title: 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.
authors.affiliation: *Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis †Cell Therapy Facility, Blood Services Group, Health Science Authority (HSA) Departments of ‡Hematology ∥Pathology, Singapore General Hospital (SGH), Singapore, Singapore §Department of Haematology, St George's Hospital and Medical School, London, United Kingdom.
authors.forename: Doreen
authors.initials: D
authors.lastname: Haase
authors.forename: Kia Joo
authors.initials: KJ
authors.lastname: Puan
authors.forename: Mireille
authors.initials: M
authors.lastname: Starke
authors.forename: Tuck Siong
authors.initials: TS
authors.lastname: Lai
authors.forename: Melissa Yan Ling
authors.initials: MY
authors.lastname: Soh
authors.forename: Iyswariya
authors.initials: I
authors.lastname: Karunanithi
authors.forename: Boris
authors.initials: B
authors.lastname: San Luis
authors.forename: Tuang Yeow
authors.initials: TY
authors.lastname: Poh
authors.forename: Nurhashikin
authors.initials: N
authors.lastname: Yusof
authors.forename: Chun Hsien
authors.initials: CH
authors.lastname: Yeap
authors.forename: Chew Yen
authors.initials: CY
authors.lastname: Phang
authors.forename: Willis Soon Yuan
authors.initials: WS
authors.lastname: Chye
authors.forename: Marieta
authors.initials: M
authors.lastname: Chan
authors.forename: Mickey Boon Chai
authors.initials: MB
authors.lastname: Koh
authors.forename: Yeow Tee
authors.initials: YT
authors.lastname: Goh
authors.forename: Sebastien
authors.initials: S
authors.lastname: Bertin-Maghit
authors.forename: Alessandra
authors.initials: A
authors.lastname: Nardin
authors.forename: Liam Pock
authors.initials: LP
authors.lastname: Ho
authors.forename: Olaf
authors.initials: O
authors.lastname: Rotzschke
mesh_terms: D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006086:Graft vs Host Disease / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D006801:Humans; D007165:Immunosuppression Therapy; D016219:Immunotherapy, Adoptive; D039441:Integrin alpha4 / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D007938:Leukemia / Q000150:complications / Q000276:immunology / Q000628:therapy*; D033581:Stem Cell Transplantation; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000637:transplantation
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053699:Interleukin-7 Receptor alpha Subunit; D039441:Integrin alpha4