A Sulfonamide Sialoside Analogue for Targeting Siglec-8 and -F on Immune Cells

Abstract

The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9-N-sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9-N-(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6-O-sulfo]-Galβ1-4GlcNAc (6'-O-sulfo ^NSANeu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'-O-sulfo ^NSANeu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F⁺ eosinophils.

Figure 1.

Binding of recombinant Siglec-8 to the α2,3-sialyl sulfonamide analog array. Arrays were screened to identify C-9 substituents of sialic acid that show increased binding compared to control glycan C1. Human Siglec-8 COMP (10 μg/ml) was pre-complexed with anti-penta-histidine IgG-Alexa Fluor 488 (5 μg/ml). The complexed proteins were overlaid onto the array. After incubation the slides were washed then scanned for fluorescence. Analogs 1-78 correspond to the groups listed in Table S1. Shown is mean fluorescence intensity of Siglec-8 binding. Each glycan was printed at 100, 20, 4, 0.8, and 0.16 μM in 4 replicates each (increasing concentration from left to right). The controls are Neu5Acα2–3Galβ1–4GlcNAc (C1) and 6′-O-sulfo Neu5Acα2–3Galβ1–4GlcNAc (C2).

Figure 2.

In vitro binding/uptake of fluorescent targeted liposomes displaying 160 or 161 to CHO cells expressing Siglec-8 and -F. Cells were treated with 20 μM fluorescent targeted liposomes (2 mol% of 160 or 161) at 37 °C for 1 hour. Liposome binding was assessed by flow cytometry. Low binding to some cells reflects loss of Siglec expression (see Figure S4).

Figure 3.

Flow cytometry analysis of in vitro binding/uptake of targeted (161, 2 mol%) or non-targeted (no ligand) fluorescent liposomes to cells expressing human and murine Siglecs. Binding is shown as mean fluorescence intensity (MFI) ± SEM (n = 3).

Figure 4.

(A) Targeted liposomes (161) bind eosinophils in vivo. WT mice were intravenously given either targeted (161, 2 mol%, n=3) or non-targeted (no ligand, n=3) fluorescent liposomes. After one hour, splenocytes were harvested, stained with antibodies and analyzed by flow cytometry. In vivo binding of targeted (black line) or non-targeted liposomes (grey) to eosinophils (CD11b⁺CCR3⁺, left) or B/T cells (CD4⁺CD8⁺CD19⁺, right) were overlaid. Representative overlays are shown. See also Figure S6. (B) Targeted liposomes (161) do not alter eosinophil frequency in vivo. WT mice (n=4) were intravenously given PBS (white), non-targeted liposomes (grey), targeted liposomes (161, 5 mol%) (black), or anti-Siglec-F (red). Eosinophil frequencies in the blood were analyzed 1- and 3-days post injection and determined by dividing cells that were CD11b⁺CCR3⁺ by live immune cells (PI⁻CD45⁺). *** P <0.001; N.S., not significant (P >0.05) determined by 1-way ANOVA followed by Tukey’s test.

Scheme 1.. Chemo-enzymatic synthesis of sulfonamide analogs.^a

^aReagents and conditions: (i) Pasteurella multocida α2,3-sialyltransferase; (ii) Photobacterium damsela α2,6-sialyltransferase; (iii) RSO₂Cl (1-156, see Table S1), DIEA (5 eq.), CH₃OH; (iv) PMe₃ (2 eq.), THF, H₂O (pH 9).