptors against other cancer types.

PROJECT NARRATIVE The limited ability to assess CAR T cell infiltration and control their activity within tumors contributes to poor anti- tumor responses against solid malignancies. This proposal seeks to develop an image-guided therapy based on thermal activation of CAR T cells to increase treatment precision and improve patient responses.