Supplementary Material for: AK002, a Humanized Sialic Acid-Binding Immunoglobulin-Like Lectin-8 Antibody that Induces Antibody-Dependent Cell-Mediated Cytotoxicity against Human Eosinophils and Inhibits Mast Cell-Mediated Anaphylaxis in Mice

<b><i>Introduction:</i></b> Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. <b><i>Methods:</i></b> The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. <b><i>Results:</i></b> AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. <b><i>Conclusion:</i></b> AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.

**引言：** 肥大细胞与嗜酸性粒细胞的病理性蓄积与活化与过敏性及炎症性疾病密切相关。唾液酸结合免疫球蛋白样凝集素 (Sialic acid-binding immunoglobulin-like lectin, Siglec)-8是一类抑制性受体，选择性表达于肥大细胞、嗜酸性粒细胞，在少量嗜碱性粒细胞中也有表达。当与抗体结合后，Siglec-8可诱导活化嗜酸性粒细胞凋亡，并抑制肥大细胞活化。AK002是一种人源化非岩藻糖基化IgG1型抗Siglec-8抗体，目前正处于过敏性、炎症性及增殖性疾病治疗的临床研究阶段。本研究考察了AK002对人体组织的细胞选择性，并评估了AK002在体外、离体及体内对嗜酸性粒细胞与肥大细胞的活性。

**方法：** 采用生物层干涉法测定AK002与Siglec-8及CD16的亲和力。通过凋亡检测与抗体依赖性细胞介导的细胞毒性 (antibody-dependent cell-mediated cytotoxicity, ADCC)实验，检测AK002对来自血液及解离人体组织的人嗜酸性粒细胞的离体活性。采用被动全身性过敏反应 (passive systemic anaphylaxis, PSA)人源化小鼠模型，评估AK002的鼠源前体(mAK002)的体内活性。

**结果：** AK002可选择性结合人体血液及组织中的肥大细胞、嗜酸性粒细胞，在低水平下结合嗜碱性粒细胞，而不与其他检测的细胞类型结合。AK002可诱导白细胞介素-5活化的血液嗜酸性粒细胞凋亡，并在自然杀伤细胞存在下对血液嗜酸性粒细胞展现出强效ADCC活性。AK002还可显著减少解离后人肺组织中的嗜酸性粒细胞数量。此外，mAK002可通过抑制肥大细胞活性，阻止人源化小鼠发生被动全身性过敏反应。

**结论：** AK002可选择性地对嗜酸性粒细胞产生强效凋亡诱导及ADCC活性，并通过抑制肥大细胞活化阻止全身性过敏反应。