Monocytes Induced to Differentiate Into Dendritic Cells After Overnight Transduction with Lentiviral Vectors Co-Expressing GM-CSF/ IL-4 and a Truncated Form of WT1: Preclinical Validation for Immunotherapy of High Risk Leukemia Patients After Allo-SCT

Abstract 2981

Wilms tumor 1 (WT1) is a transcription factor reported to be overexpressed in acute leukemia blasts and is a prognostic marker for relapse. WT1 is currently being tested in leukemia immunotherapy trials as a putative leukemia-associated antigen, but repeated WT1 peptide/adjuvant vaccination trials have failed to induce sustained high-avidity CD8+ T cell responses. Immunization against WT1 in the allogeneic stem cell transplantation (allo-SCT) setting will warrant new approaches to counteract possible tolerogenic effects of minimal residual disease and the immunesuppressed milleau of the host.

Thus, we are developing dendritic cells that co-express stimulatory cytokines and antigens to be used as vaccines for high risk acute myeloid leukemia (AML) after allo-SCT. We have previously demonstrated that DCs programmed ex vivo with lentiviral vectors (LV) expressing GM-CSF and IL-4 lead to autonomous in vivo differentiation of long-lived “SmartDCs”, which were potent vaccines with long-lasting effects in melanoma models.

Here, a tricistronic LV (LV-G242W) co-expressing GM-CSF, IL-4 and truncated form of WT1 (lacking the zinc finger domain) was produced and tested in functional assays. RESULTS: CD14+ monocytes obtained from three different healthy donors were transduced with LV-G242W at a multiplicity of infection of 20 and kept in culture without exogenous addition of cytokines. The number of integrated LV copies in differentiated SmartDCs/tWT1 was in the range of 3–9 per cell. Cytokine production persisted for 3 weeks (GM-CSF: 3–8 ng/mL and IL-4: 0.05–3.0 ng/mL). WT1 overexpression in SmartDCs/tWT1 was confirmed by RT-Q-PCR and cytospin/IHC. Typical DC morphology and expression of the DC markers CD86, HLA-DR and CD80 were stable throughout the culture. Gene expression profiling revealed that SmartDCs/tWT1 expressed higher levels of several members of the interferon stimulated genes (ISG) than conventional DCs, indicative of an activated state. Luminex analyses of cytokines secreted by SMART-DC/tWT1 revealed endogenous expression of TNF-alpha (185–500 pg/mL) and IL-6 (60–123 pg/mL), which promote DC maturation.

SmartDCs/ tWT1 and conventional DCs pulsed with WT1 class I peptides were used to stimulate autologous CD3+ T cells. SmartDCs/tWT1 promoted T cell expansion and up-regulation of the activation marker CD137. Evaluation of WT1-specific responses were performed by ELISPOT and streptamer analysis using cell samples obtained from a healthy donor and from an acute myeloid leukemia patient. For both models, SmartDCs/ tWT1 were more effective at promoting immune reactivity than SmartDCs pulsed with peptides, indicating the relevance of the intracellular antigen processing for optimal T cell activation.

SmartDCs/tWT1 genetically marked with firefly Luciferase were injected s.c. into NOD/Rag −/−/ IL2gcR−/− mice for in vivo viability and biosafety analyses. SmartDCs/tWT1 engrafted efficiently, maintained viability at the injection site for at least 3 weeks and no signs of malignancies were observed so far.

Our results support the feasibility of generation and high immunogenicity of SmartDCs/tWT1 and highlight the potential for those cellular vaccines to boost graft-versus-leukemia after allo-SCT for AML.