Abstract
Purpose: Numerous studies evidenced the potential of lentiviral vectors (LV) to induce an antitumor immune response through activation of dendritic cells (DC). However their translation to the clinic is hampered by safety concerns. Most of these are tackled by transductional targeting of LV to antigen presenting cells (APC). Therefore we developed the Nanobody (Nb) display technology.
Methods: Broad tropism LVs were produced by standard methods. For the Nb displaying LVs 293T cells, stably expressing Nb BCII10 (negative control Nb) or APC-specific Nb DC1.8 or DC2.1, were used. To analyze their transduction profile, LV encoding Thy1.1+ or Firefly luciferase were injected intranodally. Transduced cells were visualized via flow cytometry or in vivo bioluminescence (BLI) respectively. To evaluate the immune stimulatory potential of ovalbumin (OVA) encoding LV, standard in vivo immunization assays were used.
Results: We demonstrated via flow cytometry selective transduction of conventional DC or also plasmacytoid DC and macrophages with DC1.8-LV or DC2.1-LV respectively. Broad tropism LV additionally transduced fibroblasts, T and B cells while BCII10-LV did not transduce any cell type. These data were confirmed in BLI. Furthermore, broad tropism and DC2.1-LV induced higher (40-60%) OT-I proliferation compared to DC1.8 and BCII10-LV (20%). In contrast, all LV types showed similar (50-60%) OT-II proliferation percentages. As BCII10-LV are noninfectious, the activating capacity could be dedicated to remaining OVA encoding and related fragments in the LV stock. When the cytokine secretion profile of CD4+ T cells was evaluated using an enzyme-linked-immunosorbent-assay, differential targeting was reflected in a diverse cytokine profile. Finally a cytotoxic T lymphocyte assay evidenced the need for infection as only broad tropism, DC2.1 and DC1.8-LV showed target cell lysis.
Conclusions: Nb mediated targeting of LV enables the transduction of different APC subtypes. Moreover this differential transduction profile runs parallel with a different immunological outcome.
Methods: Broad tropism LVs were produced by standard methods. For the Nb displaying LVs 293T cells, stably expressing Nb BCII10 (negative control Nb) or APC-specific Nb DC1.8 or DC2.1, were used. To analyze their transduction profile, LV encoding Thy1.1+ or Firefly luciferase were injected intranodally. Transduced cells were visualized via flow cytometry or in vivo bioluminescence (BLI) respectively. To evaluate the immune stimulatory potential of ovalbumin (OVA) encoding LV, standard in vivo immunization assays were used.
Results: We demonstrated via flow cytometry selective transduction of conventional DC or also plasmacytoid DC and macrophages with DC1.8-LV or DC2.1-LV respectively. Broad tropism LV additionally transduced fibroblasts, T and B cells while BCII10-LV did not transduce any cell type. These data were confirmed in BLI. Furthermore, broad tropism and DC2.1-LV induced higher (40-60%) OT-I proliferation compared to DC1.8 and BCII10-LV (20%). In contrast, all LV types showed similar (50-60%) OT-II proliferation percentages. As BCII10-LV are noninfectious, the activating capacity could be dedicated to remaining OVA encoding and related fragments in the LV stock. When the cytokine secretion profile of CD4+ T cells was evaluated using an enzyme-linked-immunosorbent-assay, differential targeting was reflected in a diverse cytokine profile. Finally a cytotoxic T lymphocyte assay evidenced the need for infection as only broad tropism, DC2.1 and DC1.8-LV showed target cell lysis.
Conclusions: Nb mediated targeting of LV enables the transduction of different APC subtypes. Moreover this differential transduction profile runs parallel with a different immunological outcome.
| Original language | English |
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| Title of host publication | Second PhD Day of VUB, Jette, Belgium |
| Publication status | Published - 27 Mar 2012 |