Exploiting Siglec-6 for targeted anti-allergy drug delivery into human mast cells

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Project Summary Allergic diseases are IgE-dependent, mast cell-mediated conditions that affect more than 50 million Americans. Severe allergic reactions result in anaphylaxis and can be potentially life threatening, sometimes requiring hospital visits in conjunction with emergency interventions such as the use of epinephrine autoinjectors. Various pharmacotherapies have been developed for the treatment of allergic rhinitis, atopic dermatitis, asthma and food allergy, but none are capable of directly regulating mast cell behavior in a way that truly prevents mast cell reactivity to allergens. A therapeutic strategy is therefore needed to 1) selectively target mast cells for delivery of 2) a potent drug that can directly prevent IgE-dependent mast cell secretion. Sialic acid binding immunoglobulin-like lectin 6 (Siglec-6) is a cell surface receptor that is highly and preferentially expressed on all types of mast cells. While its specific function and associated mechanism of signaling is largely unexplored, its homology to other mast cell Siglecs, like CD33, Siglec-7 and Siglec-8, makes it likely to be inhibitory in function. Separate from its canonical signaling function, engagement of Siglec-6 results in its endocytosis. This supports the hypothesis that Siglec-6 can be exploited for targeted drug delivery into mast cells. Development of a Siglec-6-targeted therapy requires an appropriate delivery vehicle. Previously, the Scott Lab has developed and tested a variety of stable, scalable and customizable drug carriers that each possess distinct structure-dependent advantages for controlled delivery. For example, spherical and filamentous nanocarriers can both transport lipophilic drugs, but their differences in aspect ratio result in distinct capacity for receptor-mediated endocytosis and signaling when targeting moieties are presented on their surfaces. In terms of therapeutic agents, Acalabrutinib (AcB) and other Bruton’s kinase inhibitors (BTKi) irreversibly bind and inhibit BTK to prevent IgE-mediated activation of mast cells via FcεRI, a key driver of anaphylaxis. The Bochner Lab has just demonstrated that pretreatment with AcB in a novel humanized mast cell mouse model of anaphylaxis has profound protective effects regarding anaphylaxis severity and improved survival. In parallel, BTKi completely shut off IgE receptor signaling in both human basophils and mast cells in vitro, with IC50’s in the low to mid nanomolar range. However, the pleiotropic nature of systemic BTKi treatment results in potentially serious side effects, and thus BTKi are not approved for use outside of cancer indications. To test the hypothesis that Siglec-6-mediated targeted delivery of AcB will result in specific inhibition of allergic mast cell activation, two Specific Aims will be achieved: (1) to optimize and characterize the binding kinetics, specificity, and inhibitory activity of Siglec-6/BTKi loaded micellar and filamentous nanocarriers; and (2) to compare the specificity and inhibitory effects of these nanocarriers in Siglec-6 mast cell knock-in mice and humanized mast cell mice. The overarching goal is to generate proof-of-concept data for a subsequent R01 application to more fully develop and test drug delivery systems that selectively deliver drugs into mast cells.