Tumor infiltrating lymphocytes (TIL) are derived by excising one or more deposits of metastatic melanoma. Multiple independent TIL cultures are started from each nodule.
TIL cultures are initiated by explantation of a small (2mm3) tumor fragment or by plating 106 viable cells of a single-cell suspension of enzymatically digested tumor tissue into 2 ml of complete medium (RPMI 1640 with human serum (10%)) containing IL-2 (6000 IU/ml). The cultures are maintained at cell concentrations between 5 x105 - 2 x 106 cells/ml until several million TIL cells are available, usually within 2-4 weeks. Multiple independent cultures are screened by cytokine secretion assay, for recognition of autologous tumor cells (if available) and HLA-A2+ tumor cell lines. 2-6 independent TIL cultures exhibiting the highest cytokine secretion are further expanded until the cell number is over 5 x 107 cells (typically 3-6 weeks after tumor excision). TIL cultures that maintain specific tumor cell recognition are expanded for treatment, using one cycle of a rapid expansion protocol with irradiated allogeneic feeder cells, anti-CD3 (OKT3) antibody and IL-2 (6000 IU/ml). This results in 1000-fold expansion of cells within 14-15 days of expansion initiation.