Tumor-targeting T cells a type of white blood cells that can be harvested from cancer patients, genetically modified to recognize tumor cells, and then re-injected into the same patients have shown remarkable curative potential against advanced cancers. However, a major challenge in T-cell therapy is off-tumor toxicity, in which T cells attack healthy tissues that display the same markers as tumor cells on their surfaces. Off-tumor toxicity can result in severe side effects, including patient deaths that have been observed in several clinical trials. This research proposal aims to reduce off-tumor toxicity by enabling T cells to interrogate the interior of a target cell before making a killing decision. This will be achieved by a new class of engineered proteins that are produced and delivered by T cells into target cells, but become toxic if and only if they encounter a tumor-specific protein inside the target cell. Successful development of the proposed technology will increase the safety and efficacy of adoptive T-cell therapy for cancer by improving the specificity of tumor targeting and by expanding the types of cancers that can be targeted.
The proposed study aims develop a new class of sense-and-respond protein devices?termed Cytoplasmic Antigen Detectors and Extermination Triggers (CADETs)?that enable T cells to search inside the target cell for disease markers prior to unleashing toxicity. CADETs will be constructed by accessorizing the cytotoxic protein granzyme B with regulatory domains that respond to specific intracellular oncoproteins, such as SUMO-specific protease 1 (SENP1) and human telomerase reverse transcriptase (hTERT). A SENP1-responsive CADET has been constructed by rational design, in which an inhibitory domain blocks CADET?s enzymatic activity until it is cleaved off by SENP1. This architecture can be generalized to other tumor-associated proteases with well-defined cleavage targets. A high-throughput screening process will be developed to enable optimization of CADET molecules that respond to non-proteolytic ligands, such as hTERT. This fluorescence-based screening process will be performed in Pichia pastoris yeast cells, and top-performing CADET sequences will be characterized in primary human T cells for production and delivery efficiency as well as hTERT-activated toxicity. The optimal CADET molecule will be co-expressed with chimeric antigen receptors in T cells and evaluated for the ability to selectively kill oncoprotein-expressing tumor cells while sparing off-tumor target cells in mice. In partnership with the Boys and Girls Club of Santa Monica (SMBGC), this proposal will also develop an outreach program for children between the ages of 12 and 14 to introduce the proposed research and discuss potential paths to future careers in engineering. The proposed technology combines synthetic biology and T-cell engineering to address an important biomedical challenge, and it will serve as an exciting demonstration of the possibilities that an engineering career could offer.
