The siglec family of sialic acid-binding proteins are endocytic immune cell receptors that are recognized as potential targets for cell directed therapies. CD33 and CD22 are prototypical members and are validated candidates for targeting acute myeloid leukaemia and non-Hodgkin's lymphomas due to their restricted expression on myeloid cells and B-cells, respectively. While nanoparticles decorated with high affinity siglec ligands represent an attractive platform for delivery of therapeutic agents to these cells, a lack of ligands with suitable affinity and/or selectivity has hampered progress. Herein we describe selective ligands for both of these siglecs, which when displayed on liposomal nanoparticles, can efficiently target the cells expressing them in peripheral human blood. Key to their identification was the development of a facile method for chemo-enzymatic synthesis of disubstituted sialic acid analogues, combined with iterative rounds of synthesis and rapid functional analysis using glycan microarrays.

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.