Work

207 Off-the-shelf DLL3/CD70 dual CAR T cells with optimized site-specific integration for SCLC and neuroendocrine tumors

Year: 2026

Type: conference-abstract

Abstract: Background Delta-like ligand 3 (DLL3) is a clinically validated target in small cell lung cancer (SCLC) and other neuroendocrine tumors. Allogeneic DLL3 chimeric antigen receptor (CAR) T cells with high sensitivity, specificity, and activity have been previously described. While allogeneic DLL3 CAR T cells could offer immediate availability and scalable production compared to autologous therapies, expansion and clinical efficacy may be limited by premature immune rejection. Targeting CD70-expressing alloreactive T cells has been shown to mitigate rejection in preclinical and clinical studies, providing a rationale for incorporating an anti-rejection CD70 CAR to enhance persistence and activity of allogeneic DLL3 CAR T cells. We therefore developed allogeneic DLL3/CD70 dual CAR T cells with optimized site-specific integration (SSI) to maximize antitumor efficacy and improve resistance to immune-mediated rejection.Methods T cell receptor alpha constant (TRAC) knockout (KO) DLL3 and DLL3/CD70 CAR T cells were produced using SSI with CRISPR gene editing technology and an adeno-associated virus (AAV). In addition to SSI at the TRAC locus, multiple integration sites were evaluated. Cytotoxic activity and CAR T cell expansion were assessed in vitro and in vivo using functional assays and SCLC xenograft models. Persistence of DLL3/CD70 dual CAR T cells in the presence of alloreactive host T cells was evaluated in MLR assays.Results TRAC KO DLL3 and DLL3/CD70 CAR T cells engineered via SSI at the TRAC locus or alternative loci were successfully produced from healthy donor-derived T cells. Across knock-in (KI) designs, differences were observed in CAR expression, in vitro cytotoxicity and in vivo efficacy. Compared to SSI at TRAC, SSI at other loci associated with T cell function enhanced CAR T cell performance. Among the loci tested, SSI at SOCS1 emerged as the lead design, yielding CAR T cells with superior in vivo efficacy across DLL3-high (SHP-77) and DLL3-moderate (H82) xenograft models. Compared to DLL3 CAR T cells, DLL3/CD70 dual CAR T cells eliminated CD70+ alloreactive T cells and showed resistance to rejection.Conclusions Integration of the DLL3 and CD70 CARs into the SOCS1 locus was associated with superior efficacy compared to TRAC SSI and SOCS1 KO controls, supporting the importance of genomic context on CAR expression and function. Co-expression of an anti-rejection CD70 CAR conferred resistance to alloreactive T cells. These findings support the development of allogeneic DLL3/CD70 dual CAR T cells as a differentiated off-the-shelf design that combines optimized CAR function with intrinsic rejection resistance for the treatment of SCLC and neuroendocrine tumors.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0207