61. Higher In Vivo Maintenance, Biodistribution and Immunopotency of Dendritic Cell Vaccines Self-Differentiated through Lentiviral Vector Programming
Authors: Richard C. Koya, Takahiro Kimura, Antoni Ribas, Nori Kasahara, Renata Stripecke
Journal: Molecular Therapy (2005)

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Dendritic cell (DC) vaccines are conventionally produced ex vivo by culturing DC precursors in the presence of recombinant growth factors. Methods to facilitate the generation of DCs, to prolong their viability, to facilitate their migration to lymphoid tissues and to augment their capacity to establish and maintain antigen-specific immune responses are critical for improving their effective therapeutic potential for immunotherapy. To address these issues, we have genetically engineered purified human CD14+ monocytes with lentiviral vectors (LV) for production of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), which triggered their differentiation into cells with typical characteristics of DCs. We previously demonstrated that these lentivirus programmed DCs (DC/LVs) exhibited more potent immunostimulatory function and prolonged viability in vitro than DCs generated by conventional methods.

Using the same strategy, we have now generated murine DC/LVs in order to test the efficacy of this approach in vivo. Third generation self-inactivating LV (5 |[mu]|g/ml p24 equivalent) transduced non-adherent bone marrow (BM) cells harvested from C57BL/6 mice at >90% transduction efficiency. BM cells co-expressing GM-CSF and IL-4 after LV transduction produced these cytokines at concentrations similar to those commonly used for generation of conventionally cultured DCs by addition of exogenous cytokines (> 10ng/ml), resulting in the generation of DC/LVs as evidenced by their morphology and immunophenotype (CD11b+, CD11c high, CD80 high, MHCII high). DC/LVs maintained in culture were metabolically active and viable for 3 weeks, whereas conventional DCs showed a drastic decrease in viability in the first week of culture. In vivo bioluminescence imaging studies were then performed using luciferase as a marking gene to track DCs after s.c. injection into nude syngeneic mice. Persistent and robust bioluminescence signals were detectable from the injected DC/LVs for more than 45 days, whereas signals produced by conv. DCs faded away within the first two weeks.

To determine whether the higher viability of DC/LVs correlated with greater potency as vaccines, we next examined their antigen presentation and immunostimulatory functions in vivo. DC/LVs and conv. DCs maintained in culture for 7 days were transduced to express the melanoma associated antigen MART-1 and injected s.c. Splenocytes harvested 2 weeks after vaccination with DC/LVs showed up to 2-fold increase in activated T cells by IFN|[gamma]|- ELISPOT assay (p<0.05), and chromium lysis assays using target B16 melanoma cells showed more efficient killing by DC/LV-activated CTLs in comparison to conv. DCs (p<0.05). We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Initial experiments in a prophylactic model indicate that mice vaccinated with DC/LV show enhanced survival compared to the group vaccinated with conv.DCs.

Our results therefore indicate that DC/LVs can provide enhanced anti-tumoral therapeutic efficacy and warrant further investigation of the clinical applicability of genetically programmed DCs.