Abstract
Background
We have previously demonstrated a laboratory model for expanding autologous mononuclear cells into populations of effector killer cells. The goal of the current experiments was to develop a good manufacturing practice (GMP) method for expanding clinical-grade activated effector cells that mediate tumor cell killing through various mechanisms that could be infused into patients following high-dose chemotherapy and autologous stem cell transplant.
Methods
Mobilized mononuclear cells (MNC) from myeloma patients were placed in culture with serum-free AIM V media, interleukin-2 (1000 IU/mL) and OKT-3 (500 ng/mL) at 37°C and 5% CO2. After 7 days of expansion, the cells were analyzed for cell concentration, viability, phenotype and cytotoxicity directed against human myeloma cell lines. Expansion was compared using culture bags and flasks. Cryopreserved expanded cells were also analyzed.
Results
This clinical model of ex vivo expansion yielded polyclonal populations of cytotoxic lymphocytes, including CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD8+ CD56+ T cells and CD56+ natural killer cells. Compared with flasks, culture bags provided a 2–3-fold effector cell expansion with minimal risk of contamination. The optimal cell concentration at the time of expansion was 2.5–3.5 × 106 peripheral blood MNC/mL. Viability and cytotoxicity were maintained if the expanded cells were cryopreserved and then thawed for use.
Discussion
The results demonstrate a reproducible and reliable GMP procedure that is currently being employed in a clinical trial. These expanded cells, and their various pathways of tumor cell killing, may circumvent tumor escape mechanisms and improve outcomes.