Clinical-scale isolation of ‘minimally manipulated’ cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease

• Published in: Cytotherapy (Oxford, England) England), 2014-09, Vol.16 (9), p.1245-1256
• Main Authors: Odendahl, Marcus, Grigoleit, G. Ulrich, Bönig, Halvard, Neuenhahn, Michael, Albrecht, Julia, Anderl, Florian, Germeroth, Lothar, Schmitz, Marc, Bornhäuser, Martin, Einsele, Hermann, Seifried, Erhard, Busch, Dirk H, Tonn, Torsten
• Format: Article
• Language: English
• Subjects: adoptive T-cell transfer; Advanced Basic Science; Antigens, Viral - immunology; Cancer Vaccines; Cell Separation - methods; Cells, Cultured; CliniMACS; CMV-specific T cells; Cytomegalovirus - immunology; Cytomegalovirus Infections - immunology; Cytomegalovirus Infections - therapy; Drug Resistance; Histocompatibility Antigens Class I - chemistry; Histocompatibility Antigens Class I - metabolism; Humans; Immunotherapy, Adoptive - methods; MHC streptamer technology; Other; Protein Multimerization; Recurrence; Streptavidin - chemistry; T-Lymphocytes, Cytotoxic - pathology; T-Lymphocytes, Cytotoxic - transplantation; Virus Activation
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2014.05.023

Abstract Background aims Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity. Methods Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 1010 leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting. Results A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 106 CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection. Conclusions Clinical-scale isolation of “minimally manipulated” CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.