PROJECT SUMMARY Adoptive cell therapy using antigen receptor engineered T cells is a highly promising therapeutic approach, in which cells are genetically modified to express an antigen receptor protein and are then adoptively transferred into the patient. These cells then act as a “living drug” that can elicit potent therapeutic effects in response to sensing a target antigen on a cell anywhere in the body. This approach is revolutionizing the treatment of blood cancers and shows promise in cell therapies for treating solid tumors, auto-immune disease, and chronic viral infection. We are developing highly modular universal adaptor versions of receptors, that we termed SNAP-CAR and SNAP-synNotch. These receptors are “universal” as they can be chemically programmed to target any an- tigen of interest by a co-administered adaptor molecule consisting of an antigen binding region conjugated to a benzylguanine motif. Universal receptor systems can be used to target multiple antigens in a single patient or across several disease indications by simply changing the antigen-targeting adaptor molecules. Despite the tremendous promise of antigen receptor technologies, their implementation has been limited by difficulties in attaining diseased cell-specificity through single antigen targeting, as well as unwanted on-tar- get/off-disease toxicities and toxicities from overactive cells. We are addressing these technological gaps in antigen receptor signaling-specificity by taking a chemical biology approach and combining chemical control of biological systems through stimulus-reactive groups with receptor engineering. The objective of this proposal is to develop universal antigen receptor systems that are conditionally activated or deactivated by controller stimuli including small molecule exposure, as well as chemical changes characteristic of diseased cellular microenvironments and antigen combinations. This will be achieved through completion of the following aims: (1) Conditional control of universal antigen receptor signaling with OFF-switch adaptors. (2) Conditional control of universal antigen receptor signaling using ON-switch adaptors. (3) Combinatorial control of universal adaptor CAR T cells. The underlying principle of our approach is a covalent bond formation between the adaptor and a self-labeling SNAPtag enzyme fused to the receptor. Besides robust activation of cell signaling, the synthetic chemical linker between the antibody and the receptor allows us to dial in a wide range of stimuli responses, including chemical and biological triggers, based on distinct chemical designs of the linker molecules. Our characterization includes demonstration in vitro using primary human T cells and in vivo in human cell xenograft mouse models. Adoptive cell therapy with antigen receptor engineered cells is an active area of development and clinical use, and the unprecedented level of conditional control enabled by the proposed systems will, as a long-term goal, lead to improved patient outcomes through improved targeting and the ability to treat new disease indications.

PROJECT NARRATIVE Adoptive cell therapies using receptor engineered cells have been highly effective at curing cancers of the blood and are currently being extended to the treatment of a wide range of diseases including chronic viral infections, autoimmune diseases, and other cancers. Cell-based therapeutics are living drugs, and a major challenge to creating them for new disease indications is a lack of precise control, leading to off-target effects and overactivity, potentially causing deadly toxicities. We are developing general strategies to precisely control receptor engineered cells through chemical ON- and OFF-switches that enable temporal control by pharmacological agents and added specificity through sensing of distinct disease signals.