Fig. 2.

Validation of hCD33 Screening Hits Leads to Ligands with Suitable Affinity and Selectivity for Liposomal Targeting of hCD33-expressing Cells. (a) A solution phase inhibitor assay was used to validate and quantify the affinity gains observed for hCD33-hits identified via microarray screening and determine their relative inhibitory potency (rIP) compared to the native sialoside (rIP = 1) (b) hCD33-hits were coupled to lipids and formulated into fluorescent, ligand-displaying liposomes. Binding to the hCD33-expressing AML cell lines, U937 and HL-60, was assessed by flow cytometry. (c) A blocking antibody was used to demonstrate that binding of the best ligand-displaying liposomes, 17 and 22, to these cell lines was hCD33-dependent. (d) The selectivity of these ligand-displaying liposomes was then assessed against a panel of recombinant siglec-expressing cell lines. (e) Binding of these liposomes to white blood cells isolated from peripheral human blood further documents the selectivity of these liposomes for hCD33-expressing cells in a complex cellular mixture. Red arrow denotes monocytes and green arrow denotes neutrophils as determined by forward and side-scatter properties. In (a)–(e), representative results are shown for one of three independent experiments carried out in duplicate (a) or triplicate (b–e). The IC50 values in (a) are the average of three independent trials.