Highlights d A dual oxygen-sensing switch provides stringent hypoxiadependent regulation of a CAR d HypoxiCAR T cells deliver tumor-selective CAR expression and anti-tumor efficacy d HypoxiCAR T cells prevent on-target, off-tumor activation and cytokine release syndrome d HypoxiCAR provides a strategy to expand the CAR repertoire for solid malignancies

Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy. We evaluated FRα expression in breast cancers by genomic ( = 3,414) and IHC ( = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), , and in human TNBC xenograft models. FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth. FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. .

Summary Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

There has been significant interest in the prospects of chimeric antigen receptor (CAR) T-cell therapy in the treatment of solid malignancies, and multiple clinical trials are in progress1. However, the scope of these trials has been restricted by the lack of availability of tumorspecific targets to direct CAR binding. Tumor specificity is crucial as on-target off-tumor activation of CAR T-cells in healthy tissues can result in potentially lethal toxicities due to uncontrolled cytokine release syndrome2. Here we engineer a stringent hypoxia-sensing CAR T-cell system which achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by an inadequate oxygen supply. Using murine xenograft models, we demonstrate that despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate the unlimited expansion of the CAR T-cell target repertoire for treating solid malignancies.