Manufacturing of expanded/activated γδ T cells using the Miltenyi Prodigy® bioreactor system

Haploidentical allogeneic hematopoietic cell transplantation (HAPLO HCT) with early post-transplant cyclophosphamide-induced reduction of allogeneic activated T cells is increasing availability of HSCT for patients withhematologic malignancies but without a matched donor. However, success has been limited by an increased incidence of graft rejection and disease relapse. Post-transplant infusion of donor gamma delta (γδ) T cells could improve immune reconstitution and thereby constitute an effective prophylaxis against infection and relapse in HAPLO HCT as they facilitate engraftment, exhibit a strong graft-versus-leukemia (GvL) effect, and do not initiate graft-versus-host disease (GvHD). However, current protocols for ex vivo expansion and activation of γδ T cells are cumbersome and are generally confined to the research setting. In an effort to simplify and standardize these procedures, we adapted the CliniMACS Prodigy (Miltenyi Biotec) for γδ T cell manufacturing. Using a closed, single-use tubing set, we processed mononuclear cells from fresh apheresis products collected from healthy volunteer donors. Cells were immunophenotyped using flow cytometry and subjected to automated Ficoll separation and activated using a combination of Zoledronate (Zometa; Novartis, Basel) and Interleukin-2 (Miltenyi; Bergisch Gladbach) (ZOL/IL-2), in OpTimizer serum-free culture (Thermo Fisher, Waltham, MA). Cells were then expanded in the CentriCult-Unit of the Prodigy, under stabilized culture conditions with manual ZOL/IL-2 addition and media exchange every 2 days. The process was continuously monitored to determine kinetics of expansion and phenotype in comparison with small-scale culture in flasks. Following 14 days' culture, αβ T cells were depleted on the Prodigy® magnetic column within the closed system using a biotinylated anti-αβ monoclonal antibody and streptavidin-coated ferromagnetic microspheres (Miltenyi Biotec). We found that phenotype and function of expanded/activated γδ T cells were comparable and that T-cell yields sufficient for our planned therapeutic dosing. The automation of closed-system γδ T cell expansion should widen acceptance and applicability of this procedure in HAPLO HCT and other settings.