An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA-loaded dendritic cells for cancer vaccination

Background: Many efforts have been devoted to improve the performance of dendritic cell (DC) based cancer vaccines. Ideally, a DC vaccine should induce robust type 1 polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP) compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established classical protocol. Methods: Immunomagnetically purified monocytes were cultured in a closed system for 3 days in GMP-compliant serum-free medium and cytokines, and matured for 24 h using monophosphoryl lipid A (MPLA)+ interferon -gamma (IFN-y). Mature DCs were electroporated with messenger RNA (mRNA) encoding full-length antigen and cryopreserved. Classical DCs were cultured for 8 days in flasks, with one round of medium and cytokine supplementation, and matured with tumor necrosis factor alpha (TNF-alpha) + prostaglandin E2 (PGE2) during the last 2 days. Results: Four-day MPLA/IFN-y matured DCs were superior to 8-day TNF-alpha/PGE2 matured DCs in terms of yield, co-stimulatory/coinhibitory molecule expression, resilience to electroporation and cryopreservation and type 1 polarizing cytokine and chemokine release after cell thawing. Electroporated and cryopreserved DCs according to our protocol efficiently present epitopes from tumor antigen-encoding mRNA, inducing a strong expansion of antigen-specific CD8+ T-cells with full cytolytic capacity. Conclusion: We demonstrate using a GMP-compliant culture protocol the feasibility of generating high yields of mature DCs in a short time, with a superior immunogenic profile compared with 8-day TNF-alpha/PGE2 matured DCs, and capable of inducing vigorous cytotoxic T-cell responses to antigen from electroporated mRNA. This method is now being applied in our clinical trial program.