Efficient generation of clinical-grade genetically modified dendritic cells for presentation of multiple tumor-associated proteins.
BACKGROUND
Dendritic cells (DCs) play a central role in the initiation and regulation of immune responses. DCs for clinical applications can be generated with high yield from leukapheresis products. Using adenoviral transduction we genetically modified human DCs to produce and present melanoma-associated antigens. Coexpression of green fluorescent protein and epitope tags were used to monitor genetic modification. Generation, genetic modification, and cryoconservation of gene modified human DCs on a clinical scale in a closed system is feasible.


METHODS
CD14-positive monomuclear cells were isolated from leukapheresis products of HLA-A* 0201 positive voluntary blood donors using immunomagnetic beads. Selected cells were cultivated for 7 days. Adenovirus transduction was optimal on Day 4. Maturation was induced on Day 5. Mature DC were aliquoted and cryoconserved on Day 7. Quality control was performed using flow cytometry, expression profiling, and functional assays (ELISPOT, CBA).


RESULTS
We were able to generate sufficient genetically modified mature DCs in serum-free cultures that could be stored by cryopreservation. The use of a closed system facilitated development of methods for standardized production of clinically applicable genetically modified DCs. The adenoviral transduction system allowed simultaneous and flexible expression of tumor-associated antigens for prolonged presentation of multiple epitopes.


CONCLUSIONS
The feasibility of a closed-bag system for the cultivation of genetically modified human DCs is shown. The immature DCs were genetically modified by recombinant replication-deficient adenoviruses to express multiple epitopes of tumor-associated proteins and then differentiated to mature antigen-presenting DCs.
Garritsen
Henk S P
HS
Institute for Clinical Transfusion Medicine, Braunschweig, Germany.
Macke
Lars
L
Meyring
Wilhelm
W
Hannig
Horst
H
Pägelow
Ute
U
Wörmann
Bernhard
B
Geffers
Robert
R
Dittmar
Kurt E J
KE
Lindenmaier
Werner
W
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D018941:Antigens, CD19 / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000502:physiology*; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology*; D015789:HLA-A2 Antigen; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D009363:Neoplasm Proteins / Q000032:analysis*; D030781:Organisms, Genetically Modified / Q000502:physiology*; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology; D014611:Vaccination / Q000379:methods
D015703:Antigens, CD; D018941:Antigens, CD19; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D018950:Lipopolysaccharide Receptors; D009363:Neoplasm Proteins