Wong, Wilson W.Siddiqui, Menna Yamany2023-09-072023https://hdl.handle.net/2144/46661Adoptive chimeric antigen receptor (CAR) T cell therapy has proved a remarkable breakthrough in cancer treatment, with six FDA-approved therapies to date. This progress has motivated research efforts to expand its application to more complex cancer targets, as well as other diseases marked by cellular dysfunction. Current conventional CAR designs are limited by their ability to target only one cancer-associated antigen and suffer from a lack of tunability that results in broad toxicities and has limited their scope. To overcome these limitations, we have developed two inducible universal CAR platforms: a split, universal, programmable (SUPRA) CAR design that uses leucine zippers to decouple the targeting modality from the engineered T cell receptor, and recently a switchable CAR antibody-binding (SCARAB) platform that uses off-the-shelf antibody adapters for antigen targeting. In both platforms, separating the antigen binding domain into an added adapter allows control over the activation levels of the CAR T cells in a dose-dependent manner, reducing the risk of off-tumor toxicity. Additionally, the adapters can be switched to target multiple antigens and tackle more complex tumor phenotypes. First, we demonstrate the feasibility of using a SUPRA “OR” gate against FLT3 and CD33 as a therapeutic approach to tackle acute myeloid leukemia (AML), a heterogeneous hematopoietic malignancy marked by poor prognosis. The split platform affords tunability over activation levels and multiplexed targeting that tackles the disease heterogeneity and allowed us to identify a therapeutic dose window that eliminates cancer cell lines while sparing healthy hematopoietic stem cells. Next, we introduce the SCARAB platform that uses off-the-shelf FITC or biotin-conjugated antibodies as the targeting modality in a universal CAR design. The modular nature of this system allowed us to independently interrogate the effect of varying antigen binder choices on CAR targeting performance. We demonstrate the utility of this platform as an antigen screening tool to evaluate target combinations against ovarian cancer cells using an AND gate logic approach. Lastly, we adapt this universal platform towards other therapeutic indications, such as eliminating alloreactive or autoreactive T cell clones in transplant rejection and autoimmune disease using FITC or biotin-conjugated peptide-MHC multimer adapters. We develop in vitro models of major histocompatibility complex (MHC) mismatch in rejection and reactive T-cell receptor (TCR) targeting in Type 1 Diabetes and demonstrate that our universal CAR system can selectively target mouse and human TCRs in a pMHC-adapter dependent manner. These applications demonstrate the expansive utility of next-generation CAR T cell designs in broadening our cell therapy applications and developing the next wave of therapeutic platforms.en-USBiomedical engineeringThesis/Dissertation2023-08-300000-0001-5183-1777