No abstract

The high-affinity receptor for IgG (CD64 or FcγRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcγRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcγRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcγRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcγRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcγRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcγRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcγRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcγRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcγRI-targeted Ags as vaccines.

The recombinant adenoviral (Ad) vector is being considered as a cancer vaccine platform because it efficiently induces immune responses to tumor antigens by intradermal immunization. The aims of this study were to evaluate the potential toxicities and biodistribution after a single dose or six weekly intradermal doses of Ad2/gp100v2 and Ad2/MART-1v2, which encode tumor-associated antigens gp100 and MelanA/MART-1, respectively. The only dose-related toxicities associated with intradermal administration of these Ad vectors were inflammatory cell infiltrates in the draining lymph nodes and injection sites that persisted 83 days after administration. The biodistribution of Ad DNA as detected by real-time polymerase chain reaction was largely confined to the injection sites and draining lymph nodes of mice treated with either a single dose or multiple doses of Ad vector and in the spleens of mice treated with multiple doses of Ad vector. Adenoviral DNA was transiently detected in the bone marrow, lung, or blood of only one animal for each site and was below the limit of assay quantification (<10 copies/microg DNA). The vector persisted in the skin and lymph nodes as long as 92 days after the last dose. We conclude that Ad vectors delivered by intradermal administration provide a safe, genetic vaccine delivery platform that induces desirable immune responses at the immunization sites and the lymph nodes that, ultimately, result in immune responses specific to the tumor antigens.

The high-affinity IgG receptor, FcγRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcγRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcγRI causes receptor recycling, while Abs that cross-link FcγRI cause rapid down-modulation of surface FcγRI. Because studies performed in the absence of ligand may not be representative of FcγRI modulation in vivo, we investigated the ability of FcγRI-cross-linking Abs and non-cross-linking derivatives to modulate FcγRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcγRI on the human myeloid cell line, U937, and its high FcγRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcγRI expression and correlated with internalization of both wH22xeGFP and FcγRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcγRI, was unable to modulate FcγRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcγRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcγRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.

No abstract