The allogeneic eDN70(-) CAR-T Cells Without TCR or MHC Editing Exhibit Enhanced Persistence and Function.

Allogeneic CAR-T therapy offers an "off-the-shelf" alternative to autologous CAR-T, but faces graft-versus-host rejection (GVH) and host-versus-graft rejection (HVG). Conventional TCR/MHC elimination compromises persistence and increase NK susceptibility. Here, we developed a next-generation UCAR-T strategy that avoids TCR/MHC disruption while addressing donor-cell alloreactivity and host-mediated rejection. First, we eliminated CD4/CD8 to reduce alloreactivity while preserving TCR integrity and potentially TCR/CD3-mediated fitness, although in vivo xGvHD protection remains to be determined. In parallel, we identified CD70 as a marker selectively upregulated on alloreactive immune cells, enabling targeted removal of HVG-driving subsets. Because CD70 is also induced during T-cell activation and further enhanced following CD4/CD8 knockout, we additionally deleted CD70 to prevent fratricide, generating a CD4/CD8/CD70 triple-negative UCAR-T backbone (eDN70(-) UCAR-T). As proof-of-concept, we incorporated both CD19- and CD70-targeting CARs to generate eDN70(-)19/70CAR-T cells, which showed superior expansion, reduced exhaustion, and greater resistance to HVG pressure. Furthermore, these cells demonstrated durable tumor control and robust recall responses in vivo, with preliminary tissue-level safety from the high-dose study. Collectively, our findings establish eDN70(-) UCAR-T cells as a versatile platform capable of overcoming the persistence limitations of current allogeneic CAR-T therapies, with the CD19/CD70 dual-target configuration serving as a representative implementation of this strategy.