Cowpea mosaic virus capsid: A promising carrier for the development of carbohydrate based antitumor vaccines

Article abstract of DOI:10.1002/chem.200800203

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on differeent carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.

Full text of DOI:10.1002/chem.200800203

FULL PAPER
DOI: 10.1002/chem.200800203
Cowpea Mosaic Virus Capsid: A Promising Carrier for the Development of
Carbohydrate Based Antitumor Vaccines
Adeline Miermont,[a] Hannah Barnhill,[b] Erica Strable,[c] Xiaowei Lu,[a]
Katherine A. Wall,[d] Qian Wang,[b] M. G. Finn,*[c] and Xuefei Huang*[a]
Abstract: Immunotherapy targeting
tumor cell surface carbohydrates is a
promising approach for cancer treat-
ment. However, the low immunogenec-
ity of carbohydrates presents a formi-
dable challenge. We describe here the
enhancement of carbohydrate immuno-
genicity by an ordered display on the
surface of the cowpea mosaic virus
(CPMV) capsid. The Tn glycan, which
is overexpressed on numerous cancer
cell surfaces, was selected as the model
antigen for our study. Previously it has
been shown that it is difficult to induce
a strong T cell-dependent immune re-
sponse against the monomeric form of
Tn presented in several ways on differ-
ent carriers. In this study, we first syn-
thesized Tn antigens derivatized with
either a maleimide or a bromoaceta-
mide moiety that was conjugated selec-
tively to a cysteine mutant of CPMV.
The glycoconjugate was then injected
into mice and pre- and post-immune
antibody levels in the mice sera were
measured by enzyme-linked immuno-
sorbant assays. High total antibody
titers and, more importantly, high IgG
titers specific for Tn were obtained in
the post-immune day 35 serum, sug-
gesting the induction of T cell-depen-
dent antibody isotype switching by the
glycoconjugate. The antibodies gener-
ated were able to recognize Tn anti-
gens presented in their native confor-
mations on the surfaces of both MCF-7
breast cancer cells and the multidrug
resistant breast cancer cell line NCI-
ADR RES. These results suggest that
the CPMV capsid can greatly enhance
the immunogenicity of weak antigens
such as Tn and this can provide a
promising tool for the development of
carbohydrate based anti-cancer vac-
cines.
Keywords: antibodies · antitumor
agents · carbohydrates · vaccines ·
viruses
Introduction
Tumor associated carbohydrate antigens (TACAs) are often
strongly linked with tumor progression and malignancy.[1,2]
Cancer immunotherapy targeting TACAs is an attractive ap-
proach for elimination of circulating tumor cells and eradi-
cation of micrometastases which remain after surgery or ra-
diotherapy. However, this has been a highly challenging task
as TACAs are often weak T cell-independent antigens. To
generate long lasting effective immune surveillance and pro-
tection, T cells must be activated to induce antibody isotype
switching and immune memory.[3–14]
In order to render a T cell-dependent immune response,
TACAs can be conjugated with helper T (Th) cell epitopes
such as serum albumin from different sources, keyhole
limpet hemocyanin (KLH), or tetanus toxoid (TT).[3–14] In
this manner, the Th epitope can be presented by the B cell
specific to the TACA, which is required for a Th cell stimu-
lated humoral response.[15,16] Recently, several other novel
innovative approaches have been developed towards carbo-
hydrate based cancer vaccines. Boons and co-workers re-
[a]A. Miermont, X. Lu, Dr. X. Huang
Department of Chemistry, The University of Toledo
2801 W. Bancroft Street, MS 602, Toledo, OH 43606 (USA)
Fax : (+1)419-530-4033
E-mail: xuefei.huang@utoledo.edu
[b]H. Barnhill, Dr. Q. Wang
Department of Chemistry and Biochemistry
University of South Carolina, Columbia, SC 29208 (USA)
[c]E. Strable, Dr. M. G. Finn
Department of Chemistry and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute, 10550 N. Torrey Pines Road
La Jolla, CA 92037 (USA)
Fax : (+1)858-784-8850
E-mail: mgfinn@scripps.edu
[d]Dr. K. A. Wall
Department of Medicinal and Biological Chemistry
The University of Toledo, 2801 W. Bancroft Street, MS 606
Toledo, OH 43606 (USA)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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4939

ported that a three-component covalent construct including
TACA, Th epitope and an adjuvant can significantly im-
prove the magnitude and quality of antibodies generated
against TACAs.[17,18] Danishefsky and co-workers synthe-
sized a unimolecular hexavalent construct linking six differ-
ent TACAs into one glycopeptide, which were then coupled
to KLH.[19,20] This was evaluated as a promising means to
target microheterogeneity of TACAs expressed on tumor
cell surfaces to prevent escape of tumor cells from immuno-
surveillance. The Guo group has taken advantage of the
higher metabolic rate of tumor cells to selectively modify
the TACAs on tumor cell surfaces.[21,22] Antibodies generat-
ed against modified TACAs were found to be effective in
killing glyco-engineered melanoma cells. Besides the native
O-glycosides, several groups have reported that the usage of
S-linked and C-linked glycosides confer higher stabilities to
TACAs, which can subsequently result in higher immune re-
sponses.[23–26] Another active area of research is to examine
novel carriers for TACA, which include dendrimers,[27–29]
monoclonal antibodies,[30] influenza virosome,[31] gold nano-
particles[32] as well as direct conjugation with an adjuvant.[33]
It is known that highly patterned displays of antigens can
lead to earlier B cell amplification for potent IgM responses
as well as efficient switching to IgG.[34–37] Antigen organiza-
tion has a great influence on B cell tolerance, with B cells
unresponsive to poorly organized antigens while responding
promptly to the same antigen presented in a highly organ-
ized manner.[38] With traditional protein carriers such as
KLH and TT, it is impossible to precisely control the display
patterns of antigens on the surface. Recently, viral capsids
have emerged as a promising platform for antigen presenta-
tion.[34,39–44] Viral capsids are composed of structural proteins
that self-assemble in a highly ordered manner. Peptide epi-
topes presented on the surface of viral capsids can stimulate
an adaptive immune response by effective activation of anti-
gen presenting cells as well as stimulation of B-cell mediated
response by direct cross-linking of B cell receptors.[34,45] We
became interested in examining whether patterned display
of TACAs can also enhance their immune response. Thus
we selected the cowpea mosaic virus (CPMV) capsid, which
is highly immunogenic yet non-infectious to humans.[43,44,46]
CPMV capsid is readily available, and can be isolated in
gram quantity from cowpea plants.[47] The X-ray structure of
CPMV capsid is known to near atomic resolution with sixty
copies of protein subunits arranged in a 30 nm icosahe-
dron.[48] Extensive studies have been carried out by Finn,
Johnson, and co-workers demonstrating that CPMV capsids
can be chemically modified, which allows the attachment of
sixty copies of a compound in an icosahedral symmetry to
the external surface of the CPMV capsid.[49–53] In addition,
the CPMV capsid has been used as a carrier of small pep-
tides through genetic engineering, which was shown to acti-
vate Th cells.[46,54–57] We earlier showed that glycan display
on CPMV was effective in chickens to give highly specific
anti-carbohydrate IgY antibodies in large quantities.[58] Here
we report the results of an initial investigation in mice for
IgG generation and applications of CPMV capsids for po-
tential cancer vaccine development.
The Tn antigen (GalNAc-a-O-Ser/Thr) was chosen as the
model antigen to be conjugated to CPMV capsid. Tn anti-
gen is a TACA overexpressed on the surface of a variety of
cancer cell surfaces including breast, colon and prostate
cancer, rendering it an excellent immunotherapy target.[59,60]
Danishefsky and co-workers have reported that the conjuga-
tion of multiple copies of monomeric Tn to KLH did not
elicit a Tn-specific antibody response.[61] Instead, Tn trimer
clusters on KLH were needed to achieve immunological rec-
ognition, primarily giving rise to IgM.[62] Similar phenomena
were also observed by Lo-Man and co-workers, who report-
ed very low antibody titers when Tn monomer was used as
the antigen while Tn trimer was much more effective in
active specific immunotherapy.[27,63] We wished to determine
if the low immunogenicity of Tn monomer could be over-
come by the highly patterned display of Tn on the CPMV
capsid, presenting a new avenue to boost immune responses
to weak immunogens such as carbohydrates.
Results and Discussion
Synthesis ofTn antigens ofr bioconjugation : In order to
conjugate the Tn antigen to CPMV capsids and for ELISA
assays, we designed three Tn analogues, compound 1 with a
maleimide linker, compound 2 containing bromoacetamide
and 3 terminated with biotin at the N-terminus. The carbox-
yl ends of all Tn derivatives were capped by ethanolamine.
The Tn derivatives 1–3 were synthesized from the common
intermediate amine 4 (Scheme 1).
The preparation of amine 4 started from the reaction of
thioglycosyl donor 5 with the serine acceptor 7. The amino
group in 7 was protected by Fmoc as it is known that a car-
bamate protective group enhances the nucleophilicity of the
serine hydroxyl group. The main challenge in reaction of 5
with 7 is the formation of the a anomer. Boons and co-
workers reported the exclusive formation of an a linkage
between the thioglycoside donor 6 and a threonine accept-
or[64] using the Ph2SO and triflic anhydride promoter
system.[65] However, this reaction condition caused extensive
decomposition in our case. We found instead that N-iodo-
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Carbohydrate Based Antitumor Vaccines
FULL PAPER
greatly enhanced the product solubility. The coupling of 10
with ethanolamine proceeded smoothly when carboxylic
acid 10 was first activated with benzotriazole-1-yl-oxy-tris-
(dimethylamino)-phosphonium hexafluorophosphate (BOP)
prior to the addition of the nucleophile, giving amide 11 in
60–80% yields in a couple of hours. When BOP was added
to a mixture of 10 and ethanolamine, the reaction was very
slow, with prolonged incubation leading to Fmoc removal
under the basic reaction condition.
Compound 11 was deprotected first with catalytic sodium
methoxide at pH 9,[70] but slow addition of the base was nec-
essary to carefully control the pH of the reaction mixture in
order to prevent base-promoted elimination of the O-glyco-
side. Instead, we found it more convenient to treat 11 with
ammonia in methanol, which cleanly removed all the ace-
tates as well as the Fmoc group in 90–95% yield. The highly
polar amino alcohol 4 was purified by a short silica gel
column. It is imperative to remove all impurities in 4 in
order to facilitate purification of the final products.
Coupling of maleimide containing N-hydroxysuccinimide
ester 12 with 4 was first carried out in N-methylpyrrolidi-
none (NMP) at room temperature. Besides the desired
product 1, a significant amount of N-hydroxysuccinimide–
maleimide adduct (compound 13) was obtained, which was
Scheme 1. a) 2.8 equiv NIS, 0.2 equiv AgOTf, toluene/dioxane 1:3, MS-
AW300; b) Zn dust, AcOH, Ac2O, THF, 08C; c) H2, Pd/C, MeOH; d)
BOP, DIPEA, THF, then ethanolamine; e) NH3, MeOH.
succinimide and silver triflate[66] in diethyl ether, a solvent
well known to favor the formation of the axial anomer,[67]
provided an a/b ratio of 3:1. Changing the solvent to a mix-
ture of toluene and 1,4-dioxane (1:3)[68] led to enhancement
of the ratio to 6:1 with 50–65% yield of the desired anomer
8.
The conversion of the azido group to acetamide can be
performed under a variety of conditions including catalytic
hydrogenolysis, thioacetic acid reduction or Zn/acetic acid
reduction followed by acetylation. Previously we encoun-
tered difficulties in simultaneously reducing azide and
benzyl groups by catalytic hydrogenolysis.[67,69] Thus, we
tested the direct conversion of azide to acetamide via treat-
ment of thioacetic acid,[62] resulting in amide 9 in 85% yield.
However, the cleavage of the benzyl ester from 9 by catalyt-
ic hydrogenolysis turned out to be very inconsistent, pre-
sumably due to the poisoning of the Pd catalyst by trace
amounts of sulfur-containing impurities in 9, despite repeat-
ed purifications. Ultimately, the one pot reduction and ace-
tylation of the azide 8 by zinc dust in acetic anhydride and
acetic acid was found to give 90–95% of amide 9, which was
hydrogenated smoothly with Pd/C under atmospheric pres-
sure of hydrogen within 30 min to yield carboxylic acid 10.
The carboxylic acid was capped first with n-butylamine, but
the corresponding amide was very poorly soluble in a varie-
ty of solvents. Replacing n-butylamine with ethanolamine
very difficult to separate from 1. Extended silica gel chro-
matography led to decomposition of 1 due to the instability
of the maleimide moiety. However, performing the reaction
at ꢀ208C greatly suppressed the formation of 13 in the reac-
tion mixture, from which pure 1 was obtained in 50% yield
by selective precipitation upon completion of the reaction
and addition of diethyl ether to the mixture.[71,72]
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X. Huang, M. G. Finn et al.
Amidation of 4 by bromoacet-
amido containing N-hydroxy-
succinimide ester 14a[73] gener-
ated desired product 2 mixed
with N-hydroxysuccinimide sub-
stitution product 15 even at
ꢀ208C. Silica gel chromatogra-
phy, reversed-phase HPLC, and
size exclusion chromatography
all failed to give good separa-
tion of the desired product. Di-
cyclohexylcarbodiimide (DCC)
Scheme 2. Synthesis of Tn-S-CPMV conjugates.
or BOP promoted coupling of acid 14b[73] with 4 again pro-
duced large amounts of a-carbon substitution products by
the nucleophilic dicyclohexylurea or hydroxylbenzotriazole
side products generated in the reaction. To reduce the nucle-
ophilicity of the side product, we examined pentafluorophe-
nol activated ester 14c. Coupling of amine 4 with 14c in
NMP at 08C produced pure 2 in 59% yield without any
pentafluorophenol substitution side product following dieth-
yl ether precipitation. The biotinated Tn analog 3 was pre-
pared in 72% yield by amidation of amine 4 with biotinated
amino caproic acid N-hydroxysuccinimide ester 16.
Bioconjugation: Previously it has been reported that native
CPMV has no exposed cysteine on the exterior surface.
Therefore, the wild type CPMV is fairly inert to thiol-reac-
tive reagents.[52,53,74] A number of mutants of CPMV bearing
reactive cysteines on the exterior surface have been pro-
duced by cassette mutagenesis or site-specific mutagenesis,
and a wide range of compounds, including biotin, fluores-
cent dyes, nanogold, and carbohydrates, can be attached to
the thiol groups of such mutant CPMV particles.[52,53,74] In
this study, the T2102C/T228C chimera (residues 102 and 28
of the large subunits were replaced by cysteines, termed as
S-CPMV), was employed as a stable and thiol-reactive parti-
cle (Figure S1-S3).[75,76] Because two cysteine moieties are
located close to each other in space (approximately 21 Š
apart), it is difficult to load more than 60 molecules on the
surface of the capsid (Figure S4, Supporting Information).[75]
Both Tn derivatives 1 and 2 were immobilized on S-CPMV
through the reported procedures (Scheme 2). Briefly, puri-
fied S-CPMV was incubated with an excess of 1 or 2 over-
night at 48C in potassium phosphate buffer (PBS, pH 7.0)
with the addition of DMSO (20%) to aid in the solubility of
the Tn antigens. The unconjugated Tn antigens were re-
moved by ultracentrifugation over a sucrose gradient and
the products were analyzed by transmission electron micro-
scopy (TEM) and fast protein liquid chromatography
(FPLC) verifying the presence of intact CPMV capsids
(Figure 1). Reactions performed under the same conditions
with analogous fluorescein reagents confirmed the assump-
tion that 60ꢁ10 attachments are made to the CPMV capsid
in each case (Supporting Information).
Figure 1. Size-exclusion FPLC of S-CPMV and purified Tn-1-S-CPMV
showing intact capsids eluting at 10 mL and a small amount of disassem-
bled protein eluting at 19 mL. TEM of Tn-1-S-CPMV verifying the pres-
ence of intact capsids (inset, scale bar=100 nm).
GalNAc. An increase in absorbance monitored by UV/Vis
spectroscopy at 600 nm was observed when SBA was mixed
with Tn-S-CPMV, indicating the formation of aggregates
due to crosslinking of several Tn-S-CPMV units by SBA
leading to an increase in light scattering. In contrast, a slight
decrease in absorbance resulted from mixing S-CPMV with
SBA due to the dilution effect (Figure 2). The UV-visible
spectroscopy results were confirmed by TEM, with aggre-
gates clearly observed when SBA was mixed with Tn-1-S-
The presence of the Tn antigen on the surface of S-
CPMV was confirmed by protein-binding studies with soy-
bean agglutinin (SBA), a tetrameric plant lectin specific for
Figure 2. Binding of SBA (50 mm) and CPMV conjugates (0.2 mgmLꢀ1).
&
^
Tn-1-S-CPMV ( ), S-CPMV ( ).
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Carbohydrate Based Antitumor Vaccines
FULL PAPER
CPMV, while underivatized S-CPMV remained monomeric
with SBA (Figure 3) highlighting the specificity of this inter-
action.
antibodies generated indeed recognized the GalNAc moiety,
which is present in all Tn antigens (Figure 4a).
Finally, the immune response against the natural target,
Tn positive tumor cells, was tested. It is known that Tn anti-
Figure 3. TEM images of SBA incubated with S-CPMV (left) and Tn-1-
S-CPMV (right). Conditions identical to UV-visible light scattering assay.
Scale bars=100 nm.
Immunological studies: With the Tn-S-CPMV conjugates in
hand, immunological studies were carried out on a group of
five C57BL/6 female mice. Tn-1-S-CPMV (0.5 mg corre-
sponding to approximately 40 mg of Tn) as an emulsion in
complete Freundꢁs adjuvant was injected subcutaneously
into each mouse (day 0). Booster immunizations were per-
formed subcutaneously on days 14 and 28 with the same
amount of Tn-1-S-CPMV in incomplete Freundꢁs adjuvant
emulsion. As a control, another group of five C57BL/6 mice
received identical vaccine formulations except that S-CPMV
(0.5 mg) was used to replace Tn-1-S-CPMV. Blood was col-
lected from both groups of mice on days 0, 7 and 35 and
serum was isolated, in which the Tn specific antibody levels
were analyzed by enzyme linked immunosorbent assay
(ELISA). To exclude the immune response against the mal-
eimide linker,[77] the biotinated Tn 3 devoid of maleimide
was immobilized on a microtiter plate coated with Neutravi-
din. The sera was added to the microtiter plate at increasing
dilutions and the IgG, IgM as well as total antibody titers
were determined photometrically using secondary anti-
mouse IgG+IgM, IgG, and IgM antibodies conjugated to
horseradish peroxidase.
While day 0 and day 7 sera did not show significant anti-
body titers, the sera collected on day 35 from mice immu-
nized with Tn-1-S-CPMV showed a total titer (IgG+IgM)
of 15,000, an IgG titer of 10,500, and IgM titer of 5000
(Figure 4). Importantly, high IgG titers were obtained indi-
cating that a T cell mediated immune response was pro-
duced (Figure 4b). Mice immunized with S-CPMV only min-
imum anti-Tn antibody titer on day 35 (Figure 4a) as expect-
ed. To further ascertain that the linker does not contribute
to the immune response much, the total IgG+IgM titer of
day 35 serum was measured by ELISA in the presence of
0.1m free GalNAc. The total antibody titer detected under
these conditions dropped by more than two orders of magni-
tude, indicating that a significant amount of the polyclonal
Figure 4. a) ELISA titers of anti-Tn IgG+IgM antibody in mouse sera
prior to immunization (day 0), after immunization with Tn-1-S-CPMV
(day 35) in the absence and presence of 0.1m GalNAc. b) ELISA titers
of anti-Tn IgM and IgG antibody in mouse sera after immunization with
Tn-1-S-CPMV (day 35). Day 35 serum from mice immunized with the
carrier S-CPMV was used as the control. Antibody titers were defined as
the maximum folds of dilution resulting in 0.1 OD unit higher than the
average absorbance after greater than one hundred thousand folds of di-
lution.
gen is overexpressed on MCF-7 breast cancer cell surfa-
ces.[61] Preimmune and postimmune (day 35) sera were ana-
lyzed for binding to MCF-7 cells by Fluorescent Assisted
Cell Sorting (FACS) using a fluorescein labeled anti-IgG
secondary antibody (Figure 5a). A statistically significant in-
crease in binding to MCF-7 cells by IgG was found, which
was similar to those obtained from a trivalent vaccine con-
struct.[19] This suggests the IgG antibodies generated in our
study were able to recognize Tn in its native configurations
on the tumor cell surface. Multidrug resistant cancer cells
present a major obstacle to cancer therapy. We next evaluat-
ed the binding of our antibodies with a multidrug resistant
breast cancer cell line NCI-ADR RES. Excitingly, the day
35 postimmune serum generated in our studies showed good
affinity with NCI-ADR RES cells, suggesting that carbohy-
drate based tumor associated vaccines can be a potentially
novel therapeutic towards multidrug resistant cancer (Fig-
ure 5b). The binding of antibodies with cancer cells was
greatly reduced in the presence of free GalNAc (Figure S5,
Supporting Information). The observed differential FACS
responses of MCF-7 and NCI-ADR RES cells could be due
to different patterns of Tn displayed on these cell surfaces.
The monomeric form of Tn represents one of the weakest
forms of immunogen. Yet, through conjugation with CPMV
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X. Huang, M. G. Finn et al.
Experimental Section
General procedures: All chemical re-
actions were carried out under nitro-
gen with anhydrous solvents in flame-
dried glassware, unless otherwise
noted. All glycosylation reactions were
performed in the presence of molecu-
lar sieves, which were flame-dried
right before the reaction under high
vacuum. Glycosylation solvents were
dried using an MBraun solvent purifi-
cation system and used directly with-
out further drying. Chemicals used
were reagent grade as supplied except
where noted. Analytical thin-layer
chromatography was performed using
silica gel 60 F254 glass plates (EM Sci-
ence); compound spots were visual-
ized by UV light (254 nm) and by
Figure 5. FACS analysis of cell surface reactivity to a) MCF-7 cell and b) NCI-ADR RES cell by IgG antibod-
ies in day 35 mice sera generated against Tn-1-S-CPMV. The control samples are the mice serum from day 0
just prior to immunization. Day 35 serum from mice immunized with S-CPMV showed identical cell surface
reactivity to that of the day 0 serum on both cells.
staining with a yellow solution containing CeACHTER(NGU NH4)2ACHTRE(UGN NO3)6 (0.5 g) and
capsid, a strong humoral immune response with the desired
antibody subtype was obtained, indicating that patterned
display of Tn can enhance the immunogenicity of this weak
antigen. It is possible that the covalent linkage between Tn
and CPMV renders Tn epitopes on CPMV to be processed
and presented by the Tn-specific B cell, thus allowing B cell
stimulation by matched helper T cells. It may also be the
case that the spacing between neighboring Tn molecules on
the capsid surface (3.2–5 nm, depending on the sites of at-
tachment) can crosslink B cell receptors[78] leading to potent
activation of B cells.
(NH4)6Mo7O24·4H2O (24.0 g) in 6% H2SO4 (500 mL). Flash column chro-
matography was performed on silica gel 60 (230–400 Mesh, EM Science).
1H NMR, 13C NMR, 1H–1H gCOSY, 1H–13C gHMQC and 1H–13C
gHMBC spectra were recorded on a Varian VXRS-400 or Inova-600 in-
strument and were referenced using Me4Si (0 ppm), residual CHCl3 (d=
1H NMR 7.26 ppm, 13C NMR 77.0 ppm). Optical rotations were measured
at 25 C. ESI mass spectra were recorded on ESQUIRE LC-MS operated
in positive ion mode. High-resolution mass spectra were recorded on a
Micromass electrospray Tof II (Micromass, Wythenshawe, UK) mass
spectrometer equipped with an orthogonal electrospray source (Z-spray)
operated in positive ion mode, which is located at the Mass Spectrometry
and Proteomics Facility, the Ohio State University.
N-(9-Fluorenylmethyloxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-
a-d-galactopyranosyl)-l-serine benzylester (8): Donor 5 (1 g, 2.29 mmol)
and acceptor 7 (1.05 g, 2.51 mmol, 1.1 equiv) were first coevaporated
with anhydrous toluene and pumped under vacuum for one hour. The
mixture was then dissolved in toluene/1,4-dioxane 1:3 (40 mL) and stirred
at 08C in the presence of activated MS AW-300 (5 g) for 45 min. Silver
triflate (295 mg, 1.15 mmol, 0.5 equiv) was added followed by the addi-
tion of N-iodosuccinimide (1.03 g, 4.58 mmol, 2 equiv). The reaction mix-
ture was allowed to warm up to room temperature and was stirred for up
to 32 h when complete consumption of donor 5 was confirmed by TLC
analysis. The reaction was then quenched by filtration through Celite. Di-
chloromethane was added to the reaction mixture and extracted with a
saturated solution of sodium bicarbonate. The organic layer was dried
over anhydrous sodium sulfate, concentrated to dryness and the residue
was purified by flash chromatography (hexanes/EtOAc 3:1!2:1). The
desired a isomer was obtained in its pure form as an oil with a 50–65%
yield and a 6:1 a/b selectivity. Comparison with literature data[62] con-
firms its identity. 1H NMR (400 MHz, CDCl3): a anomer d=7.79–7.73
(m, 2H), 7.65–7.59 (m, 2H), 7.41–7.30 (m, 9H), 5.96 (d, 1H, 3J=8.0 Hz),
5.39 (d, 1H, 3J=2.4 Hz), 5.27–5.23 (m, 3H), 4.86 (d, 1H, 3J=3.6 Hz),
4.62–4.59 (m, 1H), 4.41 (d, 1H, 3J=7.2 Hz), 4.24 (t, 1H, 3J=7.2 Hz),
4.17–3.99 (m, 9H), 3.58 (dd, 1H, 3J=3.6, 11.2 Hz), 2.14 (s, 3H), 2.07 (s,
3H), 1.96 ppm (s, 3H).
CPMV capsids have been successfully adapted to express
foreign peptide epitopes on the surface and the resulting
chimera induced peptide specific antibodies, which have
been shown to provide protection against infections.[46,54–57]
However, the display of each peptide epitope requires clon-
ing and expression of chimera capsids individually. Our ap-
proach of post-expression modification gives us greater flex-
ibility in varying antigen structures.
Conclusion
We have demonstrated that by covalently conjugating a
weak carbohydrate antigen Tn to CPMV, strong humoral
immune responses were induced, which produced high titers
of IgG antibodies capable of recognizing Tn on breast
cancer cell surfaces. Although our current proof of principle
studies are focused on the Tn antigen, this technology can
be easily adapted towards the study of other carbohydrate
based anti-cancer vaccine constructs, thus presenting a gen-
eral approach towards carbohydrate based vaccine develop-
ment. Structurally well-characterized platforms such as
CPMV offer different reactive groups, such as the exterior
lysine side chains[50,53,58,79] in addition to the inserted cys-
teines used here, giving us orthogonal ways to introduce
multiple antigens onto the carrier surface. Work is in prog-
ress to construct these novel conjugates as well as to evalu-
ate in vivo the protective effects of these constructs on
tumor models.
N-(9-Fluorenylmethyloxycarbonyl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-a-d-galactopyranosyl)-l-serine benzylester (9): A mixture of com-
pound 8 (1 g, 1.37 mmol), zinc dust (2.6 g, 27.4 mmol, 20 equiv), acetic
acid (0.8 mL, 13.7 mmol, 10 equiv) and acetic anhydride (1.3 mL,
13.7 mmol, 10 equiv) in THF (30 mL) was stirred overnight at room tem-
perature. Complete reaction of compound 8 was confirmed by TLC anal-
ysis. The reaction was then quenched by filtration through Celite fol-
lowed by extraction with ethyl acetate and a saturated solution of sodium
bicarbonate. The organic layer was dried over anhydrous sodium sulfate,
concentrated to dryness and the residue was purified by flash chromatog-
raphy (hexanes/EtOAc 2:1). The desired product was obtained in its pure
form as an oil (0.99 g, 90–95%). Comparison with literature data[62] con-
4944
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 4939 – 4947

Carbohydrate Based Antitumor Vaccines
FULL PAPER
firms its identity. 1H NMR (600 MHz, CDCl3): d=7.79–7.73 (m, 2H),
7.64–7.57 (m, 2H), 7.41–7.30 (m, 9H), 5.86 (d, 1H, 3J=7.8 Hz), 5.59 (d,
1H, 3J=9.6 Hz), 5.29 (d, 1H, 3J=3.0 Hz), 5.22–5.15 (m, 2H), 5.02 (dd,
1H, 3J=2.4, 10.8 Hz), 4.75 (d, 1H, 3J=3.0 Hz), 4.59–4.57 (m, 1H), 4.54–
4.50 (m, 1H), 4.42 (d, 1H, J=7.2 Hz), 4.22 (t, 1H, J=7.2 Hz), 4.06–3.93
(m, 5H), 2.15 (s, 3H), 1.99 (s, 3H), 1.96 (s, 3H), 1.89 ppm (s, 3H).
67.8, 67.4, 61.3, 59.9, 53.9, 49.9, 41.7, 37.5, 35.3, 27.4, 25.6, 24.8, 22.1 ppm;
HRMS: m/z: calcd for C23H35N4NaO11: 567.2278; found: 567.2278
[M+Na]+.
Pentafluorophenyl 6-N-bromoacetamido hexanoate (14c): Compound
14b (0.5 g, 1.98 mmol)[73] was dissolved in CH2Cl2 (20 mL) along with N-
(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (0.43 g,
2.78 mmol, 1.4 equiv). Pentafluorophenol (0.51 g, 2.78 mmol, 1.4 equiv)
was then added and the reaction mixture was stirred at room tempera-
ture for five hours. Completion of the reaction was confirmed by TLC
analysis (CH2Cl2/methanol 90:10). The reaction was quenched by extrac-
tion with water and CH2Cl2. The organic layer was then dried over anhy-
drous sodium sulfate, concentrated to dryness and the residue was puri-
fied by flash column chromatography (hexanes/EtOAc 3:1!1:1). The de-
sired product was obtained in its pure form as a white solid (0.59 g,
71%). 1H NMR (600 MHz, CDCl3): d= (brs, 1H), 3.85 (s, 2H), 3.31–
3.28 (m, 2H), 2.66 (t, 2H, 3J=7.2 Hz), 1.81–1.76 (m, 2H), 1.62–1.57 (m,
2H), 1.47–1.42 ppm (m, 2H); 13C NMR (150 MHz, CDCl3): d=169.5,
165.6, 142.1, 140.4, 138.8, 137.2, 40.1, 33.3, 29.5, 29.1, 26.2, 24.4 ppm; ESI-
MS: m/z: calcd for C14H13BrF5NNaO3: 440.0; found: 440.1 [M+Na]+.
3
3
N-(9-Fluorenylmethyloxycarbonyl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-a-d-galactopyranosyl)-l-serine (10): A mixture of compound 9
(1 g, 1.3 mmol) and palladium on activated carbon (1 g) in methanol was
stirred under a hydrogen atmosphere for 30 min. Completion of the reac-
tion was confirmed by TLC analysis (CH2Cl2/methanol 95:5). The reac-
tion mixture was then filtered through Celite and concentrated under
vacuum. Compound 10 was obtained in its pure form with 90–95% yield
and was used without further purification. 1H NMR (600 MHz, CDCl3):
d=7.79–7.72 (m, 2H), 7.54–7.49 (m, 2H), 7.37–7.30 (m, 4H), 5.40 (brs,
1H), 5.23 (d, 1H, 3J=11.4 Hz), 4.93 (d, 1H, 3J=3.0 Hz), 4.56 (dd, 1H,
3J=3.0, 11.4 Hz), 4.28 (t, 1H, 3J=6.0 Hz), 4.18–4.09 (m, 3H), 3.95–3.87
3
(m, 2H), 3.75 (t, 1H, J=9.0 Hz), 2.18 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H),
1.99 ppm (s, 3H).
N-(9-Fluorenylmethyloxycarbonyl)-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-a-d-galactopyranosyl)-l-serine ethanolamide (11): Compound 10
(0.75 g, 1.1 mmol), BOP (973 mg, 2.2 mmol, 2 equiv) and diisopropyl-
ethylamine (0.38 mL, 2.2 mmol, 2 equiv) were dissolved in THF/CH2Cl2
1:1 (50 mL). The mixture was stirred at room temperature for 1 h and
then ethanolamine (0.33 mL, 5.5 mmol, 5 equiv) was added. The reaction
mixture was stirred at room temperature for two hours or until comple-
tion of the reaction was confirmed by TLC analysis (CH2Cl2/methanol
90:10). The reaction mixture was diluted with CH2Cl2 and extracted with
a saturated solution of ammonium chloride. The organic layer was dried
over anhydrous sodium sulfate, concentrated to dryness and the residue
was purified via flash column chromatography (CH2Cl2/methanol 90:10).
The desired product was obtained in its pure form as a white solid with
60–80% yield. 1H NMR (600 MHz, CDCl3): d=7.79–7.78 (m, 2H), 7.67–
7.65 (m, 2H), 7.42–7.40 (m, 2H), 7.35–7.32 (m, 2H), 6.37 (d, 1H, 3J=
3.0 Hz), 5.93 (d, 1H, 3J=7.2 Hz), 5.10–5.02 (m, 1H), 4.60–4.41 (m, 3H),
4.25–4.17 (m, 2H), 4.13–4.06 (m, 2H), 3.94–3.78 (m, 2H), 3.67–3.65 (m,
2H), 3.47–3.43 (m, 2H), 2.19 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H), 1.96 ppm
(s, 3H); 13C NMR (150 MHz, CDCl3): d=171.8, 170.9, 170.6, 143.8, 141.5,
128.1, 127.3, 125.1, 120.3, 98.9, 69.4, 68.5, 67.3, 67.2, 61.9, 61.3, 47.7, 47.3,
6-N-Bromoacetamido-hexanoyl-O-(2-acetamido-2-deoxy-a-d-galactopyr-
anosyl)-l-serine ethanolamide (2): Compound 4 (30 mg, 0.085 mmol) and
compound 14c (39 mg, 0.094 mmol, 1.1 equiv) were dissolved in N-meth-
ylpyrrolidinone (5 mL). The reaction mixture was stirred at ꢀ208C for
45 min. Completion of the reaction was confirmed by mass spectroscopy.
The reaction was quenched by addition of diethyl ether to the mixture in
order to precipitate the product. The solid residue was obtained by filtra-
tion. It was then redissolved in water and lyophilized. The desired prod-
uct was obtained as a white solid (30 mg, 59%). [a]2D5 = +254.8 (c = 2.1,
H2O); 1H NMR (600 MHz, D2O): d=4.70 (d, 1H, 3J=3.6 Hz), 4.38 (d,
1H, J=3.6 Hz), 3.95 (dd, 1H, J=3.6, 10.8 Hz), 3.77 (d, 1H, J=2.4 Hz),
3.73–3.69 (m, 1H), 3.61–3.58 (m, 1H), 3.55 (t, 1H, 3J=7.2 Hz), 3.46–3.43
(m, 2H), 3.05–3.00 (m, 2H), 2.14 (t, 2H, 3J=7.2 Hz), 1.84 (s, 3H), 1.45–
1.33 (m, 4H), 1.16–1.13 ppm (m, 2H);13C NMR (150 MHz, D2O): d=
177.2, 174.5, 171.7, 169.9, 97.9, 71.4, 68.5, 67.7, 67.3, 61.2, 59.9, 53.8, 49.8,
41.7, 39.7, 35.3, 28.2, 27.8, 25.5, 24.9, 22.1 ppm; HRMS: m/z: calcd for
C21H35BrN3NaO11: 607.1585; found: 607.1585 [M+Na]+.
3
3
3
6-N-Biotinamido-hexanoyl-O-(2-acetamido-2-deoxy-a-d-galactopyrano-
syl)-l-serine ethanolamide (3): Compound 4 (30 mg, 0.085 mmol) and
compound 16 (42 mg, 0.094 mmol, 1.1 equiv) were dissolve in N-methyl-
pyrrolidinone (5 mL). The reaction mixture was stirred at room tempera-
ture for 30 minutes. Completion of the reaction was confirmed by mass
spectroscopy. The reaction was quenched by addition of diethyl ether to
the mixture in order to precipitate the product. The solid residue was ob-
tained by filtration. It was then redissolved in water and lyophilized. The
41.9 ppm; ESI: m/z: calcd for C34H39N2NaO14
[M+Na]+.
:
722.2; found: 722.3
O-(2-Acetamido-2-deoxy-a-d-galactopyranosyl)-l-serine
ethanolamide
(4): Compound 11 (0.5 g, 0.71 mmol) was dissolved in a solution of am-
monia in methanol (20 mL) and the reaction was stirred at 08C for 6 h.
Completion of the reaction was confirmed by TLC analysis (CH2Cl2/
methanol 80:20). The reaction mixture was concentrated to dryness and
the residue was purified via short flash column chromatography (CH2Cl2/
methanol 80:20!20:80). The desired product was obtained in its pure
desired product was obtained as a white solid (42 mg, 72%). [a]D25
=
+26.2 (c = 1.3, H2O); 1H NMR (600 MHz, D2O): d=4.68 (d, 1H, 3J=
3.6 Hz), 4.40–4.36 (m, 2H), 4.21 (dd, 1H, 3J=4.2, 7.8 Hz), 3.93 (dd, 1H,
3J=4.2, 11.4 Hz), 3.76 (d, 1H, 3J=3 Hz), 3.71–3.65 (m, 2H), 3.59–3.51
(m, 3H), 3.44–3.41 (m, 2H), 3.16–3.10 (m, 4H), 2.98–2.95 (m, 2H), 2.7
(dd, 1H, 3J=4.8, 13.2 Hz), 2.56 (d, 1H, 3J=12.6 Hz), 2.13 (t, 2H, 3J=
7.8 Hz), 2.01 (t, 2H, 3J=7.2 Hz), 1.83 (s, 3H), 1.51–1.28 (m, 8H), 1.21–
1.11 ppm (m, 4H); 13C NMR (150 MHz, D2O): d=177.2, 176.7, 174.5,
171.7, 97.9, 71.4, 68.5, 67.7, 67.4, 62.2, 61.3, 60.4, 59.9, 55.5, 53.8, 49.9,
41.7, 39.8, 39.2, 35.6, 35.4, 28.2, 27.9, 27.8, 25.7, 25.3, 24.9, 22.1 ppm;
HRMS: m/z: calcd for C28H47N6NaO11S: 713.3156; found: 713.3158
[M+Na]+.
form as
a
white solid (0.22–0.24 g, 90–95%). 1H NMR (600 MHz,
3
3
CDCl3): d=4.76 (d, 1H, J=3.0 Hz), 4.26 (dd, 1H, J=3.0, 10.8 Hz), 3.86
(brs, 1H), 3.81 (m, 2H), 3.76–3.67 (m, 3H), 3.59 (t, 1H, 3J=5.4 Hz),
3.53–3.49 (m, 2H), 3.33–3.28 (m, 4H), 1.98 ppm (s, 3H); ESI: m/z: calcd
for C13H23N2NaO9: 374.2; found: 374.2 [M+Na]+.
N-6-Maleimido hexanoyl-O-(2-acetamido-2-deoxy-a-d-galactopyranosyl)-
l-serine ethanolamide (1): Compound 4 (30 mg, 0.085 mmol) and com-
pound 12 (29 mg, 0.094 mmol, 1.1 equiv) were dissolved in N-methylpyr-
rolidinone (5 mL). The reaction mixture was stirred at ꢀ208C for 40 min.
Completion of the reaction was confirmed by mass spectroscopy. The re-
action was quenched by addition of diethyl ether to the mixture in order
to precipitate the product. The solid residue was obtained by filtration. It
was then redissolved in water and lyophilized. The desired product was
obtained as a white solid (23 mg, 50%). [a]2D5 = +238 (c = 2.5, H2O);
1H NMR (600 MHz, D2O): d = 6.65 (s, 2H), 4.72 (d, 1H, 3J=3.6 Hz),
Synthesis ofTn-S-CPMV : The cysteine mutant of CPMV (S-CPMV) was
generated by modifying the viral RNA to mutate T2102 and T228 into
cysteines as reported.[75] S-CPMV was incubated at 1 mgmLꢀ1 with 50
molar equivalents of maleimide-Tn or 200 molar equivalents of bromo-
acetamide-Tn overnight at 48C in buffer with 20% DMSO. The reaction
was purified by ultracentrifugation over a sucrose gradient (0–40%) at
27000 rpm for 2 h at 48C using a Beckman SW28 rotor. The band corre-
sponding to intact CPMV was collected and pelleted by ultracentrifuge
at 42000 rpm using Beckman 50.2Ti rotor for 2.5 h. The pellet was resus-
pended in buffer. The conjugate was analyzed by UV-visible spectrosco-
py, TEM and FPLC.
3
3
3
4.39 (t, 1H, J=5.4 Hz), 3.98 (dd, 1H, J=3.6, 10.8 Hz), 3.79 (d, 1H, J=
2.4 Hz), 3.74–3.69 (m, 3H), 3.47–4.45 (m, 2H), 3.31 (t, 2H, 3J=7.2 Hz),
3.19–3.17 (m, 2H), 3.16 (s, 1H), 2.53 (s, 1H), 2.14 (t, 2H, 3J=7.2 Hz),
1.86 (s, 3H), 1.44–1.38 (m, 4H), 1.12–1.09 ppm (m, 2H); 13C NMR
(150 MHz, D2O): d=177.2, 174.6, 173.5, 171.7, 134.4, 97.9, 71.4, 68.5,
Chem. Eur. J. 2008, 14, 4939 – 4947
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4945

X. Huang, M. G. Finn et al.
General procedure for handling and analysis of CPMV: The virus was
stored in buffer at a concentration of about 10 mgmLꢀ1. Unless otherwise
indicated, “buffer” refers to 0.1m PBS (pH 7.0). Virus concentrations
were determined by measuring the UV-visible absorbance at 260 nm;
virus at 0.1 mgmLꢀ1 gives a standard absorbance of 0.8. Ultracentrifuga-
tion was performed at the indicated rpm values using a Beckman Opti-
maTM L-90K Ultracentrifuge equipped with either SW28 or 50.2 Ti
rotors. TEM analyses were carried out by depositing 20 mL aliquots of
each sample onto 100-mesh carbon-coated copper grids for 2 min. The
grids were then stained with 20 mL of 2% uranyl acetate and viewed with
Hitachi H-8000 electron microscope.
(0.2 mL). Samples were kept in the dark at 48C till time of analysis.
Analyses of the percentage positive cells and mean fluorescence intensity
of stained cells were done using FACScalibur (BD Biosciences).
Acknowledgement
The work was supported by National Institute of General Medical Scien-
ces, NIH (R01-GM 72667 to X.H. and R01-EB000432 to M.G.F.), an In-
terdisciplinary Research Initiation Award from the University of Toledo
(X.H. and K.A.W.), the National Science Foundation (CHEM-0748690
and BES-0508419 to Q.W.), DoD BCRP Synergy Award, DURIP and
the W. M. Keck Foundation (Q.W. and M.G.F.). We would like to thank
Profs. John E. Johnson and Tianwei Lin (The Scripps Research Institute)
for providing the initial stocks of S-CPMV, Prof. Anthony Quinn (Univ.
of Toledo) for help on FACS experiments, Brandon Slotterbeck (Univ. of
Toledo) for ELISA, Prof. Viranga Tillekeratne, Prof. Max Funk and Ms.
Nicole Ellis (Univ. of Toledo) for generously providing the MCF-7 and
NCI-ADR RES cells and other experimental help.
UV-visible binding studies with soybean agglutinin (SBA): SBA was pur-
chased from Sigma-Aldrich and used without further purification. It was
stored as a lyophilized powder at ꢀ208C until use. Tn-S-CPMV was incu-
bated at a concentration of 0.2 mgmLꢀ1 with 50 mm SBA in buffer. Bind-
ing was monitored over time by the absorbance caused by light scattering
at 600 nm. UV-visible spectroscopy measurements were made using an
Agilent 8400 UV-visible spectrometer. TEM analysis was performed
after incubation under these conditions for 30 minutes as described
above under the general procedure for handling and analysis of S-CPMV.
Immunization ofmice : Pathogen-free C57BL/6 female mice age 6–10
weeks were obtained from Jackson Laboratory (Bar Harbor, ME, USA)
and maintained in the Animal Care Facility of the University of Toledo.
All animal care procedures and experimental protocols have been ap-
proved by the Institutional Animal Care and Use Committee (IACUC)
of the University of Toledo. Groups of five C57BL/6 mice were injected
subcutaneously under the scruff on day 0 with 0.5 mg of Tn-1-S-CPMV
(containing 40 mg of the glycoconjugate 1) or 0.5 mg of S-CPMV as emul-
sions in complete Freundꢁs adjuvant (0.1 mL, Fisher). Boosters were
given subcutaneously under the scruff on days 14 and 28 with 0.5 mg of
Tn-1-S-CPMV or S-CPMV as emulsions in incomplete Freundꢁs adjuvant
(0.1 mL). Blood (~0.2 mL per mouse) was collected from both groups of
mice on days 0, 7 and 35. Sera from each group of mice were isolated
and pooled.
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Enzyme-linked immunosorbent assay (ELISA):
A 96-well microtiter
plate was first coated with a solution of Neutravidin in PBS buffer
(8 mgmLꢀ1) and then incubated overnight at 48C. The plate was then
washed four times with PBS/0.5% Tween-20 (PBST), followed by the ad-
dition of 1% (w/v) BSA in PBS to each well and incubation at room
temperature for one hour. The plate was washed again with PBST and a
solution of biotinylated Tn 3 (5 mgmLꢀ1) in 0.1% BSA in PBS was
added. The plate was incubated for one hour at 378C. The plate was then
washed and mice sera were added in a 1:3 serial dilution in 0.1% BSA/
PBS. The plate was incubated for two hours at 378C and washed. A
1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse
IgG+IgM, IgG or IgM antibody (Jackson ImmunoResearch Laboratory
IgG+IgM catalog #115-035-068, IgM #115-035-075, IgG #115-035-071) in
0.1% BSA/PBS was added to each well respectively. The plate was incu-
bated for one hour at 378C, washed and a solution of 3,3’,5,5’-tetrame-
thylbenzidine (TMB) was added. Color was allowed to develop for 20 mi-
nutes and then a solution of 0.5m H2SO4 was added to quench the reac-
tion. The optical density was then measured at 450 nm. Each experiment
was repeated at least four times and the average of the quadruplicate
was used to calculate the titer. Errors of each measurement were typical-
ly within 10%. Antibody titers were defined as the maximum folds of di-
lution resulting in 0.1 OD unit higher than the average absorbance after
greater than one hundred thousand folds of dilution.
FACS : MCF-7 and NCI-ADR RES human breast cancer cells were ob-
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NaN3) and incubated with 1:10 diluted test sera (75 mL) for 30 minutes at
48C. The cells were washed twice with FACS buffer (1 mL) and then
1:100 diluted goat antimouse IgG labeled with FITC (100 mL, Jackson
ImmunoResearch Laboratory, catalog #115-095-164) was added. The
samples were incubated for 30 minutes at 48C. The cells were washed
again twice with FACS buffer (1 mL) and re-suspended in FACS buffer
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4946
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 4939 – 4947

Carbohydrate Based Antitumor Vaccines
FULL PAPER
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Received: January 31, 2008
Published online: April 22, 2008
Chem. Eur. J. 2008, 14, 4939 – 4947
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