High-performance multiplex drug-gated CAR circuits.

Chimeric antigen receptor (CAR) T cells can revolutionize cancer medicine. However, overactivation, lack of tumor-specific surface markers, and antigen escape have hampered CAR T cell development. A multi-antigen targeting CAR system regulated by clinically approved pharmaceutical agents is needed. Here, we present VIPER CARs (versatile protease regulatable CARs), a collection of inducible ON and OFF switch CAR circuits engineered with a viral protease domain. We established their controllability using FDA-approved antiviral protease inhibitors in a xenograft tumor and a cytokine release syndrome mouse model. Furthermore, we benchmarked VIPER CARs against other drug-gated systems and demonstrated best-in-class performance. We showed their orthogonality in vivo using the ON VIPER CAR and OFF lenalidomide-CAR systems. Finally, we engineered several VIPER CAR circuits by combining various CAR technologies. Our multiplexed, drug-gated CAR circuits represent the next progression in CAR design capable of advanced logic and regulation for enhancing the safety of CAR T cell therapy.

Engineering digitizer circuits for chemical and genetic screens in human cells.

Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.

Engineering a Dual Small Molecule Gated ZAP70 Switch in T Cells.

T cells expressing tumor-specific T cell receptors are promising cancer therapeutic agents, but safety control switches are needed to manage potential side effects arising from overactivity. Here, we present the first dual small molecule-gated ZAP70 signaling switch for the regulation of T cell activity. We show that when an analogue-sensitive ZAP70 allele is fused to the engineered ligand binding domain of the estrogen receptor, ERT2, its activity can be upregulated to an extent by a metabolite of an FDA-approved tamoxifen, 4-hydroxy-tamoxifen, and downregulated by an ATP analogue, 3-MB-PP1. The strength of early T cell signaling can also be modulated by varying the concentrations of activator and inhibitor, and the switch exhibits temporal control on the time scale of minutes. Interestingly, the switch has the ability to control CD69 and calcium levels in T cells but has limited capabilities in the regulation of downstream cytokine release, suggesting further investigation is needed before it can be implemented in adoptive T cell therapy.

Synthetic Biology: Immunotherapy by Design.

Cellular immunotherapy holds great promise for the treatment of human disease. Clinical evidence suggests that T cell immunotherapies have the potential to combat cancers that evade traditional immunotherapy. Despite promising results, adverse effects leading to fatalities have left scientists seeking tighter control over these therapies, which is reflected in the growing body of synthetic biology literature focused on developing tightly controlled, context-independent parts. In addition, researchers are adapting these tools for other uses, such as for the treatment of autoimmune disease, HIV infection, and fungal interactions. We review this body of work and devote special attention to approaches that may lend themselves to the development of an "ideal" therapy: one that is safe, efficient, and easy to manufacture. We conclude with a look toward the future of immunotherapy: how synthetic biology can shift the paradigm from the treatment of disease to a focus on wellness and human health as a whole.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

Synthetic biology approaches in cancer immunotherapy, genetic network engineering, and genome editing.

Investigations into cells and their contents have provided evolving insight into the emergence of complex biological behaviors. Capitalizing on this knowledge, synthetic biology seeks to manipulate the cellular machinery towards novel purposes, extending discoveries from basic science to new applications. While these developments have demonstrated the potential of building with biological parts, the complexity of cells can pose numerous challenges. In this review, we will highlight the broad and vital role that the synthetic biology approach has played in applying fundamental biological discoveries in receptors, genetic circuits, and genome-editing systems towards translation in the fields of immunotherapy, biosensors, disease models and gene therapy. These examples are evidence of the strength of synthetic approaches, while also illustrating considerations that must be addressed when developing systems around living cells.

Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration.

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of ß1-integrin by interference with recycling following normal endocytosis of inactive ß1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces ß1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.