Programmed death-ligand 1 (PD-L1) is a promising target for cancer immunotherapy due to its ability to inhibit T cell activation; however, its expression on various noncancer cells may cause on-target off-tumor toxicity when designing PD-L1-targeting Chimeric Antigen Receptor (CAR) T cell therapies. Combining rational design and directed evolution of the human fibronectin-derived monobody scaffold, PDbody was engineered to bind to PD-L1 with a preference for a slightly lower pH, which is typical in the tumor microenvironment. PDbody was further utilized as a CAR to target the PD-L1-expressing triple negative MDA-MB-231 breast cancer cell line. To mitigate on-target off-tumor toxicity associated with targeting PD-L1, a Cluster of Differentiation 19 (CD19)-recognizing SynNotch IF THEN gate was integrated into the system. This CD19-SynNotch PDbody-CAR system was then expressed in primary human T cells to target CD19-expressing MDA-MB-231 cancer cells. These CD19-SynNotch PDbody-CAR T cells demonstrated both specificity and efficacy in vitro, accurately eradicating cancer targets in cytotoxicity assays. Moreover, in an in vivo bilateral murine tumor model, they exhibited the capability to effectively restrain tumor growth. Overall, CD19-SynNotch PDbody-CAR T cells represent a distinct development over previously published designs due to their increased efficacy, proliferative capability, and mitigation of off-tumor toxicity for solid tumor treatment.

Focused ultrasound can deliver energy safely and non-invasively into tissues at depths of centimetres. Here we show that the genetics and cellular functions of chimeric antigen receptor T cells (CAR-T cells) within tumours can be reversibly controlled by the heat generated by short pulses of focused ultrasound via a CAR cassette under the control of a promoter for the heat-shock protein. In mice with subcutaneous tumours, locally injected T cells with the inducible CAR and activated via focused ultrasound guided by magnetic resonance imaging mitigated on-target off-tumour activity and enhanced the suppression of tumour growth, compared with the performance of non-inducible CAR-T cells. Acoustogenetic control of the activation of engineered T cells may facilitate the design of safer cell therapies.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.