PROJECT SUMMARY Patients with primary refractory or relapsed T-cell lymphoma typically have a poor prognosis and limited options for effective targeted therapy. This contrasts with the clinical success of using CD19-specific chimeric antigen receptors (CARs) in immunotherapies for B-cell malignancies. Thus, to begin to achieve the long- term goal of devising a CAR-T cell platform that can be safely and effectively applied in patients with T-cell lymphoma, new Project 2 has selected CD5 as a novel target antigen for CAR-transduced cells. This common surface marker of normal T cells is also expressed by an estimated 85% of T-cell malignancies and functions as a transmembrane inhibitory receptor that attenuates signaling from the antigen receptor of T cells and a subset of B cells. Importantly, CD5-specific CAR-T cell fratricide (self-killing) is limited in our experimental model, allowing the CAR-modified cells to expand normally, after which they display potent and selective cytotoxicity against malignant T cells. Nonetheless, we reasoned that a second target antigen might be helpful, as antigen loss during treatment is a major obstacle to truly successful therapy, in patients receiving CD19-specific CAR-T cells, for example. CD7 was judged the best candidate as it is expressed at a high level on >90% of T-lymphoblastic lymphomas and >60% of mature lymphomas including those lacking CD5. Although in preliminary studies CD7-specific CARs showed strong activity against CD7+ target cells, the transduced T cells did not rapidly downregulate CD7, leading to enhanced fratricide that abrogated further expansion. This pitfall was eliminated by targeted depletion of the antigen in the CAR-modified T cells, a step that did not compromise either expansion or antitumor activity. Given these positive findings, we hypothesize that CD5-specific CAR-T cells can be safely used to target CD5+ T-cell lymphomas and induce complete remissions, and that CD7- cells will continue to expand and function in even in the presence of CD7-directed CARs by T cells. We propose to test each strategy in the following specific aims. Aim 1: Manufacture the GMP-grade vector and develop the SOPs needed for a clinical trial of CD5-specific CAR-T cells in T-cell lymphoma, and obtain all necessary local and federal regulatory approval. Aim 2: Conduct and evaluate a clinical trial using CD5 CAR-T cells to induce remissions in individuals with residual T-cell lymphoma who would then become eligible for allogeneic stem cell transplant. Aim 3: Express the CD7-specific CAR on CD7- effector T cells as a means to increase the range of targetable tumors and overcome CD5 antigen escape. Our proposed studies of CD5-specific CARs and the preclinical development of a CD7-specific CAR will do much to substantiate and advance our CAR-based platform for the treatment of T-cell malignancies and would provide a scientific basis for further optimization.