A Humanized Lym-1 CAR with Novel DAP10/DAP12 Signaling Domains Demonstrates Reduced Tonic Signaling and Increased Antitumor Activity in B-Cell Lymphoma Models.

Abstract

The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired <i>ex vivo</i> expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion <i>in vitro</i>, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for <i>ex vivo</i> expansion and <i>in vivo</i> function. Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion <i>ex vivo</i> and function <i>in vivo</i>. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both <i>ex vivo</i> and <i>in vivo</i>. DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent <i>in vivo</i> antitumor efficacy.