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.
B7-H1 Antigen , Receptors, Antigen, T-Cell , Humans , Mice , Animals , Receptors, Antigen, T-Cell/metabolism , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Ligands , Cell Line, Tumor , T-Lymphocytes , Immunotherapy, Adoptive
Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells.
Genetic Loci , Genome, Human , Molecular Imaging , Humans , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Transcription Factors/genetics
Despite its success in treating hematologic malignancies, chimeric antigen receptor (CAR) T cell therapy faces two major challenges which hinder its broader applications: the limited effectiveness against solid tumors and the nonspecific toxicities. To address these concerns, researchers have used synthetic biology approaches to develop optimization strategies. In this review, we discuss recent improvements on the CAR and other non-CAR molecules aimed to enhance CAR T cell efficacy and safety. We also highlight the development of different types of inducible CAR T cells that can be controlled by environmental cues and/or external stimuli. These advancements are bringing CAR T therapy one step closer to safer and wider applications, especially for solid tumors.
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.
Immunotherapy, Adoptive , Neoplasms , Ultrasonic Therapy , Animals , Cell- and Tissue-Based Therapy , Mice , Neoplasms/diagnostic imaging , Neoplasms/therapy , T-Lymphocytes
Recent synthetic biology advancements have shown that cells can be engineered to respond to external stimuli such as chemical compounds and light, which significantly improves the specificity and controllability of CAR T therapy. However, the lack of both spatiotemporal and depth control is still the main issue in the clinic of CAR T treatment. At the same time, mechanogenetics, capable of penetrating deep tissues with high spatiotemporal precision, is rapidly evolving and advancing to reveal its potential for cancer immunotherapy. In the past few years, researchers have demonstrated the precise and remote control of engineered cells with mechanical perturbation originated from ultrasound, which may become a new solution to circumvent the limitations of CAR T therapy in the future. This review will discuss mechanobiology and the state-of art designs of controllable CAR T cells. A specific focus of this review will be on the mechanical control of CAR T therapy.
Neoplasms , Receptors, Antigen, T-Cell , Cell Engineering , Humans , Immunotherapy , Neoplasms/genetics , Neoplasms/therapy , T-Lymphocytes
From basic studies in understanding the role of signaling pathways to therapeutic applications in engineering new cellular functions, efficient and safe techniques to monitor and modulate molecular targets from cells to organs have been extensively developed. The developmental advancement of engineering devices such as microscope and ultrasonic transducers allows us to investigate biological processes at different scales. Synthetic biology has further emerged recently as a powerful platform for the development of new diagnostic and therapeutic molecular tools. The synergetic amalgamation between engineering tools and synthetic biology has rapidly become a new front in the field of bioengineering and biotechnology. In this review, ultrasound and its generated mechanical perturbation are introduced to serve as a non-invasive engineering approach and, integrated with synthetic biology, to remotely control signaling and genetic activities for the guidance of cellular functions deep inside tissue with high spatiotemporal resolutions. This ultrasound-based approach together with synthetic biology has been applied in immunotherapy, neuroscience, and gene delivery, paving the way for the development of next-generation therapeutic tools.
