Developing Allogeneic Double-Negative T Cells as a Novel Off-the-Shelf Adoptive Cellular Therapy for Cancer

Abstract

Purpose: To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an "offthe-shelf" adoptive cellular therapy (ACT) 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 1,558 AE 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 anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8 þ T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of hostversus-graft reaction.Conclusions: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

Double-negative T Cells Inhibit Proliferation and Invasion of Human Pancreatic Cancer Cells in Co-culture

“…Previous reports have demonstrated that DNT cells have significant anti-cancer effects in patient-derived xenograft (PDX) models of lung cancer and leukemia (9)(10)(11)(12). Moreover, infusion of allogeneic DNT cells did not induce graft versus host disease (13). These unique features of DNT cells differentiate them from conventional T cells and support their potential use as a new adoptive cellular therapy for cancers (13).…”

DNT Cell-based Immunotherapy: Progress and Applications

“…NKG2D mediates cytotoxicity by combining MHC class I polypeptide-related sequence A and B with UL16-binding protein molecules, which are highly expressed on tumor cells [62,78]. DNAM-1 induces killing by recognizing CD112 and CD155 ligands, which are highly expressed on tumor cells but negligibly expressed on their normal counterparts [68,[79][80][81][82][83][84][85][86]. Lee J et al [36] demonstrated that DNTs recognize and kill acute myeloid cells mainly by activating NKG2D and DNAM-1 receptors.…”

“…The isolation and expansion of DNTs in vitro could prevent the activation of various immunosuppressive mechanisms in the tumor microenvironment. Lee JB and coworkers [86] established a system to expand clinically applicable DNTs derived from healthy volunteers ( Figure 1); specifically, they used cytokines to mediate the activation and proliferation of the DNTs in vitro, thereby preventing serious side effects caused by the application of biological agents in vivo. Infusion of allogeneic DNTs does not cause GVHD [112][113][114], and DNTs can kill cancer cells but are harmless to normal cells.…”

“…Young KJ et al [115] injected expanded DNTs with A20 lymphoma cells into immunodeficient mice, and the DNTs in the spleen of the mice were gradually increased and observed in the fifth month of treatment. Amplified DNTs express the effector memory T cell markers CD45RA, CD44, CD43, and CD49D, and DNTs have a longer duration during the immune response than do central memory T cells [86]. In summary, allogeneic DNTs satisfy the requirements for serving as off-the-shelf ACIs without genetic modification [86], making them great clinical application prospects.…”

“…Amplified DNTs express the effector memory T cell markers CD45RA, CD44, CD43, and CD49D, and DNTs have a longer duration during the immune response than do central memory T cells [86]. In summary, allogeneic DNTs satisfy the requirements for serving as off-the-shelf ACIs without genetic modification [86], making them great clinical application prospects. Zhang and coworkers carried out a clinical trial of allogeneic DNT treatment of AML patients (NCT03027102), and the effect of the DNTs on the tumor microenvironment needs confirmation.…”