Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy.

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced natural killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

Cell-based glycan arrays for probing glycan-glycan binding protein interactions.

Glycan microarrays provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. However, the construction of current glycan microarray platforms is time consuming and expensive. Here, we report a fast and cost-effective method for the assembly of cell-based glycan arrays to probe glycan-glycan-binding protein interactions directly on the cell surface. Chinese hamster ovary cell mutants with a narrow and relatively homogeneous repertoire of glycoforms serve as the foundation platforms to develop these arrays. Using recombinant glycosyltransferases, sialic acid, fucose, and analogs thereof are installed on cell-surface glycans to form cell-based arrays displaying diverse glycan epitopes that can be probed with glycan-binding proteins by flow cytometry. Using this platform, high-affinity glycan ligands are discovered for Siglec-15-a sialic acid-binding lectin involved in osteoclast differentiation. Incubating human osteoprogenitor cells with cells displaying a high-affinity Siglec-15 ligand impairs osteoclast differentiation, demonstrating the utility of this cell-based glycan array technology.

Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling.

The study of cellular glycosylation presents many challenges due, in large part, to the nontemplated nature of glycan biosynthesis and glycans' structural complexity. Chemoenzymatic glycan labeling (CeGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CeGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CeGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CeGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.