Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

• Published in: Journal of translational medicine Vol. 9; no. 1; p. 198
• Main Authors: Chiang, Cheryl L-L, Maier, Dawn A, Kandalaft, Lana E, Brennan, Andrea L, Lanitis, Evripidis, Ye, Qunrui, Levine, Bruce L, Czerniecki, Brian J, Powell, Jr, Daniel J, Coukos, George
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 14-11-2011; BioMed Central; BMC
• Subjects: Cancer; Cancer cells; Cell Culture Techniques - methods; Cell Differentiation - drug effects; Cell Extracts - pharmacology; Cell Line, Tumor; Cell Survival - drug effects; Cryopreservation; Culture Media - pharmacology; Dendritic cells; Dendritic Cells - cytology; Dendritic Cells - drug effects; Dendritic Cells - secretion; Good Manufacturing Practice; Humans; Hypochlorous Acid - pharmacology; Immunology; Interleukin-12; Interleukin-12 - biosynthesis; Interleukin-12 - secretion; Lymphocyte Culture Test, Mixed; Medical research; Ovarian cancer; Oxidation-Reduction - drug effects; Phenotype; Physiological aspects; Time Factors; Trypsin - metabolism; Vaccines; United States
• ISSN: 1479-5876; 1479-5876
• DOI: 10.1186/1479-5876-9-198

Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.
Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.
DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning(®) tissue-culture treated surface and Corning(®) ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.
This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).