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.
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.
Orchard
Paul J
PJ
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
Blazar
Bruce R
BR
Burger
Scott
S
Levine
Bruce
B
Basso
Lisa
L
Nelson
David M K
DM
Gordon
Keith
K
McIvor
R Scott
RS
Wagner
John E
JE
Miller
Jeffrey S
JS
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
D018106:CD28 Antigens; D017252:CD3 Complex; D020800:Receptor, Nerve Growth Factor; D013937:Thymidine Kinase