CD19-targeted chimeric antigen receptor (CARs) modified autologous T cells is a potent cancer therapeutic modality that has achieved a greater than 80% response rate in Relapse/Refractory (R/R) B-cell lymphomas. For those patients with tumors that are CD19 negative at initial diagnosis or relapsed due to the loss of CD19 expression, we are proposing to use Lym-1, a well-characterized antibody which preferentially binds to B-cell malignancies, as the CAR binding domain for this therapy. To reduce the potential immunogenicity of murine Lym-1 single-chain variable fragments (ScFv), two humanized Lym-1 antibodies (huLym-1-A and huLym-1-B) have been developed with distinct binding capabilities. To date. the therapeutic value of huLym-1-A/B CAR consisting of the 4- 1BB3z (BB3z) second generation intracellular signaling domains (ICDs) has been demonstrated in vivo in which complete regression of Lym-1 positive human lymphomas have been seen in NSG mouse models. In an exciting and novel set of experiments, we now have shown that replacing the ICDs in the huLym-1-B-BB3z with the cytoplasmic domains from DAP10 and DAP12 (DAP) mediated even more potent anti-tumor efficacy but dramatically less toxicity in vivo. In this proposal, we characterize the newly developed humanized Lym-1 antibodies in B-cell lymphoma tissues and identify the huLym-1CAR construct with the best efficacy and safety for clinical trials based upon our previous findings. These studies will advance the CAR structure design, as well as offer additional therapeutic strategies for B-cell malignancies to augment current treatment modalities.