Analysis of Expanded Human Cytokine Induced Killer Cells for Cellular Therapy.

Abstract

We have previously reported on the ex vivo expansion of cytotoxic effector T cells termed Cytokine Induced Killer (CIK) cells. CIK cells demonstrate both in vivo and in vitro anti-tumor activity in murine models with limited potential for GVHD induction. Human CIK cells are readily expanded from peripheral blood mononuclear cells (PBMC) when cultured with IFNg, CD3 and IL-2 and show cytotoxic activity against a variety of tumor cell lines and fresh autologous tumor cells through an NKG2D dependent mechanism. Clinical application of CIK cells requires expansion of effector cells in GMP-compliant manner. In this study, PBMC were collected by apheresis from 9 healthy non-mobilized donors and placed in closed, continuous perfusion cultures using Aastrom RepliCell@biochambers for 21–28 days. Cultures were inoculated with 3x108–7.5x108 nucleated cells containing a median of 46% CD3+ cells (range 29%–71%). At culture harvest, the median cell count was > 4x109 (range 2.6x109–8.7x109) with 97% CD3+ (86%–99%) and an average cell viability of 84.8% (range, 75%–99.6%). The average expansion of CD3+ cells in culture was > 30-fold (range13–91). CD3+CD56+ are the subpopulation of cells that demonstrated the highest cytotoxicity against tumor cell lines in culture. The mean percentage of these cells was 1.8% however at harvest, the mean CD3+CD56+ was 14% representing an average 34-fold expansion (range 19–515). The majority of CD3+ cells were CD8+ (range, 33–82%), NKG2D was predominately expressed by cells recovered from most cultures (range 47–76%). CD56+ was expressed by 1% – 36% of cells in different cultures and CD8+NKG2D+ expression ranged from 21%–50% of harvested cells. There was a moderate up-regulation of the activation markers CD25 and CD69 and the NK markers CD16, CD161 and CD94. In all cultures, the majority of CD8+ and CD56+ cells co-expressed the selectin marker a4b7. Furthermore the majority of the CD3+CD8+ cells were effector memory cells expressing CD62L−CD44+ (50%–71%) while the central memory cells CD62L+CD44+ ranged from 10%–40%, almost no naïve cells were detected in time of harvest. Cytotoxicity assays against a panel of tumor cell lines (B and T cell lymphomas, uterine cancer, erythroleukemia, ovarian cancer and plasmacytoma) showed significant yet variable killing. This system allows for the rapid expansion of cytotoxic cell in GMP-compliant manner suitable for clinical application.