Developing allogeneic double negative T cells as a novel off-the-shelf adoptive cellular therapy for cancer

Purpose: To expand clinical-grade healthy donor-derived DNTs to a therapeutically relevant number and characterize their potential to be used as an "off-the-shelf" adoptive cellular therapy against cancers. Experimental Design: We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry-based high throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo. Results: Clinical-grade DNTs expanded 1558±795.5 fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anti-cancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of allo-reactive CD8+ T cells in vitro, and co-infusion of DNTs with PBMC from a different donor into mice resulted in co-engraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity towards DNTs, suggesting the lack of host versus graft reaction. Conclusion: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

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