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

(2018) CYTOTHERAPY. 20(9). p.1164-1181
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Abstract
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.
Keywords
MONOPHOSPHORYL-LIPID-A, T-CELLS, EFFECTOR-CELLS, IN-VITRO, IMMUNOTHERAPY, MATURATION, RECEPTOR, DIFFERENTIATION, EXPRESSION, IMMUNITY, dendritic cell, immunotherapy, interferon-gamma, messenger RNA, monophosphoryl lipid A, prostaglandin E2, tumor necrosis factor-alpha, vaccination

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Chicago
Brabants, Elisabeth, Kelly Heyns, S. De Smet, P. Devreker, J. Ingels, Nancy De Cabooter, Veronique Debacker, et al. 2018. “An Accelerated, Clinical-grade Protocol to Generate High Yields of Type 1-polarizing Messenger RNA Loaded Dendritic Cells for Cancer Vaccination.” Cytotherapy 20 (9): 1164–1181.
APA
Brabants, E., Heyns, K., De Smet, S., Devreker, P., Ingels, J., De Cabooter, N., Debacker, V., et al. (2018). An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA loaded dendritic cells for cancer vaccination. CYTOTHERAPY, 20(9), 1164–1181.
Vancouver
1.
Brabants E, Heyns K, De Smet S, Devreker P, Ingels J, De Cabooter N, et al. An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA loaded dendritic cells for cancer vaccination. CYTOTHERAPY. Oxford: Elsevier Sci Ltd; 2018;20(9):1164–81.
MLA
Brabants, Elisabeth et al. “An Accelerated, Clinical-grade Protocol to Generate High Yields of Type 1-polarizing Messenger RNA Loaded Dendritic Cells for Cancer Vaccination.” CYTOTHERAPY 20.9 (2018): 1164–1181. Print.
@article{8601284,
  abstract     = {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 {\textacutedbl}classical{\textacutedbl} 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. {\textacutedbl}Classical{\textacutedbl} 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.},
  author       = {Brabants, Elisabeth and Heyns, Kelly and De Smet, S. and Devreker, P. and Ingels, J. and De Cabooter, Nancy and Debacker, Veronique and Dullaers, Melissa and Van Meerbeeck, J. P. and Vandekerckhove, B. and Vermaelen, Karim},
  issn         = {1465-3249},
  journal      = {CYTOTHERAPY},
  language     = {eng},
  number       = {9},
  pages        = {1164--1181},
  publisher    = {Elsevier Sci Ltd},
  title        = {An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA loaded dendritic cells for cancer vaccination},
  url          = {http://dx.doi.org/10.1016/j.jcyt.2018.06.006},
  volume       = {20},
  year         = {2018},
}

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