Bifunctional CD22 ligands use multimeric immunoglobulins as protein scaffolds in assembly of immune complexes on B cells

Abstract

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.

Figure 1. Design of a bi-functional ligand to drive self-assembly of IgM-CD22 complexes

(a) Structure of the bi-functional ligand, ^BPCNeuAc-NP. (b) Schematic of the ligand-driven complex formation between the decavalent scaffold, anti-NP IgM, and the B cell surface lectin CD22 on either B cells or on solid supports decorated with the extracellular domain of CD22.

Figure 2. Bi-functional ligand 11 drives complex formation of IgM with immobilized recombinant CD22-Fc and on native B cells

(a) Using ELISA, anti-NP IgM binding to CD22-Fc chimeras immobilized onto protein A coated wells was shown to be dose dependent with ^BPCNeuAc-NP, but not NP. (b) Flow cytometry was used to measure the anti-NP IgM dose dependence using CD22-Fc chimeras immobilized onto Protein A conjugated magnetic beads in the presence of 1 μM ^BPCNeuAc-NP and. The x-axis represents the degree of IgM binding in relative fluorescence units. (c) ^BPCNeuAc-NP dependent anti-NP IgM binding occurs on native BJAB cells as analyzed by flow cytometry. BJAB cells were incubated with 3 μM ligand and 10 μg/mL Alexa488-anti-NP IgM. (d) Immunofluorescence microscopy was used to visualize binding of Alexa488-anti-NP IgM to native BJAB cells.

Figure 3. Influence of linker structure in bi-functional ligand driven assembly of IgM-CD22 complexes on B cells

Ligands 11-15 or NP (2 μM) were tested in the self-assembly of IgM-CD22 complexes on native BJAB cells. Binding of anti-NP IgM (10 μg/mL) was analyzed by flow cytometry following staining with anti-IgM (FITC).

Figure 4. Analysis of antibody valency in complex formation

(a) Anti-NP antibodies of different valency were assessed for their binding to native BJAB cells in the presence of 2 μM ^BPCNeuAc-NP (11), using 10 μg/mL IgM (n=10; top), IgA (n=4; middle), or IgG (n=2; bottom). Ig binding was assessed by flow cytometry after staining with secondary antibodies (FITC). (b) Avidities of complex formation with anti-NP antibodies of different valency were assessed by titrating ^BPCNeuAc-NP (11) in the presence of CD22-immobilized magnetic beads and 5 μg/mL anti-NP IgM, IgA, or IgG, and measuring bead fluorescence by flow cytometry. Mean channel fluorescence (MCF) is plotted against ligand concentration.

Figure 5. Self-assembly of antibody-CD22 complexes on native B cells is in competition with cis ligands

Titrations of ^BPCNeuAc-NP (11) in the presence of 5 μg/mL anti-NP IgM or IgA and BJAB cells cultured in the absence (a) or presence (b) of sialic acid reveal differences in concentrations of ligand required for complex formation.

Figure 6. Temperature dependence of complex formation

Bi-functional ligand 11 was compared for its ability to induce complex formation of of 5 μg/mL anti-NP IgM (a) or IgA (b) on CD22-immobilized magnetic beads at the indicated temperatures. (c) Bi-functional ligands 13-15 (top to bottom, respectively) were similarly compared for their ability to induce complex formation of anti-NP IgM on CD22 immobilized magnetic beads. Ig binding was assessed by flow cytometry following staining with secondary antibodies (FITC).

Figure 7. Specificity of ^BPCNeuAc-NP formation of immune complexes on B cells

White blood cells isolated from peripheral human blood were (a) stained with Alexa488-anti-NP IgM in the presence of 2 μM NP or ^BPCNeuAc-NP (11) at 37 °C, or (b) subjected to double staining with Alexa488-anti-NP IgM (2 μM 11 included) and PE-labeled anti-CD3 (pan T cell), anti-CD4 (CD4+ T cells), anti-CD8 (CD8+ T cells), anti-CD19 (B cells), or anti-CD56 (natural killer cells). Binding was analyzed by flow cytometry.

Scheme 1. Synthesis of bi-functional ligands of CD22

a) GalT-GalE, UDP-Gal; b) hST6Gal I, CMP-9-N-biphenylcarboxyl-9-deoxy-Neu5Ac; c) HBTU, HOBt, Et₃N (for compound 7); d) MeOH, Et₃N (for compounds 6 and 10); e) Cu₂SO₄, sodium ascorbate, water : t-BuOH = 1 : 1 (for compounds 8 and 9).