Large-scale Isolation of Highly Pure “Untouched” Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy

Abstract

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

“…Moreover, ex vivo expansion also allowed us to obtain a purer product than that obtained by direct isolation. In our experience, largescale T reg cell selection using the CliniMACS isolation system from leukapheresis yielded a CD4 + CD25 + T-cell purity of 55% (range 42.6-62%), the majority of which expressed FoxP3, in keeping with the reported data from healthy subjects [40,46,47].In response to recent studies showing the negative effect of cryopreservation on T reg cell function [41,42], we have previously demonstrated that expanded T reg cells after thawing can effectively prevent the onset of xenogeneic GvHD as well as improve acute GvHD and survival in a mouse model of GvHD using immunosuppressed mice (i.e., NOD-SCID-gamma knockout mice) [7]. Herein, the in vitro data reported also confirmed our previous results for T reg cell expansion according to our GMP-compliant process from patients with end-stage LD or KD.…”

Regulatory T Cell Therapy of Graft-versus-Host Disease: Advances and Challenges

“…Although magnetic bead sorting for CD4 + /CD25 hi cells may incorporate CD127 lo , purity may remain inadequate because activated naive and especially memory Tcons express CD25 and are therefore difficult to separate by MACS from CD25 hi Treg population. Including more surface markers (e.g., CD45RA + , CD49d − , and CD39 hi ), can result in a better defined and purer Treg population; however, when using MACS, there is a limit to the number of markers that can be included [ 199 , 200 , 201 , 202 ].…”

Minimally manipulated murine regulatory T cells purified by reversible Fab Multimers are potent suppressors for adoptive T‐cell therapy

“…The infusion of either freshly isolated or ex vivo expanded Tregs can improve survival of recipients in acute GVHD, but it is generally believed that very large numbers of Treg cells are required to achieve therapeutic effects . Due to the relatively low frequency of nTregs in circulation (1–2% of total CD4 + T cell population in humans ) the purification of sufficiently large numbers of Tregs has been challenging or might not even be feasible in clinical practice without in vitro expansion . We established a protocol enabling the purification of Tregs and the subsequent liberation from isolation reagents in an one‐step enrichment process.…”