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. 2017 Sep 13;139(36):12450-12458.
doi: 10.1021/jacs.7b03208. Epub 2017 Aug 31.

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells

Wenjie Peng  1 , James C Paulson  1
Affiliations

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells

Wenjie Peng et al. J Am Chem Soc. .

Abstract

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 N-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.

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Conflict of interest statement

Notes

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Schematic illustration of liposome, Antibody Drug Conjugate (ADC)– and N-glycan scaffold for _targeting CD22-positive B-lymphomas cells
Red circle with letter T represents toxin. Magenta diamond represents high affinity ligands for CD22. N-Glycan structure is displayed using the Symbol Nomenclature for Gly-comics.
Figure 2
Figure 2. A library of glycan scaffolds bearing hCD22 synthetic ligands via chemoenzymatic synthesis
A) Chemical structures of sialic acid analog ligands of CD22 (1a–c). BPC: biphenyl carbonyl; MPB: m-phenoxybenzamide. B) Synthetic ligands of hCD22 based on linear, or branched O- or N-glycan scaffolds. Glycan structures are displayed using the Symbol Nomenclature for Glycomics.
Figure 3
Figure 3. Determination of the binding affinity and selectivity of synthetic ligands for hCD22
A) Assessment of the ability of the hCD22 ligands to block binding of a multivalent ligand to hCD22-CHO cells using a flow cytometry-based competitive binding assay. B) Summary of IC50 and relative inhibitory potency (rIP) values of ligands in Figure 2B assessed in the same assay described in panel A. Triantennary analogue ligands are highlighted in red. C) Selective binding of monovalent ligand 3a and triantennary N-glycan 3j for hCD22 assessed on a panel of Siglec-expressing CHO cells. Binding affinities were determined by flow cytometry.
Figure 4
Figure 4. Bi- and tri-antennary ligands of hCD22 are endocytosed by CD22-CHO cells
A) Serial dilutions (400-0.4 nM) of ligand–FITC (SGP-, 3a-, 3b-, and 3j-FITC) conjugates were incubated with hCD22-CHO or WT-CHO cells at 37 °C for 60 min. Cells were washed and endocytosed ligand was assessed by flow cytometry. B) Ligand–FITC conjugates (50 nM) were incubated with hCD22-CHO or WT-CHO cells at 37 °C for various times. Conjugates 3b- and 3j-FITC were efficiently endocytosed by hCD22-CHO cells, but not WT-CHO cells. C) Endocytosis of the triantennary hCD22 N-glycan ligand by hCD22-CHO cells. hCD22-CHO cells (top and middle) or WT-CHO (bottom) cells on coverslips were overlayed with the hCD22 ligand 3j-FITC in culture media for 3 h at 37°C. Cells were then washed, fixed, and counterstained with nuclei (blue), ligand (green), hCD22 (yellow) and Tf receptor or lysosome (red) followed by confocal fluorescence microscopy. DIC: differential interference contrast. Scale bar is 10 μm.
Figure 5
Figure 5. The triantennary glycan scaffold can be endocytosed and deliver conjugated toxins to Daudi B lymphoma cells
A) Daudi cells were incubated with serial dilutions of ligand–FITC conjugates in pure mouse serum at 37 °C for 60 mins. Endocytosed ligands were assessed by flow cytometry. B) Serial dilutions of saporin-streptavidin complex with biotinylated-glycan ligands were mixed with Daudi cells for 72h, followed by assessment of viability using the chromogenic MTT assay. The absorbance was measured at 570 nm. Ligands were LacNAc-biotin (2a-biotin) with no sialic acid, and MBPNeu5FAc bearing monovalent (3h-biotin) and tri-valent N-glycan (3j-biotin). C) Serial dilutions of ligand-MMAF conjugates were mixed with Daudi cells for 72h, followed by assessment of viability using the chromogenic MTT assay. The absorbance was measured at 570 nm. Ligands were MBPNeu5FAc bearing monovalent (3h) and tri-valent N-glycan (3j). MMAF alone was used as a control.
Figure 6
Figure 6. Determination of the binding affinity of synthetic ligands for mCD22 and mSn
A) Assessment of the ability of the mCD22 ligands to block binding of a multivalent ligand to mCD22-CHO cells using a flow cytometry-based competitive binding assay. B) Assessment of the ability of the mSn ligands to block binding of mSn to the magnetic beads coated with multivalent natural ligand using a flow cytometry-based competitive binding assay. C) Synthetic ligands of mCD22 and mSn based on linear, or branched N-glycan scaffolds. Glycan structures are displayed using the Symbol Nomenclature for Glycomics. IC50 values were listed in parenthesis behind the compound numbers. BPA: Biphenyl acetyl. TCC: 4H-thieno[3,2-c]chromene-2-carbamoyl.
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