ABSTRACT Adoptive cell therapy (ACT) with autologous T cells is a nascent but potentially transformative modality of cancer immunotherapy. Redirection of the patient’s own T cells to target tumor antigens is achieved by ex vivo lentiviral transduction and stable expression of tumor-targeting chimeric antigen receptors (CAR). While CAR- T cells have shown promise in treating some leukemias and lymphomas, their ex vivo expansion, in vivo survival, functional phenotype, and response to tumor antigens remain unpredictable, sometimes resulting in treatment failures or serious adverse events – including life-threatening cytokine release syndrome (CRS). Our overall objective is to apply well-established mechanistic principles of antigen receptor signal transduction to design CARs with improved safety profiles that more reliably target tumor antigens. Second generation CARs are comprised of an extracellular antibody-like antigen-binding domain, fused to a diverse range of hinge/spacer sequences, followed by transmembrane and cytoplasmic signaling domains that are derived from T cell costimulatory receptors and the T cell antigen receptor (TCR) zeta chain. Although it is well known that the choice of spacer can critically affect CAR function, no clear mechanistic principles have been identified that can account for the exquisite sensitivity of CARs to spacer properties. Our preliminary investigations place a well-understood mechanism of TCR triggering, the kinetic-segregation (K-S) mechanism, at the center of CAR signaling, and establishes that spacer size is the key characteristic of the ectodomain that determines CAR triggering. We show, using well-characterized xenogeneic spacers of varying size, that the K-S mechanism can be exploited to biophysically modulate CAR-T cell signaling. A range of CAR-T cell activation phenotypes can be produced through size-based tuning, including CAR-T cells that retain high cytolytic activity without inflammatory cytokine production. In this proposal, we aim to exploit K-S principles to tune the size of CARs using novel humanized spacers for effective recognition of tumor antigens and high tumor cell killing, but without excessive inflammatory cytokine production. To achieve this, we will systematically vary the ectodomain size of humanized CARs using novel syngeneic spacers to identify signaling thresholds that engage T cell cytolytic function, but not the cytokine release machinery. For these mechanism-oriented investigations, we will employ in vitro biochemical and co-culture assays of T cell function, along with super- resolution fluorescence microscopy. We will then proceed to test in vitro-tuned CAR designs for in vivo tumor killing efficacy and cytokine release profiles using murine liquid and solid tumor models. We anticipate that these novel CARs, designed using K-S principles, will be useful for improving the safety and efficacy of ACT regimens.