Abstract
Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.
Sarcoma IL-12 overexpression facilitates NK cell immunomodulation