Introduction. Despite the overwhelming success of anti-CD19 CAR T cell therapies in recent years, it is still impossible to date to replicate comparable results in patients with solid tumors. Such obstacle is due to many factors, but the three most relevant ones are: a) the complex and immune-suppressive tumor microenvironment, b) the lack of optimal tumor targets that are not also expressed on normal cells and c) the use of autologous, patient-derived cells, which are frequently sub-optimal due to chemotherapy. Here we investigated a novel strategy to overcome these three barriers by using unconventional, MHC-independent gamma delta T cells and a chimeric PD-1 protein that was previously validated in murine conventional alpha-beta T cells. We tested the hypothesis that our strategy could specifically target cancer cells without a CAR for a specific tumor-associated antigen by turning PD-1 immune suppression into T-cell activation and using donor-derived, “off-the-shelf” effector cells. Experimental procedures. We built a chimeric PD-1 receptor consisting of the extracellular portion of PD-1 fused to the intracellular domains of DAP10 and CD3z. Gamma delta T cells from healthy donors were expanded ex vivo and stably transduced with a gamma-retrovirus-derived viral vector carrying the chimeric construct. Cells were tested for killing efficiency, cytokines secretion, and memory differentiation in vitro. We then evaluated the in vivo efficacy and tolerability in murine xenografts. Results. chPD1-gdT cells selectively killed PDL-1+ tumor cells, with minimal on-target/off-tumor toxicities and without off-target toxicities. Cells were well tolerated in mice, without damage to normal PD-L1+ cells. Upon contact with PD-L1+ cancers, chPD1-gdT cells, but not untransduced gdT cells, expressed markers of memory phenotype and secreted inflammatory cytokines. In vivo, chPD1-gdT cells, but not untransduced gdT cells, cleared PD-L1+ tumors without significant elevation of CRS-related cytokines nor off-tumor toxicities. Conclusions. chPD1-gdT cells are potent and safe in vitro and in vivo and will be assessed in a Phase I/II clinical trial.

Citation Format: Amorette Barber, Xiaohong Wang, Anupama Gopisetty, Leonardo Mirandola, Maurizio Chiriva-Internati. Gamma delta T cells engineered with a chimeric PD-1 receptor effectively control PD-L1 positive tumors in vitro and in vivo with minimal toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB148.