Generation of dendritic cells from positively selected CD14+ monocytes for anti-tumor immunotherapy.
Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78+/-10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.
Curti
Antonio
A
Institute of Hematology and Medical Oncology L & A Seràgnoli, University of Bologna, Bologna, Italy.
Isidori
Alessandro
A
Ferri
Elisa
E
Terragna
Carolina
C
Neyroz
Paolo
P
Cellini
Claudia
C
Ratta
Marina
M
Baccarani
Michele
M
Lemoli
Roberto M
RM
D019496:Cancer Vaccines / Q000627:therapeutic use*; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation; D003713:Dendritic Cells / Q000166:cytology* / Q000637:transplantation; D015232:Dinoprostone / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D018189:Immunomagnetic Separation; D016219:Immunotherapy, Adoptive; D007275:Injections, Intravenous; D007279:Injections, Subcutaneous; D007378:Interleukins / Q000494:pharmacology; D007937:Leukapheresis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D009101:Multiple Myeloma / Q000473:pathology / Q000628:therapy*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014611:Vaccination
D019496:Cancer Vaccines; D007378:Interleukins; D018950:Lipopolysaccharide Receptors; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015232:Dinoprostone