Engineering lentiviral vectors to target dendritic cells: the Nanobody display technology

Introduction: Inspired by the discovery of dendritic cells (DCs) as specialized antigen-presenting cells (APCs), antitumor immunotherapy by genetic manipulation of DCs to present tumor antigens has been explored. Lentiviral vectors (LVs) have been shown to be highly efficacious in DC transduction thereby providing a powerful tool to deliver tumor antigens to DCs. Moreover, direct administration of tumor antigen encoding LVs has highlighted their potential as an off-the-shelf vaccine.
Aim: The translation of LV-vaccines to the clinic has been hampered by concerns about their safety. Most of these can be tackled by transductional targeting of LVs to DCs. Therefore we developed the Nanobody (Nb) display technology.
Methods: The Nb display technology is an innovative approach which exploits the budding mechanism of LVs to incorporate a bindingdefective but fusogenic form of VSV.G (VSV.GS) together with an APC-specific Nb. The Nbs under investigation are Nb R3_13, DC1.8 and DC2.1, respectively specific for human DCs, mouse DCs and APCs in general. Nb BCII10, specific for ?-lactamase, was used as a control throughout the study. Broad tropism LVs were applied for comparison.
Results: We generated producer cells that stably express a membrane bound form of the aforementioned Nbs. As shown by the reverse transcriptase assay this enabled us to produce Nb displaying LVs at similar titers as broad tropism LVs. Incorporation of Nbs and VSV.GS on the viral surface was demonstrated using an in-house developed ELISA and by western blot. Selective in situ transduction of DCs (Nb DC1.8), or DCs and macrophages (Nb DC2.1) was demonstrated by flow cytometry in which Thy1.1+ cells, obtained from lymph nodes injected with Thy1.1 encoding LVs, were characterized in detail. In vivo bioluminescence imaging on mice of which the inguinal lymph node was injected with Firefly Luciferase (FLuc) encoding LVs, was used to evaluate the biokinetics of Fluc+ cells. We observed long-term (20 days) presence of transduced cells after injection of broad tropism LVs in contrast to short-term (3 days) presence of transduced cells when Nb DC1.8 or Nb DC2.1 displaying LVs were administered. These data were confirmed by PCR and can be explained by the short half-life of transduced APCs. Nb R3_13 and Nb DC2.1 displaying LVs were further used to specifically transduce human lymph node DCs in vitro, highlighting the promise of the technology. Currently we are evaluating the potential of Nb displaying LVs encoding the antigen ovalbumin (OVA) to induce OVA-specific CD4+ and CD8+ T cells. Preliminary data show the induction of qualitatively different immune responses upon immunization with the LVs under investigation.
Conclusion: We report on the Nb display technology to target LVs to specific APCs, a strategy that can be exploited for fundamental research exploring the stimulatory capacity of APC types and to facilitate the translation of LV-vaccines from the bench to the clinic.