Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells

Adaeze Precious Ekwe; Raymond Au; Ping Zhang; Benjamin A McEnroe; Mei Ling Tan; Alda Saldan; Andrea S Henden; Cheryl J Hutchins; Ashleigh Henderson; Kari Mudie; Keri Kerr; Madonna Fuery; Glen A Kennedy; Geoffrey R Hill; Siok-Keen Tey

doi:10.1016/j.jcyt.2024.02.023

Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells

Cytotherapy. 2024 Mar 5:S1465-3249(24)00066-5. doi: 10.1016/j.jcyt.2024.02.023. Online ahead of print.

Authors

Affiliations

¹ Translational Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia; School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Kelvin Grove, Queensland, Australia.
² Translational Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.
³ Translational Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA.
⁴ Translational Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia; Department of Haematology and Bone Marrow Transplantation, Cancer Care Services, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia; Faculty of Medicine, University of Queensland, St Lucia, Queensland, Australia.
⁵ Department of Haematology and Bone Marrow Transplantation, Cancer Care Services, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia.
⁶ Department of Haematology and Bone Marrow Transplantation, Cancer Care Services, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia; Faculty of Medicine, University of Queensland, St Lucia, Queensland, Australia.
⁷ Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA.
⁸ Translational Cancer Immunotherapy Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia; School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Kelvin Grove, Queensland, Australia; Department of Haematology and Bone Marrow Transplantation, Cancer Care Services, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia; Faculty of Medicine, University of Queensland, St Lucia, Queensland, Australia. Electronic address: Siok.Tey@qimrberghofer.edu.au.

PMID: 38530690
DOI: 10.1016/j.jcyt.2024.02.023

Abstract

Background aims: Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity.

Methods: We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs.

Results: We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×10⁶ gene-marked cells, sufficient for a cell dose of at least 2 × 10⁶ cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A.

Conclusions: This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Keywords: adoptive T cell transfer; gene-marking; good manufacturing practice; multiparametric closed-system fluorescence-activated cell sorting; regulatory T cells; retroviral transduction.