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Abstract Immunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T product containing early memory cells, especially stem cell memory (TSCM). It is hypothesized that a stem cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment. We developed a novel Centyrin-based CAR, referred to as a CARTyrin, that is specific for human B cell maturation antigen (BCMA). Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules. We produced a plasmid DNA transposon encoding the CARTyrin that was flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing. The piggyBac™ (PB) Transposon System was used for stable integration of anti-BCMA CARTyrin into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme, called Super piggyBac™ (SPB), in a single electroporation reaction. Delivery of piggyBac™ transposon into untouched, resting primary human pan T cells resulted in 20-30% of cells with stable integration and expression of PB-delivered genes. Surprisingly, we observed that a majority of these cells were positive for expression of CD62L and CD45RA, markers commonly associated with TSCM cells. To see if this phenotype was retained upon CAR-T cell stimulation and expansion, we activated the cells via stimulation of CD3 and CD28, and later show that > 60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells were fully capable of expressing potent anti-tumor effector function. To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, we compared the phenotype of CAR-T cells generated either by PB transposition or lentiviral (LV) transduction. To do this, we constructed a new vector by subcloning the CARTyrin transgene into a common LV construct for production of virus. Following introduction of the CARTyrin to untouched resting T cells either by PB-transposition or LV-transduction, we expanded the CARTyrin+ cells and then allowed them to return to a resting state. A variety of phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatic cytokine or tumor-associated Ag, cytokine production, and lytic capability in response to BCMA+ tumor cells. Unlike the PB-transposed CARTyrin+ T cells, we found that the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of BCMA+ tumor cells. Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly TSCM cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these CARTyrin+ T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion. Disclosures Barnett: Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.