James J. Onuffer

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

Keeping a leash on cancer-killing cells Redirecting the immune system to attack tumor cells is proving to be an effective therapy against cancer. However, when patients are exposed to T cells engineered to recognize and attack cancer cells, there is a risk of runaway or excessive activity or of off-target effects, both of which can themselves be deadly. Wu et al. designed T cells expressing chimeric antigen receptors that recognize and attack cancer cells with an additional control system. This mechanism would allow a doctor administering the therapy to turn the engineered T cell “on” or “off” by administering a small molecule that is required along with cancer cell antigen to stimulate the T cells and activate their tumor cell–killing properties. Science, this issue p. 10.1126/science.aab4077 Engineering a fail-safe control mechanism in cancer-targeted T cells. INTRODUCTION Cell-based therapies have emerged as a promising treatment modality for diseases such as cancer and autoimmunity. T cells engineered with synthetic receptors known as chimeric antigen receptors (CARs) have proven effective in eliminating chemotherapy-resistant forms of B cell cancers. Such CAR T cells recognize antigens on the surface of tumor cells and eliminate them. However, CAR T cells also have adverse effects, including life-threatening inflammatory side effects associated with their potent immune activity. Risks for severe toxicity present a key challenge to the effective administration of such cell-based therapies on a routine basis. RATIONALE Concerns about the potential for severe toxicity of cellular therapeutics primarily stem from a lack of precise control over the activity of the therapeutic cells once they are infused into patients. Exogenously imposed specific regulation over the location, duration, and intensity of the therapeutic activities of engineered cells would therefore be desirable. One way to achieve the intended control is to use small molecules to gate cellular functions. Small molecules with desired pharmacologic properties could be systemically or locally administered at varying dosages to achieve refined temporal and spatial control over engineered therapeutic cells. RESULTS We developed an ON-switch CAR that enables small molecule–dependent, titratable, and reversible control over CAR T cell activity. ON-switch CAR T cells required not only a cognate antigen but also a priming small molecule to activate their therapeutic functions. Depending on the amount of small molecule present, ON-switch CAR T cells exhibited titratable therapeutic activity, from undetectable to as strong as that of conventional CAR T cells. The ON-switch CAR was constructed by splitting key signaling and recognition modules into distinct polypeptides appended to small molecule–dependent heterodimerizing domains. The ON-switch CAR design is modular; different antigen recognition domains and small-molecule dimerizing modules can be swapped in. CONCLUSION The ON-switch CAR exemplifies a simple and effective strategy to integrate cell-autonomous decision-making (e.g., detection of disease signals) with exogenous, reversible user control. The rearrangement and splitting of key modular components provides a simple strategy for achieving integrated multi-input regulation. This work also highlights the importance of developing optimized bio-inert, orthogonal control agents such as small molecules and light, together with their cellular cognate response components, in order to advance precision-controlled cellular therapeutics. Titratable control of engineered therapeutic T cells through an ON-switch chimeric antigen receptor. A conventional CAR design activates T cells upon target cell engagement but can yield severe toxicity due to excessive immune response. The ON-switch CAR design, which has a split architecture, requires a priming small molecule, in addition to the cognate antigen, to trigger therapeutic functions. The magnitude of responses such as target cell killing can be titrated by varying the dosage of small molecule to mitigate toxicity. scFv, single-chain variable fragment; ITAM, immunoreceptor tyrosine-based activation motif. There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed “ON-switch” CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control.

Bacterial Virulence Proteins as Tools to Rewire Kinase Pathways in Yeast and Immune Cells

Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection. Although these effectors contribute to pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behaviour. Here we exploit two effector proteins, the Shigella flexneri OspF protein and Yersinia pestis YopH protein, to rewire kinase-mediated responses systematically both in yeast and mammalian immune cells. Bacterial effector proteins can be directed to inhibit specific mitogen-activated protein kinase pathways selectively in yeast by artificially targeting them to pathway-specific complexes. Moreover, we show that unique properties of the effectors generate new pathway behaviours: OspF, which irreversibly inactivates mitogen-activated protein kinases, was used to construct a synthetic feedback circuit that shows novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to tune the T-cell response amplitude precisely, or as an inducible pause switch that can temporarily disable T-cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.

The first World Cell Race

Summary Motility is a common property of animal cells. Cell motility is required for embryogenesis [1], tissue morphogenesis [2] and the immune response [3] but is also involved in disease processes, such as metastasis of cancer cells [4]. Analysis of cell migration in native tissue in vivo has yet to be fully explored, but motility can be relatively easily studied in vitro in isolated cells. Recent evidence suggests that cells plated in vitro on thin lines of adhesive proteins printed onto culture dishes can recapitulate many features of in vivo migration on collagen fibers [5,6]. However, even with controlled in vitro measurements, the characteristics of motility are diverse and are dependent on the cell type, origin and external cues. One objective of the first World Cell Race was to perform a large-scale comparison of motility across many different adherent cell types under standardized conditions. To achieve a diverse selection, we enlisted the help of many international laboratories, who submitted cells for analysis. The large-scale analysis, made feasible by this competition-oriented collaboration, demonstrated that higher cell speed correlates with the persistence of movement in the same direction irrespective of cell origin.

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

Significance Directed migration of diverse cell types is critical in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to specifically and easily direct, manipulate, and study cell migration in vitro and in vivo are currently limited. We conceived of a strategy to directly control cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. In this work, we demonstrate that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide. This technology provides a generalizable tool to systematically control cell migration in vitro and in vivo and could be a valuable module for engineering future therapeutic cellular devices. Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNO-responsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.

Actin dynamics rapidly reset chemoattractant receptor sensitivity following adaptation in neutrophils

Neutrophils are cells of the innate immune system that hunt and kill pathogens using directed migration. This process, known as chemotaxis, requires the regulation of actin polymerization downstream of chemoattractant receptors. Reciprocal interactions between actin and intracellular signals are thought to underlie many of the sophisticated signal processing capabilities of the chemotactic cascade including adaptation, amplification and long-range inhibition. However, with existing tools, it has been difficult to discern actins role in these processes. Most studies investigating the role of the actin cytoskeleton have primarily relied on actin-depolymerizing agents, which not only block new actin polymerization but also destroy the existing cytoskeleton. We recently developed a combination of pharmacological inhibitors that stabilizes the existing actin cytoskeleton by inhibiting actin polymerization, depolymerization and myosin-based rearrangements; we refer to these processes collectively as actin dynamics. Here, we investigated how actin dynamics influence multiple signalling responses (PI3K lipid products, calcium and Pak phosphorylation) following acute agonist addition or during desensitization. We find that stabilized actin polymer extends the period of receptor desensitization following agonist binding and that actin dynamics rapidly reset receptors from this desensitized state. Spatial differences in actin dynamics may underlie front/back differences in agonist sensitivity in neutrophils.

Redesign of aspartate aminotransferase specificity to that of tyrosine aminotransferase

The values of kcat/Km are strongly correlated with chain length for the reactions of E. coli tyrosine aminotransferase, but are nearly independent of this variable for aspartate aminotransferase. Both enzymes exhibit nearly equal reactivity with dicarboxylic acid substrates. Six key amino acid differences were identified that were found to be responsible for 80% of the specificity difference. It is postulated that a major role for Arg292 in aspartate transaminase is to exclude nonspecific substrates by keeping the enzyme in an open inactive form. The free energy to close the enzyme into its active conformation derives from association with specific ligands.