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

项目摘要 过敏性疾病是一种依赖IgE的肥大细胞介导的疾病，影响着5000多万人 美国人。严重的过敏反应会导致过敏反应，有时还可能危及生命 需要在紧急干预的同时去医院就诊，例如使用肾上腺素自动注射器。 已经开发出各种药物疗法来治疗过敏性鼻炎、特应性皮炎、哮喘 和食物过敏，但没有一种能够直接调节肥大细胞的行为，从而真正防止肥大 细胞对过敏原的反应性。因此，需要一种治疗策略来1)选择性地针对肥大细胞进行输送 2)一种可以直接阻止依赖IgE的肥大细胞分泌的强效药物。 唾液酸结合免疫球蛋白样凝集素6(Siglec-6)是一种细胞表面受体，高度和 在所有类型的肥大细胞上优先表达。而其特定的功能和相关的机制 信号转导在很大程度上还没有被探索，它与其他肥大细胞Siglecs的同源性，如CD33，Siglec-7和Siglec-8，使 它很可能在功能上是抑制的。与其规范的信号功能分开，Siglec-6的接合结果 在其内吞作用中。这支持了这样的假设，即Siglec-6可以被用于靶向药物输送 肥大细胞。开发Siglec-6靶向疗法需要合适的递送工具。此前， Scott Lab开发并测试了各种稳定、可扩展和可定制的药物载体，每种载体 在受控输送方面具有明显的结构依赖性优势。例如，球形和丝状 纳米载体都可以运输亲脂性药物，但它们在长径比上的差异导致了不同的能力 受体介导的内吞作用和靶向部分时的信号出现在它们的表面。 在治疗药物方面，阿克拉布替尼(ACB)和其他Bruton‘s Kinase抑制剂(BTKi)不可逆转 通过FcεRI结合和抑制BTK，以防止Ig E介导的肥大细胞激活，Fc Ri是过敏反应的关键驱动因素。这个 Bochner Lab刚刚在一种新的人源化肥大细胞小鼠模型中证明了ACB的预处理。 过敏反应对严重的过敏反应和提高存活率有深远的保护作用。同时， BTKi在体外完全阻断人嗜碱性粒细胞和肥大细胞上的Ig E受体信号，IC50的S在 低至中纳米分子范围。然而，全身BTKi治疗的多效性导致潜在的 严重的副作用，因此BTKi不被批准用于癌症适应症以外的用途。 为了验证Siglec-6介导的ACB靶向递送将导致特异性抑制 过敏性肥大细胞的激活，将达到两个特定的目标：(1)优化和表征结合 载Siglec-6/BTKi胶束和丝状纳米载体的动力学、特异性和抑制活性；以及(2) 比较这些纳米载体在Siglec-6肥大细胞敲入小鼠和小鼠体内的特异性和抑制作用。 人源化肥大细胞小鼠。首要目标是为后续的R01生成概念验证数据 应用于更全面地开发和测试药物输送系统，选择性地将药物输送到肥大细胞。

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.