Mouse Anti-Human CD19 Hybridoma [DBU-24.2F21]

Purpose: 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 CD3 zeta (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. Experimental Design: 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 in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. Results: 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 ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. Conclusions: 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 in vivo antitumor efficacy.

RSK2 is a serine/threonine kinase downstream signaling mediator in the RAS/ERK signaling pathway and may be a therapeutic target in mantle cell lymphoma (MCL), an almost incurable disease subtype of non-Hodgkin lymphoma. In this study, serine-227 (RSK2(Ser227)) in the N-terminal kinase domain (NTKD) of RSK2 was found to be ubiquitously active in five MCL-derived cell lines and in tumor tissues derived from five MCL patients. BI-D1870, an inhibitor specific to RSK2-NTKD, caused RSK2(Ser227) dephosphorylation, and thereby, induced dose-dependent growth inhibition via G(2)/M cell cycle blockade and apoptosis in four of the five cell lines, while one cell line showed only modest sensitivity. In addition, RSK2 gene knockdown caused growth inhibition in the four BI-D1870-sensitive cell lines. Comparative gene expression profiling of the MCL-derived cell lines showed that inhibition of RSK2(Ser227) by BI-D1870 caused downregulation of oncogenes, such as c-MYC and MYB; anti-apoptosis genes, such as BCL2 and BCL2L1; genes for B cell development, including IKZF1, IKZF3, and PAX5; and genes constituting the B cell receptor signaling pathway, such as CD19, CD79B, and BLNK. These findings show that targeting of RSK2(Ser227) enables concomitant blockade of pathways that are critically important in B cell tumorigenesis. In addition, we found favorable combinatory growth inhibitory effects of BI-D1870 with inhibitors of BTK (ibrutinib), AKT (ipatasertib), and BCL2 (venetoclax) in cell characteristic-dependent manners. These results provide a rationale for RSK2(Ser227) in the NTKD as a potential therapeutic target in MCL and for future development of a novel bioavailable RSK2 NTKD-specific inhibitor.