Optimization of cGMP purification and expansion of umbilical cord blood–derived T-regulatory cells in support of first-in-human clinical trials

Abstract

Background Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft versus host disease (GVHD). We previously completed a “first-in-human” clinical trial using in vitro expanded umbilical cord blood (UCB) derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. Methods To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPC) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. Results aAPC increased UCB tTreg expansion >8-fold over CD3/28 stimulation. Re-stimulation with aAPC increased UCB tTreg expansion an additional 20–30 fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following cGMP validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared to that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. Discussion Expansion culture with cGMP aAPC and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, since HLA matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Umbilical cord blood–derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect

“…Enrichment of CD25 1 cells was accomplished by positive selection with directly conjugated anti-CD25 magnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) and the CliniMACS device. 9 Our validation run used an open selection system using a conjugated anti-CD25 magnetic microbeads concentration of 1:350. At the start of the clinical trial, we changed to a closed selection system and initially used the same concentration.…”

Engineering cord blood to improve engraftment after cord blood transplant

“…Given the minimal HLA disparity, thirdparty VSTs can be used in non-HSCT setting for oncology patients or for those with primary immune deficiencies. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB Treg may be a useful strategy for prevention of organ rejection and autoimmune diseases (127). For therapeutic use, a banking system for UCB Treg would be able to greatly reduce cost and time to transplant.…”

“…For therapeutic use, a banking system for UCB Treg would be able to greatly reduce cost and time to transplant. Mckenna et al have optimized a convenient banking protocol to thaw Tregs that have been frozen down at the end of their first stimulation and restimulate just prior to infusion (127).…”

Methods to manufacture regulatory T cells for cell therapy

“…Another approach is to expand T regs with artificial antigen‐presenting cells transduced with co‐stimulatory molecules and an Fc receptor which can be used to present antibodies on the cell membrane. For example, K562 cells expressing CD86 and CD64 are considerably better than CD3 and CD28‐coated beads at expanding UCB T regs . In these cells, expression of CD64, a high‐affinity Fc receptor, allows the cells to be ‘loaded’ with a monoclonal antibody that targets the CD3 receptor.…”