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The Journal of Immunology, 2006, 177: 2994-3003.
Copyright © 2006 by The American Association of Immunologists, Inc.

High-Affinity Ligand Probes of CD22 Overcome the Threshold Set by cis Ligands to Allow for Binding, Endocytosis, and Killing of B Cells1

Brian E. Collins*, Ola Blixt*, Shoufa Han*, Bao Duong*, Hongyi Li*, Jay K. Nathan*, Nicolai Bovin{dagger} and James C. Paulson2,*

* Departments of Molecular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92024; and {dagger} Shemyakin and Ovchinnokov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia{alpha}2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A regulator of BCR signaling, CD22 is a member of the siglec subgroup of the Ig superfamily (1, 2, 3) that is highly expressed on mature B cells and B cell lymphomas (4, 5). Its extracellular domain contains seven Ig domains, of which the outermost N-terminal domain recognizes sialic acid containing glycan ligands (6, 7). The cytoplasmic domain of CD22 contains positive and negative regulatory elements that mediate its activity as a modulator of B cell signaling (2, 4, 5, 8, 9, 10). In particular, negative responses are mediated by ITIMs, which become phosphorylated following Ag ligation, resulting in recruitment of the Src2 homology phosphatase-1 (5, 11, 12, 13).

The tyrosines in the ITIM motifs also serve as critical residues in another structural motif, YXXØ (where Ø is a hydrophobic residue), which mediates binding of the AP50 subunit of the AP-2 adaptor complex driving association with clathrin (14). As a consequence, cell surface CD22 is predominantly localized in clathrin containing microdomains where it undergoes constitutive endocytosis (15, 16). Ab-mediated cross-linking of CD22 increases the rate of internalization (14, 17, 18, 19), and this fact has made CD22 a target for immunotherapy of B cell lymphomas using anti-CD22 Abs (20, 21, 22). The localization of CD22 in clathrin domains is also relevant to its role in regulation of B cell signaling since "activated" BCR moves to fused raft-clathrin domains before endocytosis (16, 23, 24). Although not studied in as much detail, other Siglecs including sialoadhesin, Siglec-5, and Siglec-H have also been demonstrated to undergo endocytosis (25, 26, 27, 28).

The ligand-binding domain of CD22 is highly specific for the glycan sequence Sia{alpha}2-6Gal, a common termini of N-linked glycans on B and T cells (29, 30, 31, 32, 33). Although the affinity of CD22 for this sequence is low (Kd ~100–200 µM), the high concentration of the glycan on the B cell surface (~50–100 mM) (34) results in constitutive binding of CD22 to B glycoproteins in cis. Thus, CD22 appears "masked" to synthetic sialoside probes unless cis ligands are first destroyed by treatment with sialidase or periodate (35, 36, 37, 38). In this respect, CD22 is not unique, because masking of the binding of sialoside probes by cis ligands is documented as a general property of the siglec family, the only exception being sialoadhesin (Siglec-1) (2, 3, 35, 39, 40). Recent results suggest that glycans of neighboring CD22 molecules act as the predominate cis ligands, resulting in formation of homomultimeric complexes (41). Such cis interactions play important roles in the regulation of B cell activation as evidenced by aberrant B cell activation, and turnover following loss of cis ligands or deletion of the lectin-binding domain of CD22 (3, 4, 8, 16, 42, 43).

CD22 was originally described as a cell adhesion molecule (44, 45), and despite being masked by cis ligands has been proposed to mediate important biology via binding to glycoprotein ligands on adjacent cells. Contact of B cells with Ag-bearing target cells expressing CD22 ligands results in suppression of B cell activation, suggesting that recruitment of CD22 by trans ligands on Ag-bearing cells may be an important mode of its recognition of self and regulation of B cell signaling (46). Binding of CD22 to glycoprotein ligands on T cells also modulates T cell signaling (47), and B cell homing to bone marrow is proposed to occur by interaction of CD22 with endothelial cells in bone marrow capillaries (48, 49). This interaction of CD22 with trans ligands on opposing cells has been suggested to occur as a result of the differential expression of cis ligands on B cells, leaving subsets that are unmasked (48, 49), or by activation of B cells to cause partial unmasking of CD22 (35). However, mere contact of resting B cells with other B cells or T cells induces redistribution of CD22 to the site of cell contact as a result of interactions with trans ligands on the opposing cells (34), suggesting that trans ligands can compete for CD22 without alteration of cis ligands.

Few reports have investigated the relationship between the ligand-binding activity of siglecs and their capacity to undergo endocytosis. Although the surface expression of CD22 is reduced 50% on B cells of mice deficient in its cis ligand, no change in the distribution of CD22 in clathrin domains compared with wild-type mice was observed (16). Moreover, mutations in the ligand-binding domain of CD22 did not affect the rate of constitutive endocytosis of CD22 (18). Thus, cis ligands do not appear to have a major impact on the endocytosis of CD22. However, in an interesting report, Jones et al. (27) found that CHO cells expressing Siglec-5 could mediate sialic acid-dependent binding and uptake of Neisseria meningitides, a bacteria with a sialylated LPS, despite the fact that Siglec-5 was masked from binding sialoside-polyacrylamide probes. Taken at face value, this result suggests that recognition of trans ligands in the presence of masking cis ligands may not be unique to CD22, and that once bound may be endocytosed.

To better understand the equilibrium relationship between cis and trans ligand interactions of CD22, we reasoned that synthetic multivalent ligands of sufficient avidity should bind to CD22 on native B cells in trans despite the presence of cis ligands, and would likely prove useful for ligand-based targeting of CD22-expressing cells. To create high-affinity probes for B cell targeting, we combined molecular features previously reported to enhance the affinity for CD22. Accordingly, 9-biphenyl substituents of sialic acid shown by Zaccai et al. (50) to increase the affinity of CD22 were incorporated into the preferred sialoside ligands of human and murine CD22, and subsequently attached to a high m.w. polyacrylamide backbone to provide a highly multivalent construct. The resulting probes effectively compete with cis ligands on both human and murine B cells, and bind to native cells without prior sialidase treatment. Once bound, the probes are efficiently endocytosed demonstrating that CD22 is capable of carrying ligand-based "cargo" into the cell. Conjugates of the sialoside probe with the toxin saporin were able to target and kill CD22-bearing lymphoma cells in a sialic acid-dependent manner. The results show that the concept of cis ligand masking of CD22 and other siglecs reflects a dynamic equilibrium with cis ligands that block binding of low-affinity ligands, but do not prevent interaction with trans ligands presented with sufficient affinity or avidity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and cell and mouse lines

Wild-type and CD22null (51) mice were maintained under pathogen-free conditions at The Scripps Research Institute breeding facility and were used in accordance to the guidelines of the Institutional Animal Care Committee at the National Institutes of Health. BJAB, Raji, and Daudi cells were maintained in RPMI 1640/5% FCS or in RPMI 1640 supplemented with Neutradoma SP (Invitrogen Life Technologies). Murine spleens were ground between two frosted glass slides and passed over a cotton-plugged Pasteur to obtain single-cell suspensions of splenocytes. RBC were lysed by incubation in 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA (pH 7.2) for 5 min followed by washing with HBSS supplemented with 5 mg/ml BSA (HBSS/B). Abs used were anti-B220 (BD Pharmingen clone RA3-6B2), anti-CD22 (BD Pharmingen clone Cy34.1, and Southern Biotechnology Associates clone 2D6).

Synthesis of {alpha}-methyl-5-acetamido-9-azido-3,5,9-trideoxy-D-glycero-{alpha}-D-galacto-nonulo-pyranoside-2-nonulosate

The synthesis of the methyl glycosides were synthesized according to Brossmer et al. (52).

Synthesis of 9-azido-9-deoxy-sialyloligosaccharides

The synthesis of the 9-azido-9-deoxy-trisaccharides was prepared similarly as described previously (76). Briefly, the appropriate mannose derivative 6-azido-N-acteamido-2,6-dideoxy-mannopyranose or 6-azido-N-glycolyl-2,6-dideoxy-mannopyranose (1 equivalent (eq.)),3 sodium pyruvate (3 eq.), and cytidine 5'-triphosphate (CTP) (1.2 eq.) was dissolved in Tris-HCl (100 mM, 40 ml/mM CTP) (pH 9.5) containing MgCl2 (20 mM) and pH was adjusted to 8.5 Neu5Ac-aldolase (Nal-311 (Toyobo), 400 U/mM mannose derivative), and N. meningitidis ST3Gal-CMPNeu5Ac synthetase fusion protein (20 U/mM CTP) were added, kept at 37°C, and the pH was adjusted with 1 M NaOH to pH 8.3–9.0 as needed. After 14 h, the reaction was terminated by passing through a Prep/Scale-TFF Cartridge cellulose membrane filter molecular mass cut-off 10 kDa (Millipore). To the filtrate, oligosaccharide acceptor LacNAcbetaOMCP (0.5 eq.), MnCl2 (20 mM), alkaline phosphatase (500 U/mM), and ST6Gal I (1 U/mM acceptor) were added and the pH was adjusted to 7.0. After 14 h, the product was purified by repeated size exclusion chromatography (Sephadex G15, 2.5 x 160 cm, equilibrated in 5% n-BuOH) and the 9-azido-NeuAc{alpha}2-6LNbetaOMCP compounds 9-azido-NeuGc{alpha}2-6LNbetaOMCP were isolated in typical yields 75–85% with a purity >90% as analyzed by silica gel thin layer chromatography (eluent: ethylacetate:methanol:acetic acid:water; 6:3:3:2, by volume). Alternatively, the LacNAcbetaOMCP acceptor was replaced by CBz protected 2-aminoethyl glycoside (LacNAcbetaOCH2CH2NH-CBz) acceptor, for polyacrylamide (PAA) conjugation (see below).

General procedure for N-biphenyl-4-carbonyl chloride (BPC) acylation

Each of the sialoside derivatives 9-azido-NeuAcbetaOMe and 9-azido-NeuAc{alpha}2-LacNAcbetaOMCP (1 eq., 25–50 µM) were dissolved in methanol-water (9:1 by volume, 1–2 ml) and hydrogenolysed over palladium on carbon (10%, 20–30 mg) in H2 atmosphere at room temperature. Insoluble catalyst was removed by centrifugation and to the supernatant, diisopropylethylamine (4 eq.) and BPC (2 eq.) was added. When the reaction showed complete reduction to amine (analyzed by silica gel TLC, eluent: ethylacetate:methanol:acetic acid:water; 10:3:3:2, by volume), the mixture was concentrated, reconstituted into water, and applied onto a silica reversed-phase SepPak-C18 column (10 g size) pre-equilibrated in water. The compounds were eluted with a gradient of methanol:water (0–80%) over 50 ml and appropriate fractions containing the product were pooled, concentrated, and further purified by gel filtration chromatography (Sephadex G15, 1 x 70 cm, equilibrated in 5% n-BuOH). Fractions containing the BPC-derivative were pooled and lyophilized to generate a white fluffy solid (65–75%) with >95% purity as analyzed by silica gel thin layer chromatography (eluent: ethylacetate:methanol:acetic acid:water; 10:3:3:2, by volume) and [1H] nuclear magnetic resonance. The compound with the alternative spacer 9-azido-NeuAc{alpha}2-6LacNacOCH2CH2NH-CBz was treated with triphenylphosphine to give the desired 9-amine for BPC acylation and purified as described above.

General procedure for N-biphenyl-4-acetic acid (BPA) acylation

Each of the sialoside derivatives 9-Az-NeuAcbetaOMe and 9-Az-NeuGc{alpha}2-LacNAcbetaOMCP (1 eq., 25–50 µM) were dissolved in methanol-water (9:1 by volume, 1–2 ml) and hydrogenolysed over palladium on carbon (10%, 20–30 mg) in H2 atmosphere at room temperature. Insoluble catalyst was removed by centrifugation and to the supernatant a premixture of diisopropylcarbodiimide (1.2 eq.) and BPA (1.2 eq.) in methanol was added. After 30 min at room temperature, the reaction was diluted with 10 volumes of water and applied onto a silica reversed-phase SepPak-C18 column (10 g size) pre-equilibrated in water. The compounds were eluted with a gradient of methanol:water (0–80%) over 50 ml and appropriate fractions containing the product were pooled, concentrated further, and purified by gel filtration chromatography (Sephadex G15, 1 x 70 cm, equilibrated in 5%-n-BuOH). Fractions containing the BPA derivative were pooled and lyophilized to generate a white fluffy solid (65–75%) with >95% purity as analyzed by silica gel TLC, eluent: ethylacetate:methanol:acetic acid:water; 10:3:3:2, by volume) and [1H] nuclear magnetic resonance. The compound with the alternative spacer 9-azido-NeuGc{alpha}2-LacNAcbetaOCH2CH2NH-CBz was treated with triphenylphosphine to give the desired 9-amine for BPA acylation and purified as described above.

BPC and BPA-sialoside polyacrylamide conjugates

Sialoside analogs containing the CBz-protected 2-aminoethyl spacer were hydrogenolysed to give the amine and further conjugated to the biotinylated PAA of either 30 or 1000 kDa as described previously (53).

ELISA inhibition assays

For inhibition assays, high-binding 96-well microtiter plates (Corning Costar) precoated with neutravidin (50 µl of 1 mg/ml neutravidin (Pierce Biotechnology) in 50 mM sodium bicarbonate (pH 9.5) overnight at 4°C) were washed with PBS and blocked with PBS containing 5 mg/ml BSA for 1 h at 37°C. Fifty microliters of the indicated biotinylated sialoside (0.33 µM) was aliquoted into each well, and allowed to bind for 1 h at room temperature and free sialoside was washed away. The Siglec-Fc chimera, produced as described previously (33), was diluted to 0.1 mg/ml into HBSS/B (with 1 mM EDTA). F(ab')2 goat anti-human IgG (0.066 mg/ml; Jackson ImmunoResearch Laboratories) was allowed to complex with peroxidase conjugated F(ab')2 rabbit anti-goat F(ab')2 specific (0.010 mg/ml) for 15 min. CD22-Fc (1.5 ml) was added to 1 ml of the complexed solution and incubated for 15 min. The inhibitor of interest was diluted into HBSS and then 25 µl of the inhibitor and 25 µl of the CD22Fc complex were added to each well and allowed to bind for 30 min at 37°C before washing four times with 200 µl of HBSS. Wells were developed with 50 µl of 1 mg/ml o-phenylenediamine (Sigma-Aldrich), 0.03% H2O2 in 50 mM phosphate/citrate (pH 5.0), the reaction stopped with 50 µl of 2.5 M H2SO4 and the OD was measured. For PAA ELISA-type assays, wells coated with protein A (1 µg/well in 50 mM sodium bicarbonate (pH 9.0) overnight at 4°C) were washed and blocked with HBSS/B for 1 h at 37°C. Siglec-Fc diluted into HBSS/B (50 µl at the indicated concentration) was allowed to adhere for 1 h at 37°C before washing 2 x 200 µl with HBSS/B. The PAA probe was diluted into HBSS/B (50 µl at the indicated concentration) and allowed to bind for 2 h at 37°C. Wells were washed 3 x 200 µl with HBSS/B and then 50 µl of alkaline phosphatase conjugated-streptavidin (Sigma-Aldrich) was added to each well and allowed to bind for 1 h at 37°C before washing 3 x 200 µl with HBSS/B. Wells were developed with p-nitrophenylphosphate (pNPP) developing solution (Sigma-Aldrich) and read at OD405.

PAA probe binding to cells

Cells were resuspended in chilled HBSS/B (2 x 106 cells/ml). If needed, cells were treated with Arthrobacter ureafaciens sialidase (200 mU/ml) for 30 min at 37°C and washed 3 x 1 ml to remove any remaining sialidase. Cells (100 µl) were incubated with the PAA probe (1 µg) for 1.5 h on ice, with or without added inhibitor. After a wash of 1 ml of HBSS/B, bound probe was detected with streptavidin-CyChrome or streptavidin-PE (BD Pharmingen 1 µl/tube) for 45 min on ice, washed, and analyzed by flow cytometry. As needed, 0.2 µl of anti-B220 was added during the streptavidin labeling to detect B cells.

Cell adhesion to PAA probes and magnetic beads

Dynal microbeads were washed with HBSS/B and then coated with the PAA probe of interest at 6 µg of probe and 5 µl of magnetic beads in 500 µl of HBSS/B. After 1 h room temperature incubation, the beads were washed two times to remove unbound probe, and added to the cells in a 10:1 ratio of beads:cell. The beads were allowed to bind to the cells for 1 h at 4°C with end-over-end rotation and then exposed to a magnet before fixing onto a glass slide and visualizing by microscopy. Alternatively, 20 µg of the PAA probe was added and allowed to bind to 1 ml of the cell suspension (2 x 107 cells/ml in HBSS/B) for 2 h on ice. Cells were washed, resuspended in 90 µl of HBSS/B solution, and incubated at 4°C for 30 min with 10 µl of streptavidin-coated beads (Miltenyi Biotec). Cells were washed, applied to a magnetic column, and eluted according to manufacturer’s guidelines. Cells were stained with anti-B220 as above and analyzed by flow cytometry.

Measurement of probe internalization and cell killing

Cells (2 x 106 cell/ml) were labeled with the PAA probe and streptavidin as above. Following staining, 100-µl aliquots of the cell suspension were placed on ice or at 37°C for the indicated period of time. Cells were then washed with 400 µl of RPMI 1640 (pH 2.5) to remove surface-bound probe, and then washed 2 x 1 ml of HBSS/B. For potassium depletion, cells were washed with DMEM:water (1:1), then incubated with potassium-depleted medium at 37°C for 1 h, then chilled; the probe was added and incubated as above but in either HBSS/B or in potassium-depleted medium (with BSA) for 1.5 h. For killing, cells were labeled with the PAA probe as above and then with the streptavidin-saporin conjugate (Advanced Targeting Systems) at the indicated concentrations. Cells were washed, plated at 0.2 x 106 cells/well in 200 µl of RPMI 1640/10%FCS/penicillin-streptomycin, cultured for 48 h, and viable cells were quantified.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Development of high-affinity sialoside ligands of CD22

Although both human CD22 (hCD22) and murine CD22 (mCD22) have preferred specificity for the sequence Sia{alpha}2-6Galbeta1-4GlcNAc, mCD22 strongly prefers the sialic acid N-glycolyl-neuraminic acid (NeuGc) found abundantly in murine tissues. In contrast, hCD22 binds the same sialoside with either NeuGc or N-acetyl-neuraminic acid (NeuAc), only the latter of which is found in human tissues (29, 33, 54, 55). This key difference must be taken into account in the rational design of inhibitors for each ortholog of CD22. Modeling studies of Zaccai et al. (50) indicated a hydrophobic pocket in CD22 adjacent to the 9-position of sialic acid, and further identified biphenyl substituents at that position, BPC and BPA, that increased the affinity of NeuAc for hCD22 and mCD22, respectively (50, 56). Using a chemoenzymatic approach (33, 41), we assembled the specificity elements preferred by hCD22 and mCD22 into the sequences 9-BPC-NeuAc{alpha}2-6Galbeta1-4GlcNAcbeta-ethylamine (BPC-NeuAc-LN) and 9-BPA-NeuGc{alpha}2-6Galbeta1-4GlcNAcbeta-ethylamine (BPA-NeuGc-LN), resulting in inhibitory potencies (IC50 values) of 1–2 µM for hCD22 and mCD22, respectively (Fig. 1 and Table I). Thus, giving a potency >1000-fold higher than NeuAc alone. The potency of the 9-biphenyl substituent over NeuAc alone is also clearly seen in the inhibition of sialoside probe binding to asialo murine B cells and the human Daudi B cell line (Fig. 2).


Figure 1
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  		FIGURE 1. Biphenyl substituents at C-9 dramatically enhance the affinity of sialoside glycans for CD22. A, Compounds used in this study. B, Inhibition curves of selected sialoside inhibitors. Inhibitors were added with CD22-Fc chimeras to block the chimera binding to immobilized NeuAc{alpha}2-6LacNAc (hCD22) or NeuGc{alpha}2-6LacNAc (mCD22). Data are the average ± SD of triplicate determinants and representative of two independent experiments.

 

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  		Table I. Inhibitory potency of sialosides

 

Figure 2
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  		FIGURE 2. Inhibition of multivalent-probe binding to cell surface CD22 by biphenyl sialoside ligands. A, Murine splenocytes were mock (gray shaded) or A. ureafaciens sialidase treated to unmask surface CD22 and then incubated with NeuGc{alpha}2-6LacNAc-PAA alone (black line) or in the presence of 10 µM (orange line), 200 µM (pink line), or 2 mM (dark blue line) of the indicated inhibitor for 1.5 h on ice, then washed. Bound probe was detected with streptavidin-PE, and B cells stained with anti-B220. Data are of B220+ cells only. B, Daudi cells were mock (gray shaded) or A. ureafaciens sialidase treated and then incubated with NeuAc{alpha}2-6LacNAc-PAA alone (black line) or in the presence of 10 µM (orange line), 30 µM (light blue line), 200 µM (pink line), or 2 mM (dark blue line) of the indicated inhibitor. Bound probe was detected with streptavidin-PE and measured by flow cytometry.

 
Combining high affinity and multivalency to increase avidity

To assess the combined effects of increased affinity and multivalency on the performance of probes for CD22, we focused on the PAA backbone used in the commercially available sialoside constructs used in other studies (34, 36, 57, 58, 59, 60, 61). In this system, sialosides are pendantly attached to the backbone along with 5% biotin to allow for detection with streptavidin. Accordingly, the sialosides were attached to PAA backbones of ~30 kDa, as used in commercially available probes, and 1000 kDa. Using an ELISA-type assay involving binding of the PAA probes to immobilized mCD22-Fc chimera, maximal binding of the high molecular mass NeuGc-LN-PAA (NeuGc-PAA) was achieved at >10-fold lower concentration relative to lower molecular mass NeuGc-PAA (Fig. 3A). Direct comparison of the high molecular mass BPA-NeuGc-LN-PAA (BPA-NeuGc-PAA) with NeuGc-PAA showed much higher binding to the BPA analog in keeping with the increased affinity (Fig. 3B). In contrast, the BPA substituent had a negative effect on binding of Siglec-10 another B cell siglec.


Figure 3
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  		FIGURE 3. High valency PAA probes have increased avidity for CD22. A, mCD22-Fc chimera was immobilized onto protein A-coated microtiter plates and overlaid with 1 µg (top) or the indicated amounts (bottom) of the high m.w. or low m.w. PAA30kDa probe. After washing, bound probe was detected with alkaline phosphatase-labeled streptavidin and developed with pNPP measured at OD405. Data are the average ± triplicate determinants and are representative of three independent experiments. B, mCD22 or siglec-10 chimeras (1 µg/well) were immobilized onto protein A-coated microtiter plates and overlaid with the indicated amounts of the PAA probes. After washing, bound probe was detected with alkaline phosphatase-labeled streptavidin and developed with pNPP measured at OD405. Data are the average ± triplicate determinants.

 
Competition with cis ligands on native B cells

The 1000-kDa BPC-NeuAc-LN-PAA (BPC-NeuAc-PAA) and NeuAc-LN-PAA (NeuAc-PAA) probes were assessed for their ability to compete with cis ligands by comparing binding to BJAB cells treated with or without sialidase. Both probes bound to the sialidase treated B cells, as compared with LacNAc-PAA (LN-PAA) control (Fig. 4A). Significantly, although the NeuAc-PAA only bound to the asialo BJAB cells, the BPC-NeuAc-PAA bound to native BJAB cells nearly as well as to "asialo" cells, demonstrating that it competes with cis ligands for binding to CD22. Similar results were obtained evaluating binding of NeuGc-PAA and BPA-NeuGc-PAA to native primary murine B cells (Fig. 4B). Irrespective of the probe, no binding to B cells from CD22null mice was observed demonstrating that binding was specific for CD22. Moreover, none of the corresponding 30-kDa probes bound to native BJAB or murine B cells (data not shown), indicating that both the high monovalent affinity and high multivalency was required to achieve high enough avidity to compete with the cis ligands that normally mask CD22 on these cells.


Figure 4
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  		FIGURE 4. High-avidity PAA probes bind to masked CD22 on native B cells. A, BJAB cells were mock (solid line) or A. ureafaciens sialidase treated (dotted line and shaded) to unmask CD22 and incubated on ice with the indicated probe for 1.5 h (in all cases shaded is the control LN-PAA, and dotted and solid lines the indicated probe). Bound probe was detected with PE-streptavidin and measured by flow cytometry. B, Splenocytes from CD22null (shaded) or wild-type mice (solid and dotted lines) were mock (solid line) or A. ureafaciens sialidase treated (dotted line and shaded) to unmask CD22 and then incubated on ice with the indicated probe for 1.5 h. After washing, bound probe was detected with PE-labeled streptavidin and B cells detected with anti-B220. Data are of B220+ cells only.

 
Enrichment of B cells with CD22 ligand-coated magnetic beads

Because CD22 is expressed specifically on B cells, we assessed the ability of ligand-coated magnetic beads for targeting and purifying B cells. Because the PAA probes contain biotin, "ligand-coated" magnetic beads were readily prepared by simply mixing streptavidin-coated magnetic beads (1.5-µm diameter; Dynal Biotech) with either NeuGc-PAA, BPA-NeuGc-PAA, NeuAc-PAA, or BPC-NeuAc-PAA and then incubated with native murine B cells (Fig. 5A) or BJAB cells (Fig. 5B). In all cases, beads coated with probes containing sialosides without the biphenyl (BPA or BPC) substituents failed to bind to native B cells. In contrast, beads containing probes with the BPA or BPC substituents bound avidly to native primary murine B cells or BJAB cells, respectively. In contrast to the flow cytometry data, beads coated with either 30 or 1000 kDa BPA-NeuGc-PAA bound equally well to murine B cells (Fig. 5A). Presumably this is due to the additional valency afforded by adsorption of the probes to the magnetic beads, because only the 1000-kDa BPA-NeuGc-PAA was able to support bead binding if the probes were added to the cells before addition of the beads (data not shown).


Figure 5
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  		FIGURE 5. Magnetic beads coated with high-avidity sialoside probes bind to native B cells. Streptavidin-coated magnetic beads were mixed with the indicated PAA probe, unbound probe washed away, and the beads added to enriched murine B cells (A) or the human B cell line BJAB (B). The beads were allowed to bind to the cells for 30 min, exposed to a magnet and fixed onto a glass microscope slide. Data are representative of two experiments. C, Murine splenocytes from WT or CD22null mice were incubated with BPA-NeuGc-PAA for 1.5 h, washed, and then incubated with biotinylated magnetic beads. The cells and beads were applied to a magnetic column and then washed and eluted. Total (before purification) and eluted cells were quantitated and assessed for purity by flow cytometry as indicated by the percent B220/CD22 positive. D, Recovery of B cells from wild-type and CD22–/– mice following purification as described for C. Data are representative of two distinct experiments.

 
Streptavidin-coated micromagnetic beads commonly used for Ab-mediated cell purification (~50-nm diameter; Miltenyi Biotec) also allowed enrichment of B cells from mixed splenocytes (Fig. 5C). Splenocytes from wild-type and CD22null mice were allowed to bind to the BPA-NeuGc-PAA probe and streptavidin-coated micromagnetic beads. The cells/beads mixture was loaded onto a magnetic column, washed, and then eluted. B cells were recovered in good yield with a purity of 95%, equivalent to Ab-based purification. By contrast, no enrichment and poor recovery of B cells was obtained from splenocytes from CD22null mice (Fig. 5C).

Increasing the affinity of cis ligands prevents binding of high-avidity probes

Stable binding of the high-avidity synthetic probes was achieved by increasing the affinity of the sialoside ligand and the degree of multivalency. We hypothesized that binding resulted from effective competition of the probes with cis ligands to act as ligands in trans. This would predict that increasing the affinity of the cis ligands would also prevent binding of the high-avidity probes. To test this, we used the K20 subclone of the BJAB cell line, which is deficient in a key step in sialic acid biosynthesis. Although the K20 cells are normally sialic acid deficient, they can incorporate exogenously added sialic acid precursors, sialic acids and modified sialic acids into glycans of cell surface glycoproteins (41, 62). Accordingly, NeuAc and 9-BPC-NeuAc were added to the culture medium of K20 cells to create cis ligands with the corresponding sialic acid. As observed previously for other sialic acid analogs (41, 63) (S. Han and J. C. Paulson, unpublished results), the cells incorporated both NeuAc and the BPC-NeuAc to levels approximating those of wild-type cells as measured by flow cytometry with the lectin Sambucus nigra agglutinin (data not shown). These cells were then challenged for binding of magnetic beads coated with NeuAc-PAA and BPC-NeuAc-PAA ligands, with the level of binding scored as "beads per cell". As before, the BPC-NeuAc-PAA-coated beads, but not the NeuAc-PAA-coated beads, bound to cells containing NeuAc-bearing cis ligands (Fig. 6). However, with the cells bearing the BPC-NeuAc cis ligands, not even the BPC-NeuAc-PAA-coated beads bound. Thus, increasing the affinity of the cis ligands with the BPC substituent shifted the equilibrium in favor of binding cis ligands, reinforcing the dynamic interaction between cis and trans ligands.


Figure 6
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  		FIGURE 6. Increasing the affinity of cis ligands blocks binding of high-affinity sialoside probes. BJAB K20 cells were allowed to incorporate NeuAc or BPC-NeuAc into cell surface glycoproteins for 72 h. Cells were then incubated with the indicated PAA probe-coated beads as above. Data are the average number of cells/bead ± SD of 6–14 determinants.

 
Endocytosis of PAA probes

Because CD22 undergoes constitutive and Ab-induced endocytosis (14, 15), we investigated the ability of CD22 to endocytose or "carry" bound BPC-NeuAc-PAA probes to intracellular compartments. Accordingly, BJAB cells were stained with the fluorescently labeled BPC-NeuAc-PAA at 4°C, and allowed to internalize CD22 at 37°C for up to 60 min. Residual cell surface BPC-NeuAc-PAA was removed by a brief low pH wash (RPMI 1640; pH 2.5), and the degree of endocytosis is reflected by the amount of cell-associated probe after low pH wash. Detectable internalization of the BPC-NeuAc-PAA probe occurred within the first 10 min of incubation, and reached maximal within 30 min (Fig. 7A). No internalization or binding was observed with a control LN-PAA probe (data not shown). Endocytosis of the probe was inhibited following potassium depletion, consistent with clathrin-mediated endocytic mechanism, but was not affected nystatin treatment, which inhibits caveolin-mediated internalization (Fig. 7B).


Figure 7
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  		FIGURE 7. Endocytosis of BPC-NeuAc-PAA by CD22. BJAB cells were incubated with the high m.w. BPC-NeuAc-LN-PAA (dotted line, solid line) or LN-PAA (shaded) probes for 1.5 h on ice. Then detected with fluorescently labeled streptavidin for 45 min on ice. After washing, the cells were then moved to 37°C for the indicated period of time. Following the incubation, cells were washed with RPMI 1640 (pH 2.5) (solid = intracellular) to remove surface-bound probe or HBSS/BSA (shaded, dotted = total probe) as a control. Bound or internalized probe was measured by flow cytometry. B, For potassium depletion, cells were washed with RPMI 1640:water (1/1) and then incubated for 1 h in medium with ({diamondsuit}) or without ({square}) potassium. After prechilling on ice, cells were incubated in the corresponding medium with the BPC-NeuAc-PAA probe and internalization was measured as above. Data are the average ± SD of two independent experiments.

 
Targeting and killing of B cells with sialoside probes

Because CD22 is a target for Ab-based immunotherapy of B cell lymphomas, and because CD22 mediates efficient endocytosis of bound ligands, we sought to illustrate the ability of CD22 to mediate B cell killing via a ligand-based toxin conjugate. To this end, we used the toxin saporin, a potent inhibitor of protein synthesis, commercially available as a conjugate to streptavidin. Because the BPC-NeuAc-PAA is biotinylated, the saporin-ligand conjugate was obtained by simply mixing the two preparations in the desired ratio. Using the BJAB cell line, derived from Burkitt’s lymphoma, we observed high levels of B cell killing within 48 h of culture with the saporin-conjugated BPC-NeuAc-PAA (Fig. 8A). BJAB killing required both the targeting probe and the toxin, as no killing was observed in the absence of either, nor was killing observed if LN-PAA was used in place of the sialylated ligand. Similar results were observed with the additional lymphoma cell lines Raji and Daudi (Fig. 8B).


Figure 8
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  		FIGURE 8. Targeting and killing of lymphoma cells by BPC-NeuAc-PAA conjugated with toxin. A, Streptavidin-conjugated saporin at the indicated concentration was complexed with the BPC-NeuAc-PAA probe for 15 min and then mixed with BJAB lymphoma cells. After 48 h in culture, cells were harvested and viable cells were quantified. Data are the average ± SD of quadruplicate determinants and are representative of three independent experiments. B, Streptavidin-conjugated saporin at the indicated concentration was complexed with the BPC-NeuAc-PAA ( Figure 8) or LN-PAA ({square}) and added to the human lymphoma cell lines BJAB, Raji, and Daudi as above. Live cells were quantified after 48-h culture, and data are the average ± SD of quadruplicate determinants and are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Because CD22 and most members of the siglec family are masked by cis ligands in their native state, cis ligand interactions are presumed to play a predominate role in modulation of siglec function (1, 2, 3, 35, 36, 40). In support of this, there is abundant evidence that cis ligands of CD22 modulate its activity as a regulator of B cell signaling and proliferation following BCR ligation (3, 5, 8, 16, 43). However, the role of cis ligands in regulating interactions with trans ligands is less clear. Here, we demonstrate that the masking by cis ligands is not a static property of CD22, but is instead probe dependent. Although low-affinity sialoside-PAA probes fail to bind to CD22 unless B cells are pretreated to destroy cis ligands, higher avidity probes bind to CD22 on native cells. Increasing the affinity of cis ligands (e.g., by incorporating 9-BPC-NeuAc into B cell glycoproteins) also results in masking of the high-avidity probes. Thus, the concept of masking reflects a simple competition between cis and trans ligands, and is a dynamic property of CD22 that describes the effect of cis ligands on the binding of the trans ligand or sialoside probe used as a reference.

We propose that cis ligands effectively regulate the binding of siglecs to biologically relevant trans ligands by setting a competitive threshold (3). In the case of CD22, the intrinsic affinity for its preferred glycan sequence, NeuAc{alpha}2-6Galbeta1-4GlcNAc- is relatively weak (Kd {approx}100–200 µM), regardless of whether it is presented as a free trisaccharide or as a terminal sequence on glycans of a glycoprotein (32, 33, 57). Thus, natural cis and trans ligands do not differ significantly in their intrinsic affinity for CD22. The concentration of {alpha}2-6-linked sialic acids on the surface of the B cell is estimated to be ~25–50 mM (34), accounting for the masking of low-affinity trans ligands, including synthetic multivalent-PAA probes containing the native sialoside sequence, as reported earlier (35, 36). Although serum glycoproteins are rich in {alpha}2-6-linked sialic acids, and are potentially trans ligands of CD22 (31, 64, 65), the total sialic acid concentration in serum is only ~2 mM (66), well below the threshold needed to compete with cis ligands on the native B cell. By contrast, contact of two B cells or T cells results in redistribution of CD22 to sites of cell contact as a result of the interaction with glycoprotein ligands in trans (34). At a first approximation, the concentration of trans ligands at the interface between two B cells is equivalent to those in cis. However, it is likely that the effective concentration of trans ligands is greater (e.g., positioned for better access to the CD22-binding site), providing a driving force for redistribution.

Using a model system for activation and proliferation of B cells exposed to Ag-bearing cancer cells, Lanoue et al. (46) demonstrated that transfection of the target cells with the gene of the sialyltransferase (ST6Gal I) that produces the ligand of CD22 resulted in suppression of BCR signaling, presumably due to recruitment of CD22 to the site of Ag engagement of the BCR. This result emphasizes that not all cells carry trans ligands sufficient to overcome masking by cis ligands. It also has direct implications for a role of CD22 in suppressing B cell response to self Ags on cells expressing trans ligands (1, 2, 34, 46). Because both T cells and B cells are rich in trans ligands, it would also follow that B-B and B-T cell interactions (e.g., in spleen or lymph nodes) could also sequester CD22 away from the BCR and promote activation by exogenous Ag.

Endocytosis of CD22 occurs constitutively (15, 17, 18), and is presumed to occur via a clathrin-mediated mechanism (14, 16). Previous studies have documented that anti-CD22 Abs increase the rate of endocytosis and reduce the surface expression of CD22 (14, 17, 18). In this report, we show that the high-avidity sialoside-PAA probes bound by CD22 are also endocytosed, demonstrating that CD22 can carry ligand-based "cargo" to endocytic compartments. Once internalized, the probes were retained in intracellular compartments and/or degraded because no recycling of the probes back to the surface was observed (data not shown). The rate of endocytosis of the probes was similar to endocytosis induced by anti-CD22, but no decrease in CD22 surface staining was observed in contrast to a 50–70% reduction of CD22 observed following Ab-mediated internalization (data not shown). The results suggest that CD22 carrying ligand-based cargo is either more rapidly recycled to the cell surface, or represents only a small fraction of the total CD22, even at the saturating levels of probe used for these experiments. Interestingly, internalization of the ligand-based cargo is dramatically decreased following BCR cross-linking (data not shown), as previously shown for internalization of anti-CD22 (14, 18). This effect was proposed to result from phosphorylation of the ITIM motifs following BCR ligation, resulting in disruption of clathrin association by blocking the binding of the AP50 adaptor protein that recognizes the same nonphosphorylated tyrosine motifs (14).

CD22 has been exploited as a B cell-specific receptor for immunotherapy of B cell lymphomas resulting in significant clinical success even when standard chemotherapies had failed (20, 21, 22, 67, 68). But not all patients respond completely to Ab-mediate therapies, tumor cells can have heterogeneous expression of the target epitopes (69), and Ab-mediated therapies are much costlier than small molecule therapeutics (70). As demonstrated here, targeting CD22 using a high-avidity sialoside ligand-saporin conjugate-mediated effective killing of B cell lymphoma cells, suggesting an alternative approach to Ab-mediated therapy. In principle, any other toxin or radioisotope could be targeted to B cells by attaching it to high-avidity ligands of CD22. Mixtures of Abs recognizing distinct epitopes result in more efficient treatments as compared with the individual Abs by themselves (71). Analogously, administration of the probe in conjugation with a nonblocking anti-CD22 Ab, may further increase the rate of internalization of the probe and result in greater B cell killing. Although the polyacrylamide backbone is nontoxic in vivo (72, 73), other carrier backbones with the sugar derivative presented in high valency may prove more useful in therapeutic applications. Because other siglecs are expressed in many leukocyte types (e.g., NK cells, macrophages, dendritic cells, eosinophils), and several siglecs have been demonstrated to mediate endocytosis (1, 2, 3, 61), it will be of interest to see whether analogous high-affinity ligands of other siglecs can be produced to carry ligand-based payloads into other cell types.

Although the synthetic high-avidity probes reported here are efficiently endocytosed by CD22, are there natural trans ligands that are endocytosed by CD22? Based on the considerations discussed above, soluble glycoproteins carrying glycans recognized by CD22 do not have sufficient valency to compete with cis ligands for binding. However, many bacterial and protozoan pathogens display surface sialic acids on cell surface glycans, including the NeuAc{alpha}2-6Gal linkage recognized by CD22 (74, 75). Based on the observation that macrophages bearing Siglec-5 or sialoadhesin were able to mediate endocytosis of N. meningitides-bearing LPS-containing sialylated glycans with the NeuAc{alpha}2-3Gal linkage, Jones et al. (27) proposed that siglecs could play a role in innate immunity. Thus, it will be of interest to determine whether CD22 can similarly mediate endocytosis of pathogens carrying sialylated glycans, and provide a bridge to adaptive immunity by processing pathogen proteins for presentation of Ags to T cells.


   Acknowledgments
 
We thank Drs. Lars Nitschke for the CD22null mice, Ajit Varki for the construct encoding for hCD22-Fc chimera, Paul Crocker for the cells expressing mCD22-Fc and siglec 10-Fc chimeras, and Michael Pawlita for the BJAB K20 cell line. In addition, we thank Kirk Allin for his assistance in enzyme production, and Anna Tran-Crie for her help in manuscript preparation. The LacNAc-PAA was provided by the Consortium for Functional Glycomics.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants GM60938 and AI050143 (to J.C.P.) and GM25042 (to B.E.C.), GM62116 (to The Consortium for Functional Glycomics), and the Physico-Chemical Biology Program, RAS (to N.B.). Back

2 Address correspondence and reprint requests to Dr. James C. Paulson, 10550 North Torrey Pines Road, MEM L71, La Jolla, CA 92037. E-mail address: jpaulson{at}scripps.edu Back

3 Abbreviations used in this paper: eq., equivalent; CTP, cytidine 5'-triphosphate; BPC, N-biphenyl-4-carbonyl chloride; LacNAc, Galbeta1-4GlcNac; BPA, N-biphenyl-4-acetic acid; PAA, polyacrylamide; hCD22, human CD22; mCD22, murine CD22; LacNAc, Galbeta1-4GlcNAc; pNPP, p-nitrophenylphosphate. Back

Received for publication April 4, 2006. Accepted for publication June 6, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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