1. Introduction

Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that contain sialic acid-binding N-terminal V-set domains^[1][2] [1,2]. These receptors are predominately found on immune cells and most have immunoreceptor tyrosine-based motifs (ITIMs) that are involved in inhibitory cell signaling. The Siglec family of cell surface receptors have emerged as attractive therapeutic targets due to their restricted expression profile on immune cells and their immunomodulatory activities^[3][4][5][6] [3–6].

Mast cells and eosinophils are effector cells in the pathogenesis of many allergic and non-allergic diseases^[7][8] [7,8]. Of all the Siglecs, Siglec-8 is the only one found selectively on mast cells, eosinophils, and to a lesser extent, basophils^[9][10] [9,10]. Siglec-8 was discovered more than 20 years ago in a cDNA library generated from a subject with hypereosinophilic syndrome (HES)^[9][10] [9,10].

2. Siglecs and the Discovery of Siglec-8

Siglecs, formerly called sialoadhesins or sialoadhesin factors (SAF) among other names, is a term that was adopted in 1998 to describe a subset of I-type lectins within the immunoglobulin gene superfamily that bind sialylated glycans and share certain structural motifs within their N-terminal and C-2 set domains^[11] [11]. At the time this nomenclature was adopted, only a few Siglecs were known. Since then, the list has expanded to include 10 human and 5 mouse Siglecs, belonging to Siglec-3 (CD33)-related subgroups, and 4 Siglecs were conserved between humans and mice (Siglec-1, Siglec-2 (CD22), Siglec-4, and Siglec-15). Besides the shared extracellular structural characteristics that define them as Siglecs, most possess tyrosine-based signaling motifs in the form of immunoreceptor tyrosine-based inhibitory or switch motifs (ITIMs and ITSMs, respectively). A small number of Siglecs lack such signaling domains because they have very short cytoplasmic tails. Examples include Siglec-14 and Siglec-16 in humans, Siglec-H in mice, and Siglec-15 in mice and humans. Each of these Siglecs lacking ITIM or ITSM domains contain a charged amino acid in their transmembrane domain that allows them to co-associate in the cell membrane with adapter molecules, such as DAP-12, to mediate SYK-dependent signaling.

Siglec-8 was discovered as a result of a joint effort between the Bochner and Schleimer laboratories at The Johns Hopkins University School of Medicine, along with scientists in the Department of Immunology, SmithKline Beecham Pharmaceuticals, and at Human Genome Sciences, Inc. Based on random, high-throughput EST (expressed sequence tag) sequencing, a clone from a cDNA library generated from a donor in the Bochner lab with a very high eosinophil blood count due to a form of HES was identified that possessed novel sequences with high homology to Siglec-7, Siglec-6, Siglec-5 and CD33. Simultaneously, the Crocker lab, via a separate collaboration with scientists at Human Genome Sciences, designed to identify novel Siglecs, discovered the same human eosinophil clone and sequence. Parallel investigations in both labs resulted in the publication of two papers in 2000 describing Siglec-8 (or sialoadhesin factor-2, SAF-2) as highly and selectively expressed on eosinophils [9], but also on human mast cells and very weakly on basophils [10]. As initially discovered, Siglec-8 contained a short cytoplasmic tail devoid of signaling motifs. This so-called short form was subsequently shown to result from a premature stop codon; the full-length mRNA encoded a form of Siglec-8 with a membrane-proximal ITIM and a membrane-distal ITSM that was subsequently shown to be the more predominant form of Siglec-8^[12][13] [12,13].

Soon after Siglec-8 was discovered, mouse mAbs were generated^[9][10] [9,10] and the 2E2 Siglec-8 mAb clone was licensed in 2012 to Allakos, Inc. These have been the foundation of efforts at Allakos, Inc. to develop mAbs against Siglec-8 for clinical use.

3. Expression Pattern of Siglec-8

Siglec-8 is selectively expressed on human eosinophils, mast cells, and to a lesser extent, basophils, with ~18,000–22,000 Siglec-8 receptors/cell on eosinophils and mast cells and ~500 receptors/cell on basophils^[14] [14]. While it appears that the transcription factor Olig2 (Oligodendrocyte transcription factor 2) may participate in the control of Siglec-8 expression^[15] [15], more needs to be learned about how Siglec-8 expression is regulated. Unlike other receptors expressed on eosinophils, such as IL-5R⍺, the expression of Siglec-8 is stable between blood and tissue compartments, suggesting Siglec-8 remains targetable by antibodies on both blood and tissue eosinophils^[14] [14]. Several groups have recently examined the expression pattern of Siglec-8 on eosinophils and mast cells during inflammatory disease states, including HES, asthma, eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), and systemic mastocytosis (SM). The expression of Siglec-8 on blood eosinophils is similarly prominent in patients with eosinophilic diseases, including multiple variants of HES and EoE^[16] [16]. In addition, the levels of Siglec-8 remain stable on blood eosinophils after patients received treatment with prednisone or imatinib, despite significant decreases in absolute eosinophil counts [16]. Airway tissue eosinophils from bronchoalveolar lavage and sputum from subjects with asthma have similar levels of surface Siglec-8 as their blood eosinophils^[17][18] [17,18]. Likewise, Siglec-8 expression is comparable on gastrointestinal tissue eosinophils and mast cells from patients with EoE or EG, compared to tissues from control subjects without these diseases, despite elevated numbers and an activated phenotype of these cells in these diseases^[19] [19]. Similarly, mature bone marrow mast cells from patients with SM expressed Siglec-8 at levels similar to those observed in other tissues^[20][21] [20,21]. The expression of Siglec-8 remained low on basophils from patients with and without EoE or EG. Interestingly, a soluble form of Siglec-8 can be detected in the serum of some individuals using an ELISA, but the clinical significance and origin of this form of Siglec-8 remains unknown^[16] [16]. While flow cytometry is a powerful method for evaluating the expression of Siglec-8, as described above, we have found specificity differences between commercially available Siglec-8 antibodies due to the high sequence identity between some of the CD33-related family members. Using a Siglec-based cross-reactive ELISA, the Siglec-8 mAb clones FAB7975 (R&D Systems, Minneapolis, MN, USA) and 347104 (Biolegend, San Diego, CA, USA) were found to bind specifically to Siglec-8, whereas HPA012556 (Sigma and Atlas) was found to cross-react with multiple Siglecs, including Siglec-9, -7, and -12 (unpublished observations). These studies suggest that Siglec-8 expression is robust and stable on eosinophils and mast cells independent of disease state and tissue of origin.

Siglec-8 is not expressed by hematopoietic stem cells, or eosinophil or mast cell precursors. Based on in vitro studies in which cells were grown from precursors, Siglec-8 is only expressed during late stages of mast cell and eosinophil maturation^[21][22] [21,22]. Siglec-8 was not expressed by any eosinophil cell line tested and was expressed at low levels by the LAD2, LUVA, and HMC 1.2 mast cell lines^[10][21][22] [10,21,22] (unpublished observations). Like many CD33-related Siglecs, Siglec-8 is only expressed at the human and ape level, and is not detected on rhesus or cynomolgus monkey eosinophils^[21] [21]. Furthermore, Siglec-8 does not have a true mouse ortholog—the closest functional paralog in mice is Siglec-F, which is expressed on eosinophils (and other cells such as alveolar macrophages, tuft cells, granulocyte-macrophage progenitors and sometimes on certain tissue neutrophils that co-express Siglec-E) but not mast cells^{[23][24][25][26][27][28][29][30]} [23–30] (see Table 1 for Siglec-F and Siglec-8 comparison). Researchers in the Bochner laboratory and at Allakos, Inc. have developed strains of mice that selectively express Siglec-8 in the eosinophil compartment (SIGLEC8Eo), in the mast cell compartment (Mcpt5-Siglec8 and Cpa3-Siglec8), or on eosinophils, mast cells, and basophils (Siglec-8 transgenic)^[19][31][32] [19,31,32]. Strains that express Siglec-8 on specific immune cells rely on cell-specific or cell-selective Cre expression to remove a STOP cassette and allow for the discrimination between the effects of Siglec-8 on each cell population. Siglec-8 transgenic mice that express the human SIGLEC8 gene, including the putative promoter and regulatory elements, most accurately mimic the expression of Siglec-8 in humans. The SIGLEC8Eo strain has been crossed with the Siglec-F null strain, to create mice that express Siglec-8 but not Siglec-F^[33] [33]. These mice are useful for testing antibodies (see below) and glycomimetics that preferentially bind to Siglec-8, and for determining their specificity of targetin^[34]g [34]. In addition, mice with humanized immune systems (engrafted with human thymus, liver, or hematopoietic stem cells) that express SCF, GM-CSF, and IL-3 (NSG-SGM3) generate human mast cells and eosinophils that express functional Siglec-8^[35] [35].

Table 1.

Comparison of Siglec-8 and Siglec-F.

Siglec, sialic acid-binding immunoglobulin-like lectins; DMBT1, deleted in malignant brain tumors 1; Gal, galactose; ROS, reactive oxygen species; MAP, mitogen-activated protein.