Chimeric Antigen Receptors (CAR) are synthetic immune-receptors, which can redirect T cells to selectively kill tumor cells. Despite the recent FDA approval of Kymriah and Yescarta, the CAR constructs in current use have several limitations. These limitations include i) toxicities (e.g., Cytokine Release Syndrome or CRS, neurological toxicity and deaths); ii) CAR-T cell exhaustion and lack of persistence; iii) immunogenicity and iv) disease relapse. A number of these limitations can be traced back to the design of current CAR constructs which is prone to tonic signaling due to self-aggregation of antigen binding and costimulatory domains. Over the past 3 years, our laboratories have developed a number of innovative and sensitive assays to measure the expression and activity of CAR constructs. We have also developed novel prototype CAR constructs which lack an integrated costimulatory domain and rely on the selective NF-せB signaling activity of a signaling protein K13 for co-stimulation. The overall objective of the current project is to use the above assays and techniques to develop next generation CAR constructs targeting CD19 with improved efficacy and safety. In aim 1, we will test a panel of CAR constructs containing different humanized single chain variable fragments (scFv) and signaling backbones using in vitro assays of tonic signaling, cytokine production, T cell exhaustion, cytotoxicity and immunological synapse formation. In aim 2, we will test selected candidate CAR constructs in the in vivo assays. Collectively, the above studies will help in the selection of novel CAR-T constructs for the treatment of B cell lymphomas and acute and chronic leukemia that are less toxic and more efficacious than the currently available therapies.