Characterization of monoclonal antibody-induced internalization of human and mouse sialoadhesin on macrophages and the effect on phagocytosis
20 December 2016
UAntwerp, Campus Drie Eiken, Building Q, Promotiezaal - Universiteitsplein 1 - 2610 Wilrijk (Antwerp) (route: UAntwerpen, Campus Drie Eiken
4:30 PM - 6:30 PM
Marjorie De Schryver
Peter Delputte, Paul Cos
PhD defence Marjorie De Schryver - Department of Biomedical Sciences
Sialoadhesin (CD169; Siglec-1) is a macrophage-specific receptor that is upregulated during inflammation, auto-immune diseases and infections. Furthermore, sialoadhesin has the capacity to internalize upon monoclonal antibody (mAb) binding, which should allow delivery of a cargo or modulation of macrophages. Due to this expression pattern and the capacity to internalize, this receptor represents a potential target for macrophage-specific immunotherapy. Equivalence of human sialoadhesin characteristics with other species must be demonstrated to allow studies in animal models. Interestingly, sialoadhesin has no signalization motif and the amino acid sequence of the cytoplasmic tail, which usually transduces intracellular signals, is not conserved between species. Therefore, it is difficult to predict functional similarities between human sialoadhesin and the sialoadhesins of two other well studied species, mouse and swine. In addition, only species-specific anti-sialoadhesin antibodies are available which does not allow a direct comparison between species. Novel tools and insights are needed to directly compare the functionality of sialoadhesin in different species.
In this doctoral thesis, new mAbs were developed for mouse and human sialoadhesin and for the first time, also mAbs recognizing both human and porcine sialoadhesin. Unfortunately, no mAbs could recognize mouse sialoadhesin and another species, probably due to the low sequence similarity.
Because the cytoplasmic tail of sialoadhesin, which regulates internalization, is not conserved, the mechanism of the mAb-induced internalization of sialoadhesin and the intracellular routing could be different. Since such differences can drastically affect the outcome of sialoadhesin targeting, a direct comparison between species was made using the newly developed mAbs. No major differences were observed between species using either cross-reactive or species-specific antibodies.
Immunotargeting using mAbs can potentially have a detrimental outcome by unwanted effects on the immune system. Targeting sialoadhesin on macrophages could also lead to unwanted side-effects. Since phagocytosis is a key property of macrophages, we evaluated the effect of anti-sialoadhesin antibodies. The obtained results were conflicting, depending on the type of phagocytosis that was analyzed. Uptake of fluorescent polystyrene beads was drastically reduced, but macrophages recovered over time. Fc-mediated phagocytosis was unaffected. Interestingly, phagocytosis of bacteria remained unaffected for human monocyte-derived macrophages, while it was increased in mouse macrophages.
The results obtained in this doctoral thesis give more insight in the mechanism of antibody-induced internalization and intracellular routing of human and mouse sialoadhesin and confirm that sialoadhesin is a potential target for immunotherapy. More studies are however needed to address the effect on phagocytosis.