Targeting Siglec-8/Siglec-F Reduce Allergic Responses In Vitro and In Vivo

描述(申请人提供)：Siglec-8是CD33唾液酸结合免疫球蛋白样凝集素(Siglec)家族的成员，仅在人类肥大细胞、嗜酸性粒细胞和嗜碱性粒细胞上发现。这种跨膜蛋白具有识别特定碳水化合物分子(多糖)的胞外结构域，以及基于酪氨酸的细胞内抑制基序，可以将受体结合转化为免疫抑制。小鼠Siglec-F和人类Siglec-8是功能趋同的对虾。小鼠Siglec-F主要表达在小鼠嗜酸性粒细胞上。人的嗜酸性粒细胞与特定的Siglec-8抗体孵育，或小鼠的嗜酸性粒细胞与Siglec-F抗体孵育，诱导细胞凋亡，给小鼠注射Siglec-F抗体可减少嗜酸性粒细胞的数量。相反，Siglec-8在体外被抗体结合到人肥大细胞上不会诱导细胞凋亡，但会抑制通过FceRI激活而诱导的介质释放。Siglec-8和Siglec-F都结合一种独特的糖链配体，称为6‘-磺酸-SLeX或NeuAca2-3(6-O-Sulo)Ga1S1-4[Fuca1-3]G1cNAc。因此，通过Siglec-8/Siglec-F的天然糖链配体，或通过抗体或拟糖激动剂激活Siglec-8/Siglec-F，可能提供一种新的方法来特异性地抑制和/或耗尽嗜酸性粒细胞和肥大细胞，从而减少过敏性炎症反应。因此，我们假设：(I)过敏性炎症细胞表面的Siglec-8/Siglec-F与其在组织上表达的天然碳水化合物配体结合，通过激活负信号通路来限制过敏性炎症；(Ii)体内的过敏性炎症可以通过调节天然Siglec-8配体在组织中的表达来控制；以及(Iii)Siglec-8/Siglec-F单抗或基于其配体结构的合成分子(糖类模拟)将能够阻断过敏性炎症。这些概念将通过四个具体目标进行探讨。目的1鉴定天然Siglec-8/Siglec-F配体并测定其组织表达。目的2确定Siglec-8/Siglec-F配体模拟物在体外限制嗜酸性粒细胞和肥大细胞过敏反应的能力。目的3将探讨单抗或配体模拟物在体内限制过敏反应的能力，目的4将确定Siglec-8参与诱导嗜酸性粒细胞凋亡和抑制肥大细胞介质释放的机制。我们关于Siglec-8/Siglec-F的拟议工作应该确定新的治疗方法，用于治疗以嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞数量增加或介体增加为特征的疾病。

DESCRIPTION (provided by applicant): Siglec-8 is a member of the CD33 sialic acid-bind immunoglobulin-like lectin (Siglec) family found only on human mast cells, eosinophils and basophils. This transmembrane protein has an extracellular domain that recognizes specific carbohydrate molecules (glycans), and intracellular tyrosine-based inhibition motifs that putatively converts receptor binding into immune suppression. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs. Mouse Siglec-F is expressed predominantly on mouse eosinophils. Incubation of human eosinophils with specific Siglec-8 antibodies, or mouse eosinophils with Siglec-F antibodies, induces apoptosis, and dosing of mice with Siglec-F antibodies reduces eosinophil numbers. In contrast, Siglec-8 engagement on human mast cells by antibodies in vitro does not induce apoptosis but does inhibit mediator release induced via FceRI activation. Both Siglec-8 and Siglec-F bind a unique glycan ligand referred to as 6'-sulfo-sLex, or NeuAca2-3(6-O-sulfo)Ga1S1-4[Fuca1-3]G1cNAc. Therefore, activation of Siglec-8/Siglec-F through its natural glycan ligand, or through antibodies or glycomimetic agonists, may provide a novel means to specifically inhibit and/or deplete eosinophils and mast cells, thereby reducing allergic inflammatory responses. We thus hypothesize that: (i) Siglec-8/Siglec-F on the surface of allergic inflammatory cells binds to its natural carbohydrate ligands, expressed on tissues, to limit allergic inflammation by activating a negative signaling pathway; (ii) Allergic inflammation in vivo can be controlled by regulating expression of natural Siglec-8 ligands in tissues; and (iii) Siglec-8/Siglec-F mAbs or synthetic molecules based on the structure of their ligands (glycomimetics) will be capable of interrupting allergic inflammation. These concepts will be explored through four specific aims. Aim 1 will characterize natural Siglec-8/Siglec-F ligands and determine their tissue expression. Aim 2 will determine the ability of Siglec- 8/Siglec-F ligand mimetics to limit allergic responses in eosinophils and mast cells in vitro. Aim 3 will explore the ability of mAbs or ligand mimetics to limit allergic responses in vivo, and Aim 4 will identify mechanisms by which Siglec-8 engagement induces eosinophil apoptosis and inhibits mast cell mediator release. Our proposed work on Siglec-8/Siglec-F should identify new therapeutic approaches for the treatment of diseases characterized by increased numbers of, or mediators from, eosinophils, basophils and mast cells.