title: Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor.
abstract: Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.
authors.affiliation: Program in Blood and Marrow Transplantation, Department of Pediatrics, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA. orcha001@maroon.tc.umn.edu
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Orchard
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Blazar
authors.forename: Scott
authors.initials: S
authors.lastname: Burger
authors.forename: Bruce
authors.initials: B
authors.lastname: Levine
authors.forename: Lisa
authors.initials: L
authors.lastname: Basso
authors.forename: David M K
authors.initials: DM
authors.lastname: Nelson
authors.forename: Keith
authors.initials: K
authors.lastname: Gordon
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
authors.forename: John E
authors.initials: JE
authors.lastname: Wagner
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Miller
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018106:CD28 Antigens / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D002498:Centrifugation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008213:Lymphocyte Activation; D008280:Magnetics; D051379:Mice; D008863:Microspheres; D020800:Receptor, Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D020800:Receptor, Nerve Growth Factor; D013937:Thymidine Kinase