title: Identity, potency, in vivo viability, and scaling up production of lentiviral vector-induced dendritic cells for melanoma immunotherapy.
abstract: SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.
authors.affiliation: Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Mudita
authors.initials: M
authors.lastname: Pincha
authors.forename: Bala Sai
authors.initials: BS
authors.lastname: Sundarasetty
authors.forename: Gustavo
authors.initials: G
authors.lastname: Salguero
authors.forename: Ralf
authors.initials: R
authors.lastname: Gutzmer
authors.forename: Henk
authors.initials: H
authors.lastname: Garritsen
authors.forename: Laura
authors.initials: L
authors.lastname: Macke
authors.forename: Andreas
authors.initials: A
authors.lastname: Schneider
authors.forename: Daniela
authors.initials: D
authors.lastname: Lenz
authors.forename: Constanca
authors.initials: C
authors.lastname: Figueiredo
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.forename: Eliana
authors.initials: E
authors.lastname: Ruggiero
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Christina
authors.initials: C
authors.lastname: Puff
authors.forename: Ute
authors.initials: U
authors.lastname: Modlich
authors.forename: Heiko
authors.initials: H
authors.lastname: von der Leyen
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Wicke
authors.forename: Arnold
authors.initials: A
authors.lastname: Ganser
authors.forename: Renata
authors.initials: R
authors.lastname: Stripecke
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D015925:Cryopreservation; D016207:Cytokines / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D003713:Dendritic Cells / Q000276:immunology* / Q000821:virology; D005434:Flow Cytometry; D005452:Fluoresceins; D016654:Genes, RAG-1 / Q000235:genetics; D005822:Genetic Vectors / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008545:Melanoma / Q000276:immunology / Q000628:therapy*; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D010446:Peptide Fragments; D013388:Succinimides
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D016207:Cytokines; D017931:DNA Primers; D005452:Fluoresceins; C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D008565:Membrane Proteins; D010446:Peptide Fragments; D013388:Succinimides; C500290:peptide SVYDFFVWL