Generation of Dendritic Cells from Positively Selected CD14 + Monocytes for Anti-tumor Immunotherapy

• Published in: Leukemia & lymphoma Vol. 45; no. 7; pp. 1419 - 1428
• Main Authors: Curti, Antonio, Isidori, Alessandro, Ferri, Elisa, Terragna, Carolina, Neyroz, Paolo, Cellini, Claudia, Ratta, Marina, Baccarani, Michele, Lemoli, Roberto M
• Format: Journal Article
• Language: English
• Published: United States Informa UK Ltd 01.07.2004; Taylor & Francis
• Subjects: Cancer Vaccines - therapeutic use; CD14 + cells; Cell Differentiation; Cells, Cultured; Cellular immunotherapy; Cryopreservation; Dendritic cells; Dendritic Cells - cytology; Dendritic Cells - transplantation; Dinoprostone - pharmacology; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Humans; Immunomagnetic Separation; Immunotherapy, Adoptive; Injections, Intravenous; Injections, Subcutaneous; Interleukins - pharmacology; Leukapheresis; Lipopolysaccharide Receptors - analysis; Monocytes - cytology; Monocytes - drug effects; Multiple myeloma; Multiple Myeloma - pathology; Multiple Myeloma - therapy; Tumor Necrosis Factor-alpha - pharmacology; Tumor-associated antigens; Vaccination
• ISSN: 1042-8194; 1029-2403
• DOI: 10.1080/10428190310001653682

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.