Development and Characterization of New Species Cross-Reactive Anti-Sialoadhesin Monoclonal Antibodies

Abstract

Sialoadhesin (Sn) is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs) to target Sn-positive (Sn + ) cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC) could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species.

Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages

“…Similarly, the surface charge of macrophages may influence phagocytosis. In our study, we used anti-Sn mAbs that interfere with the binding of sialic acids to Sn [21]. Previous studies have shown that Siglecs on cell surface can interact with negatively charged sialic acids also present on the same cell surface in cis interactions [44].…”

“…Anti-hSn mAb 12A4 and anti-mSn mAb SySy94 were produced and purified as previously published [21]. F(ab') 2 fragments were produced as previously described [34].…”

“…Studies with the porcine Arterivirus and HIV-1, show that Sn can mediate uptake of virus into early endosomes and in the case of HIV-1 can also mediate trans-infection [13][14][15][16][17][18]. Further research then showed that in different species, anti-Sn mAbs are able to induce internalization of Sn [18][19][20][21]. Interestingly, unlike most other Siglecs, no known signalization motif has yet been identified for Sn, even more, the short cytoplasmic tail of Sn is not conserved between species [22].…”

Isolation and characterization of a new population of nasal surface macrophages and their susceptibility to PRRSV-1 subtype 1 (LV) and subtype 3 (Lena)

“…To identify the Sn positive and negative cells in the CD163 positive and negative cell populations in the nasal mucosa, a double IF staining was performed using goat polyclonal antibody (pAb) against human CD163 (R&D Systems, Mineapolis, MN, USA) and mouse monoclonal antibody (mAb) against porcine sialoadhesin (Sn) (clone 41D3) [27] (Table 1). For the additional characterization of the macrophages in the nasal mucosa, a frozen ventral turbinate section was stained by a triple immunofluorescence with a mouse mAb against porcine Sn (clone 41D3) and a goat pAb against human CD163 together with a mouse mAb against porcine MHCII (clone MSA3, Kingfisher Biotech, St. Paul, MN, USA) or a mouse mAb against porcine CD14 (clone MIL2) [28]; or by a triple immunofluorescence with a mouse mAb against human Sn (clone 26B2) [29] and a goat pAb against human CD163 together with a mouse mAb against human CD1c (clone L161, Biolegend, San Diego, CA, USA) ( Table 1).…”

Interferon Alpha Favors Macrophage Infection by Visceral Leishmania Species Through Upregulation of Sialoadhesin Expression

“…The binding capacity of myc-tagged Biv4.40 to Sn was assessed by immunofluorescent staining. Biv4.40 was added to Snexpressing CHO cells and non-transfected CHO cells that served as negative controls (30). Cells were seeded onto coverslips and incubated at 37 • C for 24 h. Biv4.40 was added to the cells on ice for 60 min at 1 µg/mL.…”