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

性状（由申请方提供）：Siglec-8是仅在人肥大细胞、嗜酸性粒细胞和嗜碱性粒细胞上发现的CD 33唾液酸结合免疫球蛋白样凝集素（Siglec）家族的成员。这种跨膜蛋白具有识别特定碳水化合物分子（聚糖）的细胞外结构域和基于胞内酪氨酸的抑制基序，所述抑制基序将受体结合转化为免疫抑制。小鼠Siglec-F和人Siglec-8是功能趋同的旁系同源物。小鼠Siglec-F主要在小鼠嗜酸性粒细胞上表达。将人嗜酸性粒细胞与特异性Siglec-8抗体孵育，或将小鼠嗜酸性粒细胞与Siglec-F抗体孵育，诱导细胞凋亡，并且用Siglec-F抗体给药小鼠减少嗜酸性粒细胞数量。相比之下，Siglec-8在体外通过抗体对人肥大细胞的接合不诱导细胞凋亡，但确实抑制经由FceRI活化诱导的介质释放。Siglec-8和Siglec-F均结合一种独特的聚糖配体，称为6 '-磺基-sLex或Neuron 2 -3（6-O-磺基）Ga 1 S1 -4[Fuca 1 -3] G1 cNAc。因此，Siglec-8/Siglec-F通过其天然聚糖配体或通过抗体或拟糖激动剂的活化可提供特异性抑制和/或消耗嗜酸性粒细胞和肥大细胞的新手段，从而减少过敏性炎症反应。因此，我们假设：（i）变应性炎症细胞表面上的Siglec-8/Siglec-F与其在组织上表达的天然碳水化合物配体结合，以通过激活负信号传导途径来限制变应性炎症;和（iii）Siglec-8/Siglec-F mAb或基于其配体结构的合成分子（糖模拟物）将能够中断变应性炎症。将通过四个具体目标探讨这些概念。目的1将表征天然Siglec-8/Siglec-F配体并确定其组织表达。目的2将确定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中的过敏反应的能力。目的3将探索mAb或配体模拟物限制体内过敏反应的能力，目的4将鉴定Siglec-8接合诱导嗜酸性粒细胞凋亡和抑制肥大细胞介质释放的机制。我们提出的Siglec-8/Siglec-F工作应确定新的治疗方法，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质增加为特征的疾病。

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.