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

Article abstract of DOI:10.1021/ja802008q

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 NeuAcα2-6Gal. 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 afunctional 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.

Full text of DOI:10.1021/ja802008q

Published on Web 05/28/2008
Bifunctional CD22 Ligands Use Multimeric Immunoglobulins
as Protein Scaffolds in Assembly of Immune Complexes on B
Cells
Mary K. O’Reilly,† Brian E. Collins,† Shoufa Han,† Liang Liao,† Cory Rillahan,†
Pavel I. Kitov,‡ David R. Bundle,‡ and James C. Paulson*,†
Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines
Road, La Jolla, California 92037, and Department of Chemistry, Alberta Ingenuity Centre for
Carbohydrate Science, UniVersity of Alberta, Edmonton AB, T6G 2G2 Canada
Received March 25, 2008; E-mail: jpaulson@scripps.edu
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
NeuAcR2-6Gal. 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.
Introduction
protein scaffolds have shown promise due to the opportunity
for defined architecture that can, in some cases, benefit from
Glycan-binding proteins (GBP) participate in the communica-
tion of cells with their extracellular environment, mediating
diverse biology including host-pathogen interactions, cell-cell
adhesion, and modulation of cell signaling receptors.1–3 GBPs
typically exhibit low affinity (e.g., Kd ∼ 10-1000 µM) for their
ligands and require multivalent interactions to mediate biological
effects.4,5 Because of the low intrinsic affinity, the design of
ligand-based probes of GBPs has largely relied on highly
multivalent display of ligands on synthetic polymers or ‘neogly-
coproteins’ to provide the avidity needed to stabilize binding.4–7
Rational design of lower valency constructs or dendrimers has
met with limited success, with several notable exceptions.8–12
To achieve sufficient avidity while maintaining lower valency,
symmetrical oligomerization. Exemplary is the demonstration
that bivalent glycan ligands tethered to a central core with
pentameric radial symmetry could bridge two pentameric Shiga
toxins. Optimal spacing of low affinity glycan ligands (IC50
)
2-5 mM) gave up to 107-fold enhancement of affinity.8,10 Using
a similar strategy, bifunctional ligands were found to drive
assembly of a face-to-face complex of a toxin with another
pentameric protein, serum amyloid protein (SAP).13,14 Complex
formation could be achieved with either covalently tethered or
nontethered bifunctional ligands. In the latter case, SAP
effectively becomes a pentameric scaffold for presentation of
low affinity monomeric glycan ligands to the pentameric
toxin.13–15
† The Scripps Research Institute.
Use of a bifunctional ligand-antigen molecule to ‘decorate’
a target cell or microbe with an antigen has been proposed by
‡ University of Alberta.
(1) Crocker, P. R.; Paulson, J. C.; Varki, A. Nat. ReV. Immunol. 2007, 7
(4), 255–66.
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(3) Paulson, J. C.; Blixt, O.; Collins, B. E. Nat. Chem. Biol. 2006, 2 (5),
238–48.
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617–25.
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F. T.; Liskamp, R. M.; Gabius, H. J. ChemBioChem 2001, 2 (11),
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L. L. J. Am. Chem. Soc. 2002, 124 (50), 14922–33.
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J. M.; Bundle, D. R. Org. Lett. 2005, 7 (20), 4369–72.
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J. M.; Grant, G. A.; Mulvey, G. L.; Kitova, E. N.; Klassen, J. S.;
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7736 J. AM. CHEM. SOC. 2008, 130, 7736–7745
10.1021/ja802008q CCC: $40.75
2008 American Chemical Society

Bifunctional CD22 Ligands
A R T I C L E S
Figure 1. Design of a bifunctional ligand to drive self-assembly of IgM-CD22 complexes. (a) Structure of the bifunctional 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.
several groups for targeting the immune response,12,16–18 and
has been successfully demonstrated for targeting mammalian
cells that contain the folate receptor or Rvꢀ3 integrin.16,17 These
receptors bind with high affinity to their ligands, folate and RGD
peptide, respectively (Kd ) 10-9-10-10). Thus, the correspond-
ing bifunctional ligand-antigen conjugates bind tightly to the
surface of the cell, which display the conjugated antigen for
subsequent recognition and binding by antibody. This powerful
approach has not yet been exploited for mammalian GBPs,
largely due to the constraints of the low affinity for their ligands.
Members of the siglec family of GBPs are expressed on cells
of the immune system, exhibiting roles in cell signaling and
cell adhesion that are modulated by interaction with their sialic
acid containing glycan ligands.1 Exemplary is CD22, a known
regulator of B cell signaling that has been established as a target
for immunotherapy of B cell lymphomas and autoimmune
disease.19,20 CD22 exhibits low intrinsic affinity for its ligands
(Kd ) ∼0.2 mM), and their abundance on B cells provides cis
(on the same cell) interactions that effectively mask the ligand-
binding site.21,22 To date, only highly multivalent synthetic
ligands have been demonstrated to compete with cis ligands
and bind to native B cells.23
investigated the possibility of designing such a ligand that would
dock decavalent IgM to CD22. Although CD22 is a monovalent
GBP and does not share decameric symmetry with IgM, it is
concentrated in clathrin rich microdomains and forms multimers
in situ,23–26 suggesting that the radially symmetric IgM, with
binding sites spread over a diameter of 350 Å, might bridge
the distance between clustered CD22 monomers. Accordingly,
we constructed a bifunctional ligand that would simultaneously
bind to CD22 and a monoclonal antibody specific for the hapten
nitrophenol (NP; Figure 1). The CD22 ligand comprises the
preferred glycan sequence recognized by CD22 (NeuAcR2-
6Galꢀ1-4GlcNAc) with a 9-biphenyl carbonylamido (BPC)
substituent on the sialic acid, known to increase affinity for
CD22 by 100 fold.23,27 We show herein that this ligand is able
to efficiently and specifically assemble complexes between anti-
NP antibodies with CD22 on the surface of a B cell.
Experimental Section
General Methods for Synthesis of Bifunctional Ligands. 2-(4-
Hydroxy-3-nitrophenyl)acetic acid, N-hydroxysuccinimide ester,
was purchased from Biosearch Technologies. 2-Acetamido-3,4,6-
tri-O-acetyl-2-deoxy-ꢀ-D-glucopyranosyl azide was purchased from
TCI North America. All other reagents and solvents were purchased
from Aldrich Chemicals and used without further purification.
Enzymes including the ꢀ-1-4-galactosyltransferase-/UDP-4′-Gal
epimerase fusion protein28 (GalE-GalT), Nesseria meningitidis
CMP-NeuAc synthetase, and human ST6Gal I sialyltransferase
(hST6Gal 1) were prepared as previously reported.28–30 Solvent
concentration was performed under reduced pressure at <40 °C
Motivated by reports of bifunctional ligand-mediated targeting
of multivalent protein scaffolds to proteins of interest, we
(16) Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.; Kiessling,
L. L. ACS Chem. Biol. 2007, 2 (2), 119–27.
(17) Lu, Y.; Low, P. S. Cancer Immunol. Immunother. 2002, 51 (3), 153–
62.
(18) Shokat, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 1861–
1862.
(19) Qu, Z.; Griffiths, G. L.; Wegener, W. A.; Chang, C. H.; Govindan,
S. V.; Horak, I. D.; Hansen, H. J.; Goldenberg, D. M. Methods 2005,
36 (1), 84–95.
(23) Collins, B. E.; Blixt, O.; Han, S.; Duong, B.; Li, H.; Nathan, J. K.;
Bovin, N.; Paulson, J. C. J. Immunol. 2006, 177 (5), 2994–3003.
(24) Han, S.; Collins, B. E.; Bengtson, P.; Paulson, J. C. Nat. Chem. Biol.
2005, 1 (2), 93–7.
(20) Steinfeld, S. D.; Tant, L.; Burmester, G. R.; Teoh, N. K.; Wegener,
W. A.; Goldenberg, D. M.; Pradier, O. Arthritis Res. Ther. 2006, 8
(4), R129.
(25) Varki, A.; Angata, T. Glycobiology 2006, 16 (1), 1R–27R
(26) Grewal, P. K.; Boton, M.; Ramirez, K.; Collins, B. E.; Saito, A.; Green,
R. S.; Ohtsubo, K.; Chui, D.; Marth, J. D. Mol. Cell Biol. 2006, 26
(13), 4970–81.
(21) Bakker, T. R.; Piperi, C.; Davies, E. A.; Merwe, P. A. Eur. J. Immunol.
2002, 32 (7), 1924–32.
(22) Razi, N.; Varki, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (13), 7469–
74.
(27) Kelm, S.; Gerlach, J.; Brossmer, R.; Danzer, C. P.; Nitschke, L. J.
Exp. Med. 2002, 195 (9), 1207–13.
9
J. AM. CHEM. SOC. VOL. 130, NO. 24, 2008 7737

A R T I C L E S
O’Reilly et al.
bath temperature. All H and 13C NMR spectra were recorded at
30 °C using a Varian Unity Inova 400 or a Bruker DRX 600
Spectrometer. Chemical shifts are reported in parts per million from
high to low field and referenced to the solvents. Standard abbrevia-
tions indicating multiplicity were used as follows: s ) singlet, d )
doublet, t ) triplet, q ) quadruplet, and m ) multiplet. MALDI-
FTMS spectrometry was recorded with an IonSpec Ultima FTMS
(IonSpec Corp., Irvine, CA) using dihydroxybenzoic acid as the
matrix and carried out by services at The Scripps Research Institute.
Thin layer chromatography was performed on silica gel 60F from
Fisher. After development with appropriate eluents, spots were
visualized by dipping in 5% sulfuric acid in ethanol, followed by
charring. Flash chromatography was performed on 230-400 mesh
silica gel under positive air pressure.
was dissolved in DMF (7.2 mL). To this were added HBTU (0.69
g, 1.82 mmol), HOBt (245 mg, 1.82 mmol), and 2-(4-hydroxy-3-
nitrophenyl)acetic acid (197 mg, 1 mmol). The mixture was stirred
at room temperature for 5 min, and Et3N (11 µL, 1.82 mmol) was
added. The reaction mixture was stirred at room temperature for
1 h. The solvent was removed under reduced pressure. Flash
chromatography on the crude material (chloroform:methanol 4:1)
yielded the desired product as a yellow powder (360 mg, 99%).
1H NMR (CD3OD, 600 MHz) δ 7.91 (d, J ) 1.8 Hz, 1H), 7.43
(dd, J ) 3.6, 8.4 Hz), 7.07 (t, 1H, J ) 3.0, 5.4 Hz), 4.93 (d, 1H,
J ) 10.2 Hz), 3.76 (dd, 1H, J ) 1.8, 12.6 Hz), 3.65 (m, 2H), 3.52
(2d, J ) 15 Hz), 3.46 (t, J ) 8.4 Hz), 3.38 (m, 2H), 1.70 (s, 3H);
13C NMR (CD3OD, 175 MHz) δ 175.34, 175.21, 153.44, 139.02,
134.98, 127.86, 126.46, 120.94, 120.80, 79.33, 78.54, 74.96, 70.22,
61.35, 55.14, 41.96, 22.67. MS (C16H21N3O9) Calculated (MH+)
400.1356, found 400.1355.
1
Synthesis of 5-Acetamido-9-(biphenyl-4-carboxamido)-3,5,9-
trideoxy-D-glycero-r-D-galacto-non-2-ulopyranosonic Acid So-
dium Salt (9-BPC-Neu5Ac). 5-Acetamido-9-amino-3,5,9-trideoxy-
D-glycero-D-galacto-2-nonulopyranosyl-onate (9-amino-NeuAc)24
(200 mg) in methanol was kept basic with triethylamine, and to
the solution was added dropwise biphenylacetyl chloride dissolved
in methylene chloride (2 M) until the reaction was complete as
monitored by TLC. The solvents were evaporated, and the residue
was purified by flash chromatography on silica gel eluted with
methylene chloride/methanol (10:1 to 1:2) to afford 9-BPC-Neu5Ac
4-O-{6-O-[5-Acetamido-9-(biphenyl-4-carboxamido)-3,5,9-
trideoxy-D-glycero-r-D-galacto-non-2-ulopyranosyl-2-onic acid
sodium salt]-ꢀ-D-galactopyranosyl}-2-acetamido-2-deoxy-ꢀ-D-
glucopyranosyl Azide (8). 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
ꢀ-D-glucopyranosyl azide (1 g, 2.7 mmol) was dissolved in
methanol (100 mL). To this solution was added sodium (30 mg,
1.3 mmol). The reaction was stirred at room temperature for 30
min. The pH was adjusted to 7 with Dowex H+ resins. Solvent
was then removed under reduced pressure to give deprotected
glycosyl azide as a white powder (630 mg, 95%). This material,
compound 3, (25 mg, 0.1 mmol) was dissolved in sodium
cacodylate buffer (20 mL, 100 mM) containing MgCl2 (20 mM),
MnCl2 (20 mM), CTP (112.8 mg), 9-BPC-Neu5Ac (92 mg), and
UDP-glucose (100 mg). To this solution, GalE-GalT (1 U), CMP-
NeuAc synthetase (600 U), and hST6Gal 1 (1.6 U) were added.
The reaction was stirred at room temperature for 24 h while the
pH was constantly kept at 7.5. The reaction mixture was then
centrifuged at 3000 rpm for 15 min, and the supernatant was
separated and lyophilized. The product was then first purified from
a G15 size exclusion column (1 × 75 cm, 5% n-butanol in water)
1
(180 mg) in 75% yield. Rf: 0.5 (4:3:2 i-PrOH:MeCN:MeOH). H
NMR (CD3OD) δ 7.92 (d, 2H, J ) 8.4 Hz), 7.71 (d, 2H, J ) 8.4
Hz), 7.64 (d, 2H, J ) 7.2 Hz), 7.45 (t, 2H, J ) 7.5 Hz), 7.37 (m,
1H), 4.05 (m, 2H), 3.91 (m, 2H), 3.55 (t, 1H, J ) 6.3 Hz), 3.52
(m, 2H), 2.60 (m, 1H), 2.36 (t, J ) 6.9 Hz), 2.16 (dd, 1H, J ) 9.0,
4.2 Hz), 1.95 (s, 3H), 1.87 (t, 1H, J ) 12.0 Hz); MS: (C24H28N2O9):
calculated (MH+): 489.1867, found: 489.1876.
Synthesis of 2-Aminoethyl 4-O-{6-O-[5-Acetamido-9-(biphe-
nyl-4-carboxamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-non-
2-ulopyranosyl-2-onic acid sodium salt]-ꢀ-D-galactopyranosyl}-
2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (6). To a solution of
sodium cacodylate buffer (50 mL, 100 mM, pH 8.0) containing
MgCl2 (20 mM), MnCl2 (20 mM), CTP (150 mg), 9-BPC-Neu5Ac
(50 mg), UDP-glucose (150 mg), and 2-aminoethyl 2-acetamido-
2-deoxy-ꢀ-D-glucopyranoside31 (1, 40 mg) were added GalE-GalT
(20 U), CMP-NeuAc synthetase (10 U), and hST6Gal 1 (2 U). The
reaction mixture was slowly stirred at room temperature for 3 days.
Enzymes were removed by passing the reaction mixture through a
Centricon filter (MWCO 10 000 Da). The filtrate was lyophilized,
and the residue was dissolved in a minimum amount of water,
loaded onto a P-2 gel chromatography column, and eluted with
water. Appropriate fractions were pooled and lyophilized to afford
1
to give compound 8 as a white powder (50 mg, 56%). H NMR
(CD3OD, 600 MHz) δ 7.79 (m, 3H), 7.39 (m, 5H), 7.21 (m, 4H),
4.58 (d, 1H, J ) 8.4 Hz), 4.22 (d, 1H, J ) 8.4 Hz), 3.92 (m, 3H),
3.77 (m, 5H), 3.62 (m, 9H), 3.49-3.38 (m, 7H), 2.53 (bd, 1H, J )
8.4 Hz), 1.93 (s, 3H), 1.85 (s, 3H), 1.71 (t, 1H, J ) 12 Hz); 13C
NMR (CD3OD, 175 MHz) δ 176.02, 172.32, 171.23, 144.72,
139.63, 133.02, 129.89, 129.09, 128.55, 127.76, 104.31, 100.14,
89.15, 81.05, 77.19, 74.33, 73.60, 73.24, 73.14, 71.44, 70.85, 70.39,
69.18, 68.30, 64.20, 60.96, 55.12, 52.62, 44.11, 39.89, 23.08, 22.91.
MS: (C38H50N6O18): calculated (MH+): 879.3254, found: 879.3245.
1
the desired product (55 mg) in 70% yield. H NMR (CD3OD) δ
Synthesis of 2-(6-Aminohexanoylamide)-ethyl 4-O-{6-O-[5-
acetamido-9-(biphenyl-4-carboxamido)-3,5,9-trideoxy-D-glycero-
r-D-galacto-non-2-ulopyranosyl-2-onic acid sodium salt]-ꢀ-D-
galactopyranosyl}-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside (10).
Compound 1 (200 mg, 0.76 mmol), 6-(Fmoc-amino)caproic acid
(320 mg, 0.9 mmol, 1.2 equiv), HBTU (576 mg, 1.52 mmol, 2
equiv), and HOBt (205.2 mg, 1.52 mmol, 2 equiv) were mixed in
4 mL of DMF. To this was added triethylamine (220 uL, 1.52 mmol,
2 equiv). The reaction mixture was stirred at room temperature
overnight. TLC showed a complete conversion to the product. The
solvent was removed under reduced pressure. The product was
purified by flash chromatography (chloroform:methanol 4:1) to give
Fmoc-protected hexanoic acid derivative as a white powder (326
mg, 72%). Part of this material (25 mg, 0.042 mmol) was treated
with 20% piperidine solution in DMF (416 uL) for 4 h, and the
solvent was removed under reduced pressure. The residue, com-
pound 5, was suspended in sodium cacodylate buffer (10 mL, 100
mM) containing MgCl2 (20 mM), MnCl2 (20 mM), CTP (56.4 mg),
9-BPC-Neu5Ac (46 mg), and UDP-glucose (49 mg). To this were
7.82 (d, 2H, J ) 8.4 Hz), 7.61 (d, 2H, J ) 8.4 Hz), 7.55 (d, 2H,
J ) 7.2 Hz), 7.36 (t, 2H, J ) 7.8 Hz), 7.27 (m, 1H), 4.46 (d, 1H,
J ) 8.4 Hz), 4.23 (d, 1H, J ) 7.2 Hz), 3.56-4.1 (m, 10H), 3.30
(d, 1H, J ) 7.8 Hz), 3.20 (m, 4H), 3.01 (m, 1H), 2.64 (dd, 1H),
1.91 (s, 3H), 1.88 (s, 3H), 1.57 (t, 1H, J ) 12 Hz); 13C NMR
(CD3OD) δ: 174.87, 174.63, 169.83, 145.41, 141.18, 134.42,
130.07, 129.11, 129.04, 128.11, 128.02, 105.04, 102.36, 101.54,
81.80, 76.47, 75.67, 74.66, 74.07, 73.42, 72.57, 72.33, 71.65, 70.32,
69.77, 66.51, 64.91, 62.01, 59.60, 52.28, 56.73, 53.93, 52.80, 51.23,
44.64, 42.35, 40.98, 23.49, 22.88, 17.66, 15.89; MS(C40H56N4O19)
Calculated (MNa+) 919.3436, found 919.3473
Synthesis of N-(2-Acetamido-2-deoxy- ꢀ-D-glucopyranosyl)-
2-(4-hydroxy-3-nitrophenyl)acetamide (7). Glycosylamine 2,
prepared according to a published procedure,32 (200 mg,0.91 mmol)
(28) Blixt, O.; Brown, J.; Schur, M. J.; Wakarchuk, W.; Paulson, J. C. J.
Org. Chem. 2001, 66 (7), 2442–8.
(29) Blixt, O.; Allin, K.; Pereira, L.; Datta, A.; Paulson, J. C. J. Am. Chem.
Soc. 2002, 124 (20), 5739–46.
(30) Blixt, O.; Paulson, J. C. AdV. Synth. Catal. 2003, 345, 687–690.
(31) Eklind, K.; Gustafsson, R.; Tiden, A. K.; Norberg, T.; Arberg, P. M.
J. Carbohydr. Chem. 1996, 15, 1161–1178.
(32) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaya, V. A.; Kochet-
kov, N. K. Carbohydr. Res. 1986, 146 (1), C5–C1.
9
7738 J. AM. CHEM. SOC. VOL. 130, NO. 24, 2008

Bifunctional CD22 Ligands
A R T I C L E S
added GalE-GalT (0.5 U), CMP-NeuAc synthetase (300 U), and
hST6Gal 1(0.8 U). The reaction was stirred at room temperature
for 24 h while the pH was constantly kept at 7.5. The reaction
mixture was then centrifuged at 3000 rpm for 15 min, and the
supernatant was separated and lyophilized. The product was then
purified from a G15 size exclusion column (1 × 75 cm, 5%
n-butanol in water) to give compound 10 as a white powder (25
mg, 60%). 1H NMR (CD3OD, 600 MHz) δ 7.69 (m, 8H), 7.42 (m,
3H), 7.35 (m, 1H), 4.33 (d, 1H, J ) 8.4 Hz), 4.30 (d, 1H, J ) 8.4
Hz), 3.98-3.80 (m, 5H), 3.76-3.64 (m, 6H), 3.59 (dd, 1H, J )
7.2, 13.8 Hz), 3.53 (m, 3H), 3.47-3.38 (m, 6H), 3.20 (t, 1H, J )
4.8 Hz), 2.85 (t, 1H, J ) 7.2 Hz), 2.55 (dd, 1H, J ) 4.8, 12.6 Hz),
2.10 (m, 2H), 1.97 (s, 3H), 1.87 (s, 3H), 1.58 (t, 1H, J ) 12.6 Hz),
1.55 (m, 2H), 1.47 (m, 2H), 1.22 (m, 1H); 13C NMR (CD3OD,
175 MHz) δ 178.12, 174.98, 173.40, 171.87, 144.41, 141.27,
133.04, 125.92, 125.85, 118.57, 112.11, 104.23, 101.73, 101.14,
81.28, 75.27, 74.51, 73.34, 73.31, 73.26, 71.54, 71.11, 70.98, 70.87,
70.64, 69.87, 69.18, 69.01, 68.95, 68.08, 64.27, 63.30, 61.15, 55.62,
53.15, 52.70, 44.20, 43.81, 40.97, 40.24, 40.10, 36.21, 27.23, 25.90,
25.55, 23.17, 22.86. MS: (C24H28N2O9): calculated (MH+):
1010.4458, found: 1010.4438.
71.11, 70.94, 69.17, 68.98, 54.33, 52.71, 44.17, 42.27, 41.20, 22.84,
22.65. MS: (C46H57N5O22): calculated (MH+): 1032.3568, found:
1032.3556.
Synthesis of N1-{4-O-{6-O-[5-Acetamido-9-(biphenyl-4-car-
boxamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-non-2-ulopy-
ranosyl-2-onic acid sodium salt]-ꢀ-D-galactopyranosyl}-2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl}-4-[2-(4-hydroxy-3-
nitrophenyl)acetamidomethyl]-[1,2,3]-triazole (13). Compound
8 (∼44 mg, 0.05 mmol) was dissolved in water (1 mL). 4-Hydroxy-
3-nitrophenylethyloylpropargylamine (15 mg, 0.06 mmol) in t-
butanol (1 mL) was added. To this were added sodium ascorbate
(9.1 mg) and copper sulfate (3.8 mg), and the reaction mixture was
stirred at room temperature overnight. The solvent was then
lyophilized, and the residue solid was purified by a G15 size
exclusion column (1 × 75 cm, 5% n-butanol in water) to give
compound 13 as yellow crystals (39 mg, 70%). 1H NMR (CD3OD,
600 MHz) δ 7.81 (d, 1H, J ) 2.4 Hz), 7.74 (m, 2H), 7.61 (d, 2H,
J ) 8.4 Hz), 7.55 (d, 2H, J ) 7.8 Hz), 7.38 (m, 3H), 6.98 (d, 2H,
J ) 9 Hz), 5.66 (d, 1H, J ) 10.2 Hz), 4.37 (d, 1H, J ) 7.8 Hz),
4.30 (m, 2H), 4.06 (t, 1H, J ) 10.2 Hz), 3.96-3.91 (m, 2H), 3.86
(d, 1H, J ) 3 Hz), 3.81-3.71 (m, 5H), 3.68-3.58 (m, 5H), 3.44
(m, 4H), 2.55 (dd, 1H, J ) 4.8, 12.6 Hz), 1.89 (s, 3H), 1.69 (t, 1H,
J ) 12.0 Hz), 1.65 (s, 3H); 13C NMR (CD3OD, 175 MHz) δ 175.81,
174.83, 174.20, 172.46, 171.63, 153.52, 146.16, 144.98, 139.98,
139.24, 134.56, 133.15, 129.98, 129.22, 128.61, 128.09, 127.91,
127.82, 126.18, 122.84, 120.75, 118.57, 116.84, 104.06, 100.07,
86.85, 79.95, 78.26, 74.29, 73.62, 73.25, 72.89, 71.56, 70.96, 70.45,
69.01, 68.40, 64.09, 60.77, 55.49, 52.59, 43.99, 41.54, 39.86, 35.00.
MS: (C49H60N8O22): calculated (MH+): 1113.3895, found: 1113.3895.
Synthesis of N1-{4-O-{6-O-[5-Acetamido-9-(biphenyl-4-car-
boxamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-non-2-ulopyra-
nosyl-2-onic acid sodium salt]-ꢀ-D-galactopyranosyl}-2-acetamido-
2-deoxy-ꢀ-D-glucopyranosyloxyethyl}-4-[2-(4-hydroxy-3-
nitrophenyl)acetamidomethyl]-[1,2,3]-triazole (14). Compound
9 (synthesized as described previously33) (20 mg, 0.02 mmol) and
N-propargyl-2-(4-hydroxy-3-nitrophenyl)acetamide (5.2 mg, 0.022
mmol) were mixed in water (1 mL) and t-butanol (1 mL). To this
sodium ascorbate (6.8 mg) and copper sulfate (2.8 mg) were added.
The reaction mixture was stirred at room temperature overnight.
The solvent was then lyophilized and the residue solid was purified
by a G15 size exclusion column (1 × 75 cm, 5% n-butanol in water)
Synthesis of (4-Hydroxy-3-nitrophenyl)methylcarbamidoet-
hyl 4-O-{6-O-[5-acetamido-9-(biphenyl-4-carboxamido)-3,5,9-
trideoxy-D-glycero-r-D-galacto-non-2-ulopyranosyl-2-onic acid
sodium salt]-ꢀ-D-galactopyranosyl}-2-acetamido-2-deoxy-ꢀ-D-
glucopyranoside (11). To a flask with methanol (5 mL) were added
2-(4-hydroxy-3-nitrophenyl)acetic acid, N-hydroxysuccinimide ester
(50 mg), and compound 6 (20 mg). The solution was kept basic
with addition of triethylamine and stirred at room temperature for
two days. The solvent was evaporated, and the residue was purified
by flash chromatography on silica gel to afford the desired product
1
(18 mg) in 75% yield. H NMR (CD3OD) δ7.85 (d, 1H, J ) 2.4
Hz), 7.86 (d, 2H, J ) 8.4 Hz), 7.60 (d, 2H, J ) 8.4 Hz), 7.54 (d,
2H, J ) 7.2 Hz), 7.35 (dd, 1H, J ) 6.3, 1.8 Hz), 7.32 (t, 2H, J )
7.2 Hz), 7.25 (m, 1H), 6.93 (d, 1H, J ) 8.7 Hz), 4.40 (d, 1H, J )
8.1 Hz), 4.20 (d, 1H, J ) 6.9 Hz), 3.95 (m, 2H), 3.34 (s, 2H),
3.80-3.40 (m, 8H), 3.15-3.3 (m, 4H), 2.64 (dd, 1H, J ) 4.5, 12.0
Hz), 1.87 (s, 3H), 1.86 (s, 3H), 1.60 (t, 1H, J ) 12.0 Hz), 13C
NMR (CD3OD) δ: 174.83, 174.50, 173.51, 170.03, 145.52, 141.26,
138.71, 134.38, 130.10, 130.01, 129.98, 129.05, 129.00, 128.10,
128.03, 126.47, 105.18, 102.49, 101.54, 76.42, 73.98, 72.42, 69.10,
64.89, 62.13, 42.09, 40.90, 26.11, 23.44, 9.51; MS (C48H61N5O23)
Calculated (MNa+) 1098.3649, found 1098.3649.
1
to give compound 14 as yellow crystals (20 mg, 80%). H NMR
(CD3OD, 600 MHz) δ 7.73 (s, 1H), 7.67 (t, 2H, J ) 7.8 Hz), 7.54
(m, 3H), 7.32 (m, 3H), 6.92 (d, 1H, J ) 8.4 Hz), 4.37-4.26 (m,
4H), 4.17 (d, 1H, J ) 7.8 Hz), 4.03 (dd, 1H, J ) 1.8, 12.0 Hz),
3.93 (m, 1H), 3.88 (t, 1H, J ) 8.4 Hz), 3.82 (s, 1H), 3.74 (m, 3H),
3.66 (d, 1H, J ) 13.2 Hz), 3.60-3.35 (m, 9H), 3.29 (m, 1H), 2.52
(dd, 1H, J ) 1.2, 12.0 Hz), 1.88 (s, 3H), 1.73 (s, 3H), 1.63 (t, 1H,
J ) 12.6 Hz); 13C NMR (CD3OD, 175 MHz) δ 175.72, 175.04,
174.24, 173.13, 171.28, 153.23, 144.94, 144.67, 139.91, 139.24,
134.48, 133.01, 130.01, 129.94, 129.20, 128.56, 126.22, 125.01,
120.75, 120.68, 104.20, 103.70, 101.72, 101.65, 100.35, 81.13,
75.28, 74.29, 73.55, 73.32, 73.22, 73.13, 73.06, 71.51, 71.10, 70.32,
70.21, 69.06, 68.56, 68.51, 64.06, 61.07, 55.38, 55.28, 52.58, 51.01,
44.19, 44.10, 41.42, 40.36, 35.44, 22.87. MS: (C51H64N8O23):
calculated (MH+): 1157.4157, found: 1157.4127.
Synthesis of 6-[2-(4-hydroxy-3-nitro-phenyl)acetamino]-hex-
anoic acid {N1-{4-O-{6-O-[5-Acetamido-9-(biphenyl-4-carboxa-
mido)-3,5,9-trideoxy-D-glycero-r-D-galacto-non-2-ulopyranosyl-
2-onic acid sodium salt]-ꢀ-D-galactopyranosyl}-2-acetamido-2-
deoxy-ꢀ-D-glucopyranosyloxy-ethyl}-amide (15). Compound 10
(11 mg, 0.0109 mmol) was dissolved in DMF (0.28 mL). To this
were added 2-(4-hydroxy-3-nitrophenyl)acetic acid N-hydroxysuc-
cinimide ester (3.85 mg, 0.013 mmol) and Et3N (3.85 uL, 0.028
mmol). The reaction was stirred at room temperature for 2 h. The
Synthesis of N-{4-O-{6-O-[5-Acetamido-9-(biphenyl-4-carboxa-
mido)-3,5,9-trideoxy-D-glycero-r-D-galacto-non-2-ulopyranosyl-2-
onic acid sodium salt]-ꢀ-D-galactopyranosyl}-2-acetamido-2-deoxy-
ꢀ-D-glucopyranosyl} 2-(4-Hydroxy-3-nitrophenyl)acetamide (12).
Compound 7 (20 mg, 0.05 mmol) was dissolved in sodium
cacodylate buffer (10 mL, 100 mM) containing MgCl2 (20 mM),
MnCl2 (20 mM), CTP (56 mg), 9-BPC-Neu5Ac (46 mg), and UDP-
glucose (50 mg). To this were added GalE-GalT (0.5 U), CMP-
NeuAc synthetase (300 U), and hST6Gal 1 (0.8 U) . The reaction
was stirred at room temperature for 24 h while the pH was kept
constant at 7.5. The reaction mixture was then centrifuged at 3000
rpm for 15 min, and the supernatant was separated and lyophilized.
The product was then first purified from a G15 size exclusion
column (1 × 75 cm, 5% n-butanol in water) to give compound 12
1
as a yellow powder (34 mg, 65%). H NMR (CD3OD, 600 MHz)
δ 7.75 (2d, 4H, J ) 9 Hz), 7.66 (dd, 2H, J ) 1.2, 7.2 Hz), 7.60 (d,
1H, J ) 2.4 Hz), 7.44 (t, 2H, J ) 7.2 Hz), 7.04 (dd, 1H, J ) 2.4,
9.0 Hz), 6.63 (d, 1H J ) 9.0 Hz), 4.88 (d, 1H, J ) 9.6 Hz), 4.30
(d, 1H, J ) 7.8 Hz), 3.95 (m, 1H), 3.86-3.40 (m, 12H), 2.55 (dd,
1H, J ) 4.2, 12 Hz), 1.89 (s, 3H), 1.70 (s, 3H), 1.58 (t, 1H, J ) 12
Hz). 13C NMR (CD3OD, 175 MHz) δ 183.41, 176.44, 175.81,
175.44, 174.18, 171.61, 144.85, 140.50, 137.63, 137.32, 133.42,
130.03, 129.23, 128.73, 128.07, 127.99, 127.07, 126.85, 104.23,
101.14, 80.73, 79.11, 77.04, 74.46, 73.54, 73.38, 73.23, 71.58,
(33) Kaltgrad, E.; O’Reilly, M. K.; Liao, L.; Han, S.; Paulson, J. C.; Finn,
M. G. J. Am. Chem. Soc. 2008, 130 (14), 4578-4579.
9
J. AM. CHEM. SOC. VOL. 130, NO. 24, 2008 7739

A R T I C L E S
O’Reilly et al.
solvent was then removed under reduced pressure. The residue solid
was purified with a G15 size exclusion column (1 × 30 cm, 5%
n-butanol in water) to give compound 15 as a yellow powder (8
of the extracellular portion of CD22, was immobilized onto Protein
A-conjugated magnetic beads (Dynal Biotech) by adding 5 mL of
culture supernatant containing hCD22-Fc chimera to 20 µL of bead
slurry and incubating overnight at 4 °C with end-over-end rotation.
Beads were isolated by exposure to a magnet, washed three times
with HBSS/BSA, and then resuspended in 200 µL of HBSS/BSA.
Aliquots of 2 µL of beads were added to the indicated concentra-
tions of anti-NP IgM diluted into HBSS/BSA and containing 1 µM
of either NP or BPCNeuAc-NP, for a final volume of 100 µL.
Binding proceeded for 1 h at 4 °C. Beads were then isolated and
washed with 300 µL of HBSS/BSA containing 50 nM of NP or
BPCNeuAc-NP. Beads were then incubated for 30 min at 4 °C in
100 µL of HBSS/BSA containing 50 nM NP or BPCNeuAc-NP and
FITC-anti-IgM to detect bound IgM. Beads were isolated and
washed with 300 µL of HBSS/BSA containing 50 nM NP or
BPCNeuAc-NP and then resuspended in 200 µL of the same buffer
and read by flow cytometry using a BD FACS Calibur Flow
Cytometer, using CellQuest software for analysis. For ligand
titration experiments, 2-fold dilutions of ligand were prepared in
96-well plates (50 µL/well). To each well were added 50-µL
aliquots of a solution containing 10 µg/mL anti-NP Ig and
approximately 1-2 × 105 beads/mL. Plates were incubated for at
least 14 h at 4 °C, 21 °C, or 37 °C to allow complex formation to
reach equilibrium. Using a magnetic block to immobilize the beads
at the bottoms of the wells (Dynal MPC-96 S, Invitrogen), beads
were washed with HBSS/BSA containing 100 nM BPCNeuAc-NP
and then stained with FITC-labeled anti-IgG, IgA, or IgM. Beads
were analyzed from the plates on a BD FACS Calibur HTS, and
data was analyzed using FlowJo software. With the exception of
data in Figure S1, data points for all ligand titrations represent the
average ( SD of duplicates. Curves were generated by nonlinear
regression analysis of the data using the equation for log (agonist)
vs response-variable slope in GraphPad Prism software, version
5.0a.
IgM Binding to BJAB Cells. BJAB cells, cultured with or
without exogenous sialic acid (4 mM), were resuspended in HBSS/
BSA or RPMI medium containing 10% FBS and 50 µM 2-mer-
captoethanol at 2 × 106 cells/mL, and the indicated amount of anti-
NP IgM, IgA, or IgG and NP or BPCNeuAc-NP were added to the
cells and allowed to bind for 1 h at 4 °C. Cells were then washed
with 0.2 mL of HBSS/BSA containing 100 nM ligand and then
incubated with FITC-labeled anti-IgM, IgA, or IgG for 30 min on
ice. After washing again, the cells were read by flow cytometry.
For ligand titrations, 2-fold dilutions of BPCNeuAc-NP were
prepared in the RPMI medium above at 50 µL/sample. BJAB cells
were suspended at 4 × 106 cells/mL, in the same medium, also
containing anti-NP Ig at 10 µg/mL. Aliquots of 50 µL of the cells
were added to the ligand dilutions, followed by incubation for 7 h
at 4 °C. Data were analyzed as described above for titrations on
CD22-Fc beads. For immunomicroscopy, BJAB cells were incu-
bated on ice for 1 h with 4 µg of Alexa488-anti-NP IgM in the
presence of either 4 µM NP or BPCNeuAc-NP. After washing, cells
were fixed, stained for nuclei with Hoechst dye, and visualized by
fluorescence microscopy.
1
mg, 62%). H NMR (CD3OD, 600 MHz) δ 7.80-7.59 (m, 6H),
7.38 (m, 4H), 6.97 (m, 2H), 4.35 (d, 1H, J ) 7.8 Hz), 4.29 (d, 1H,
J ) 7.2 Hz), 3.95 (m, 1H), 3.90-3.35 (m, 16H), 3.25-3.03 (m,
5H), 2.54 (dd, 1H, J ) 2.4, 12 Hz), 2.04 (m, 1H), 1.90 (s, 3H),
1.88 (s, 3H), 1.62 (t, 1H, J ) 12 Hz), 1.38 (m, 3H), 1.13 (m, 3H);
13C NMR (CD3OD, 175 MHz) δ 177.61, 175.65, 175.28, 174.28,
173.59, 171.29, 153.24, 144.78, 139.14, 134.58, 133.14, 130.04,
129.34, 128.61, 127.95, 125.95, 120.82, 104.22, 101.60, 81.20,
75.37, 74.44, 73.52, 73.36, 71.75, 71.04, 70.66, 69.17, 68.59, 64.11,
61.14, 55.74, 52.56, 47.49, 44.02, 43.66, 41.80, 41.74, 40.68, 40.13,
40.02, 38.09, 36.38, 29.27, 28.72, 28.62, 26.32, 25.73, 24.03, 23.47,
23.16, 22.85. MS: (C54H72N6O24): calculated (MH+): 1189.4671,
found: 1189.4698.
Antibodies and Cells. Cells expressing the B1-8 anti-NP IgM
were kindly provided by R.G. Corley and maintained in RPMI
(Gibco) with 5% low IgG sera (HyClone). Anti-NP IgG and IgA
were purchased from AbD Serotec. Secondary antibodies to anti-
NP antibodies were FITC-conjugated F(ab′)2 goat antimouse IgM
+ IgG, goat antihuman IgA (R-chain specific), and goat antihuman
IgG (Fcγ-specific) (Jackson Immunoresearch). Antibodies for
lymphocyte labeling were phycoerythrin (PE)-conjugated mouse
antihuman CD3, CD4, CD8, CD19, and CD56 (BD Pharmingen).
CD22-Fc was produced by transient transfection of COS cells with
a plasmid encoding hCD22-Fc, a gift of Ajit Varki (UCSD).34 Cells
maintained in DMEM were transfected by lipofectamine (Invitro-
gen) and then cultured for 3 additional days in OptiMEM. Culture
supernatant was used without purification. Native BJAB cells were
maintained in RPMI 1640 with 10% FBS and 50 µM 2-mercap-
toethanol. Asialo BJAB cells were prepared by culturing BJAB
cells in serum-free HYQ-SFM4MAb media (HyClone) for several
days. Peripheral blood from healthy human donors was obtained
from Scripps Normal Blood Donor Services after collection into
heparanized tubes.
Purification of anti-NP IgM and Labeling for Immunomi-
croscopy. The secreted anti-NP IgM produced by B1-8 hybridoma
cells was isolated by dialysis of the culture supernatant against water
overnight with two changes of water. Precipitated IgM was pelleted
by centrifugation at 13 000g for 1 h at 4 °C and resuspended in
phosphate-buffered saline. Anti-NP IgM was further purified using
a Hi Trap IgM purification HP column (GE Healthcare). IgM was
then labeled using the Alexa Fluor 488 Protein Labeling Kit
(Invitrogen-Molecular Probes) according to manufacturer’s instruc-
tions. In short, the 1 mg/mL anti-NP IgM was combined with 10
mg of labeling reagent and 200 µM 2-(4-hydroxy-3-nitropheny-
l)acetic acid (NP) to protect the binding site. After the labeling
was allowed to proceed for 1 h, protein was purified by gel filtration.
ELISA Assays. ELISA type assays used high binding microtiter
plates (Nunc Brand, Fisher) coated with Protein A (1 µg/well in
50 µL sodium bicarbonate pH 9.5) overnight at 4 °C. Wells were
washed with 200 µL of Hank’s buffered saline solution containing
5 mg/mL bovine serum albumin (HBSS/BSA), and culture super-
natant containing hCD22Fc chimera was incubated for 2 h at room
temperature. Wells were then washed with 2 × 200 µL of HBSS/
BSA. A mixture of anti-NP IgM (5 µg) and the indicated
concentration of bifunctional ligand was added in 50 µl and allowed
to bind for 30 min. Wells were washed with 2 × 200 µl of HBSS/
BSA and then incubated with a 1:3000 dilution of the alkaline
phosphatase conjugated anti-IgM for 30 min at RT. Wells were
washed 3 × 200 µL and developed with p-nitrophenyl phosphate.
Data are the average ( SD of triplicates and are representative of
three independent experiments.
IgM Binding to Lymphocytes from Human Peripheral
Blood. Lymphocytes were isolated by overlaying four 14-mL
batches of freshly drawn heparinized human blood diluted 1:1 with
Dulbecco’s phosphate-buffered saline (DPBS) onto 10.5-mL ali-
quots of Ficoll-Paque Plus (GE Healthcare) and processing accord-
ing to the manufacturer’s protocol. Briefly, lymphocytes were
collected at the interface by centrifugation (400g, 30 min, 18-20
°C) and then washed and resuspended in DPBS at 2 × 106 cells/
mL for staining. Cells were aliquoted at 2 × 105 cells/tube and
labeled with 8 µL of PE-conjugated anti-CD3, CD4, CD8, CD19,
or CD56 to identify cell type, or with 10 µg/mL Alexa488-anti-
NP IgM and 2 µM BPCNeuAc-NP for complex formation. Al-
exa488-anti-NP IgM and BPCNeuAc-NP were also combined with
cell type-specific antibodies for double labeling. Labeling proceeded
for 1 h at 37 °C. Cells were washed with HBSS/BSA containing
CD22-Fc Bead Binding. hCD22-Fc, which comprises a chimera
of the constant fragment of a human IgG fused to the C-terminus
(34) Brinkman-Van der Linden, E. C.; Sjoberg, E. R.; Juneja, L. R.;
Crocker, P. R.; Varki, N.; Varki, A. J. Biol. Chem. 2000, 275 (12),
8633–40.
9
7740 J. AM. CHEM. SOC. VOL. 130, NO. 24, 2008

Bifunctional CD22 Ligands
A R T I C L E S
Scheme 1. Synthesis of bifunctional 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).
100 nM BPCNeuAc-NP and fluorescence was measured using a
FACS Calibur flow cytometer with CellQuest Pro software for
analysis.
In Vitro Detection of Bifunctional Ligand-Driven
IgM-CD22 Complex Formation. The ability of the BPCNeuAc-
NP (11) to assemble a complex between anti-NP IgM and CD22
was initially tested in ELISA format with CD22-Fc chimera
adsorbed to protein A-coated ELISA wells (Figure 2a). Binding
of IgM was observed withBPCNeuAc-NP in a dose-dependent
manner, with no binding over background observed in the
presence of 2-(4-hydroxy-3-nitrophenyl)acetic acid (NP) alone.
The bifunctional ligand (1 µM) also assembled a complex with
anti-NP IgM on CD22-Fc-coated magnetic beads that could be
observed by flow cytometry (Figure 2b), whereby varying the
concentration of anti-NP IgM revealed detectable binding at 0.07
µg/mL and maximal binding at 5-10 µg/mL (∼7-13 nM IgM).
CD22-Fc beads were also used to titrate the ligand concentration
with a fixed concentration of anti-NP IgM (Figure S1, see also
Figures S3, S4, 4, and 6). We found that the bifunctional ligand
11 becomes inhibitory at concentrations above ∼10 µM,
presumably a result of the bifunctional ligand becoming a competi-
tive inhibitor as it saturates the binding sites of both CD22 and the
anti-NP IgM, preventing a bridging of the two proteins.
Results and Discussion
Synthesis of the Bifunctional Ligands. The exemplary CD22
ligand, BPCNeu5AcR2-6-Galꢀ1-4-GlcNAc-ethylamine was
synthesized in a one-pot enzymatic reaction employing glyco-
syltransferases to sequentially transfer galactose and 9-BPC-
Neu5Ac to N-acetylglucosamine conjugated to an ethylamine
linker (Scheme 1). The product was then coupled directly to a
commercially available N-hydroxysuccinamide activated 2-(4-
hydroxy-3-nitrophenyl)acetic acid to yield the desired bifunc-
tional ligand (BPCNeuAc-NP 11). Ligands with alternate linkers
were synthesized using related strategies modified as appropriate
to merge the chemical and enzymatic steps. The shortest linker
was constructed by first coupling the glycosylamine 2 to 2-(4-
hydroxy-3-nitrophenyl)acetic acid under standard conditions to
give compound 7, which underwent one-pot enzymatic trans-
formations to afford bifunctional ligand 12. Compound 15, with
the longest linear linker, was prepared by first coupling
compound 1 to Fmoc-protected 6-aminohexanoic acid. Removal
of the Fmoc protecting group and enzymatic transformation
yielded the trisaccharide 10, which was then coupled with 2-(4-
hydroxy-3-nitrophenyl)acetic acid N-hydroxysuccinimide ester
to give desired ligand 15. Alternate ligands with a 1,2,3-triazole
linker modification were prepared by using a copper(I)-catalyzed
azide-alkyne cycloaddition reaction35 to install the triazole
linkers (Scheme 1). CD22 ligands with an azide group at the
reducing end were either readily prepared from commercially
available 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-D-glucopy-
ranosyl azide (ligand 8) or synthesized as previously described
(ligand 9).33 The nitrophenyl moiety was first coupled with
propargylamine to install an alkyne group that was then coupled
with the azide group of the corresponding CD22 ligands.
Assembly of CD22-IgM Complexes on B Cells. To evaluate
the ability of IgM to bind to CD22 on B cells, Alexa488-anti-
NP IgM and BPCNeuAc-NP (11), BPCNeuAc, or NP were
incubated with native human B lymphoma cells (BJAB K20)
and assessed for binding by flow cytometry (Figure 2c). Only
in the presence of the bifunctional ligand is anti-NP IgM highly
associated with BJAB cells. Complex formation was also
visualized by immunofluorescence microscopy. Bright staining
of the cells in the presence of the ligand (11), but not NP,
demonstrated that the desired complex does indeed form on
BJAB cells (Figure 2d). These results were somewhat surprising
since cis ligands of CD22 are well documented to block the
binding of all but highly multivalent polymeric ligands of
CD22.22,23 To demonstrate the specificity of complex formation
for CD22, IgM was shown to bind to CHO cells transfected
with a plasmid for CD22 expression only in the presence of
ligand 11, while no binding was observed to nontransfected cells
(Figure S2).
(35) Kaltgrad, E.; Sen Gupta, S.; Punna, S.; Huang, C. Y.; Chang, A.;
Wong, C. H.; Finn, M. G.; Blixt, O. ChemBioChem 2007, 8 (12),
1455–62.
9
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A R T I C L E S
O’Reilly et al.
Figure 2. Bifunctional 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 indicated concentration of anti-NP IgM. 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.
complexes, and accommodation of the relatively rigid triazole
ring validates the use of the copper-catalyzed azide-alkyne
cycloaddition reaction (“click chemistry”)36 for the facile
assembly of bifunctional ligands.
Assembly of Immune Complexes of Lower Valency. To better
understand the importance of valency in complex formation,
we evaluated the ability of the bifunctional ligand 11 (BPC
-
NeuAc-NP) to induce complex formation between anti-NP
antibodies of the IgM (decavalent), IgA (tetravalent), and IgG
(bivalent) subclasses and CD22 on B cells and recombinant
CD22 expressed on CHO cells.37 All of these antibodies were
able to form complexes on native B cells (Figure 4), and on
CHO cells in both a ligand and CD22-dependent manner (data
not shown, results similar to Figure S2). The binding of the
lower valency antibodies were particularly surprising since cis
ligands of CD22 are well documented to block the binding of
all but highly multivalent polymeric ligands of CD22.22,23
For a more quantitative analysis, BPCNeuAc-NP titrations
were carried out to examine Ig complex formation on CD22-
immobilized magnetic beads. Clear differences in the concentra-
tion dependence for ligand-mediated complex formation is seen,
with apparent Kd values of 7, 17, and 23 nM for IgM, IgA, and
IgG, respectively. The Hill plots of the data in Figure 4 reflect
cooperativity in complex assembly, with Hill coefficients that
are greater than 1. Bifunctional ligands 13-15 were also able
to drive CD22-IgA complex formation with correspondingly
lower avidity compared to IgM (Figure S4).
Figure 3. Influence of linker structure in bifunctional 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).
Effect of Linker Structure on Complex Assembly. To
investigate the influence of linker structure on complex forma-
tion, bifunctional ligands with linkers of different length and/
or with a heterocyclic triazole ring were also tested (ligands
12-15). With the exception of the shortest linker, 12, which
may impose steric constraints between IgM and CD22, complex
formation on native B cells was supported by each of the ligands
(Figure 3) and thus is relatively tolerant to changes in the linker
structure. These findings were confirmed in experiments exam-
ining complex formation of anti-NP IgM on CD22 beads upon
titration of each of the bifunctional ligands (Figure S3). This
analysis revealed very similar capacity of the ligands bearing
medium and long linear and short triazole linkers to form
Of particular note, the apparent Kd values are g100 fold lower
than the respective Kd values of the monovalent ligands for
CD22 (Kd = 2 µM)23 and the B1-8 anti-NP IgM (Kd = 1
(36) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40 (11), 2004–2021.
(37) Tateno, H.; Li, H.; Schur, M. J.; Bovin, N.; Crocker, P. R.; Wakarchuk,
W. W.; Paulson, J. C. Mol. Cell Biol. 2007, 27 (16), 5699–710.
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Bifunctional CD22 Ligands
A R T I C L E S
and CD22-Fc beads also suggests that lateral mobility is not
necessary to achieve complex formation. The effect of cis
ligands is even more pronounced for the lower-valency IgA,
suggesting that increased valency is particularly important for
competition with the high concentration of sialic acid bearing
cis ligands at the cell surface.
These observations provide strong evidence that the bifunc-
tional ligand drives dynamic assembly of the IgM-CD22
complex on the surface of the B cell, and does not require a
preformed complex of the ligand with either CD22 or IgM. The
CD22-binding portion of the bifunctional ligand, with a Kd of
∼2 µM,21,23 is not by itself able to compete with cis ligands on
BJAB cells to occupy a significant fraction of CD22 binding
sites, since the same ligand in a multivalent polyacrylamide
construct (n ) 15) fails to stably bind to B cells unless cis
ligands are destroyed.23 Thus, the IgM scaffold is required for
theBPCNeuAc-NP ligand to displace cis ligands and form a stable
complex. As observed with complex formation on magnetic
beads, the apparent dissociation constant of complex formation
on cells is orders of magnitude lower than that of either of the
individual binding interactions,21,38 suggesting that both IgM
and CD22 mutually benefit from the multivalency afforded by
one another.
The efficiency of the bifunctional ligand-mediated complex
formation between anti-NP IgM and CD22 is surprising not only
because of the weak affinity of the CD22 ligand and the presence
of cis ligands but also because of the low copy number of CD22
on the B cell (∼10-20 000).40 Thus, the documented clustering
of CD22 in clathrin rich microdomains may be a critical factor
for bifunctional ligand mediated docking of IgM by providing
a local high density of CD22 monomers.24,26,41,42
Thermodynamics of Complex Assembly. Once bound, IgM
rapidly dissociates from the B cell upon repeated washing at 4
°C but is retained on the cell surface if at least 50 nM
BPCNeuAc-NP ligand 11 is included in the wash buffer (Figure
S5). These observations demonstrate that upon formation, the
complex is in rapid equilibrium with the ligands in solution.
The dissociation of IgM upon washing is even more pronounced
at elevated temperatures, which is consistent with the temper-
ature dependence demonstrated for complex formation on
magnetic beads with IgM (Figure 6a) or IgA (Figure 6b).
Complex formation obeys a temperature dependence character-
ized by decreased affinity with increasing temperature, which
is expected since the change in entropy (∆S ) upon complex
formation is anticipated to be negative, and in fact this trend
has been observed previously for CD22 binding to a
glycoprotein-ligand.21 Complexes formed using bifunctional
ligands with alternate linkers (13-15) also obey this temperature
dependence (Figure 6c).
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.
µM).38 Thus, at a concentration of 10-20 nM BPCNeuAc-NP,
there is negligible occupancy of the binding sites of the free
CD22 or anti-NP IgM, providing strong evidence that the
complexes self-assemble at the surface of the bead.
Effects of Cis Ligands in Complex Assembly on B Cells. To
establish the degree to which cis ligands modulate the assembly
of Ig complexes with CD22 on B cells, we took advantage of
the fact that BJAB K20 B cell line is well suited to evaluate
the effect of cis ligands, since it is deficient in the epimerase/
kinase step of sialic acid biosynthesis.39 Thus, when the cells
are cultured in the absence of sialic acids (asialo-BJAB), there
are no cis ligands, but when grown in the presence of serum or
sialic acid their glycoproteins are sialylated equivalently to wild
type BJAB cells.24 The effect of cis ligands was clearly evident
for CD22 complex formation with both IgM and IgA upon
titration of BPCNeuAc-NP (11), revealing that complex formation
on asialo-BJAB cells (Figure 5a) was achieved at much lower
concentration than needed for fully sialylated BJAB cells (Figure
5b), as evidenced by the differences in apparent Kd values. All
binding at 4 °C is a result of surface complex formation, and
not internalization, since it is readily displaced by brief treatment
with buffer of low pH (data not shown). The avidities of
complex formation on asialo-BJAB cells agree very well with
those on CD22-Fc beads, which also do not bear cis ligands.
The similarity between ligand-induced binding to asialo B cells
Specificity of IgM Binding to Primary B Lymphocytes. B cell
depletion therapy is now recognized to have therapeutic benefit
for B cell leukemia as well as a number of chronic inflammatory
diseases such as rheumatoid arthritis and systemic lupus
erythamatosis.43 CD22 is currently being evaluated in human
clinical trials as a target for B cell depletion therapy because of
its restricted expression on B cells and efficient uptake of toxin-
conjugated anti-CD22 antibody.19,20 Driving the formation of
(40) D’Arena, G.; Musto, P.; Cascavilla, N.; Dell’Olio, M.; Di Renzo, N.;
Carotenuto, M. Am. J. Hematol. 2000, 64 (4), 275–81.
(41) Collins, B. E.; Smith, B. A.; Bengtson, P.; Paulson, J. C. Nat. Immunol.
2006, 7 (2), 199–206.
(38) Dal Porto, J. M.; Haberman, A. M.; Shlomchik, M. J.; Kelsoe, G.
J. Immunol. 1998, 161 (10), 5373–81.
(39) Keppler, O. T.; Hinderlich, S.; Langner, J.; Schwartz-Albiez, R.;
Reutter, W.; Pawlita, M. Science 1999, 284 (5418), 1372–6.
(42) Zhang, M.; Varki, A. Glycobiology 2004, 14 (11), 939–49.
(43) Browning, J. L. Nat. ReV. Drug DiscoVery 2006, 5 (7), 564–76.
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A R T I C L E S
O’Reilly et al.
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. Bifunctional ligand 11 was compared for its ability to induce complex formation of 5 µg/mL
anti-NP IgM (a) or IgA (b) on CD22-immobilized magnetic beads at the indicated temperatures. (c) Bifunctional 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).
immune complexes using bifunctional ligands comprising a
ligand for CD22 coupled to an antigen, as reported here, may
provide an alternative approach to targeting B cells. Haas et al.
have shown that treatment of mice with an antibody targeted
to the ligand-binding site of CD22, but not antibodies targeting
other regions of the protein, resulted in depletion of both normal
and malignant B cells.44 The mechanism for depletion by
targeting only the ligand binding site remains unclear but may
be addressed and exploited by IgM-mediated targeting of the
CD22 ligand binding site with the native ligand. IgM antibodies
are also well-known activators of complement fixation, resulting
in cell killing. To assess the specificity of targeting B cells using
the bifunctional CD22 ligand, we measured the ability of anti-
NP IgM to form complexes on mixed white blood cells from
peripheral human blood. When cells were treated with Al-
exa488-anti-NP IgM in the presence of either NP or BPCNeuAc-
NP (11), a population of IgM-binding cells was detected only
in the presence of the bifunctional ligand (Figure 7a). To identify
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Bifunctional CD22 Ligands
A R T I C L E S
Figure 7. Specificity ofBPCNeuAc-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.
the cell type represented by this IgM-binding population,
antibody markers for B cell, T cell, and natural killer cell subsets
were used in a dual labeling experiment. Figure 7b shows that
the cells that exhibit BPCNeuAc-NP (11)-dependent binding of
anti-NP IgM are B220+ B cells, and that all B cells bind the
anti-NP IgM. Conversely, NK cells, CD4+ T cells and CD8+
T cells do not exhibit binding.
density of the receptor, and linking chemistry of the bifunctional
ligand. It is anticipated that design parameters for each variable
will be better defined with each successful combination of
protein scaffold and cell surface glycan-binding protein.
Acknowledgment. We thank M. Pawlita, A. Varki, and R.B.
Corley for generous gifts of the BJAB cell line, plasmid encoding
hCD22, and the B1-8 IgM anti-NP hybridoma, respectively. We
thank Dr. Ramya T. N. Chakravarthy for providing asialo BJAB
cells. The authors wish to thank Ms. Anna Tran-Crie for her help
with manuscript preparation. This work was funded in part by NIH
grants GM060938 and AI050143 (J.C.P.), and Natural Science and
Engineering Research Council and Alberta Ingenuity grants (D.R.B.).
M.K.O. is supported by a postdoctoral fellowship from the
American Cancer Society.
Conclusions
Here we report the use of small molecule bifunctional ligands
of CD22 that use antibodies as protein scaffolds to assemble
antibody-CD22 complexes of low valency (n ) 2-10) on B
cells. This is in contrast to the failure of higher valency polymer-
based ligands of CD22 to bind to native B cells due to masking
by cis ligands.22,23,45 We attribute the success of this approach
to the unique geometry afforded by the antibody scaffold,
allowing assembly of the complex with optimal spacing to allow
simultaneous contact with multiple CD22 molecules. The results
suggest a general approach in the rational design of ligand-
based probes for glycan-binding proteins and other low-affinity
receptors using soluble multivalent proteins as a scaffold, as
an alternative to polymers, dendrimers or neo-glycoproteins. The
relevant variables include ligand affinity, valency of the scaffold,
Supporting Information Available: Titration curve for com-
pound 11 driving CD22-IgM complex formation, showing
inhibition at higher concentrations. Flow cytometry data for IgM
binding to CHO cells expressing CD22 and not to wild type
CHO. Materials and methods for CHO cell binding assay.
Titration curves for compounds 11 and 13-15 with anti-NP
IgM and IgA on CD22 beads. Flow cytometry data demonstrat-
ing the maintenance of the IgM complex upon washing in the
presence, but not in the absence, of compound 11. This material
is available free of charge via the Internet at http://pubs.acs.org.
(44) Haas, K. M.; Sen, S.; Sanford, I. G.; Miller, A. S.; Poe, J. C.; Tedder,
T. F. J. Immunol. 2006, 177 (5), 3063–73.
(45) Yang, Z. Q.; Puffer, E. B.; Pontrello, J. K.; Kiessling, L. L. Carbohydr.
Res. 2002, 337 (18), 1605–13.
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