Human antibodies to dendritic cells : generation, analysis and use in vaccination

Abstract

Dendritic cells (DCs) are widely recognized as professional antigen presenting cells (APCs) that play a pivotal role in directing the immune response. DCs are a heterogeneous cell population that continuously derive from bone marrow cells and reside as sentinels in an immature stage in the periphery. Upon encounter of an antigen, immature DCs migrate to the lymphoid organs. During this migration, DCs process the antigens they captured and express costimulatory molecules. Mature DCs present antigens captured in the periphery to T cells, inducing an adaptive immune response. The subject of the research described in this thesis is the identification of human antibodies that bind to human DCs, the use of such antibodies for further examination of DC subsets and the evaluation of their potential as targeting vehicles for the delivery of tumor antigens to DCs for use in DC-based cancer vaccination strategies. The importance of DCs in generating an immune response has stimulated the exploration of these cells in cancer vaccines. Current DC-based vaccine approaches mostly rely on the ex vivo generation of DCs from precursor cells, which are subsequently loaded with tumor antigens and re-infused into patients to evoke an anti-tumor response. An alternative approach is provided by human antibodies as vectors for in vivo targeted delivery of tumor antigens to DCs. In the search for suitable cell surface molecules expressed by DCs in peripheral blood and tonsil, we employed a phage antibody display approach to generate phage antibodies binding to cell surface molecules on DCs found in human peripheral blood and human tonsil. We obtained several phage antibodies with desirable characteristics, recognizing both DCs and their precursors the monocytes, which have been used as tools in further delineation of DC subsets. These phage antibodies were converted to fully human monoclonal antibodies (huMabs) of the IgG4 isotype. Different DC subsets have been described based on phenotype, function and anatomical localization. The diversity of DC subsets and their potential role in polarization of the immune response, prompted us to determine in detail the reactivity patterns of our huMabs on in vivo DC subsets found at different anatomical localizations and in vitro generated DCs . In light of the potential application of our huMabs as tools to target antigens to DCs, we analyzed whether engagement of the molecules recognized by the huMabs exerted functional effects. In a first step toward in vivo targeting of DCs in cancer immunotherapy, we explored whether antibody-mediated targeting of tumor antigens to DCs using a huMab as vector could provide a novel antigen delivery strategy. This strategy involves the construction of a eukaryotic expression vector encoding a huMab, genetically fused to a tumor antigen. We established proof of principle of this approach in an in vitro setting. The phage antibodies were also converted to lipid-tagged scFv antibody fragments. In a cell-based approach to cancer vaccination, we explored whether these lipid-tagged scFvs incorporated in tumor cells could induce receptor-mediated endocytosis by monocytes. In conclusion, phage display antibody technology was successfully used to isolate four novel huMabs directed against DCs. In addition, a first step was made to evaluate the potential of modified forms of these huMabs as targeting vehicles for the delivery of tumor antigens. Targeting the antibody-tumor antigen fusion protein to DCs resulted in the induction of strong T helper and CTL responses. Future research on these huMabs in in vitro and in vivo models could open new and exciting avenues for cancer vaccination