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Using Siglec-8 Endocytic Mechanisms To Target Human Eosinophils And Malignant Mast Cells | Science Trends

Using Siglec-8 Endocytic Mechanisms To Target Human Eosinophils And Malignant Mast Cells

Pharmacological selectivity remains a critical goal in medicine. Most drugs act on a number of molecular targets and are also delivered equally well to cells involved in the illness and those that are innocent bystanders, consequently exerting both desired and undesirable effects.

The latter off-target effects may be severe enough to limit the dosage of the administered medication to levels that are below the optimal range for its intended effects. Even if ideal targets are identified to treat a given disease and a drug is developed that effectively hits that target, the effects of the drug on other molecules and on other populations of cells may preclude its use in the clinic. This is especially limiting for drugs that halt cell replication or induce cell death, regardless of malignant status, or those that target proteins that, in addition to their disease-causing effects, for example, play key roles in healthy cells. However, the selective delivery of such agents only to relevant cell populations may overcome these barriers.

One potential means to deliver drugs only to the intended cells involves the use of receptor-mediated internalization of these therapeutic agents through endocytic receptors only found on certain cell types, such as the siglecs. Members of the siglec (sialic acid-binding immunoglobulin-like lectin) family of receptors bind to complex sugars (or glycans) that include sialic acid at the terminal position of a chain of sugars. These glycans can be linked to proteins or lipids and may be secreted or associated with a cell membrane.

Each member of the siglec family binds to its own particular sugar structure — although some of these binding partners may interact with more than one siglec — and many recognize glycan structures that are produced by the body and are therefore involved in discrimination of innocuous “self” molecules from those that are potentially dangerous or harmful foreign molecules. In response to recognizing “self,” these siglecs help to shut off immune responses.

More importantly, however, each member of the siglec family is only found on the surface of a subset of cells, mostly on cells of the immune system. The receptor known as Siglec-8, for example, has been found to be expressed selectively on immune cells involved in asthma and allergic disease: eosinophils, mast cells, and basophils. This restricted pattern of expression allows it to be used as a marker of allergic immune effector cells and potentially allows for targeted delivery of drugs to these cell types in allergic diseases and also malignancies and myeloproliferative disorders such as chronic eosinophilic leukemia, hypereosinophilic syndromes, and systemic mastocytosis.

In our recent study, “Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells,” published in the Journal of Allergy and Clinical Immunology, we explored the possibility that Siglec-8 endocytosis could be exploited to selectively deliver agents to these cell types. Specifically, antibodies and a glycan ligand of Siglec-8 were used to selectively engage and mark the receptor to measure its internalization. Pharmacological inhibitors were used to determine how this endocytic process is regulated. Interactions between Siglec-8 and other competing binding partners were disrupted to assess the effects of those interactions on receptor endocytosis.  Finally, a lethal toxin was targeted to Siglec-8 as a demonstration of the efficacy of this pathway for drug delivery to these cell types.

After binding the antibody or ligand to Siglec-8 on the cell surface and incubating the cells to permit receptor endocytosis, the remaining antibody or ligand on the cell surface was detected and compared to the levels that were initially bound to the cell. As the receptor was gradually internalized over time, the bound antibody or ligand was internalized along with it and less was detected at the cell surface. However, when directly labeled antibody was bound to Siglec-8, no loss of antibody signal was observed over the same time periods, ruling out a competing explanation that, rather than being internalized, the receptor is shed from the cell.

These data indicated the presence of a promising pathway by which therapeutic agents may be delivered into these cells. While this movement into the cell proceeded at a relatively slow pace compared to some other receptors, most of the cell-surface Siglec-8 was internalized within two hours, and new Siglec-8 molecules were moved to the cell surface, enabling repetitive targeting, if necessary.

Following endocytosis, receptors can be trafficked through various compartments or recycled back to the cell surface. In the case of Siglec-8, the intracellular compartment to which it localizes following endocytosis was identified by confocal microscopy as the lysosomal compartment using a probe that fluoresces only following proteolytic digestion in this organelle. Pharmacological inhibitors of signaling molecules and cytoskeletal components were used during the Siglec-8 endocytosis assay to demonstrate that its internalization is dependent on the enzymatic activities of tyrosine kinases and protein kinase C (PKC), actin cytoskeletal rearrangement, and, interestingly, both clathrin- and caveolae-mediated endocytic pathways.

One feature of siglec receptors is that they may bind to glycans displayed on the surface of the same cell. These so-called cis interactions likely play a role in preventing interactions of the siglec family member with low-affinity ligands or permitting siglec interactions in trans only under certain conditions when such input is beneficial. In order to determine whether Siglec-8 binds to sialylated cis ligands, eosinophils were treated with an enzyme that cleaves sialic acids from glycans and the extent of glycan ligand binding in trans was measured. Approximately 50% more glycan ligand was found to bind to the surface of the treated eosinophils, demonstrating the presence of a sialylated cis ligand that competed with the trans ligand for binding and limited receptor internalization commensurately.

Finally, and perhaps most importantly, we aimed to determine whether the endocytosis of Siglec-8 could be exploited to deliver agents specifically to eosinophils and mast cells. As a proof of concept, a toxin called saporin was conjugated to an anti-Siglec-8 antibody in the hope that this strategy would deliver the toxin into the appropriate cells. This toxin, known as a ribosome-inactivating protein (a.k.a. RIP), is incapable of entering cells by itself but, if internalized through other means, causes the cell to die by shutting off protein synthesis.

In the presence of the cytokine IL-5, Siglec-8 ligation normally causes eosinophils to undergo cell death, and the toxin-conjugated antibody replicated this effect. However, in the absence of IL-5, Siglec-8 ligation does not regularly cause this effect, but the saporin-conjugated anti-Siglec-8 antibody treatment consistently killed these cells as well. An isotype control antibody conjugated to the toxin did not recreate this effect in eosinophils, and neutrophils, which do not express Siglec-8, were unscathed by treatment with the toxin-conjugated anti-Siglec-8 antibody, indicating the selectivity of this approach. Even more impressively, malignant mast cells, the cell viability of which was unaffected by Siglec-8 ligation alone or saporin-conjugated control antibody treatment, underwent extensive cell death when treated with the saporin-conjugated anti-Siglec-8 antibody.

Future studies are planned that will focus on the delivery of therapeutic agents to Siglec-8-expressing cells in mouse models using both human mast cell lines and mouse strains that are genetically engineered to express human Siglec-8 on the surface of their mast cells and/or eosinophils. This work also sets the stage for the use of other receptors with restricted expression patterns, such as other siglec family members, in the selective delivery of therapeutic agents to enhance clinical outcomes and minimize off-target effects.

These findings are described in the article entitled Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells, recently published in the Journal of Allergy and Clinical Immunology. The work was conducted by Jeremy A. O’Sullivan, Daniela J. Carroll, and Yun Cao from Northwestern University, Adriano N. Salicru from the Johns Hopkins University School of Medicine, and Bruce S. Bochner from Northwestern University and the Johns Hopkins University Scool of Medicine.

About The Author

Jeremy A. O’Sullivan
My lab focuses on the regulation of immune responses, especially those involving eosinophils, mast cells, and basophils including asthma, allergic diseases, and systemic mastocytosis, by members of the siglec family of cell-surface receptors as well as the use of these receptors as therapeutic targets through which to deliver agents selectively to these cell types.