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INTRODUCTION |
The sialoadhesin CD22 is a member of a group of cell adhesion
molecules within the immunoglobulin superfamily that display binding to
glycans with terminal sialic acid residues (1-3). It structurally
resembles the carcinoembryonic antigen subfamily, with members like the
adhesion molecules myelin-associated glycoprotein and CD33
(4-7).
Two isoforms of CD22 have been identified: 110- and 130-kDa proteins
termed CD22
and CD22
, respectively. CD22
contains seven
extracellular immunoglobulin-like domains, a short transmembrane sequence, and a 78-amino acid cytoplasmic tail. CD22
lacks two of
the seven immunoglobulin-like domains, but is otherwise identical to
CD22
(8, 9).
CD22 is a cell-surface glycoprotein that is uniquely located on B-cells
and B-cell-derived tumor cells (10, 11). Upon activation of B-cells,
the expression level of cell-surface CD22 initially increases, but is
subsequently down-regulated upon differentiation into
antibody-producing cells. The essential role of CD22
in B-cell
activation offers an excellent possibility for the development of
agents that interfere with B-cell-mediated immune responses. CD22
antagonists may prove valuable in preventing unwanted immune responses
like allergy and chronic inflammatory processes, whereas CD22
agonists may be used to trigger the B-cell immune system in vaccination
therapy (3, 7, 12, 13). Alternatively, ligands for CD22
may function
as homing devices for the specific delivery of radionuclides and
cytostatics to B-cell-derived tumors, e.g. in the case of
acute leukemia.
These interesting perspectives have prompted us and others to map the
binding characteristics of CD22
(3, 8, 14-19). From these studies,
it has appeared that CD22
binds
2,6-sialylated glycoproteins,
whereas it does not recognize
2,3-sialylated ligands (14, 8, 4). The
basic monovalent binding motif in endogenous ligands for CD22
has
been identified as
Neu5Ac(
2,6)-N-acetyllactosamine1
(15). Further studies by Powell et al. (16) suggested that this basic monovalent binding motif for human CD22
could be further stripped to the disaccharide Neu5Ac(
2,6)Hex(NAc) without loss of
affinity, in which Hex may be
N-acetyl-
-D-galactosamine (GalNAc),
-D-galactose (Gal), or
N-acetyl-
-D-glucosamine (GlcNAc). In fact,
the most potent disaccharide displayed a 3-fold higher affinity for
CD22
compared with the reference trisaccharide structure Neu5Ac(
2,6)Lac (9).
Although the binding characteristics of murine and human CD22
are
essentially similar, murine CD22
tends to have a slight preference
for 5-glycolylneuraminic acid, whereas human CD22
prefers Neu5Ac. A
second remarkable difference between human and murine CD22
is that
only halogenated Neu5Ac derivatives are recognized by human CD22
(3).
Summarizing the above data, it appears that (a) the presence
of an intact sialic acid moiety is essential for binding to CD22
; (b) modification of the sialic acid group at C-5 does not
dramatically affect ligand binding; and (c) the affinity for
CD22
may be enhanced by further optimization of the sialic
acid-terminated glycoside.
The aim of this study was to identify structural features of a CD22
ligand that determine the interaction with its receptor. A library of
mono-, di-, and trisaccharide analogs of NeuAc(
2,6)Lac was
synthesized and evaluated to elucidate the basic structural elements
for high affinity recognition. In addition, we have addressed the
effect of saccharide valency on the affinity for CD22
; based on the above criteria, we have devised a potent inhibitor of CD22
binding that may be of use in CD22
-directed immune therapy. We postulate that the most potent synthetic ligands can also serve as
homing devices in B-cell tumor-specific targeting of cytostatics.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine serum albumin (BSA; fraction V,
delipidated), Dulbecco's modified Eagle's medium, RPMI 1640 medium,
fetal calf serum, and penicillin/streptomycin were obtained from
BioWhittaker Europe (Verviers, Belgium). Glasgow minimum essential
medium (First Link UK); 5% IgG-poor fetal calf serum (Life
Technologies, Inc., Breda, The Netherlands); sodium butyrate,
DL-methionine DL-sulfoxide, fluorescein
5-isothiocyanate (FITC), goat anti-human IgG Fc fragment (referred to
below as anti-Fc), and human orosomucoid (Sigma, Zwijndrecht, The
Netherlands); horseradish peroxidase (Baker-grade, Deventer,
The Netherlands); dimethyl sulfoxide (Baker-analyzed Deventer, NL); and multiwell plates (Costar 3590 flat-bottomed high
binding, Corning Inc., Corning NY) were obtained from the indicated
manufacturers. Anti-CD22 antibody (Ab1) was kindly provided by
Dr. P. A. Van der Merwe (21). Anti-CD22 antibody (Ab2) was purified from ascites kindly provided by the Central Laboratory for
Blood Transfusion (Amsterdam).
A library of mono-, di-, and trisaccharides was synthesized, as will be
described elsewhere.2 Synthesis of multivalent saccharides
will be published elsewhere.
Production of Chimeric Murine CD22D3-IgG--
Murine CD22D3-IgG
was purified from a stable transfected Chinese hamster ovary cell line
kindly provided by Dr. P. R. Crocker (5). Cells were grown
in Glasgow minimum essential medium containing 5% IgG-poor fetal calf
serum, 20 units of penicillin/streptomycin, and 400 µM
DL-methionine DL-sulfoxide. After the cell
culture was 80% confluent, production of CD22 was induced by addition of 2 mM sodium butyrate, and the culture medium was
collected for 3 weeks. Murine CD22 was purified from the pooled media
by protein A-Sepharose chromatography at a yield of ±200 µg/ml as previously described by Nath et al. (5). Purity was
established by polyacrylamide gel electrophoresis analysis.
FITC Labeling of Ab2--
Ab2 was dissolved in 0.5 M
sodium carbonate buffer (pH 9.5). FITC (1 mg) dissolved in 0.5 ml of
Me2SO was added to Ab2 (80 µg of FITC/mg of Ab2), and the
mixture was incubated for 1 h at room temperature. FITC-labeled
Ab2 was purified by protein A-Sepharose chromatography and stored in 10 mM Tris and 150 mM NaCl (pH 8.2).
Erythrocyte Isolation--
Porcine erythrocytes were isolated as
follows. 70 ml of citrate buffer (210 mM citric acid, 0.9 M sodium citrate, 210 mM
NaH2PO4, and 1.3 M glucose) was
added to 500 ml of porcine blood. The blood was then centrifuged at
3000 rpm for 10 min at 4 °C; the plasma was removed; and
erythrocytes were washed three times with PBS. 6 volumes of
erythrocytes were stored in 1 volume of buffer containing 750 mM NaCl, 145 mM mannitol, 230 mM
glucose, and 6 mM adenine for 4 weeks at 4 °C
Erythrocyte Solid-phase Binding Assay--
The solid-phase
binding assay is based on the binding of porcine erythrocytes to murine
CD22D3-IgG, which is immobilized on anti-Fc-coated multiwell plates.
Erythrocyte binding to CD22 was monitored by measuring the peroxidase
activity of erythrocyte-entrapped hemoglobin. In short, multiwell
plates were coated by overnight incubation at 4 °C with anti-Fc in
PBS (0.75 µg/well); subsequently, the wells were washed with PBS + 0.25% BSA and blocked by incubation for 1 h at 37 °C with 5%
(w/w) skimmed milk. After washing, the wells were incubated for 3 h at 4 °C with CD22 at a concentration of 0.1 µg/ml in PBS. After
washing, porcine erythrocytes (1% dilution in PBS + 0.25% BSA) were
incubated for 1 h at room temperature. The competition studies of
erythrocyte binding to CD22 were performed by incubating the
erythrocytes in the presence of a variable concentration of the
inhibitor (1-1500 µM). After washing the wells with PBS + 0.25% BSA, the bound erythrocytes were visualized, and erythrocytes were first lysed in H2O and subsequently stained with
o-phenylenediamine hydrochloride for the presence of
erythrocyte-entrapped hemoglobin. CD22-specific binding was defined as
the differential binding in the absence and presence of an excess of
orosomucoid (3 µM). From the competition curves, the
IC50 (the concentration of inhibitor giving 50% reduction
of specific erythrocyte binding) could be calculated by nonlinear
regression analysis (PRISM, GraphPAD Software for Science, San Diego,
Ca). pIC50 values were obtained from three to
four independent experiments of 12 triplicate data points.
Flow Cytometric Analysis--
Ab2 was diluted in Hanks' buffer
(136.9 mM NaCl, 5.4 mM KCl, 0.8 mM
MgSO4·7H2O, 0.3 mM
Na2HPO4·2H2O, 0.4 mM
KH2PO4, 6.7 mM HEPES, and 5.6 mM glucose (pH 7.4)). For the FACS analysis, Ramos cells
(105 cells/ml) were used. Cells (1 × 105)
were resuspended in Dulbecco's modified Eagle's medium + 2% BSA and
incubated with FITC-Ab2 (60-5 nM) for 1 h at 37 °C
in Dulbecco's modified Eagle's medium + 2% BSA under gentle
agitation. Subsequently, cells were centrifuged for 2 min at 1800 rpm,
incubated for 5 min at 4 °C with 0.1 M glycine (pH 3),
washed three times with ice-cold Hanks' buffer (0.5 ml), and
resuspended in Hanks' buffer. Cells were analyzed for fluorescence and
forward scatter using a FACScan (FL1 = 530 nm, FL2 = 575 nm,
and FL3 = 640 nm).
Confocal Laser Scan Microscopy Studies--
Ramos cells (1 × 106 cells/sheet of glass (25-mm diameter, No. 1, Nutacom
B. V., Leimuiden The Netherlands)) were incubated with or
without FITC-Ab2 for 0-2 h at either 4 or 37 °C in RPMI 1640 medium. Binding and uptake of FITC-Ab2 by Ramos cells were detected via
confocal laser scan microscopy on a Bio-Rad Slow-scan MRC 500 apparatus
equipped with a helium-neon triple emitter (543 nm). Image analysis was
performed with a Kalman collection filter (10 scans).
 |
RESULTS |
Synthesis--
The aim of this study was to investigate the
contribution of structural features of CD22
(henceforth CD22) ligand
binding to its receptor and to develop a synthetic high affinity ligand for CD22 for use as a homing device in B-cell tumor-specific targeting of cytostatics. For this purpose, the molecular structure of the ligand
should warrant optimal and selective binding to CD22, but should also
be easily accessible from a synthetic point of view.
To this end, we systematically stripped the presumed basic motif
Neu5Ac(
2,6)Lac (Fig. 1, compounds 1 and 2) by synthesizing a series of tri-, di-, and monosaccharide
analogs. At the trisaccharide level, we tested the effect of a methoxy
(compound 1) or an azido (compound 2) group at the anomeric position.
Compound 1 was structurally identical to the basic binding motif
NeuAc(
2,6)Lac(NAc) and served as a reference for subsequent binding
studies.

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Fig. 1.
Chemical structures of the monomeric
glycosides used in this study. All structures are based on the
presumed basic binding motif NeuAc( 2,6)Lac. Structures are
classified as mono-, di-, and trisaccharide variations. Listed are the
compounds with their respective number; the modifications are displayed
under X and Y. SMe,
thiomethoxy; N3, azide; Bz,
benzoyl; Naph, naphthoyl;
NO2Bz, 4-nitrobenzoyl;
OPhNO2, 4-nitrophenyl; Oct, octanoyl;
ThioPh, thiophenoyl. Asterisks indicate that OH
at C-4 has an equatorial orientation (instead of axial).
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In addition, we devised a series of disaccharides, in which Neu5Ac was
coupled to a modified glucosyl or galactosyl group (Fig. 1, compounds
3-11 and compounds 12 and 13, respectively), that enabled us
to assess the effect of groups at C-1, C-2, and C-4 of the anomeric
sugar. (For more details about the synthesis, see Footnote 2.)
Finally, we synthesized a number of Neu5Ac-derived monosaccharides with
structural elements at C-2 and C-5. The acetyl group at C-5 was
replaced by an octanoyl (compound 14), benzoyl (compound 15), naphthoyl
(compound 16), thiophenoyl (compound 17), or nitrobenzoyl
(compound 18) group (see Fig. 1). In addition, the effect of
substitution of the C-2 methoxy group by a C-2 thiomethoxy group on the
binding affinity for CD22 was investigated (Fig. 1, compound 20).
Analysis of the Monomeric Ligands in the Erythrocyte Solid-phase
Binding Assay--
The monovalent ligands 1-20 were tested for their
affinity for CD22 in an in vitro competition assay, which is
based on the binding of porcine erythrocytes to immobilized CD22 (5).
First, the binding assay was optimized in terms of the coating level of
anti-Fc, CD22 concentration, and erythrocyte species and concentration. Fig. 2 shows the optimization of the CD22
concentration. The erythrocyte binding signal was clearly dependent on
the CD22 concentration and increased steadily with increasing CD22
concentrations, leveling off at >1.1 µg/ml CD22. For subsequent
competition studies, a concentration of 0.1 µg/ml CD22 appeared to be
optimal. The optimization studies also showed that human and murine
erythrocytes bound less avidly to CD22 compared with porcine
erythrocytes (data not shown).

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Fig. 2.
Effect of CD22 coating concentration on
erythrocyte binding. Porcine erythrocytes were incubated in wells
that have been coated with 0-3 µg/ml CD22. Binding and inhibition
were determined as described under "Erythrocyte Solid-phase Binding
Assay" under "Experimental Procedures." From the curve, the
apparent IC50 for erythrocyte binding to CD22 can be
calculated as 0.12 µg/ml. Data points are means from two independent
experiments of nine data points in triplicate.
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To calibrate the assay, competition studies were performed using two
established substrates for CD22, i.e. human orosomucoid and
bovine thyroglobulin, and a specific anti-CD22 monoclonal antibody
(Ab1) (15, 21). Both CD22 substrates were able to inhibit erythrocyte
binding to CD22 in a monophasic fashion and to a similar extent as Ab1
(Fig. 3). From the competition curves, the IC50 could be calculated by nonlinear regression
analysis. For proper evaluation, specific binding to CD22 was defined
as the differential binding in the absence and presence of an excess of
orosomucoid (3 µM).

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Fig. 3.
Competition of erythrocyte binding to murine
CD22 by human orosomucoid ( ), bovine
thyroglobulin ( ), and Ab1 ( ). Porcine erythrocytes were
incubated for 1 h at room temperature with CD22 (0.1 µg/ml) in
the absence or presence of the inhibitor. Erythrocyte binding was
determined as described under "Experimental Procedures." The
IC50 values for orosomucoid, thyroglobulin, and Ab1 were
77, 181, and 0.28 nM, respectively. Data points are
means ± S.D. from three independent experiments of 10 data points
in triplicate.
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Next, competition experiments were performed using the synthetic CD22
ligands from the ligand library. All competition curves were monophasic
and showed >90% inhibition of erythrocyte binding at higher ligand
concentration (Fig. 4).
Neu5Ac(
2,6)LacOMe (compound 1), the 1-methoxy analog of the basic
binding motif, was used as a reference compound and displayed an
average IC50 of 244 µM (Fig. 4). The affinity
of compound 1 was indexed as 1, and the IC50 values of all
other ligands were expressed relative to that of compound 1 (Table
I).

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Fig. 4.
Typical inhibition curve of the reference
compound 1. Porcine erythrocytes were incubated for 1 h at
room temperature with CD22 (0.1 µg/ml) in the absence or presence of
the inhibitor. Erythrocyte binding was determined as described under
"Experimental Procedures." The IC50 calculated for the
reference compound 1 was 244 µM. Data points are
means ± S.D. from four independent experiments of 10 data points
in triplicate.
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Table I
Affinity of oligosaccharides 1-20
pIC50 values were calculated by log IC50
(M). The relative IC50 value was calculated by
pIC50compound 1 pIC50compound x = 10relative IC50. A relative IC50 >1
represents a more avid binding to CD22 than the reference compound 1. pIC50 values are means of three to four independent
inhibition curves of eight data points in triplicate (n = 9 or 12).
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From Table I, it can be seen that, in line with the results of Powell
et al. (16), the
-D-glucosamine-derived
disaccharides 3-11 all displayed similar to higher affinity for CD22
compared with trisaccharide 1. Substitution of the anomeric methoxy by an azido group reduced the affinity effect at the disaccharide level
(compound 3 versus 10), but did not significant affect the binding at the trisaccharide level (compound 1 versus 2)
However, addition of a single electron-rich nitro group to the aromatic benzoyl ring led to a strongly increased affinity for CD22 (compound 9 versus 3 and compound 9 versus 6). In fact, the
nitrobenzoyl disaccharide 9 displayed an almost 10-fold higher affinity
than the reference compound 1 (p < 0.0005),
establishing compound 9 as the most potent ligand of the monovalent
carbohydrates tested.
To investigate the effect of modifications of the NeuAc group on ligand
recognition, the 5-acetyl group was substituted by various structural
elements, including naphthoyl, octanoyl, 4-nitrobenzoyl, and
thiophenoyl (Fig. 1). The monosialoside analogs of Neu5Ac-Lac generally
displayed IC50 values similar to that of the reference compound 1 (Table I, compounds 14-20). None of the substitutions led
to a significant change in affinity.
Effect of Valency on Ligand Recognition by CD22--
As has
previously been shown for numerous other lectins, ligand recognition
may greatly benefit from multimeric presentation (22-26). In an
attempt to map the effect of valency on ligand binding to CD22, we
synthesized multivalent ligands on the basis of glutamate. Disaccharide
9 served as a starting point for the multivalent ligands since it was
the most potent monomeric ligand (Fig.
5). These glutamate-based clusters
allowed rapid adjustment of the valency of the cluster backbone and
thus optimization of the orientation and spacing of the terminal
glycosides within the cluster. The di-, tri-, and tetravalent glutamate
clusters all displayed an ~10-fold higher affinity than the
monovalent compound 9. Apparently, an additional increase in the
valency of the glutamate clusters had little or no effect on the
affinity for CD22 (Fig.
6A).

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Fig. 5.
Chemical structures of the multivalent
saccharides used in this study. As given under R, all
multivalent compounds were derived from the monomeric glycoside 9 (the
most potent disaccharide from this study) and compound 19 (a
monosaccharide) (see Fig. 1). Compounds are classified in
glutamate-based (upper panel) and Tris-based (middle
panel) clusters and glutamate-based compounds with different
spacer lengths C4, C6, and C10
(lower panel).
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Fig. 6.
Effect of multivalent presentation on the
affinity of a sialylated saccharide for CD22.
A, comparison of the affinity of di-, tri-, and
tetravalent glutamate-based saccharides (compounds 21a, 21b, and 21c,
respectively) with that of the monovalent reference compound 9. Clearly, the multivalent compounds all displayed an ~10-fold higher
affinity than the monovalent glycosides. B, effect of the
valency of Tris-based mono- and disaccharides (compounds 19 and 9, respectively) on CD22. The multivalent compounds showed a 10-30-fold
increase in affinity. C, effect of saccharide spacing
(C4, C6, and C10) on the affinity
of the ligands for CD22. No significant effect was observed with
compounds 24-26. Data points are means ± S.D. from three
independent experiments of at least nine data points in triplicate.
Compound numbers are given on the x axis; the
y axis shows the increase in affinity relative to
that of the reference compound. The chemical structures of the
multivalent compounds 21-26 are given in Fig. 5, whereas those of the
monomeric compounds 9 and 19 are given in Fig. 1.
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To address whether the observed gain in affinity was caused by the
glutamate backbone itself, we also synthesized multivalent clusters
based on a Tris dendritic core (Fig. 5). The Tris core has already been
successfully applied in the synthesis of trivalent galactoside ligands
for the asialoglycoprotein receptor (27) and offers the advantage that
it is symmetrical and more suitable for large-scale synthesis. From
Fig. 6, it can be concluded that the glutamate- and Tris-based clusters
of compound 9 displayed a similarly enhanced affinity for CD22,
although the Tris-based cluster appeared to be slightly more potent
than the glutamate-based cluster.
To assess the significance of the above finding, we also studied
whether the extent of the clustering effect depended on the terminal
sugar moiety. To this end, a Tris-based cluster of the Neu5Ac
monosaccharide 19 was synthesized and tested. Analogs of this trivalent
Neu5Ac cluster 23 displayed a 10-fold higher affinity for CD22 compared
with the monovalent parent saccharide 19, suggesting that
the valency effect is independent of the terminal sugar.
Finally, we investigated whether the affinity of a multivalent CD22
ligand can be further improved by optimization of the spacing of the
terminal saccharides within a cluster glycoside. This would allow us to
verify that the affinity of multimeric compounds could be further
improved beyond the bivalent level by increasing the distance between
vicinal glycoside groups, which may be the limiting factor in
preventing optimal recognition of the separate glycoside groups.
Therefore, a series of tetravalent glutamate clusters was devised in
which a C4, C6, or C10 spacer was
inserted between two disubstituted glutamates. From the binding studies, it can be concluded that the spacer length did not affect the
affinity of these tetravalent compounds for CD22 (Figs. 5 and
6C).
In Vitro Studies on Murine CD22
--
To validate the
potential of the newly designed high affinity ligands for CD22-directed
targeting of drugs, we investigated substrate uptake by CD22-expressing
tumor cells (28). For the kinetic studies on Ramos cells, we used an
anti-CD22 antibody (Ab2) that is a potent and specific inhibitor of
ligand binding to CD22. Ab2 was fluorescently labeled with FITC, and
Ramos cells were incubated for 0-30 min with antibody, followed by an
acid wash step whereby membrane-associated FITC-Ab2 was removed.
Cellular fluorescence, a measure of internalized ligand, was monitored in time by FACS analysis. We concluded that cellular internalization of
Ab2 via the CD22 receptor proceeded very rapidly with a half-life time
of 6.2 min (Fig. 7).

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Fig. 7.
Uptake of FITC-labeled anti-CD22 antibody by
Ramos (CD22+) cells. Ramos cells (105
cells/ml) were incubated with FITC-labeled Ab2 (10 nM) for
up to 60 min at 37 °C. At the indicated time points, cells were
washed, and the extracellular bound antibody was then removed by
incubating the cells for 5 min at 4 °C with 0.1 M
glycine (pH 3) and analyzed for fluorescence and forward scatter using
a FACScan. The graph indicates the uptake of FITC-labeled Ab2 during
the incubation time. From the curve, the half-life time (6.2 min) can
be calculated using nonlinear regression analysis.
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To establish the actual route of CD22-mediated substrate uptake, we
analyzed the uptake kinetics of FITC-Ab2 by Ramos cells using confocal
laser scan microscopy. A punctate fluorescence staining
indicative of lysosomal uptake was observed after incubation for 30 min
at 37 °C (Fig. 8). In agreement with
the above FACS studies, internalization of Ab2 proceeded rapidly, and
after incubation for only 5 min, already a clearly punctate
fluorescence uptake was observed.

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Fig. 8.
Cellular uptake of FITC-labeled anti-CD22
antibody by Ramos (CD22+) cells. Ramos cells
(1 × 106) were incubated with or without 10 nM FITC-Ab2 at 37 °C for 5 min (B), 10 min
(C), and 20 min (D). As a control, Ramos cells
were incubated for 20 min without FITC-Ab2 (A). Ab2 was
labeled with FITC as described under "Experimental Procedures." The
arrow indicates an example of clear lysosomal uptake of
FITC-Ab2.
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All together, the data conclusively demonstrate that CD22-mediated
internalization does occur and is rapid. This implies that the
lysosomal pathway is used for uptake of CD22 ligands.
 |
DISCUSSION |
In this study, we developed new high affinity ligands for CD22
that can be utilized as homing devices in B-cell-directed delivery of
cytostatics. A library of 20 Neu5Ac-terminated mono-, di-, and
trisaccharides that carry different structural features was synthesized, and the compounds were tested for their ability to inhibit
erythrocyte binding to CD22 in an in vitro competition assay. To calibrate the competition assay, the IC50 values
of orosomucoid and thyroglobulin (natural ligands for CD22) were determined. The IC50 values were in close agreement with
values reported in the literature (16). Combined with the finding that Ab1 and the CD22 substrates (orosomucoid and thyroglobulin) inhibited erythrocyte binding to a similar extent, the results establish that
erythrocyte binding is completely mediated by CD22. The reproducibility of this assay is sufficiently high as judged from the small variation in IC50 values for orosomucoid in five independent
experiments (individual values of 77, 82, 81, 86, and 93 nM).
The library of monovalent mono-, di-, and trisaccharide structures was
tested with this assay. The reference compound Neu5Ac-LacOMe (compound
1), which is also the OMe derivative of the minimal basic motif (15),
displayed an average IC50 value of 244 µM. Taking into account that the affinity of Neu5Ac derivatives for murine
CD22 is slightly lower than that for human CD22 (18), this compares
well with the value reported by Powell and Varki (15) for human
CD22 (30-100 µM).
Comparison of the relative IC50 values of the
1-O-methyl-derivatized (compounds 3 (di), 1 (tri), and 12 (di)) saccharides showed that the orientation of the C-4 hydroxyl group
of the glycoside proximal to Neu5Ac (i.e. GlcNAc) does not
affect CD22 recognition. The observation that the GlcNAc-derivatized
disaccharide (equatorial C-4, compound 3) tends to be more potent than
the trisaccharide (equatorial C-4, compound 1) and the Gal-derivatized
disaccharide (axial C-4, compound 12) can also be attributed to an
effect of the 2-N-acetyl group (compound 3)
versus the 2-OH group (compound 12).
Azido and amino groups at C-2 of the glucosyl group of
sialyl-LeX were found to enforce the binding to E-selectin
by a factor of 4-6 (29). In the case of CD22 ligands, azido and amino
groups at C-2 of the anomeric sugar did not increase the affinity.
Substitution of the acetyl group at C-2 of the disaccharide by a more
lipophilic moiety (benzoyl, naphthoyl, or octanoyl) impaired binding to
CD22 (compounds 4-6 versus 1, respectively). The latter
results may seem somewhat unexpected in view of reports in which
insertion of lipophilic groups confers more avid binding in various
ligand-receptor systems (4, 30-34). Generally, the in vitro
competition data of di- and trisaccharides demonstrate that
(a) the affinity is enhanced by introducing 4-nitroaryl
groups at C-1 or C-2 of the reducing hexose; (b) the
affinity is not influenced by insertion of either lipophilic or
cationic groups at C-2 of the reducing end glycoside; and
(c) ligand recognition is rather tolerant toward modification of the 1- or 2-position, although an electron-rich 4-nitrobenzoyl group at C-2 significantly improves the affinity. The
most potent saccharide, the nitrobenzoyl derivative 9, displayed an
almost 10-fold higher affinity for CD22 compared with the reference compound Neu5Ac(
2,6)LacOMe. Apparently, optimization of the
penultimate carbohydrate moiety is an effective entry to improving the
affinity for CD22.
To further strip the minimal binding motif for high affinity binding to
CD22, we also synthesized and tested a series of monosaccharide analogs. Insertion of side groups at C-2 and C-5 only marginally affected the affinity for CD22 (2, 15, 16). The binding affinity of
both mono- and disaccharides tends to be increased by the presence of a
nitro aromatic group. In general, the affinity of the monosaccharides
is essentially similar to that of the di- and trisaccharides. The above
data may therefore suggest that the minimal motif required for binding
to CD22 may be the Neu5Ac monosaccharide rather than the disaccharide
Neu5Ac(
2,6)Hex, as has previously been proposed (16). The
penultimate sugar may then confer receptor specificity rather than a
gain in binding energy.
It has been established for various lectins, including the
asialoglycoprotein receptor, the mannose receptor, E-selectin, and
myelin-associated glycoprotein, that ligand recognition greatly benefits from multimeric presentation (4, 22-26). The fact that the
endogenous substrates for CD22 (orosomucoid and thyroglobulin) have
multiple terminal sialoside groups suggests that the affinity of a
ligand for CD22 may be further improved by enhancing its valency as
well. To map the effect of ligand valency, we prepared bi-, tri-, and
tetravalent glutamate-derived clusters (compounds 21a, 21b, and 21c)
based on the most potent disaccharide, compound 9. The di-, tri-, and
tetravalent clusters all displayed a 10-fold higher affinity for CD22
compared with the monomeric compound 9. Apparently, optimal recognition
by CD22 is already attained for bivalent glycosides. This contrasts
with the results of Powell and Varki (15), who observed, in a column
binding assay, that the apparent affinity for CD22 is consistently
increased upon increasing the number of sialic acid residues to up to
4. However, their study was quantitative, and no absolute affinities
were given for the tri- and tetravalent clusters. Accordingly, their study does not allow firm conclusions on the actual gain in affinity after di-, tri-, and tetravalent presentation. In addition, due to the
high flexibility of the cluster backbones used in our study, we
anticipate that the ligand configuration giving optimal receptor binding is more readily accomplished than in the case of the rigid glycoside backbone of Powell and Varki (15).
Likewise, symmetric clusters of trisaccharide 9 and monosaccharide 19 based on a Tris dendritic core (compounds 22 and 23) showed a
10-30-fold higher affinity for CD22 compared with the corresponding
monovalent motif. This conclusively establishes the presence of a
"cluster" effect for CD22 (15, 16) and excludes that the observed
gain in affinity could be attributed to the backbone itself. The
trivalent monosaccharide (compound 23), being 10-fold more potent than
the monomeric sugar sialoside 19, may represent a good compromise
between high affinity for CD22 on the one hand and synthetic
accessibility on the other. It remains to be determined how the
affinity for CD22 compares with that for other siglecs such as
sialoadhesin and P-selectin (20).
In the final stage of ligand design, we investigated whether the
affinity of multimeric compounds for CD22 can be further improved
beyond the bivalent level by proper spacing of the terminal glycosides.
Three tetravalent clusters of compound 19 were synthesized with
C4, C6, and C10 spacers
interconnecting the disubstituted glutamyls. Insertion of a flexible
elongated spacer did not further enhance the affinity. Apparently, the
observation that optimal binding to CD22 was already attained for
bivalent ligands is not caused by suboptimal spacing of the terminal
sugar moieties within a tri- or tetravalent cluster. The higher
flexibility and symmetry of the Tris cluster backbone may explain the
3-fold higher affinity of the Tris-based cluster 22 compared with the
glutamate cluster 21c.
In conclusion, multivalent ligands display a 10-fold higher affinity
for CD22 compared with the corresponding monovalent motifs regardless
of the terminal sugar moiety, the spacing of the terminal glycosides,
and the nature of the backbone. Optimal recognition was attained for
bivalent structures. To our knowledge, compound 22 is the most potent
antagonist for CD22 yet synthesized. Further evaluation of the
specificity of compound 22 for CD22 versus other sialoside-recognizing receptors such as myelin-associated glycoprotein, sialoadhesin, and the selectins is currently in progress.
Finally, our FACS and confocal laser scan microscopy studies confirm
that CD22 ligands are rapidly internalized, establishing the potential of compound 22 as a homing device for immunomodulatory
therapeutics and cytostatics to CD22-expressing cells.