Discovery of Multifold Modified Sialosides as <i>Human</i> CD22/Siglec‑2 Ligands with Nanomolar Activity on B‑Cells

Sialic acids are abundant in higher domains of life and lectins recognizing sialosaccharides are heavily involved in the regulation of the human immune system. Modified sialosides are useful tools to explore the functions of those lectins, especially members of the Siglec (sialic acid binding immunoglobulin like lectin) family. Here we report design, synthesis, and affinity evaluation of novel sialoside classes with combined modification at positions 2, 4, and 9 or 2, 3, 4, and 9 of the sialic acid scaffold as <i>human</i> CD22 (<i>human</i> Siglec-2) ligands. They display up to 7.5 × 10⁵-fold increased affinity over αMe Neu5Ac (the minimal Siglec ligand). CD22 is a negative regulating coreceptor of the B-cell receptor (BCR). <i>In vitro</i> experiments with a human B-lymphocyte cell line showed functional blocking of CD22 upon B-cell receptor (BCR) stimulation in the presence of nanomolar concentrations of the novel ligands. The observed increased Ca²⁺ response corresponds to enhanced cell activation, providing an opportunity to therapeutically modulate B-lymphocyte responses, e.g., in immune deficiencies and infections

Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec‑7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion

Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar <i>K</i>_d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds

Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec‑7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion

Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar <i>K</i>_d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds

Characterization of the pDCre x RFP reporter line shows terminal targeting of pDCs.

<p>(<b>A, B</b>) BM and spleen cells from pDCre x RFP mice were stained for Siglec-H, CD11c, MHCII, CD3ε, CD19 and NK1.1 (<b>A</b>) Characterization of the reporter expression (RFP) by different cell types: pDCs were gated as Siglec-H⁺, B cells as Siglec-H⁻ CD19⁺ CD3ε⁻ NK1.1⁻, T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁻, NK-T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁺, and NK cells as Siglec-H⁻ CD19⁻ CD3ε⁻ CD11c^int NK1.1⁺. FACS plots are representative for two individual experiments. (<b>B</b>) Characterization of the RFP reporter expression by CD11c^hi MHCII^hi cDCs in spleen and CD11c^int CD19⁻ CD3ε⁻ NK1.1⁻ Siglec-H⁻ cells in BM. (<b>C</b>) Quantification of (A, B) showing pooled data from 2 independent experiments using 4–5 mice/group. (<b>D</b>) Phenotypic comparison of pDC markers expressed by RFP⁻ and RFP⁺ pDCs from BM (top panel) and spleen (bottom panel). pDCs were gated as Siglec-H⁺ CD11c^int. Histogram overlays display the isotype controls as dashed line, and the marker expression by RFP⁻ pDCs as grey filled histogram and by RFP⁺ pDCs as bold line. Data shown are from one representative experiment out of two using 4 mice/group. (<b>E</b>) Splenic <i>ex vivo</i> pDCs were purified by FACS sorting from B16-Flt3L treated pDCre x RFP mice and incubated with the indicated MOIs of MCMV <i>in vitro</i>. IFNα/TNFα concentrations were quantified in the supernatants after 24 h incubation by ELISA or cytometric bead assay. Data shown are from one representative experiment out of two using a pool of 3 mice. The differences between RFP⁻ and RFP⁺ pDCs were not significant as calculated by Students t-test. Data displayed in (D, E) are from one out of two individual experiments with similar results. Data are displayed as mean ± SD.</p

IFNα serum levels are elevated in the absence of Siglec-H upon MCMV infection.

<p>Lethally irradiated CD45.1⁺ wt mice were reconstituted with CD45.2⁺ wt or Siglec-H^−/− BM followed by infection with 5×10⁴ PFU of wt MCMV. (<b>A</b>) FACS plots show efficient donor reconstitution in the blood eight weeks after BM transfer (upper panel). Siglec-H and B220 stainings of splenocytes confirm the lack of Siglec-H expression in wt mice reconstituted with Siglec-H^−/− BM (lower panel). (<b>B</b>) The kinetics of serum IFNα levels 1.5, 3 and 6 days p.i. compared between Siglec-H^−/− and wt mice, n = 4–5 mice/group. Data are from one of two individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p

MCMV activates Ly49H⁺ NK cells and expands Ag-specific CD8⁺ T cells from Siglec-H^−/− and wt mice with similar viral clearance in the primary and secondary organs.

<p>Siglec-H^−/− and wt chimeric mice were infected with 5×10⁴ PFU of wt MCMV or mock treated with PBS. (<b>A, B</b>) IFNγ serum concentrations were determined at 36 h p.i. and the MFI of CD69 expression on NK1.1⁺ blood NK cells was quantified by flow cytometry. (<b>C, D</b>) Representative histogram overlays and quantification of KLRG-1 expression on splenic NK1.1⁺ Ly49H⁺ cells at day 8 p.i. (<b>E</b>) Representative FACS plots for H-2D^b M45 tetramer staining and CD62L expression among CD8⁺ splenocytes. (<b>F, G</b>) Quantification of frequencies and absolute numbers of tetramer⁺ CD8⁺ T cells from the tetramer staining shown in (E). (<b>H</b>) Shows frequencies of IFNγ⁺ cells among CD8⁺ T cells after restimulation with the indicated concentrations of H-2D^b M45 peptide. (<b>I</b>) Viral load was measured in the spleen, liver and salivary glands of MCMV infected Siglec-H^−/− and wt mice at day 3, 6 and 8 p.i. Dashed line indicates the limit of detection. Data shown are pooled from 2–3 individual experiments. **<i>p</i><0.01 Students t-test, ns = not significant, nd = not detectable.</p

Siglec-H is downregulated upon MCMV infection <i>in vivo</i>.

<p>pDCre x RFP mice were infected with 5×10⁴ PFU MCMV <i>in vivo</i>. (<b>A</b>) Representative FACS plots show CD11c versus Siglec-H of live splenocytes at 36 h p.i. (<b>B</b>) Gating strategy showing exclusion of B-, T-, NK-T, and NK cells by CD19, CD3ε, NK1.1 in a default channel. RFP⁺ pDCs were gated as CD11c^int B220⁺ MHCII^int to exclude MHCII⁻ DC precursors. Histogram overlays show RFP⁺ pDCs from mock (bold line) and MCMV infected (dashed line) pDCre x RFP mice at 36 h p.i. Isotype staining is displayed as grey histogram. Data are representative of 2 independent experiments using 4–5 mice with comparable results.</p

Siglec-H receptor does not play a role in pDC infection.

<p>Flt3-L derived CD11c⁺ B220⁺ pDCs and CD11c⁺ B220⁻ cDCs were sorted from BMDC cultures of wt and Siglec-H^−/− mice. DCs were mock treated with PBS or MCMV-GFP infected at MOI 2. (<b>A</b>) Representative FACS plots show Siglec-H expression and MCMV-GFP expression at 24 h p.i. (<b>B</b>) Quantification of the viral titers per 10⁶ DCs or MEFs at 24 h p.i. (<b>C</b>) Representative histogram plots of CD86 expression on wt and Siglec-H^−/− sorted pDCs (live cell gate) at 24 h p.i. (gray bar = isotype control, gray dashed line = mock treatment, gray solid line = CpG-A treatment, dark bold line = MCMV infection). Data are from one of three individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p