66. Development of a novel methodology for cryopreservation of melanoma cells applied to the CSF470 therapeutic vaccine

Abstract

CSF470 therapeutic vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is being tested in a Phase II/III clinical trial in stage II/III melanoma patients. Currently, irradiated cells that comprise CSF470 vaccine are frozen using dimethyl sulfoxide (Me2SO) as a cryoprotectant and stored in liquid nitrogen (LN2) until its use. Prior to inoculation, each dose must be thawed under sterile conditions, washed to remove Me2SO and finally resuspended until clinical i.d. administration. In order to facilitate CSF470 pharmaceutical production and distribution we need to develop a methodology that allows the preservation of CSF470 in a freeze-dried formulation, avoiding storage in LN2 while keeping its biological and immunogenic properties. To achieve freeze-drying of eukaryotic nucleated cells they should be frozen in the absence of the currently used cryoprotectant Me2SO, this being the critical step. We tested diverse freezing conditions, employing cryoprotectants suitable to be used as excipients in the final product. Since the freeze and thaw process represent a stress for the cells, we also studied hsp 27, 70 and 90 pattern of expression in the four cell lines that comprise the vaccine. We investigated trehalose (TRE) as cell cryoprotectant, both before and after gamma irradiation. We also analyzed TRE uptake by melanoma cells and investigated the Fluid-Phase endocytosis kinetics, employing the fluorescent dye Lucifer Yellow (LYCH). We assayed different compositions of freezing media with TRE and human serum albumin (HSA). We compared samples frozen with TRE and control Me2SO, evaluating cellular integrity (trypan blue exclusion), persistence of vaccine antigens (MART-1, gp100, GD2, GD3 and HLA-A0201, FACS), proliferative capacity (MTT assay) and in case of irradiated cells, the proportion of apoptotic/necrotic cells, by Annexin-V/IP staining. TRE uptake was measured using the anthrone-sulfuric assay. Cells incubated with 0.2 M TRE after 5 h of fluid-phase endocytosis at 37 °C reached an intracellular TRE concentration that varied between 100 and 170 mM for each cell line. Optimal freezing conditions were 0.2 M TRE and 30 mg/ml HSA at − 80 °C. After freezing and subsequent thawing, the number of recovered cells, cellular integrity as well as the presence of specific antigens obtained for CSF470 frozen in TRE were not statistically different to the Me2SO control condition. Furthermore, the apoptosis/necrosis profile after gamma irradiation was similar in both cases. The non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryoprotection procedures. However, some differences were observed in the separated cell lines in terms of growth kinetics after cryopreservation. Differences in the hsps expression pattern, as detected by Western blot, were found between cell lines, suggesting a possible role for these proteins in their freezing performance. Using the TRE-freezing medium we are able to cryopreserve CSF470 vaccine in the absence of Me2SO at − 80 °C, and under these conditions we could start the vaccine freeze-drying assays.