PROJECT SUMMARY Allergies are the 6th most common cause of chronic illness in the US, but effective treatment options remain limited. Recently discovered, cell-specific, inhibitory immune receptors, such as sialic acid-binding immunoglobulin-type lectin (Siglec) receptors, are promising therapeutic targets to suppress these unwanted immune responses, but have been difficult to target because they specifically bind sialic-acid containing carbohydrates, or glycans. Using two newly developed tools: a bacterial glycan-building platform for constructing modifiable Siglec receptor glycan ligands and a glycan-displaying nanogel capable of varying glycan density, we aim to generate optimized Siglec receptor ligands towards the development of therapeutic candidates for the treatment of allergy and prevention of anaphylaxis. We will use Siglec 3 (CD33) as a model to validate this approach since it is expressed on both mast cells and basophils, two cell types implicated in allergy and anaphylaxis. In the first aim, we will generate a library of chemically modified glycan variants that are candidate Siglec receptor ligands to assess for high affinity interactions with ELISA. We will then load high affinity glycan ligands on nanogels at varying densities, apply to sensitized primary human skin-derived mast cells, and assess metrics of mast cell activity including release of granule components, surface markers, and cytokine release. In the second aim, we will supplement the field with a new tool for the characterization of Siglec receptors. Genomic incorporation of glycosylation machinery in engineered Escherichia coli will yield a stable strain for generating modifiable sialoglycoproteins that are candidate Siglec ligands. Ultimately, using bacterial glycoengineering, we will develop a customizable platform for generating Siglec receptor ligands that can be readily ported to other labs and broadly applied for the characterization of other lectins. Once complete, the proposed aims will not only yield therapeutic candidates capable of high affinity Siglec 3 (CD33) binding, but also a new approach and tool that can be applied to identify therapeutic candidates for other Siglec receptors and lectins. This project spans glycobiology, cell biology, nanomaterials, and using allergy models of disease. As such, it will provide a rich training experience bolstered by research mentors with synergistic expertise and ample support from Northwestern University, the Center for Synthetic Biology, the Medical Scientist Training Program, and the Interdisciplinary Biological Sciences Graduate Program. In addition to providing a robust training experience, this will work will also advance the therapeutic potential of targeting Siglec receptors for the treatment of allergy and prevention of anaphylaxis, among other relevant diseases.