Comblike dendrimers containing Tn antigen modulate natural killing and induce the production of Tn specific antibodies

Article abstract of DOI:10.1021/jm050741g

Comblike glycodendrimers were prepared by the chemoselective ligation of cysteine-modified glycopeptides (1-7) with a 3-maleimidopropionate-modified linear synthetic carrier (8). Glycodendrimers bearing mono-, di-, or tri-Tn clusters (9-11) were tested as inhibitors using plant and mammalian lectins. In the former group, the Codium fragile lectin showed moderate discrimination among 9, 10, and 11. In the latter group, A and B isoforms of rat NKR-P1 lectin strongly discriminated between 9 and 10. 10 caused a 4-fold increase in killing of the NK resistant tumor cell lines at concentrations as low as 10^-8 M. Surprisingly, 11 interacted exclusively with the rat NKR-P1B isoform and inhibited efficiently natural killing in both rats and humans, even in the presence of the activating compounds 9 and 10. Dinitrophenol haptenization or influenza virus hemagglutinin T-cell epitope conjugation increased the immunogenicity of the parent compounds and resulted in the production of Tn specific antibodies.

Full text of DOI:10.1021/jm050741g

6
400
J. Med. Chem. 2006, 49, 6400-6407
Comblike Dendrimers Containing Tn Antigen Modulate Natural Killing and Induce the
Production of Tn Specific Antibodies
,†
‡,§
‡
‡
|
⊥ ,∞
Pavel Vep rˇ ek,* Mari a´ n Hajd u´ ch, Petr D zˇ ub a´ k, Rostislav Kukl ´ı k, Jana Pol a´ kov a´ , and Karel Bezou sˇ ka
Group of Glycoconjugates, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, FlemingoVo n a´ m. 2,
1
66 10 Prague 6, Czech Republic, Laboratory of Experimental Medicine, and Department of Oncology, Faculty of Medicine, Palacky´
UniVersity and Faculty Hospital in Olomouc, 775 20, Czech Republic, Department of Organic Chemistry and Department of Biochemistry,
Faculty of Science, Charles UniVersity, HlaVoVa 8, 128 40 Prague 2, Czech Republic, and Institute of Microbiology, Academy of Sciences of
the Czech Republic, V ´ı de o` sk a´ 1083, 142 20 Prague 4, Czech Republic
ReceiVed July 29, 2005
Comblike glycodendrimers were prepared by the chemoselective ligation of cysteine-modified glycopeptides
(1-7) with a 3-maleimidopropionate-modified linear synthetic carrier (8). Glycodendrimers bearing mono-,
di-, or tri-Tn clusters (9-11) were tested as inhibitors using plant and mammalian lectins. In the former
group, the Codium fragile lectin showed moderate discrimination among 9, 10, and 11. In the latter group,
A and B isoforms of rat NKR-P1 lectin strongly discriminated between 9 and 10. 10 caused a 4-fold increase
-8
in killing of the NK resistant tumor cell lines at concentrations as low as 10 M. Surprisingly, 11 interacted
exclusively with the rat NKR-P1B isoform and inhibited efficiently natural killing in both rats and humans,
even in the presence of the activating compounds 9 and 10. Dinitrophenol haptenization or influenza virus
hemagglutinin T-cell epitope conjugation increased the immunogenicity of the parent compounds and resulted
in the production of Tn specific antibodies.
Introduction
branched, synthetic, low-molecular weight scaffolds have been
1
3
introduced. These molecules can generally be prepared by a
solid-phase synthesis. However, with the increasing molecular
weight of the target compound, incomplete sequences ac-
cumulate, and difficulties in isolation of the target compound
may arise. To provide precision and chemical unambiguity, a
chemoselective ligation based on assembly of unprotected
building blocks under mild aqueous conditions has been
Malignant transformation of cells often results in dramatic
changes in the abundance and structure of their surface
carbohydrates.
expressed carbohydrate antigens have been identified. Tn (R-
D-GalNAc-Ser/Thr) and T (R-D-Gal-1f3-R-D-GalNAc-Ser/Thr)
are of particular interest because they are overexpressed by the
largest group of malignant tumors. The Tn antigen is overex-
pressed in about 90% of human adenocarcinomas and also in
more then 70% of lung, colon, and stomach carcinomas, and
1
,2
A number of tumor-associated surface-
3,4
5
14-16
introduced.
Systematic clinical trials comparing the efficacy
of all these preparations have only recently been performed:
trimeric Tn clusters were found to be more efficient for
vaccination than Tn monosaccharide conjugates, and conjugates
to KLH were more effective than conjugates to BSA.17
Moreover, MUC1 glycosylated with Tn at three to five sites
per 20 amino acids and conjugated to KLH induced the strongest
antibody response against tumor cells expressing this antigen.17
the extent of its expression correlates with carcinoma dif-
ferentiation and aggressiveness.6
Since their original discovery, the potency of carbohydrate
antigens for vaccination against tumors has been evaluated using
natural sources of carbohydrate antigens such as OSM,a,7 semi-
synthetic conjugates of carbohydrate or glycopeptide antigens
8
,9
10
with KLH or BSA, semienzymatically produced glycosy-
To explore the potential of additional types of fully synthetic
compounds, we prepared tetrameric comblike dendrimers with
one to three copies of the serine Tn antigen (as a B-cell epitope)
on each branch. To maximize yields and purity, we used a
chemoselective ligation strategy, an approach based on the
assembly of modified building blocks in aqueous buffered
solution. We used a nucleophilic addition of cysteine-modified
glycopeptides to a 3-maleimidopropionate-modified linear KG
11
12
lated mucin sequences, and lipidated glycopeptides. To
eliminate some of the disadvantages of these preparations,
1
3
*
Corresponding author. Telephone: + 420 220 183 429. Fax: + 420
2
24 310 090. E-mail: veprek@uochb.cas.cz.
†
Group of Glycoconjugates, Academy of Sciences of the Czech Republic.
Laboratory of Experimental Medicine, Palack y´ University and Faculty
‡
Hospital in Olomouc.
§
18
Department of Oncology, Palack y´ University and Faculty Hospital in
tetramer. The binding capacity of the dendrimers was evaluated
Olomouc.
in lectin binding assays using four plant and two mammalian
lectins. To modulate the immunogenicity of these compounds,
we modified the parent compounds by N-terminal haptenization
|
|
Department of Organic Chemistry, Charles University.
⊥
Department of Biochemistry, Faculty of Science, Charles University.
Institute of Microbiology, Academy of Sciences of the Czech Republic.
Abbreviations: BSA, bovine serum albumin; BSM, bovine submaxillary
∞
a
19
with 2,4-dinitrophenol (DNP) or by C-terminal conjugation
mucin; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC, N,N′-Dicyclohexy-
lcarbodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; DNP, 2,4-dinitrophenol; ELISA, enzyme-linked immunosorbent
assay; ELLA, enzyme-linked lectin assay; HCTU, O-(1H-6-chlorobenzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOSu, N-
hydroxysuccinimide; HRP, horseradish peroxidase; IgA, immunoglobulin
A; IgG, immunoglobulin G; IgM, immunoglobulin M; IVH, influenza virus
hemagglutinin; KLH, keyhole limpet hemocyanin; NK, natural killer; OSM,
ovine submaxillary mucin; RP-HPLC, reverse phase high-performance liquid
chromatography; tR, retention time; TBTU, 2-(1H-benzotriazole-1-yl)-
with the T-cell epitope derived from the influenza virus
2
0
hemagglutinin (HA306-318, PKYVKQNTLKLAT). In the
present work, we show that our compounds are not only able
to elicit the production of specific antibodies but also act as
specific modulators of the activity of NK cells, which are
essential for efficient elimination of tumors.
Synthetic Strategy. Building blocks (1-7) were synthesized
following standard protocols of solid-phase synthesis using
1
,1,3,3-tetramethyluronium tetrafluoroborate.
1
0.1021/jm050741g CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/16/2006

Comblike Dendrimers Containing Tn Antigen
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6401
Table 1. Physicochemical Data of Comblike Glycopeptide Dendrimers (9-15)
puritya
(%)
molecular weight
tR
yield
(%)
compd
sequence
(min)b
found
calcdc
9
(Ac-Ser(Tn)-Gly3-Cys-NH2)4LCd
(Ac-[Ser(Tn)]2-Gly3-Cys-NH2)4LC
(Ac-[Ser(Tn)]3-Gly3-Cys-NH2)4LC
(Dnp-Ser(Tn)-Gly3-Cys-NH2)4LC
(Dnp-[Ser(Tn)]2-Gly3-Cys-NH2)4LC
(Dnp-[Ser(Tn)]3-Gly3-Cys-NH2)4LC
96.7
97.7
97.8
98.5
96.8
97.5
98.2
12.5
13.1
13.6
14.3
14.6
15.1
14.2
72
75
77
60
62
65
85
3896.35
5057.98
6218.07
4393.10
5554.15
6714.12
10999.98
3899.03
5060.15
6221.23
4395.37
5556.39
6717.47
11003.35
1
1
1
1
1
1
0
1
2
3
4
5
e
(Ac-[Ser(Tn)]2-Gly3-P -Cys-NH2)4LC
a
Determined by RP-HPLC. b Gradient 0-100% in 30 min with 2 min lag after sample injection (solvent A, 0.1% TFA in water; solvent B, 0.1% TFA
in MeOH; column used, Vydac 208TP53 C8 3.2 × 250 mm; flow rate ) 0.75 mL/min, λ ) 218 nm. Monoisotopic mass. d LC ) linear tetravalent
c
e
comblike carrier (compound 8). P ) T-cell epitope (PKYVKQNTLKLAT).
Fmoc/tBu chemistry. Amino acids were coupled in a 5-fold
molar excess using TBTU/DIEA activation with the exception
of Fmoc-Ser(3,4,6-tri-O-Ac-R-D-GalNAc)-OH, which was coupled
in 1.3-fold molar excess in order to save this valuable glyco-
sylated amino acid. In all cases the coupling reaction proceeded
to completeness in 2 h. O-Acetyl groups protecting the hydroxy
groups of the GalNAc residues were removed by treatment of
the glycopeptide resin with a 15% solution of hydrazine hydrate
in methanol (2 × 40 min). Cleavages from the resin and side
chain deprotections were achieved simultaneously using cleav-
age cocktails that depended on the amino acid composition.
Building block 8 was prepared according to the following
procedure. First, a linear KG peptide tetramer was synthesized
using Boc/Bn chemistry. Amino acids were coupled in 5-fold
molar excess using TBTU/DIEA activation. Cleavage from the
resin and side-chain deprotection were achieved simultaneously
by 1 h treatment with HF:anisole 9:1 (v/v). Second, the peptide
was then coupled in a solution of DMF and 10% NaHCO3 1:1
2D). This lectin discriminated between 9 and 10; compound
11 showed the same binding properties as compound 10. The
C. fragile lectin (Figure 2B) differentiated among all three tested
dendrimers and also showed the strongest binding affinity of
all lectins tested. The ability of the latter two lectins to
discriminate between the tested compounds was related to the
density of the carbohydrate coating, an effect that was most
evident for compounds 9 and 10 (Figure 2B,D). Altogether, the
C. fragile lectin displayed the best ability to discriminate
between the tested compounds and showed the highest differ-
ence between the binding of the monosaccharide and the
5
dendrimers (11 was 10 times more effective than D-GalNAc
monosaccharide).
To provide a preliminary biochemical assessment of the
ability of compounds 9-11 to raise the production of anti-Tn
antibodies, we tested their abilities to inhibit the binding of the
anti-Tn monoclonal antibody 83D4 to plates coated with asialo-
OSM. We were able to prove the dependence of the inhibitory
potencies of these glycodendrimers on the degree of sugar
substitution; while there has been very little inhibition by the
monosaccharides in this experimental system, the dendrimers
turned out to be efficient inhibitors with IC50 values ranging
(v/v) with freshly prepared succinimido 3-maleimidopropanoate
in 1.1-fold molar excess.
Glycopeptide dendrimers (9-15) were prepared by a chemose-
lective ligation strategy. This approach offers efficient and
effective preparation of high molecular weight compounds of
high homogeneity and is based on the assembly of purified
unprotected building blocks in buffered aqueous solution. For
the preparation of our comblike dendrimers we used a nucleo-
philic addition of the thiol group of the cysteine-modified
antigens (compounds 1-7) to an activated double bond of the
-5
-8
from 10 M for compound 9 up to 10 M for compound 11
(Figure 3A).
The endogenous mammalian lectins are proteins that are very
relevant to the potential in vivo application of compounds 9-11.
These receptors may be responsible for the therapeutic efficacy
of glycodendrimers due to their ability to activate the cells of
3
-maleimidopropionate-modified linear tetrameric KG carrier
2
2
the immune system and may influence their biodistribution
(compound 8). This procedure well-known from protein chem-
2
2
in the organism. Therefore, a major receptor of natural killer
cells critical for the activation of cytotoxic tumor killing
istry, which is compatible with the stability of the O-glycosidic
bond, worked well in our hands and provided target compounds
in good purity and yields (60-85%, Table 1). Structures of
prepared comblike dendrimers 9-11 are given in Figure 1.
During dialysis of the crude products we noticed that a portion
of compounds 12-14 remained irreversibly bound to the
membrane (probably due to the strong electrostatic interactions
of DNP groups with the material of the membrane), resulting
in lower yields.
2
2,23
lymphocytes, NKR-P1,
has been selected as the relevant
test system. Compounds 9-11 were tested as inhibitors of
binding of two isoforms of rat NKR-P1, NKR-P1A and NKR-
P1B, to their high affinity ligand â-D-GlcNAc23BSA neogly-
2
4
coprotein. In assays with rat NKR-P1A, compound 9 had
inhibitory activities comparable with the standard monosaccha-
ride ligand, D-GlcNAc (Figure 3B). However, compound 10 was
-
10
a much better inhibitor with IC50 of 10
M, ranking this
Biochemical Assessment. The binding properties of com-
pounds 9-11 were first evaluated using four commercially
available plant lectins with specificity for the D-GalNAc
compound itself among the high-affinity ligands for NKR-P1A.
Notably, the compound 11 was a very poor inhibitor for NKR-
P1A with an inhibitory activity even weaker than the negative
control, D-mannose (Figure 3B). When similar tests were
performed with rat NKR-P1B receptor, the results for com-
pounds 9 and 10 were very similar to those obtained with the
A isoform: compound 9 had activity comparable to that of
D-GlcNAc monosaccharide, while compound 10 had a very high
residue: Bandeiraea simplicifolia, Codium fragile, Dolichos
biflorus, and Vicia Villosa B4.21 The results of binding assays
are presented in Figure 2. We observed that the binding activities
of the comblike dendrimers were superior to those of the
standard carbohydrate inhibitor, D-GalNAc. Moreover, the
individual lectins differed in their ability to discriminate between
the tested dendrimers. B. simplicifolia and D. biflorus lectins
-10
inhibitory activity (IC50 of 3 × 10 M). However, the binding
of compound 11 to NKR-P1B was entirely different: not only
was there specific binding, but the IC50 was about 10 times lower
(Figure 2A,C) showed no or negligible discrimination. Some
ability to discriminate was found for the V. Villosa lectin (Figure

6
402 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Vep rˇ ek et al.
-
11
than measured for compound 10 (IC50 of 11 ) 3 × 10
M;
see Figure 3C).
Immunological Evaluation. Tn-antigen-containing structures
-15 were tested for their ability to elicit an immune response
9
in Balb/c mice (Table 2). The results indicate that Tn antigen
conjugated with a small and defined immunodominant molecule
(DNP) or a T-cell peptide epitope (HA306-318) induces a
humoral immune response. Interestingly, the highest titers of
anti-Tn IgG antibodies were detected in the serum of IVH
presensitized animals treated with compound 15. Presensitization
of these animals with IVH did not substantially increase anti-
Tn IgG antibody levels but rather increased the production of
anti-Tn IgM and IgA antibodies. Even more interestingly,
presensitization of animals with DNP resulted in a dominant
increase of anti-Tn IgA antibodies, which has not been previ-
ously reported. Furthermore, anti-Tn IgG and IgM antibodies
were tested for their ability to bind to Tn-positive Jurkat cells
at equivalent concentrations (50% of their maximum antibody
titer as determined by ELISA). The results are presented in Table
3
. Cell-staining capacity was generally higher for IgM anti-Tn
antibodies and further increased in the animals presensitized
with DNP/IVH and vaccinated with DNP/IVH-conjugated Tn
antigen.
Since the dendrimers 9 and 10 both had a high affinity for
NKR-P1A (activating) and NKR-P1B (inhibitory) receptors, it
was necessary to perform a test of cytotoxicity to evaluate the
possible in vivo activities. For this purpose, two rodent leukemic
cell lines, an NK-sensitive line (YAC-1) and NK-resistant line
(
P815), were employed. When compared to the control,
compound 9 stimulated the natural killing of both YAC-1 and
P815 targets (Figure 4,parts A and D, respectively) in a dose-
dependent manner, but the enhancement of natural killing was
more profound in the case of the NK-resistant P815 cell line
-
6
-8
(
nearly 3-fold increase in natural killing using 10 -10
M
of compound 9 compared to control, Figure 4D). The effects
of compound 10 on natural killing were very similar, except
that the activation potential was even higher (Figure 4B,E) and
there was a nearly 4-fold increase in natural killing in the case
of the NK-resistant cell line P815 (Figure 4E). However, the
effect of compound 11 on natural killing was exactly the
opposite; this compound efficiently inhibited natural killing in
both tested cell lines (Figure 4C,F).
Because of the different impacts our compounds had on
natural killing, we investigated their combined effects. We
therefore tested the influence of the individual compounds and
their combinations on natural killing of the NK-resistant rat cell
-
8
line P815 at an effector:target cell ratio of 30:1 and 10
M
concentrations. It is evident that the inhibitory effect of
compound 11 is predominant (Figure 4G). This dendrimer
inhibited natural killing efficiently in the presence of equimolar
concentration of activating dendrimer 9 or 10 and even in the
presence of a combination of 9 and 10 at equimolar concentra-
tions. Furthermore, to evaluate the immunological effects of the
dendrimers from the standpoint of their potential use in human
therapies, the latter test was repeated using the human NK-
resistant cell line RAJI, and very similar results were obtained
for this human cell line (Figure 4H).
Discussion
We have shown previously that â-N-acetyl-D-hexosamine-
Figure 1. Structure of comblike dendrimers: 9 (R ) acetyl) and 12
R ) 2,4-dinitrophenyl) (A); 10 (R ) acetyl), 13 (R ) 2,4-
dinitrophenyl), and 15 (R ) acetyl, P ) PKYVKQNTLKLAT peptide
sequence) (B); 11 (R ) acetyl) and 14 (R ) 2,4-dinitrophenyl) (C)
prepared by chemoselective ligation.
coated dendrimers have the potential to be efficient tools in
(
2
3
experimental tumor therapies. These compounds are targeted
to the tumor neovascular epithelial linings and efficiently recruit
2
3
the killer lymphocytes into the tumor sites. We have also

Comblike Dendrimers Containing Tn Antigen
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6403
Figure 2. Inhibitory activities of the comblike dendrimers using model plant lectins. Dendrimers 9-11 were used together with a standard carbohydrate
D-GalNAc) as inhibitors of binding of B. simplicifolia (A), C. fragile (B), D. biflorus (C), and V. Villosa (D) lectins to microtiter plates coated with
desialylated bovine submaxillary mucin. Results are presented as average values ( range from duplicate experiments.
(
Table 2. Production of Serum Anti-Tn Antibodies Evaluated Using
ELISA
2
log of antibody titer of anti-Tn antibodies ((SD)
vaccination
antigen
sensitizationa
IgG
IgM
IgA
-
-
-
9
-
5.83 ( 0.43
5.52 ( 0.57
6.08 ( 0.61
8.22 ( 0.73
9.57 ( 1.04
10.12 ( 1.13
7.83 ( 0.81
8.17 ( 0.85
10.30 ( 0.74
12.68 ( 1.08
14.16 ( 1.17
15.67 ( 1.37
16.83 ( 1.52
18.26 ( 1.93
4.87 ( 0.33
4.99 ( 0.51
4.38 ( 0.36
5.30 ( 0.59
5.74 ( 0.62
5.90 ( 0.65
5.41 ( 0.58
5.96(0.53
6.29 ( 0.64
6.03 ( 0.59
6.61 ( 0.72
7.32 ( 0.71
5.74 ( 0.61
15.27 ( 1.63
3.27 ( 0.29
3.64 ( 0.35
3.84 ( 0.40
4.17 ( 0.39
4.49 ( 0.48
4.63 ( 0.41
5.01 ( 0.43
5.20 ( 0.46
5.89 ( 0.53
12.27 ( 1.34
13.84 ( 1.30
17.62 ( 1.85
5.30 ( 0.57
8.16 ( 0.83
DNP
IVH
-
10
11
12
13
14
12
13
14
15
15
-
-
-
-
-
DNP
DNP
DNP
-
IVH
a
Prior to the immunization, animals were injected with five doses of
0
.15% DNP at 1-week intervals or two doses of 50 µL of a commercial
influenza virus at 2-week intervals.
critical role of the density and mode of presentation of the
carbohydrate ligand in polyamidoamine-type dendrimers.23 In
the light of these results, it appeared interesting to evaluate
alternative ways of presentation of the active carbohydrate
epitopes, and comblike dendrimers seemed an attractive pos-
sibility.
For initial evaluation of the biological activities of glyco-
dendrimers, binding assays with plant lectins of the appropriate
specificity are often used. The technique employed is usually a
plate binding or plate inhibition assay. The latter assay is
preferred because it allows testing of a wide range of chemical
compounds independently of their molecular size and polydis-
persity. We employed an inexpensive assay in which the plates
were coated with asialo-OSM to provide the binding surface
for selected plant lectins. The synthesized comblike dendrimers
served as the inhibitors in this assay. To the best of our
knowledge, this is the first study in which binding activities of
carbohydrate-coated dendrimers have been extensively evalu-
ated. Even though the inhibitory activities of our compounds
were higher than those of the standard monosaccharide inhibitors
for all four plant lectins, only two of the lectins had the ability
Figure 3. Inhibitory activities of the comblike dendrimers using
monoclonal anti-Tn antibody 83D4 (A) and two isoforms of the
recombinant soluble rat NK cell receptors, NKR-P1A (B) and NKR-
P1B (C). GlcNAc and Man were used as a positive and a negative
control, respectively. Results are presented as average values ( range
from duplicate experiments.
proven the ability of these compounds to significantly activate
NK cells and natural killing through their high-affinity binding
to the NKR-P1A (activating) receptors at the surface of NK
cells. In vivo tumor treatment experiments using mouse
melanoma and rat colorectal carcinoma models proved the

6
404 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Vep rˇ ek et al.
Table 3. Flow Cytometric Analysis of Anti-Tn Antibodies Bound to the Surface of Tn-Positive Jurkat Cells
Anti-Tn antibodies FACS (% of gated cells) ((SD)
IgG IgM
vaccination
antigen
sensitizationa
pretreatment
post-treatment
pretreatment
post-treatment
-
-
-
9
-
10.2 ( 3.21
9.9 ( 2.84
10.6 ( 2.93
11.8 ( 4.27
9.8 ( 3.57
10.4 ( 2.11
10.7 ( 3.80
10.1 ( 2.21
9.3 ( 2.04
10.0 ( 3.77
10.1 ( 2.07
9.75 ( 2.02
10.5 ( 2.67
9.2 ( 2.93
9.7 ( 2.43
11.4 ( 3.08
8.9 ( 3.87
9.2 ( 1.81
10.9 ( 2.40
10.5 ( 4.12
10.33 ( 2.4
10.4 ( 2.15
9.63 ( 2.19
11.0 ( 1.97
9.7 ( 3.15
10.5 ( 3.25
11.4 ( 2.32
10.7 ( 2.95
11.0 ( 1.94
10.7 ( 1.88
10.6 ( 3.06
10.1 ( 3.48
9.2 ( 3.52
DNP
IVH
-
11.7 ( 3.35
23.1 ( 6.19
28.9 ( 8.33
27.1 ( 7.73
26.0 ( 6.58
31.8 ( 9.83
33.9 ( 10.22
41.3 ( 9.44
44.8 ( 11.75
43.4 ( 11.55
25.3 ( 7.20
30.8 ( 6.88
17.9 ( 4.10
29.3 ( 7.38
25.8 ( 6.87
22.4 ( 8.41
27.7 ( 7.74
28.6 ( 7.12
35.2 ( 7.83
38.4 ( 12.25
33.6 ( 8.96
35.8 ( 9.62
38.9 ( 9.36
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
2
3
4
5
5
-
-
-
-
-
DNP
DNP
DNP
-
IVH
a
Prior to the immunization animals were injected with five doses of 0.15% DNP at 1-week intervals or two doses of 50 µL of a commercial influenza
virus at 2-week intervals.
We assume that this is due to the structural complementarity
2
6
of the Tn dimer and lectin binding groove. Similarly, our
results obtained with the V. Villosa lectin seem to agree with
the affinities of Tn1-Tn3 glycopeptides measured previously
using surface plasmon resonance; the affinity increased with
increasing degree of substitution, but the increase was more
dramatic from the monosubstituted to the disubstituted com-
pound than from the disubstituted to the trisubstituted com-
2
7
pound.
In addition to plant lectins, the comblike dendrimers were
also tested using a mammalian lectin system, namely the NKR-
P1 receptor of natural killer cells. In rodent natural killer cells,
these receptors occur as several isoforms: NKR-P1A, -B, and
-
D. These isoforms are functionally interesting, since NKR-
P1A is the major activating receptor of rodent (rat and mouse)
NK cells, while NKR-P1B has inhibitory effects.28 We have
shown previously that these isoforms have distinct binding
specificities toward both simple and complex oligosaccharide
2
8
structures. According to our findings, we did not expect that
these receptors would interact with the R-D-GalNAc-containing
dendrimers. However, we observed surprising binding affinities
of these compounds toward both A and B isoforms of NKR-
P1. While compounds 9 and 10 both bound strongly to both
NKR-P1 isoforms, compound 11 displayed a unique and
selective affinity for NKR-P1B, making it one of the best ligands
identified for this NKR-P1 isoform to date. These results are in
sharp contrast to data obtained previously with polyamidoamide-
based dendrimers that reacted only with the A isoform.28 Thus,
while compounds 9 and 10 cannot selectively target any specific
isoform of NKR-P1, compound 11 is the best reagent available
so far for the selective targeting of NKR-P1B. The exact manner
in which the comblike dendrimers described here interact with
the carbohydrate-binding groove of NKR-P1 receptors will
require further investigation, but the R-anomeric configuration
of D-GalNAc is quite possibly an important component of this
recognition mechanism. We speculate that while the extended
oligosaccharide sequences require â-linked sugar units in order
to fit horizontally into the binding grooves of these receptors,
the R-linked D-GalNAc units of the tested dendrimers may
Figure 4. Comblike dendrimers 9 (panels A and D) and 10 (panels B
and E) enhance and 11 (panels C and F) inhibits natural killing of NK-
sensitive tumor cell line YAC-1 (A-C) and NK-resistant tumor cell
line P815 (D-F) by rat NK cells. Compounds were dissolved in PBS
(
pH 7.4) and tested at the indicated concentrations together with the
control sample (PBS only) at an effector:target cell ratio given in each
panel. The combined effects of the dendrimers were also tested using
NK-resistant mouse tumor cell line P815 (G) and human tumor cell
-
8
line RAJI (H) at an E:T ratio of 30:1. Compounds were tested at 10
M concentrations.
2
3
interact with the groove vertically. This mode of interaction
would be less sensitive to amino acids interrupting the integrity
of the groove, and as shown in Figure 3, may produce interesting
to discriminate between our compounds with varying degrees
of carbohydrate substitution. The results obtained for assays with
the C. fragile lectin correlate with previous findings about the
ability of this lectin to bind an R-D-GalNAc residue positioned
2
3
selectivity.
Vaccines designed to induce an optimal antibody response
have several components, all of which must be optimized. The
2
5
on the reducing or nonreducing end of the saccharide moiety.

Comblike Dendrimers Containing Tn Antigen
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6405
first component is the antigen itself, which must closely
resemble its native form on the target cell, in this case, Tn
expression on tumor mucins. While the Tn antigen is a
monosaccharide covalently linked to serine or threonine, mono-
clonal antibodies and sera selected for preferential reactivity
bination of the effective humoral immune response with
induction of NK cell activity, as demonstrated for compound
10, should translate into an effective antitumor immune response
in further in vivo experiments.
Our unique findings with regard to compound 11, which not
only reacted selectively and strongly with the inhibitory receptor
NKR-P1B but also efficiently inhibited natural killing in both
rodent and humans, open new avenues in the development of
effective inhibitors of NK and NK/T cells in pathological
situations connected with overactivation of these cells. In
particular, recent findings demonstrate that iVR14 NK/T cells
29
with cancer cells usually interact with clusters of such antigens.
The second component is the carrier molecule, usually an
immunogenic protein, like KLH or BSA. In our study this part
of the vaccine was substituted by linking one to three copies of
Tn antigen to a comblike synthetic peptide backbone using a
completely synthetic approach, which resulted in the preparation
of fully immunocompetent antigens. As anticipated, the best
immune response was obtained using constructs with a cluster
of three Tn antigens, while vaccine immunogenicity was lower
when one or two Tn molecules were used (Tables 2 and 3).
The third component of the vaccine is an immunologic adjuvant
molecule. A synthetic vaccine containing Tn antigen linked to
a T-cell epitope peptide derived from ovalbumin and able to
activate T cells has only recently been described.3 For this
reason, we developed an alternative approach that combines a
tumor-associated Tn antigen with the hapten DNP (12-14) or
with an immunodominant T cell epitope from IVH designated
HA306-318 (15). Conjugation of the Tn antigen with HA306-
3
2
are responsible for allergen-induced airway inflammation,
a
33
cardinal feature of allergic asthma. Thus, the effective inhibi-
tion of NK cells by compound 11 may be exploited under these,
and potentially other, pathological conditions.
Conclusions
The compounds described in this report display surprising
reactivities with rat NKR-P1A and NKR-P1B receptors. These
findings were corroborated by the unique effects of these
compounds on natural killing. The major factors that influenced
the effective immune response against these compounds in-
clude: the number of Tn repetitive sequences, spatial arrange-
ment of Tn antigen in comblike dendrimers, and conjugation
of Tn antigen with immunoadjuvant molecules. The chemical
syntheses of complex glycopeptide conjugates described here
make these intriguing compounds available for the development
of vaccines against tumors and as potential antiallergic com-
pounds.
0
318 itself resulted in production of substantially higher antibody
titers compared to unconjugated Tn vaccines. Interestingly, IVH
sensitization further increased the anti-Tn antibody titer, par-
ticularly in the IgA and IgM classes (Table 2).
Our rationale for the haptenization approach was based on
positive results of DNP-modified tumor vaccines in human
31
melanoma clinical trials. These trials indicated that DNP
modification is a clinically feasible, safe, and effective procedure
that induces significant patient antitumor response. This ap-
proach relies on the availability of the autologous tumor cells
as a source of tumor rejection antigens and the ability of
haptenization to render immunogenic proteins to which the host
is otherwise unresponsive. To the best of our knowledge, our
results provide the first example of DNP conjugation to a Tn
antigen. Consistent with the published data, DNP sensitization
followed by application of haptenized Tn vaccine significantly
increased anti-Tn antibody production, especially within the IgG
and IgA classes (Table 2).
Experimental Section
Chemicals and Materials. Acetonitrile, dichloromethane, N,N-
dimethylformamide, methyl tert-butyl ether, trifluoroacetic acid,
H-â-Ala-OH, N-hydroxysuccinimide, maleic anhydride, and 2,4-
dinitrophenylfluoride were purchased from Fluka. Fmoc-Ala-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Pro-
OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Val-OH, Fmoc-γAbu-OH, and Fmoc-Lys(Boc)-OH were
obtained from IRIS Biotech GmbH (Marktredwitz, Germany). Boc-
Gly-OH, Boc-Lys(2-Cl-Bzl)-OH, and Rink amide MBHA resin
2
(0.57 mmol of NH /g) were purchased from Calbiochem-Nova-
biochem. B. simplicifolia, C. fragile subspecies tomentosides, D.
biflorus, V. Villosa B4, OSM, and BSA were purchased from Sigma-
Aldrich. Iodine-125 (IMS30, 10 mCi, 370 MBq) was purchased
from Amersham Biosciences (UK, London). Anti-Tn monoclonal
antibody 83D4 (IgM, κ)34 was a gift from Dr. Luca Vannucci of
the Institute of Microbiology, Prague, Czech Republic.
Amino acid analyses were performed on a BIOCHROM-20
PHARMACIA amino acid analyzer (Biotech). Samples were
hydrolyzed in sealed glass tubes under nitrogen using 6 M HCl at
The ability of our carbohydrate dendrimers to elicit antibody
production is promising for the development of a therapeutic
anticancer vaccine. However, activation of cellular immune
responses, especially by T cells, NK/T cells, and NK cells, is
of importance for permanent protection against cancer. There-
fore, we tested the potency of our compounds to activate the
killer lymphocytes. In the short-time cytotoxicity assays that
measure mostly the activities of NK cells, both compounds 9
and 10 exerted significant activation effects (Figure 4). The
ability of these compounds to activate natural killing of the
resistant tumor cell lines in both rodents and humans (P815 and
RAJI, parts G and H of Figure 4, respectively) is of particular
importance. While the molecular mechanism for this activation
in rodents and in humans may differ (in humans, the NKR-P1
is not an activating NK cell receptor, and the comblike
dendrimers tested here did not react with human NKR-P1; data
not shown), these results point to the potential of the newly
synthesized compounds not only for use in animal experimental
therapy models but also for use in human cancer therapies. These
findings open new doors for biological testing of this compound
in vivo, which is interesting in the light of the previously
reported significant antitumor properties of GlcNAc-coated
polyamidoamide-type dendrimers.23 We also expect that com-
1
10 °C for 20 h. Analytical RP-HPLC was performed on a Waters
Alliance HPLC system (PDA 996 detector, software Millenium
Chromatography Manager) using a Vydac C
8
(208TP53, 3.2 × 250
mm) column. Conditions were as follows: gradient 0-100% in
30 min with 2 min lag after sample injection (solvent A, 0.1% TFA
in water; solvent B, 0.1% TFA in MeOH) flow rate ) 0.75 mL/
min, λ ) 218 nm. The structures were confirmed by ESI-MS [LCQ
ion-trap mass spectrometer (Finnigan Corp.), 4000 controlled unit
(
Thermo Separation Product, Inc.), Xcalibur software (Finnigan
Corp.); positive mode] and MALDI-TOF MS (Reflex IV, Bruker
Daltonics; positive mode, R-cyano-4-hydroxycinnamic acid used
as a matrix).
Asialo-OSM was prepared by 1 h treatment of OSM (100 mg
in 1 mL of distilled water with 1% TFA). The reaction mixture
was then lyophilized from water three times.
Fmoc-Ser(3,4,6-tri-O-Ac-r-D-GalNAc)-OH was prepared ac-
cording to the described procedure to yield 1.1 g (7%) of white

6
406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Vep rˇ ek et al.
lyophilizate.35 1H NMR (DMSO-d
): δ 1.80 (s, 3H), 1.89 (s, 3H),
vaccine (Begrivac, Boehringer Mannheim) at 2-week intervals.
Vaccination with synthetic Tn antigens was performed 1 week after
the last dose of DNP or IVH. Control groups received the vehicle
and aluminum hydroxide (control) or were presensitized with IVH/
DNP as described above. Serum was obtained from the treated mice
2 weeks after the second dose of the synthetic Tn vaccine.
6
1
.94 (s, 3H), 2.10 (s, 3H), 3.76 (dd, J ) 10.8, 5.2 Hz, 1H), 3.82
(
(
dd, J ) 10.8, 4.0 Hz, 1H), 3.96 (dd, J ) 11.6, 7.2 Hz, 1H), 4.04
dd, J ) 11.2, 6.0 Hz, 1H), 4.15-4.38 (m, 6H), 4.84 (d, J ) 3.7
Hz, 1H), 5.04 (dd, J ) 11.6, 3.2 Hz, 1H), 5.29 (d, J ) 2.4 Hz,
1
H), 7.30-7.92 (m, 10 H). Anal. Calcd for C32
36 2
H N O13: C, 58.53,
H, 5.53, N, 4.27. Found C 58.28, H 5.56, N 4.11.
Analysis of Serum Anti-Tn Antibodies. Antibody levels were
Synthesis of Building Blocks 1-8. Compounds 1-7 were
synthesized on RINK amide MHBA resin using the 9-fluorenyl-
methoxycarbonyl/tert-butyl (Fmoc/tBu) strategy, and the compound
38,39
determined by ELISA as described elsewhere.
animal sera were diluted with phosphate-buffered saline (PBS, pH
.4) and incubated with BSM-coated plates (Sigma-Aldrich).
Briefly, individual
7
8
was prepared on MHBA resin using the tert-butyloxycarbonyl/
benzyl (Boc/Bn) strategy. Protected amino acids were coupled in
-fold molar excess using TBTU/DIEA activation. Fmoc-Ser(3,4,6-
Specific classes of immunoglobulins were identified using the
different goat anti-mouse IgG/IgM/IgA antibodies conjugated to
HRP (Sigma-Aldrich). Titers of specific antibodies were determined
as the last serum dilution where the absorbance was higher than 2
5
tri-O-Ac-R-D-GalNAc)-OH was coupled in 1.3-fold molar excess
using TBTU/DIEA activation. The O-acetyl groups protecting the
hydroxy groups of the GalNAc residues were removed by treatment
of the glycopeptide resin with a 15% solution of hydrazine hydrate
in methanol (2 × 40 min). Cleavage from the resin and side chain
deprotection were achieved simultaneously using cleavage cocktails
that depended on the amino acid composition. Pure compounds
were obtained after RP-HPLC in yields ranging from 28% to 48%.
Identity and purity of the compounds were confirmed by amino
acid analysis, RP-HPLC, and ESI mass spectrometry. For more
details see the Supporting Information.
Preparation of Comblike Dendrimers 9-15. Compounds 1-7
were dissolved in 500 µL of a 50% solution of 15 mM phosphate
buffer (pH 6.7) in DMF. Linear carrier 8 was dissolved in 250 µL
of DMF and added in three aliquots to the solution of corresponding
building block 1-7 (effective molar ratio 4.05:1 of the respective
compound to the linear KG carrier 8). Progress of the ligation
×
SD (standard deviation) relative to control wells.
Cytotoxicity Assays. The standard short-term (4 h) cytotoxic
assays were performed and evaluated essentially as previously
described.40
Flow Cytometric Analysis. Fluorescence-activated cell sorting
(
FACS) was performed using a modification of the method of
41
Slovin et al. to demonstrate antibody binding to the cell surface
of Jurkat cells, a human T lymphoid leukemic cell line known to
express Tn.42 Pre- and postimmunization sera were diluted to 50%
of their maximum antibody titer as determined by ELISA and
6
incubated with 10 Jurkat cells for 1 h on ice. The cells were
washed, treated with rat monoclonal anti-mouse IgG/IgM antibody
labeled with fluorescein isothiocyanate (BD Bioscience), and
analyzed by flow cytometry. Pretreatment sera-treated samples were
gated to the reactivity of 10% positive cells and compared with
post-treatment positivity.
36
reaction was monitored by RP-HPLC (12-14) or by Ellman’s test
9-11 and 15). The reaction was typically completed within 12-
4 h. The reaction mixture was then poured into 5 volumes of water
(
2
Acknowledgment. Dedicated to Prof. Jan Kocourek on the
and lyophilized. Crude products were dissolved in 5% acetic acid,
dialyzed using SpectraPor tubes 1,000 MWCO (“molecular weight
cutoff”; Spectrum Laboratories, Inc. Rancho Dominguez, CA) for
occasion of his 80th birthday. We are grateful to H. Hoffmann
(Institute of Organic Chemistry and Biochemistry, Czech
Republic) and J. Neuzil (Griffith University, Australia) and to
the reviewers for their critical reading of the manuscript and
stimulating suggestions. We thank Anna Jano sˇ t’ a´ kov a´ for
technical assistance in the analysis of serum Tn-antibodies. The
work on this project was supported by the Ministry of Education
of the Czech Republic (MSM 6198959216, MSM 113100001,
MSM 21620808), by the Grant Agency of the Academy of
Sciences of the Czech Republic (A4020213 and A5020403),
Grant Agency of the Czech Republic (304/06/1691), and by an
Institutional Research Concept for the Institute of Organic
Chemistry and Biochemistry (Z4 055 0506) and the Institute
of Microbiology (AVOZ50200510).
2
× 12 h, and lyophilized. Identity and purity were confirmed by
amino acid analysis, RP-HPLC, and MALDI-TOF mass spectrom-
etry (see the Supporting Information).
Binding Assays. Plant lectins and soluble recombinant NKR-
P1A and -B24 were radiolabeled with Na125I using Iodogen (Pierce,
Rockville, IL). Binding assays were performed as described
previously.24 Briefly, 96-well poly(vinyl chloride) microplates
(
Titertek Immuno Assay-Plate, ICN Flow, Irvine, Scotland) were
coated overnight at 4 °C with 50 µL of ligand (100 µg/mL) in 10
mM Tris-HCl, 150 mM NaCl, 1mM CaCl , and 1 mM NaN . The
plates were blocked with 1% BSA in the above buffer for 2 h at 4
C, incubated with the radiolabeled protein at a concentration
2
3
°
corresponding to half of the saturating amount and various dilutions
of the inhibitors (total reaction volume of 100 µL), washed three
times with Tris buffer (see above), and drained. A scintillation
solution (100 µL) was added, and the radioactivity in the individual
wells was assessed using a â-counter (Microbeta, Wallac, Turku,
Finland). All experiments were performed in duplicate and the
degree of inhibition was calculated with respect to the control wells
Supporting Information Available: Synthetic procedures for
the preparation of building blocks (1-8) and analytical data of the
prepared glycopeptides and glycodendrimers (1-15). This material
is available free of charge via the Internet at http://pubs.acs.org.
(wells containing no inhibitor). Binding assays with the monoclonal
References
antibody 83D4 were performed using the same methodology.
Animals and Immunization Procedures. Eight week old female
Balb/c mice (AnLab) were used for immunization. Animals were
bred in a pathogen-free animal facility with food and water ad
libitum and treated with two doses of synthetic vaccines (100 µg/
dose at 2-week intervals) adsorbed on 1 mg of aluminum hydroxide
(
1) Byrd, J. C.; Bresalier, R. S. Mucins and mucin binding proteins in
colorectal cancer. Cancer Metastasis ReV. 2004, 23, 77-99.
(2) Hakomori, S. I. Tumor malignancy defined by aberrant glycosylation
and sphingo(glyco)lipid metabolism. Cancer Res. 1996, 56, 5309-
5318.
(
3) Toyokuni, T.; Singhal, A. K. Synthetic carbohydrate vaccines based
on tumor-associated antigens. Chem. Soc. ReV. 1995, 24, 231-242.
4) Springer, G. F. T and Tn pancarcinoma markers: Autoantigenic
adhesion molecules in pathogenesis, prebiopsy carcinoma-detection,
and long-term breast carcinoma immunotherapy. Crit. ReV. Oncog.
37
(Sigma-Aldrich) per dose as an adjuvant. To investigate the effect
(
of DNP haptenization of the Tn vaccines, mice were sensitized to
DNP by five weekly administrations of a 0.15% DNP solution in
acetone and olive oil (3:1) applied to the skin of both ears. The
majority of animals responded to the last dose of DNP with a local
inflammatory reaction. The significance of Tn antigen conjugation
with the IVH T-cell epitope (HA306-318) was also evaluated on
IVH-sensitized animals. For this purpose, mice were immunized
with two doses (50 µL per dose) of a commercial influenza virus
1995, 6, 57-85.
(
5) Kumar, S. R.; Sauter, E. R.; Quinn, T. P.; Deutscher, S. L. Thomsen-
Friedenreich and Tn antigens in nipple fluid: Carbohydrate bior-
narkers for breast cancer detection. Clin. Cancer Res. 2005, 11,
6868-6871.
(6) Springer, G. F. T and Tn, general carcinoma auto-antigens. Science
1984, 224, 1198-1205.

Comblike Dendrimers Containing Tn Antigen
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6407
(
7) O’Boyle, K. P.; Coatsworth, S.; Anthony, G.; Ramirez, M.; Green-
wald, E.; Kaleya, R.; Steinberg, J. J.; Dutcher, J. P.; Wiernik, P. H.
Effects of desialylation of ovine submaxillary gland mucin (OSM)
on humoral and cellular immune responses to Tn and sialylated Tn.
Cancer Immun. 2006, 6, 5-13.
(26) Wu, A. M.; Song, S. C.; Hwang, P. Y.; Wu, J. H.; Chang, K. S. S.
Binding-studies on the combining site of a GalNAcR1f-specific
lectin with Thomsen-Friedenreich activity prepared from green
marine-algae Codium fragile subspecies tomentosoides. Eur. J.
Biochem. 1995, 233, 145-151.
(27) Osinaga, E.; Bay, S.; Tello, D.; Pritsch, O.; Assemat, K.; Cantacuzene,
D.; Nakada, H.; Alzari, P. Analysis of the fine specificity of Tn-
binding proteins using synthetic glycopeptide epitopes and biosensor
based on surface plasmon resonance spectroscopy. FEBS Lett. 2000,
469, 24-28.
(28) Plihal, O.; Byrtusova, P.; Pavlicek, J.; Mihok, L.; Ettrich, R.; Man,
P.; Pompach, P.; Havlicek, V.; Husakova, L.; Bezouska, K. The
isoforms of rat natural killer cell receptor NKR-P1 display a distinct
binding of complex saccharide ligands. Collect. Czech. Chem.
Commun. 2004, 69, 631-644.
(29) Nakada, H.; Numata, Y.; Inoue, M.; Tanaka, N.; Kitagawa, H.;
Funakoshi, I.; Fukui, S.; Yamashina, I. Elucidation of an essential
structure recognized by an anti-GalNAc-aplha-Ser}Thr] monoclonal
antibody (MLS-128). J. Biol. Chem. 1991, 266, 12402-12405.
(30) Peri, F.; Cipolla, L.; Rescigno, M.; La Ferla, B.; Nicotra, F. Synthesis
and biological evaluation of an anticancer vaccine containing the
C-glycoside analogue of the Tn epitope. Bioconjugate Chem. 2001,
12, 325-328.
(31) Sojka, D. K.; Felnerova, D.; Mokyr, M. B. Anti-metastatic activity
of hapten-modified autologous tumor cell vaccine in an animal tumor
model. Cancer Immunol. Immunother. 2002, 51, 200-208.
(32) Lisbonne, M.; Diem, S.; Keller, A. D.; Lefort, J.; Araujo, L. M.;
Hachem, P.; Fourneau, J. M.; Sidobre, S.; Kronenberg, M.; Taniguchi,
M.; Van Endert, P.; Dy, M.; Askenase, P.; Russo, M.; Vargaftig, B.
B.; Herbelin, A.; Leite-de-Moraes, M. C. Cutting edge: Invariant V
alpha 14 NKT cells dare required for allergen-induced airway
inflammation and hyperreactivity in an experimental asthma model.
J. Immunol. 2003, 171, 1637-1641.
(
8) Danishefsky, S. J.; Allen, J. R. From the Laboratory to the clinic: A
retrospective on fully synthetic carbohydrate-based anticancer cac-
cines. Angew. Chem., Int. Ed. 2000, 39, 836-863.
(
9) Sandmaier, B. M.; Oparin, D. V.; Holmberg, L. A.; Reddish, M. A.;
MacLean, G. D.; Longenecker, B. M. Evidence of a cellular immune
response against sialyl-Tn in breast and ovarian cancer patients after
high-dose chemotherapy, stem cell rescue, and immunization with
theratope STn-KLH cancer vaccine. J. Immunother. 1999, 22, 54-
66.
(
(
(
10) Kunz, H.; Birnbach, S. Synthesis of O-glycopeptides of the tumor-
associated Tn- and T-antigen type and their binding to bovine serum
albumin. Angew. Chem., Int. Ed. Engl. 1986, 25, 360-362.
11) Freire, T.; Lo-Man, R.; Piller, F.; Piller, V.; Leclerc, C.; Bay, S.
Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for
antitumor vaccination. Glycobiology 2006, 16, 390-401.
12) Buskas, T.; Ingale, S.; Boons, G. J. Towards a fully synthetic
carbohydrate-based anticancer vaccine: Synthesis and immunological
evaluation of a lipidated glycopeptide containing the tumor-associated
Tn antigen. Angew. Chem., Int. Ed. 2005, 44, 5985-5988.
13) Veprek, P.; Jezek, J. Peptide and glycopeptide dendrimers. Part I. J.
Pept. Sci. 1999, 5, 5-23.
(
(
(
14) Tam, J. P.; Yu, Q.; Miao, Z. Orthogonal ligation strategies for peptide
and protein. Biopolymers 1999, 51, 311-332.
15) Langenhan, J. M.; Thorson, J. S. Recent Carbohydrate-based
chemoselective ligation applications. Curr. Org. Synth. 2005, 2, 59-
81.
(
16) Niederhafner, P.; Sebestik, J.; Jezek, J. Peptide dendrimers. J. Pept.
Sci. 2005, 11, 757-788.
(
17) Kagan, E.; Ragupathi, G.; Yi, S. S.; Reis, C. A.; Gildersleeve, J.;
Kahne, D.; Clausen, H.; Danishefsky, S. J.; Livingston, P. O.
Comparison of antigen constructs and carrier molecules for augment-
ing the immunogenicity of the monosaccharide epithelial cancer
antigen Tn. Cancer Immunol. Immunother. 2005, 54, 424-430.
18) Nefzi, A.; Sun, X. C.; Mutter, M. Chemoselective ligation of
multifunctional peptides to topological templates via thioether
formation for TASP synthesis. Tetrahedron Lett. 1995, 36, 229-
(33) Dombrowitz, D. Exploiting the innate immune system to control
allergic asthma. Eur. J. Immunol. 2005, 35, 2786-2788.
(34) Osinaga, E.; Pancico, G.; Porchet, N.; Mistro, D.; Aubert, J. P.;
Roseto, A. Analysis of the carcinoma-associated epitope defined by
monoclonal antibody 83D4. Breast Cancer Res. Treat. 1994, 32,
139-152.
(
(35) Toyokuni, T.; Hakomori, S.; Singhal, A. K. Synthetic carbohydrate
vaccines: Synthesis and immunogenicity of Tn antigen conjugates.
Bioorg. Med. Chem. 1994, 2, 1119-1132.
230.
(
(
(
19) Berd, D.; Sato, T.; Maguire, H. C.; Kairys, J.; Mastrangelo, M. J.
Immunopharmacologic analysis of human autologous hapten-modi-
fied human melanoma vaccine. J. Clin. Oncol. 2004, 22, 403-415.
20) Lamb, J. R.; Eckels, D. D.; Lake, P.; Woody, J. N.; Green, N. Human
T-cell clones recognize chemically synthesized peptides of influenza
hemagglutinin. Nature 1982, 300, 66-69.
21) B. simplicifolia, binding affinity for R-D-Gal (major affinity), R-D-
GalNAc (secondary affinity); C. fragile, binding affinity for R-D-
GalNAc (Tn) at the terminal reducing or nonreducing end; D. biflorus,
binding affinity for R-D-GalNAc (Tn) at the terminal nonreducing
end; V. Villosa B4, binding affinity for R-D-GalNAc at the terminal
reducing end.
(36) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys.
1959, 82, 70-77.
(37) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring
Harbor Laboratory Press: Plainview, NY, 1998.
(
38) Slovin, S. F.; Raguapathi, G.; Adluri, S.; Ungers, G.; Terry, K.; Kim,
S.; Spassova, M.; Bornmann, W. G.; Fazzari, M.; Dantis, L.;
Olkiewicz, K.; Lloyd, K. O.; Livingston, P. O.; Danishefsky, S. J.;
Scher, H. I. Carbohydrate vaccines in cancer: Immunogenicity of a
fully synthetic globo hexasaccharide conjugate in man. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 5710-5715.
(
39) Ragupathi, G.; Slovin, S. F.; Adluri, S.; Sames, D.; Kim, I. J.; Kim,
H. M.; Spassova, M.; Bornmann, W. G.; Lloyd, K. O.; Scher, H. I.;
Livingston, P. O.; Danishefsky, S. J. A fuly synthetic globo H
carbohydrate vaccine induces a focused humoral response to prostate
cancer patients: A proof of principle. Angew. Chem., Int. Ed. 1999,
(
(
(
(
22) Rohlenova, A.; Ledvina, M.; Saman, D.; Bezouska, K. Synthesis of
linear and branched regioisomeric chitooligosaccharides as potential
mimetics of natural oligosaccharide ligands of natural killer cells
NKR-P1 and CD69 lectin receptors. Collect. Czech. Chem. Commun.
3
8, 563-566.
2004, 69, 1781-1804.
(
40) Bezouska, K.; Yuen, C.-T.; O’Brien, J.; Childs, R. A.; Chai, W.;
Lawson, A. M.; Drbal, K.; Fiscaronerova, A.; Pospisil, M.; Feizi, T.
Oligosaccharide ligands for NKR-P1 protein activate NK cells and
cytotoxicity. Nature 1994, 372, 150-157.
23) Vannucci, L.; Fiserova, A.; Sadalapure, K.; Lindhorst, T. K.; Kuldova,
M.; Rossmann, P.; Horvath, O.; Kren, V.; Krist, P.; Bezouska, K.;
Luptovcova, M.; Mosca, F.; Pospisil, M. Effects of N-acetyl-
glucosamine-coated glycodendrimers as biological modulators in the
B16F10 melanoma model in vivo. Int. J. Oncol. 2003, 23, 285-96.
24) Bezouska, K.; Vlahas, G.; Horvath, O.; Jinochova, G.; Fiserova, A.;
Giorda, R.; Chambers, W. H.; Feizi, T.; Pospisil, M. Rat natural-
killer-cell antigen, NKR-P1, related to C-type animal lectins is a
carbohydrate-binding protein. J. Biol. Chem. 1994, 269, 16945-
(
41) Slovin, S. F.; Ragupathi, G.; Adluri, S.; Ungers, G.; Terry, K.; Kim,
S.; Spassova, M.; Bornmann, W. G.; Fazzari, M.; Dantis, L.;
Olkiewicz, K.; Lloyd, K. O.; Livingston, P. O.; Danishefsky, S. J.;
Scher, H. I. Carbohydrate vaccines in cancer: Immunogenicity of a
fully synthetic globo H hexasaccharide conjugate in man. Proc. Natl.
Acad. Sci. U.S.A 1999, 96, 5710-5715.
16952.
(
42) Inoue, M.; Nakada, H.; Tanaka, N.; Yamashina, I. Tn antigen is
25) Wu, A. M.; Song, S. C.; Chang, S. C.; Wu, J. H.; Chang, K. S. S.;
Kabat, E. A. Further characterization of the binding properties of a
GalNAc specific lectin from Codium fragile subspecies tomen-
tosoides. Glycobiology 1997, 7, 1061-1066.
expressed on leukosialin from T-lymphoid cells. Cancer Res. 1994,
54, 85-88.
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