Generation of DC-based vaccine for therapy of B-CLL patients. Comparison of two methods for enriching monocytic precursors.
BACKGROUND
The generation of Ag-loaded DC under good manufacturing practice (GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes.


METHODS
We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and evaluated the feasibility of generating DC from B-CLL patients and the effects of cryopreservation.


RESULTS
Monocyte yield from a single leukapheresis product of a B-CLL patient varied from 1 x 108 to 10 x 108 total cells, from which 40-200 x 106 mature DC could be produced. Adequate numbers of monocytes could not be enriched from one patient with 0.2% monocytes in the leukapheresis product, and the target of 50 x 106 DC was barely achieved in another patient with 0.9% monocytes in the pheresed cells. These results suggested that successful production of DC is dependent on a minimum frequency of 1% CD14(+) monocytes in the leukapheresis product. Cryopreservation of tumor cell-loaded DC yielded a recovery rate of 86+/-4.4% upon thawing, with a total viability of 90+/-2.8%. Most importantly, cryopreserved Ag-loaded DC retained their morphology, phenotype and function.


CONCLUSIONS
The results demonstrate that adequate numbers of functional DC required for clinical therapy can be generated from patients who have >1% of CD14(+) monocytes in the leukapheresis product. Moreover, Ag-loaded DC can be cryopreserved and recovered without significant change in phenotype or function.
Kokhaei
P
P
Immune and Gene Therapy Lab, CCK, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
Adamson
L
L
Palma
M
M
Osterborg
A
A
Pisa
P
P
Choudhury
A
A
Mellstedt
H
H
D019496:Cancer Vaccines / Q000276:immunology* / Q000627:therapeutic use; D002470:Cell Survival; D015925:Cryopreservation; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D018189:Immunomagnetic Separation; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D015448:Leukemia, B-Cell / Q000276:immunology / Q000628:therapy; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000628:therapy; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D010641:Phenotype; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
D019496:Cancer Vaccines; D018664:Interleukin-12; D007371:Interferon-gamma