Siglec-15-mVENUS and Siglec-15-Fc (WT and R143A mutant) constructs were transiently transfected into HEK293F/S suspension cells. Cells were split in 200 mL cultures at 0.8 × 10^6 cells/mL. The DNA: FectoPRO complexe was then added directly to the cells, and cells were incubated at 37 °C, 130 rpm, 8% CO2 and 70% humidity for 6–7 days. Supernatants were passed through a HisTrap Ni-NTA and then separated on a Superdex 200 Increase size exclusion column in 20 mM Tris pH 9.0, 300 mM NaCl buffer to achieve size homogeneity. The heavy chain and light chain of 5G12 Fab were co-expressed at 2:1 ratio into HEK293F cells. Siglec-15–5G12 Fab complex was obtained by transiently co-transfecting Siglec-15-mVENUS with the heavy and light chains of 5G12 Fab into HEK293F suspension cells at 2:2:1 ratio.

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.