Project 1 Abstract The long-term goal of this project is to improve chimeric antigen receptor T cell (CAR-T) therapy for patients with T-cell Acute Lymphoblastic Leukemia (T-ALL) via the use of ‘off-the-shelf’ universal allogeneic CAR-T (UCART) cells. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. However, T cell malignancies pose a challenge to CAR-T cell therapy due to the shared expression of target antigens between normal and malignant T cells. CD7 is highly expressed in T- ALL and the majority of normal peripheral T cells and NK cells. To overcome this shared antigen expression problem, our group has developed CAR-T cells targeting CD7 that are genetically edited using CRISPR/Cas9 to lack both CD7 and the T cell receptor alpha chain (TRAC) in order to avoid fratricide and permit the use of allogeneic T cells for treatment of T-ALL. In our preclinical studies, these universal allogeneic CAR-T targeting CD7 (UCART7) prevented fratricide and efficiently killed human T-ALL cell lines and patient-derived primary T- ALL in vitro and in vivo without causing xenogeneic graft-versus-host disease (GVHD). We hypothesize that the administration of allogeneic UCART7 cells to patients with relapsed or refractory (r/r) T-ALL will be safe, tolerable, and provide anti-tumor activity resulting in improved clinical outcomes. We plan to test this hypothesis in Aim 1 by conducting a phase 1 trial titled “A Phase 1/2 Study of the Safety and Efficacy of Anti-CD7 Allogeneic CAR-T cells (WU-CART-007) in Patients with r/r T-ALL/LBL”. Correlative studies will characterize the phenotype and function of UCART7 and their effects on (1) serum cytokines, (2) endogenous CD7-expressing cells, (3) other cell subsets lacking CD7 and (4) T-ALL using cytokine bead arrays, flow cytometry, CITE-seq, and single cell cytokine analyses. Three obstacles to effective CAR-T cell therapy, including potentially UCART7, are (1) cytokine release syndrome (CRS), (2) suboptimal CAR-T cell cytotoxicity and persistence and (3) relapse due to antigen escape via downregulation or mutation of the CAR-T target antigen. IL-7 and IL-15 are the main regulators of peripheral homeostatic expansion of CD4 and CD8 T cells. NT-I7 and N-803 are long-acting derivatives of IL-7 and IL-15, respectively, that are safe and well tolerated in mice and man. Furthermore, duvelisib, a dual phosphoinositide 3-kinase delta/gamma (PI3Kdg) inhibitor, may provide effective prophylaxis against CRS while enhancing the persistence and efficacy of CAR-T cells. We hypothesize that NT-I7 or N-803 in combination with duvelisib can promote UCART proliferation, persistence, and T-ALL killing in mice resulting in long-term tumor-free survival in the absence of CRS. We plan to test this hypothesis in Aim 2 using xenogeneic and immunocompetent mouse models of T-ALL. Finally, multi-antigen-targeted CAR-T cells are a promising direction to overcome single antigen relapse issues fol...