Expansion of Tumor-Infiltrating Lymphocytes from Melanoma Tumors

Autologous, in vitro-expanded tumor-infiltrating lymphocytes (TIL) have been successfully used for treatment of melanoma patients. Expansion of TIL from tumors is usually performed in two steps. The details of the procedure differ between different laboratories, but the general concept remains the same. In the first step, small fragments of a tumor are placed in culture medium containing one or more T cell stimulating growth factors. Here the most common protocol using interleukin (IL)-2 is described. The length of the first step is flexible to allow generation of enough cell to start the second step of the procedure. The second step is a so-called rapid expansion protocol (REP) where harvested TIL from the first step are induced to massive proliferation via triggering of their T cell receptor (TCR) complex in presence of an excess of feeder cells and, again, T cell stimulating growth factors. This second expansion step is usually around 2 weeks in length. Here will be described a REP that uses soluble anti-CD3 antibodies for TCR triggering, irradiated peripheral blood mononuclear cells (PBMC) as feeder cells, and IL-2 as the T cell growth factor. Furthermore, the described protocol utilizes gas-permeable cell culture flasks that yield large number of cells similar to conventional bioreactors but using standard laboratory equipment.