Engineering of the tumor-cell glycocalyx with synthetic sialylated glycopolymers showed in 2014 that cell-surface hypersialylation inhibits human natural killer (NK) cell cytotoxicity through the inhibitory receptor Siglec-7, and posed several questions: the identity of the endogenous Siglec-7 ligands on tumors, why some sialylated structures that bind Siglec-7 biochemically (notably the disialoganglioside GD3) do not strongly inhibit NK cells, the in-vivo relevance of the mechanism, its breadth beyond NK cells, and its therapeutic exploitability. Later literature has resolved the in-vivo question (Siglec-7/9 act as glyco-immune checkpoints in mouse models and are therapeutically targetable) and established the mechanism's breadth across myeloid lineages, and has identified endogenous functional ligands in several individual tumor contexts (CD43 with disialylated O-glycans in leukemia, GD2 in neuroblastoma, disialyl-T on CD45/CD162 in chronic lymphocytic leukemia, tri-sialylated T on podocalyxin/MUC13 in colon cancer, sialoglycans whose synthesis depends on the sialyltransferase enzyme ST3Gal1 in prostate cancer). What remains open is the systematic ligand-prevalence map across human tumor types and, centrally, the molecular and structural rules — glycan structure, carrier-protein scaffold, density, membrane context, and synapse geometry, against the background of cis-ligand masking of Siglec-7 on NK cells — that determine whether an endogenous sialylated structure that binds Siglec-7 functionally inhibits NK-cell activation rather than merely binding.

Background

Natural killer (NK) cells are innate lymphocytes that kill transformed cells without prior sensitization, integrating signals from activating and inhibitory receptors at the contact zone with a target cell, the immune synapse. Many tumor types share a biochemical trait: an increase in sialic acid (a nine-carbon acidic sugar that caps glycan chains) on cell-surface glycans, a state called hypersialylation. Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mostly inhibitory receptors that recognize sialylated glycans; most contain an immunoreceptor tyrosine-based inhibition motif (ITIM) that, when the receptor is engaged, recruits phosphatases that dampen immune-cell activation. Siglec-7 (also called p75/AIRM1) is a CD33-family Siglec expressed abundantly on human NK cells, and also on most monocytes and subsets of CD8+ cytotoxic T lymphocytes. Like other Siglecs, Siglec-7 is largely occupied on resting NK cells by sialylated glycans on the NK cell's own surface (cis ligands), so functional engagement by glycans on a target cell (trans ligands) requires ligands of sufficient avidity to unmask the receptor.

Before 2014, candidate tumor ligands for Siglec-7 were the disialylated glycosphingolipids (gangliosides) GD3, whose expression on target cells modulates NK cytotoxicity, and DSGb5, a preferred Siglec-7 ligand in renal cell carcinoma; however, these interactions could mainly be demonstrated after enzymatically removing sialic acids from the NK cells with a sialidase, which left their biological relevance uncertain. Hudak, Canham and Bertozzi (2014) broke this impasse with glycocalyx engineering: synthetic glycopolymers end-functionalized with a phospholipid insert into cell membranes and present defined sialylated glycans at controlled density on living tumor cells. Displaying such sialoside polymers on several tumor cell lines inhibited primary human NK-cell cytotoxicity in a Siglec-7-dependent manner, reduced the efficacy of therapeutic antibodies in antibody-dependent cellular cytotoxicity, and — when used to decorate allogeneic and xenogeneic primary cells — protected those cells from NK-mediated killing, suggesting Siglec-7 as a therapeutic target in transplantation.

Two observations in that study framed the questions addressed here. First, the endogenous Siglec-7 ligands actually used by tumors were unknown: the authors explicitly hoped their glycopolymers would 'spur efforts to confirm the endogenous ligands for Siglec-7'. Second, a puzzle emerged: the polymer carrying GD3 — a glycan that glycan arrays (immobilized panels of defined glycans) and soluble Siglec-7-Fc binding assays identify as a high-affinity Siglec-7 ligand — bound Siglec-7 on cell surfaces but did not inhibit NK cytotoxicity more than other sialylated ligands, and less than expected, suggesting 'a divergence between binding preference and biological activity for Siglec-7' possibly reflecting 'ligand-induced conformational changes or geometrical requirements' or difficulty binding 'in a congested cell-cell synapse'. The years since have transformed the field's understanding: Siglec-7 and its relatives are now designated glyco-immune checkpoints, their in-vivo importance and targetability have been established, and specific endogenous ligands have been identified in several cancers — but a systematic answer to which ligand architectures functionally engage Siglec-7 in which tumors, and why biochemical Siglec-7 binding does not predict functional inhibition, is still missing.

Problem Statement

For human tumors, determine (1) the endogenous functional ligands of Siglec-7 — which combinations of glycan structure (disialylated O-glycans such as disialyl-T, the core-1 mucin-type O-glycan GalNAc-Gal capped with two sialic acids, and its tri-sialylated analogue tri-sialylated T; and disialogangliosides, that is glycosphingolipids carrying two sialic acids, such as GD2, GD3 and DSGb5) and carrier molecule (heavily O-glycosylated mucin-type glycoproteins such as CD43 (leukosialin), CD45, CD162, PODXL and MUC13, versus glycosphingolipids) actually mediate Siglec-7-dependent inhibition of NK-cell cytotoxicity in each tumor type, and how prevalent these ligand architectures are across human cancers (prevalence here meaning the fraction of primary human tumors of a given type in which the architecture is detected as a functional Siglec-7 ligand, assessed from glycomic or glycogene-expression data or from synthesis of the published ligand-identification literature); and (2) the structural, biophysical and cell-biological rules — glycan structure, carrier-protein scaffold, ligand density, membrane context, and synapse geometry, together with cis-ligand masking of Siglec-7 on the NK-cell surface — that determine whether a sialylated structure that binds Siglec-7 in biochemical assays functionally inhibits NK-cell activation, thereby explaining the observed divergence between binding and biological activity (for example, GD3 binding Siglec-7 without producing strong inhibition). Both parts serve one objective: to replace the current case-by-case ligand discoveries with a predictive account of what makes an endogenous sialylated structure a functional Siglec-7 checkpoint ligand on a tumor cell. The two parts are jointly posed, and the rules of part (2) jointly cover glycan structure, carrier-protein scaffold, ligand density, membrane context and synapse geometry, together with cis-ligand masking of Siglec-7 on the NK-cell surface. Part (1) is to state, for every tumor type for which functional Siglec-7 ligand data exist — and at minimum for the tumor contexts named in this statement (leukemia, chronic lymphocytic leukemia, neuroblastoma, colon cancer, prostate cancer and renal cell carcinoma) — which of the named ligand architectures act there as functional Siglec-7 ligands together with those architectures' prevalence across human cancers, and part (2) is to state the rules explicitly enough to predict, for a given endogenous sialylated structure, whether Siglec-7 binding yields functional inhibition of NK-cell activation or binding without inhibition.

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