Trispecific CAR-T cells targeting CD19, CD20 and CD22 to treat B-cell malignancies

Project summary CD19 targeted chimeric antigen receptor T cells (CAR-Ts) have revolutionized cellular immunotherapy in refractory B-cell malignancies such as B-cell Non-Hodgkin’s lymphoma (B-NHL) and B-cell acute lymphoblastic leukemia (B-ALL). Despite high rates of complete remissions, relapses, 50% of which within the first year, remain a critical challenge highlighting the need for novel CAR-T cell products. Two patterns of relapse are observed: (i) Antigen-negative relapses, caused by target antigen loss and (ii) antigen-positive relapses, which are mediated by lack of persistence or loss of function of the CAR-Ts. Commercial CD19 CAR-Ts employ either a CD28 or a 4-1BB costimulatory domain however, a novel OX-40 domain has been shown to promote persistence, cytotoxicity and decrease exhaustion. Finally, increasing evidence suggests that stem-like memory T cell phenotype in the CAR-T product is associated with more durable responses. Schneider et. al developed a Trispecific CD19, CD20, CD22-targeting CAR-Ts with an OX-40 costimulatory domain and showed significant activity in preclinical lymphoma models compared to CD19 CAR-Ts. We validated these findings with in-house manufactured Trispecific CAR-Ts and confirmed their specificity, cytotoxicity, and immunophenotypic fitness in preclinical B-cell lymphoma models. Our proposal seeks to address the limitations of commercial CD19 CAR-Ts through a first-in-human in-house manufactured Trispecific CAR-Ts with an OX- 40 costimulatory domain for relapsed, refractory B-cell malignancies. These trispecific CAR-Ts are manufactured in the Ohio State University Cell therapy laboratory using the CliniMACS Prodigy device over a 6-day manufacturing process which allows for infusion of stem-like memory CAR-Ts. We hypothesize that trispecific CARTs will (i) reduce the risk of relapse mediated by antigen negative clonal escape; (ii) enhance persistence of CAR-Ts which will translate into deeper and more durable clinical responses. To address this, we propose two Aims. In Aim 1, we aim to conduct a first-in-human phase I trial with in-house manufactured trispecific CAR-Ts in patients with relapsed/refractory B-NHL, B-ALL, B-prolymphocytic leukemia, and chronic lymphocytic leukemia. Primary endpoints are feasibility, safety of trispecific CAR-Ts along with establishing a recommended phase II dose. Secondary endpoints are efficacy and duration of response. In Aim 2, we aim to identify key mechanisms of efficacy and resistance to trispecific CAR-Ts. Specifically, we plan to assess: (i) persistence of CAR-Ts and its correlation with complete remission rates, duration of response as well as incidence and severity of adverse events; (ii) immunophenotypic and transcriptional features of impaired function of CAR-Ts and other mononuclear cells using state of the art high-dimensional spectral flow cytometry and CITE-sequencing. At completion of this project, our work will have established feasibility of in-house manufacturing of trispecific CAR-Ts and provided insights into mechanisms of relapse and efficacy.