CD22 Ligands on a Natural <i>N</i>‑Glycan Scaffold Efficiently Deliver Toxins to B‑Lymphoma Cells

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural <i>N</i>-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs

In Silico-Aided Design of a Glycan Ligand of Sialoadhesin for in Vivo Targeting of Macrophages

Cell-specific delivery of therapeutic agents using ligand targeting is gaining interest because of its potential for increased efficacy and reduced side effects. The challenge is to develop a suitable ligand for a cell-surface receptor that is selectively expressed on the desired cell. Sialoadhesin (Sn, Siglec-1, CD169), a sialic acid-binding immunoglobulin-like lectin (Siglec) expressed on subsets of resident and inflammatory macrophages, is an attractive target for the development of a ligand-targeted delivery system. Here we report the development of a high-affinity and selective ligand for Sn that is an analogue of the natural ligand and is capable of targeting liposomal nanoparticles to Sn-expressing cells in vivo. An efficient in silico screen of a library of ∼8400 carboxylic acids was the key to identifying novel 9-<i>N</i>-acyl-substituted <i>N</i>-acetylneuramic acid (Neu5Ac) substituents as potential lead compounds. A small panel of targets were selected from the screen and synthesized to evaluate their affinities and selectivities. The most potent of these Sn ligands, 9-<i>N</i>-(4<i>H</i>-thieno­[3,2-<i>c</i>]­chromene-2-carbamoyl)-Neu5Acα2–3Galβ1–4GlcNAc (^TCCNeu5Ac), was conjugated to lipids for display on a liposomal nanoparticle for evaluation of targeted delivery to cells. The ^TCCNeu5Ac liposomes were found to target liposomes selectively to cells expressing either murine or human Sn in vitro, and when administered to mice, they exhibited in vivo targeting to Sn-positive macrophages

Sn-targeted liposomes bind to IFN-α stimulated bone marrow derived macrophages (BMM)s.

<p>(<b>A</b>) Mature macrophages differentiated from wild type mouse bone marrow cells were stimulated with IFN-α or left untreated before staining with anti-Sn, anti-F4/80 antibodies (<i>filled gray</i>) and Sn-targeted liposomes (<i>filled gray</i>). Cells were thoroughly washed prior to FACS analysis. Isotype antibody or naked liposome stained cells were used as negative controls (<i>black lines</i>). (<b>B</b>) BMMs derived from wild type or Sn^−/− mice were stained with fluorescent naked (blue lines) or Sn-targeted (<i>filled red</i>) liposomes prior to FACS analysis. Unstained cells (<i>black lines</i>) were used as a negative control. Data are representative of 3 independent experiments.</p

Specificity of Sn-targeted liposomes against a panel of siglec-expressing cells.

<p>FACS analysis for binding of naked or 3′-^BPCNeuAc liposomes to siglec-expressing CHO lines and TSn and BW5147 cell lines that express hSn and Siglec-G, respectively. (A) Binding of liposomes is expressed as mean channel fluorescence (MCF) ± s.d. (n = 3). Binding degree of 3′-^BPCNeuAc liposomes to TSn, CHO-mSn cells was statistically significant comparing to the same cell line that was treated with the naked liposomes (*<i>P</i><0.05). (B) Compatible amount of 3′-^BPCNeuAc liposome binding to CHO-mSn and SIglec-G-BW5147 cells is shown. Cells were stained with 3′-^BPCNeuAc (<i>Red</i>), Naked liposomes (<i>Gray</i>), or unstained (<i>Black</i>). Data are representative of 2 independent experiments.</p

Antigen delivery to BMMs using Sn-targeted liposomes led to proliferation of antigen-specific T cells.

<p>Sn-expressing BMMs (stimulated by IFN-α) were treated with free OVA proteins, naked or Sn-targeted liposomes that carry OVA for 1 hr or left untreated as a negative control. CD4⁺ T cells purified from OT-II transgenic mice were CFDA labeled and incubated with the washed BMMs that were treated with indicated conditions. After 72 hr culture <i>in vitro</i>, T cells were harvested and analyzed for CFDA dilution by flow cytometry. Percentages of the dividing CD4⁺ T cells are indicated. Shown are representative of 2 independent experiments.</p

Synthesis of the 3′-^BPCNeuAc-pegylated lipids.

<p>The sialic acid ligand with an ethylamine linker was coupled to an NHS-activated pegylated lipid.</p

Liposomes bearing 3′-^BPCNeuAc ligands bind to and internalized by Sn/CD169 expressing cells.

<p>(<b>A</b>) FACS analysis for binding of the naked (<i>blue line</i>) or 3′-^BPCNeuAc (<i>red line</i>) liposomes to TSn and Daudi cells that express surface hSn and hCD22 (Siglec-2), respectively. Unstained cells (<i>filled grey</i>) were used as a negative control. Shown are results from 1 of 3 representative experiments followed indicated treatment. (<b>B</b>) Ligand-bound liposomal cargos but not antibodies exhibited time-dependent accumulation in Sn-expressing cells. TSn cells were incubated with fluorescently labeled anti-Sn antibody (Clone 7–239 (Serotech); <i>filled red, left panel</i>) or Sn-targeted liposomes (<i>filled red, right panel</i>) for 5, 20 and 90 min before they were washed with isotonic HBSS buffer prior to FACS analysis. Isotype antibody or naked liposome stained cells were used as negative controls. (<b>C</b>) Internalization of Sn-targeted liposomes by TSn cells. Cells were incubated with fluorescent naked or 3′-^BPCNeuAc liposomes for 5, 30 or 60 min at 37°C before they were washed with isotonic HBSS buffer (pH 7) or acid buffer (pH 3.3) prior to FACS analysis to determine levels of the total liposome uptake (membrane bound plus internalized), or internalized liposomes. Cells that were not stained with liposomes were used as a negative control (<i>filled gray</i>). (<b>D</b>) Recycling of Sn between cell surface and inside the cell. <i>Top</i>; unlabeled anti-Sn Ab was incubated with Sn expressing CHO cells at 37°C. Cells were then cooled to 4°C and stained with a labeled secondary Ab after a neutral wash (<i>Red line</i>) or acid wash (<i>Orange line</i>) to detect residual cell surface bound anti-Sn Ab. Isotype control antibody staining is shown as <i>filled histogram</i>. <i>Middle</i>; acid-washed cells from the <i>Top</i> were subjected to a further incubation at 4°C and stained with labeled secondary Ab, showing that no anti-Sn Ab had returned to the cell surface. <i>Bottom</i>; acid-washed cells from the <i>Top</i> were warmed to 37°C to allow internalized anti-Sn Ab to be recycled back to the cell surface. Ab re-appearing on the surface of the cell is detected by staining the cells with labeled secondary Ab. Results shown are representative of at least two independent experiments.</p

Fluorescence microscopy analysis of the endocytosis of 3′-^BPCNeuAc liposomes in the Sn-expressing cells.

<p>CHO-mSn/CD169 cells were stained with fluorescently labeled naked or 3′-^BPCNeuAc liposomes (<i>green</i>) followed by staining with anti-Sn (<i>red</i>) or antibodies that detect early endosomes (<i>red</i>) and lysosomes (<i>red</i>). The nuclei were visualized by staining with DAPI (<i>blue</i>).</p

<i>B</i>-value analysis of Bris07 HA.

<p><b>(A-B)</b><i>B</i>-values (Ų) of Cα atoms in <b>(A)</b> Bris07 P194 and <b>(B)</b> Bris07 L194 are projected on the HA structure. Of note, on average, the <i>B</i>-values of Cα atoms in Bris07 P194 (mean ± s.d. = 58 ± 24 Ų) are lower than that of Bris07 L194 (mean ± s.d. = 64 ± 24 Ų). <b>(C)</b> The normalized <i>B</i>-values of Cα atoms in Bris07 P194 (blue) and L194 (green) are compared. The bottom panel shows their difference in HA1: (normalized <i>B</i>-values of Cα atoms in P194)–(normalized <i>B</i>-values of Cα atoms in L194). The amino-acid position is plotted along the x-axis. Positions corresponding to the residues (Res) of interest are shaded in grey.</p

Crystal structures of Bris07 P194 and L194 HAs in complex with receptor analogs.

<p>HA structures of <b>(A)</b> Bris07 P194 in complex with 3'SLNLN, <b>(B)</b> Bris07 L194 in complex with 3'SLNLN, <b>(C)</b> Bris07 P194 in complex with 6'SLNLN, and <b>(D)</b> Bris07 L194 in complex with 6'SLNLN are shown. The apo form for each structure is aligned and colored in grey. Glycan receptor analogs (3'SLNLN and 6'SLNLN) are colored in yellow and shown as stick representations. Hydrogen bonds are represented by black dashed lines.</p