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

Paul J. Orchard, Bruce R. Blazar, Scott Burger, Bruce Levine, Lisa Basso, David M.K. Nelson, Keith Gordon, R. Scott McIvor, John E. Wagner, Jeffrey S. Miller

Research output: Contribution to journalArticle

34 Scopus citations

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 × 108 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 × 1010). 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.

Original languageEnglish (US)
Pages (from-to)979-988
Number of pages10
JournalHuman gene therapy
Volume13
Issue number8
DOIs
StatePublished - May 20 2002

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