Large-Scale Production of Autologous CD14+-Monocyte Derived Dendritic Cells Co-Electroporated with Amplified Total Tumour mRNA and Human CD40L mRNA in Patients with B-Cell Chronic Lymphocytic Leukemia.
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AbstractBACKGROUND: B-cell chronic lymphocytic leukemia (CLL) is currently incurable with conventional chemotherapeutic approaches. The induction of specific anti-CLL immune responses in vitro using autologous tumour-antigen loaded dendritic cell (DC)-based approaches has been previously demonstrated. The aim of this study was to optimize and validate large-scale production of an autologous DC vaccine prior to initiation of a planned Phase I/II clinical trial for patients with previously treated CLL.STUDY DESIGN: Following informed consent, 8 patients with confirmed CLL had 11 leukapheresis collections for mononuclear cells performed between April 2005 and May 2006. Methods for vaccine production were optimized during an initial cohort of CLL patients (N=7); production methods ‘at scale’ were subsequently confirmed with a validation cohort (N=3).METHODS: Leukemic B-cells were positively selected (CD19+/CD5+) and preserved in RNAlater™ prior to overnight shipment to Argos Therapeutics Inc (Durham, NC); amplified patient specific total tumour mRNA and huCD40L mRNA were subsequently supplied by Argos for final vaccine formulation. CD14+ monocytes were positively selected using a CliniMACS device (Miltenyi Biotech, Germany). Purified monocytes were subsequently cultured for 5 days in growth medium containing IL-4 and GM-CSF; DCs were matured with addition of TNF-β, IFN-γ, and PGE2 on day 5. DCs were harvested on day 6, co-electroporated with autologous total tumour RNA and huCD40L RNA, and cryopreserved for subsequent analysis.RESULTS: Mean patient age was 62 years, 6/8 were male, and all had Rai clinical stage I, previously untreated disease; mean peripheral white blood cell count was 17.2 × 109/L (range 7.4 – 26.7 × 109/L). Mean CD14+ monocyte yield post-CliniMACS selection was 2.42 × 109 cells (range 1.51 – 3.53 × 109) and monocyte purity was high (mean 96.5%, range 80 – 99.7%). In the validation cohort, day 6 immunophenotype (mean, range), measured 4 hours post-electroporation, was consistent with mature, activated DCs: CD14+ 2.5% (1.21–5.00), CD80+ 98.2% (98.0–99.7), CD83+ 87.5% (78.0 – 94.7), and CD86+ 99.5% (99.0–99.9). Mean CD40L expression, a surrogate marker for electroporation efficiency, was 80.3% (range 70.2 – 88.0); mean CD209 expression was 97.7% (95–99.7). Absolute numbers of DCs generated post-electroporation ranged from 1.26 × 108/L to 3.24 × 108/L and post-thaw DC viability ranged from 71–94%. T-cell co-culture experiments confirmed the generation of specific, autologous cytotoxic T-lymphocyte (CTL) responses to CLL targets, demonstrated by: (1) MHC class I restricted autologous CLL induced INF-γ response (intracellular staining), and (2) statistically significant functional MHC class I restricted CTL response (chromium-release assay).CONCLUSIONS: This study confirms the feasibility of generating large numbers of autologous CD14+-derived dendritic cells co-electroporated with patient specific total tumour RNA and huCD40L RNA from CLL patients. These data provide justification for a currently accruing Phase I/II clinical trial designed to evaluate this treatment in CLL patients that have a stable, low disease burden following at least one course of systemic chemotherapy.
