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

CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me(2)SO) and stored in liquid nitrogen (liqN(2)). Prior to inoculation, doses must be thawed, washed to remove Me(2)SO and suspended for clinical administration. Avoiding the use of Me(2)SO and storage in liqN(2) would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine's biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37°C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70-140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at -84°C. Vaccine doses could be frozen in trehalose at -84°C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me(2)SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at -84°C avoiding the use of Me(2)SO and liqN(2) storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.