Computationally designed, small molecule-responsive cell receptors for treating solid tumors

Project summary Chimeric antigen receptor T (CAR T) cell cancer therapies are thwarted by the difficulty of targeting antigens on solid tumor cells. Archetypal CAR-binding tumor cell surface proteins are not unique to tumor cells and can also be found on host cells. Further, solid tumor cells express these antigens at different levels, even within one tumor. In this complex and immunosuppressive environment, the standard CAR T strategy of targeting tumor cell surface proteins is insufficient and results in off-tumor CAR T cell binding and activation, leading ultimately to T cell exhaustion. To solve this problem, we propose a modular, logic-gated split CAR format that can respond to the combination of tumor cell surface proteins and metabolites enriched in the TME by clustering intracellular domains to activate T cell signaling pathways. This format allows for CAR T cells to activate the production of cytokines, cell adhesion proteins, and other tumor destruction modalities only after localizing to the TME. The proposal has three Aims. In Aim 1, we will establish the split CAR format for targeting solid tumors, which includes two synthetic receptors that are each fused to compatible intracellular co-stimulatory domains and cytokine engagement domains. One receptor, engineered from the blueprint of the human GABAB G-protein coupled receptor (GPCR), will function as a small molecule “sensor receptor”; its “partner receptor” will bind to a tumor cell surface protein as well as the agonist-bound form of the first receptor. When the paired receptors are bound to the extracellular agonist and the tumor cell surface protein, their co-stimulatory domains will localize together inside the cell to activate NFAT signaling in Jurkat reporter cells. In Aim 2, simultaneously with Aim 1, we will adapt the sensor receptors to bind and respond to TME metabolites by developing a computational protein design protocol to reengineer their binding sites. We will screen the designed protein library to isolate TME metabolite-responsive CARs and tune their binding for physiologically relevant metabolite concentrations using established experimental protein engineering methods. In preliminary data, we have prototyped a computational design strategy and TME sensor receptor format using homologous model receptors. In the final Aim, we will combine the redesigned TME metabolite receptors with a HER2+ breast cancer-specific partner receptor to deploy the split CARs in primary human T cells co-cultured with HER2+ breast tumor cells and monitor T cell activation and tumor cell lysis as a proxy for CAR T efficacy. Following the successful application of our CAR T strategy in cell culture as described in this proposal, we will next collaborate with leading T cell biologists at our university to deploy the receptors in a preclinical humanized mouse model to evaluate CAR T efficacy in vivo. Our proposed CAR T cell therapy platform is unique in its capacity to bind to a both an endogenous TME metabolite and a tumor cell surface marker to elicit a programmable intracellular T cell activation response.