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

David H McKenna Jr; Darin Sumstad; Diane M Kadidlo; Bjorn Batdorf; Colin J Lord; Sarah C Merkel; Christine M Koellner; Julie M Curtsinger; Carl H June; James L Riley; Bruce L Levine; Jeffrey S Miller; Claudio G Brunstein; John E Wagner; Bruce R Blazar; Keli L Hippen

doi:10.1016/j.jcyt.2016.10.011

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

Cytotherapy. 2017 Feb;19(2):250-262. doi: 10.1016/j.jcyt.2016.10.011. Epub 2016 Nov 22.

Authors

David H McKenna Jr¹, Darin Sumstad², Diane M Kadidlo², Bjorn Batdorf², Colin J Lord³, Sarah C Merkel³, Christine M Koellner³, Julie M Curtsinger⁴, Carl H June⁵, James L Riley⁶, Bruce L Levine⁵, Jeffrey S Miller⁴, Claudio G Brunstein⁴, John E Wagner³, Bruce R Blazar³, Keli L Hippen⁷

Affiliations

¹ Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA. Electronic address: mcken020@umn.edu.
² Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
³ Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
⁴ Department of Medicine, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
⁵ Department of Pathology and Laboratory Medicine, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA; Abramson Family Cancer Center Research Institute, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA.
⁶ Abramson Family Cancer Center Research Institute, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA; Department of Microbiology, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA.
⁷ Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA. Electronic address: hippe002@umn.edu.

Abstract

Background aims: 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 (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture.

Results: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with 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 aAPCs 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, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Keywords: cGMP production; graft versus host disease; regulatory T cell.

MeSH terms

Animals
Antigen-Presenting Cells / cytology
Antigen-Presenting Cells / transplantation
Calibration
Cell Culture Techniques / methods
Cell Culture Techniques / standards*
Cell Proliferation*
Cell Separation / methods
Cell Separation / standards*
Cells, Cultured
Clinical Trials as Topic
Cord Blood Stem Cell Transplantation / methods
Cord Blood Stem Cell Transplantation / standards*
Female
Fetal Blood / cytology*
Fetal Blood / immunology
Graft vs Host Disease / immunology
Graft vs Host Disease / therapy
Hematopoietic Stem Cell Transplantation / methods
Hematopoietic Stem Cell Transplantation / standards
Humans
K562 Cells
Manufacturing Industry / standards
Mice, Inbred NOD
Mice, SCID
Mice, Transgenic
Practice Guidelines as Topic
Quality Control
T-Lymphocytes, Regulatory* / cytology
T-Lymphocytes, Regulatory* / physiology

Abstract

MeSH terms

Grants and funding