Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.

Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.

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

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.