A C-linked disaccharide analogue of Thomsen-Friedenreich epitope induces a strong immune response in mice

Article abstract of DOI:10.1002/chem.201200364

Anticancer vaccine: Antigen 2 made out of a tripeptide of β-D-Galp-(1→3)-CH₂-α-D-GalNAc-O-Ser and conjugated with keyhole limpet hemocyanin (KLH) protein induced an early and strong immune response (IgG antibodies) in mice. Antigen 3 made out o

Full text of DOI:10.1002/chem.201200364

COMMUNICATION
DOI: 10.1002/chem.201200364
A C-Linked Disaccharide Analogue of Thomsen–Friedenreich Epitope
Induces a Strong Immune Response in Mice
Loay Awad,*[a] Rime Madani,[b] Annabelle Gillig,[a] Maria Kolympadi,[a]
Maria Philgren,[b] Andreas Muhs,[b] Catherine Gꢀrard,[c] and Pierre Vogel*[a]
Cancer cells can express aberrant cell-surface-glycosyla-
tion patterns that makes them to be recognizable by the
immune system.[1] This phenotype gives an opportunity to
develop carbohydrate-based vaccines for cancer immuno-
therapy.[2] Thomsen–Friedenreich (TF) antigen is a disacchar-
ide (b-d-Galp-1-3-a-d-GalNAcp) oncofetal blood group-re-
lated antigen normally O-linked to serines or threonines of
mucines (MUC) expressed on cells at the secretory borders
of epithelial tissues. The disaccharide is linked to malignan-
cy and plays an important role in docking breast- and pros-
tate-cancer cells onto endothelium.[3] Patients immunized
Figure 1. Synthetic TF-antigen 1 clustered as a triglycopeptide conjugated
with KLH protein. Antigen 2 is analogue of 1, in which the TF-disacchar-
ide is a C-disaccharide (this work).
with synthetic TF-Keyhole limpet hemocyanin KLH conju-
gate vaccines, plus various adjuvants can generate high-titer
IgM and IgG antibodies.[4] Antigen 1 constructed from a dis-
accharide tripeptide cluster conjugated to KLH (+adjuvant
QS-21) has been shown by Danishefsky and co-workers to
be a relevant antigen target in a multivalent phase II-vaccine
trial in patients with high-risk minimal prostate cancer.[5] Be-
cause the disaccharide moiety of TF-epitope b-d-Galp-1-3-
a-d-GalNAcp-O-l-serine is hydrolyzed by b-galactosidases
in the body,[6] there is a need for more stable antigens than
1. Under similar conditions as reported for 1, we have now
found that the C-disaccharide analogue 2 with QS21 as adju-
vant[7] induces a strong immune response in mice after two
boosts. This suggests that C-disaccharides can be used to
construct therapeutic vaccines against cancer and other dis-
eases. Much weaker immune response was observed when
Various strategies towards the incorporation of non-natu-
ral hydrolysis-resistant carbohydrate analogues into vaccine
constructs have been explored, including the use of C-glyco-
sides[8] and S-glycosides[8b,9] analogues of O-glycosides, as
well as O-deoxyfluoroglycosides.[10] It has been proposed
also to use b3-homothreonine conjugates instead of threo-
nine or serine to construct mucin-like glycopeptides antigen
analogues.[11] In this report, we have explored whether the
replacement of the O-linked disaccharide moiety in a C-
linked disaccharide analogue would still induce an immune
response, and whether the latter would depend on the b- or
a-configuration of the d-galalactopyranoside moiety of the
artificial antigen. The exchange of one acetal oxygen atom
for a CH2 group (change from acetal to ether function)
modifies the polarity and water solubility of the disacchar-
ide, as well as the population of its conformers about the
two interglycosidic bonds. Several studies have suggested
that the energy maps of C-linked disaccharides are similar
to maps of the corresponding O-disaccharides, but there are
differences in the locations and the relative free energies of
using antigen 3 constructed from a-d-Galp-1ACTHUNTRGNE(UNG CH2)-3-a-d-
GalNAcp-O-serine (a a-C-galactoside rather than b-C-gal-
actoside as in 2; Figure 1).
[a] Prof. L. Awad, Dr. A. Gillig, Dr. M. Kolympadi, Prof. P. Vogel
Laboratory of Glycochemistry and Asymmetric Synthesis (LGSA)
Ecole Polytechnique Fꢀdꢀrale de Lausanne (EPFL)
1015 Lausanne (Switzerland)
the minima.[12] For example, with b-d-Galp-(1
ACTHNUGTRENNG(U CH2)-4)-b-d-
Fax : (+41)21-6930550 or (+41)21-6939375
E-mail: loayawad@gmail.com
GlcNAcp-OMe, the C-disaccharide populates three distinc-
tive conformational families in water solution, the major
one being the anti-Y-conformation, which is only marginally
populated for the O-linked disaccharide.[13]
The construction of antigens 2 and 3 are outlined in
Schemes 1 and 2, respectively, and are described in details in
the Supporting Information. The synthesis of the C-disac-
charide mimetic of the TF-disaccharide started with alde-
hyde 4.[14] Itoh–Nozaki condensation[15] of 4 with isolevoglu-
pierre.vogel@epfl.ch
[b] Dr. R. Madani, Dr. M. Philgren, Dr. A. Muhs
AC-Immune, Ecole Polytechnique Fꢀdꢀrale de Lausanne (EPFL)
1015 Lausanne (Switzerland)
[c] Dr. C. Gꢀrard
GlaxoSmith Kline Biologicals
1330 Rixensart (Belgium)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200364.
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Scheme 1. Synthesis of triglycopeptide L18 and the preparation of the corresponding antigen 2: Triphenylmethyl (Trt) ; 2-(1H-7-azabenzotriazol-1-yl)–
1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HAUT); trifluoroacetic anhydride (TFAA); 1,8-diazabicycloACHTNUTRGNEUNG[5.4.0]undec-7-ene
(DBU); 4-dimethylaminopyridine (DMAP); N,N-diisopropylethylamine, or Hꢂnig’s base (DIEA); phosphate buffered saline (PBS).
cosenone (5)[16] induced by Et2AlI gave a single enone,
which reacted with BnONHOMe to give 6. Water elimina-
tion from 6 produced a major enone that was reduced (1,4-
reduction) followed by ketone reduction with LiBH4 pro-
ducing 7. Further reduction of BnNOMe provided the corre-
sponding amine that was converted into an azide by diazo
transfer.[17] After exchange of the silyl group for acetate
esters, which produced 8, the 1,6-anhydrogalactose was used
in the a-O-galactosidation of l-serine derivative 9. This gave
the major galactoside 10, the azido moiety of which was re-
duced into acetamide 11. Liberation of the carboxylic acid
UV/Vis spectroscopy that 230ꢁ20 molecules of the triglyco-
peptide were grafted per molecule of KLH protein.
Antigen was constructed in the same manner
3
(Scheme 2) starting with aldehyde 19, which was reacted
with isolevoglucosenone (5) and Et2AlI to produce a single
enone that was converted into a-C-disaccharide 22 applying
the same sequence of reactions that converted 6 into 12
(Scheme 1). The free carboxylic acid 25 was coupled with
trityl resin monoprotected diamine 1,4-di(aminomethyl)ben-
zene to give 26; this linker was selected to offer chromo-
phore for better detection in HPLC. After Fmoc cleavage of
26, the free primary amine was coupled with acid 25. Re-
peating the same sequence of reactions and capping the last
amine moiety as an acetamide gave polyacetylated triglyco-
peptide 28. Its liberation from the solid polymer and subse-
quent coupling with pentafluorophenyl 4-acetylthiobuta-
noate (prepared in situ from 4-acetylthiobutyric acid and
pentafluorophenol[19]) gave 29 (22%, nine steps). Zemplen
deacetylation of 29 provided the corresponding unprotected
triglycopeptide L30, which was conjugated with MBS–
KLH[5a] (Scheme 2) by Michael addition to give antigen 3.
The degree of conjugation was assumed to be n=200ꢁ50
molecules[20] of triglycopeptide per molecule of KLH as con-
ꢀ
gave 12 that was coupled to a trityl polymer by using NH2
ꢀ
A
Removal of the Fmoc group of 13 gave an amine that was
coupled with carboxylic acid 12 to produce a dipeptide. Re-
peating the same sequence of reactions, 14 was obtained.
Removal of Fmoc group of 14 and capping the primary
amine with 4-(dimethylamino)-azobenzene-4’-carboxylic
acid 15 gave 16. Subsequent liberation of the protected tri-
glycopeptides from the polymer and coupling with penta-
fluorophenyl acetylthioacetate[5a,18] gave 17 (25% over ten
steps). Zemplen deacetylation of 17 provided the corre-
sponding deacetylated triglycopeptide L18, which was react-
ed with MBS-KLH by Michael addition to give antigen 2.
Tagging with the azocarboxamide permitted an accurate
determination of the degree of conjugation. It was found by
1
firmed by comparison of the H NMR spectra in D2O of 2
and 3 with that of MBS–KLH.
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Inducing of a Strong Immune Response in Mice by Disaccharide
COMMUNICATION
Scheme 2. Synthesis of triglycopeptide L30 and the preparation of the corresponding antigen 3.
Vaccine candidates b-V and a-V were prepared out of
25 mg of antigen 2 and antigen 3, respectively, and 125 mg of
adjuvant QS21 per 1 mL of water. The pH of these solutions
was 6.8 (first injections), but was then adjusted to pH 7.0
with 2ꢃPBS (all following injections). One blank solution
without the antigen B-QS-21 was prepared out of 125 mg of
QS-21 per 1 mL of water. These solutions were stored at
ꢀ208C. BALB/c adult mice (nine weeks old, ten females/
group) were housed at the EPFL-animal facility, and all ex-
periments were performed according to Swiss legislation
(permission obtained). Mice received subcutaneous (s.c.) in-
jections of the different vaccine preparations or the adjuvant
QS21 batch. Injections of 100 mL/dose (except the first one,
which used 200 mL) consisted of 5 mg antigen/25 mg QS21/
dose. Injections were done on five occasions with a two-
weeks interval between each immunization (at day 0, 15, 28,
42, and 56). Mice from all groups showed a normal growth
with time (the analysis of variance ANOVA repeated mea-
sure, P=0.001). Even before starting the treatment, the
group treated with b-V vaccine candidate had a slightly
higher body weight (two-ways ANOVA repeated measure,
P=0.071, interaction groupꢃtime, P=0.5). No treatment
effect on the body weight was observed (see Figure on p.58
in the Supporting Information).
the second injection at day 21 and 35 (Figure 3A). Total
IgG titers were significantly higher than titers in mice immu-
nized with the B-QS-21 (adjuvant alone) (two-ways
ANOVA, Bonferroni posttest P<0.0001) at all days after
day seven. A boost effect was observed at day 21 (two-ways
ANOVA, days effect P<0.0001), and then the response in-
creased slightly with each boosting.
In contrast, no IgG titers toward a-V were detected in the
group immunized with the a-V at all days (Figure 2B). Only
at days 49 and 63, very weak total IgG titers were observed
(2-way ANOVA, Bonferroni posttest P<0.0001), but they
were too low to be considered as an immune response. To
verify that the detected antibodies rose against b-V were
specific to antigen 2, sera were analyzed on plate coated
with pure ligand L18. Anti-L18 IgG titers were strong at all
days (Figure 3A). A boost effect was observed at day 21
and 35. As a control, sera of three bleeding days (7, 35, and
65) of the group treated with B-QS-21 were also analyzed in
the same ELISA test. In these control sera, no immune re-
sponse was detected against the adjuvant (Figure 3A). The
cross reaction of the antibodies generated by each vaccine
candidate b-V and a-V were analyzed against the different
antigens 2 and 3 (Figure 3B). Sera of day 63 of mice immu-
nized with b-V or with a-V were analyzed on plates coated
with triglycopeptide L18 or with triglycopeptide L30. Sera
of mice immunized with antigen 2 showed a big difference
in binding L18 versus L30 (P<0.005). Thus, IgG antibodies
raised against b-V did not cross react with L30. There were
very low levels of IgG antibodies against either L30 or L18
Total IgG titers from sera (bleeding at day ꢀ7, 7, 21, 35,
49, and 63) were analyzed by enzyme-linked immunosorbent
assay (ELISA) by using plates coated with b-V or a-V
(Figure 2). The sera of the mice immunized with b-V
showed a strong immune response against b-V already after
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L. Awad, P. Vogel et al.
Figure 3. A) Anti-L18 titers in mice immunized with b-V vaccine or with
adjuvant (QS-21) alone. B) Cross reaction of antibodies induced by b-V
and a-V against L18 and L30: I) Sera/b-V on L18. II) Sera/b-V on L30.
III) Sera/a-V on L18. IV) Sera/a-V on L30. Sera at day 63 were used in
these analyses. Mean of antibodies+standard deviations are shown.
Figure 2. Immune response in mice immunized with b-V or with a-V vac-
cines. Analysis of IgG antibodies raised against: A) b-V; B) a-V in the
sera of mice at day ꢀ7, 7, 21, 35, 49, and 63. Mean of antibodies+stan-
dard deviations are shown.
in the sera of mice immunized with a-V. Taken together,
these results suggest a specific binding of the b-V-derived
antibodies to antigen 2.
To further verify the specificity of the antibodies raised
against b-V and a-V, respective sera were analyzed on plates
coated with KLH alone (Figure 4). Anti-KLH IgG titers
were barely detected before day 63. At that day, the sera
showed a very weak interaction with KLH alone. The sera
of mice immunized with B-QS-21 (adjuvant alone) did not
show any interaction with KLH in this ELISA test at any
day. As a positive control, Ab12B4-specific anti-KLH anti-
body was used.
Figure 4. Anti-KLH titers in mice immunized with b-V or a-V vaccine
candidates or with QS-21. Analysis of anti-KLH IgG antibodies in the
sera of mice at day ꢀ7, 7, 21, 35, 49, and 63 of b-V and a-V treated mice.
Mean of antibodies+standard deviations are shown.
Specific IgG responses were determined by ELISA.
Plates were coated with the vaccines b-V and a-V (5 mm).
Other plates were coated with triglycopeptides L18 and L30
(5 mgmLꢀ1), or with KLH (25 mmmLꢀ1; Figure 4).
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Inducing of a Strong Immune Response in Mice by Disaccharide
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[3] a) G. D. MacLean, M. B. Bowen-Yacyshyn, J. Samuel, A. Meikle, G.
Stuart, J. Nation, S. Poppema, M. Jerry, R. Koganty, T. Wong, J. Im-
munother. 1992, 11, 292–305; b) R. R. Koganty, M. A. Reddish,
B. M. Longenecker, Drug Discovery Today 1996, 1, 190–198; c) J.
Kanitakis, I. al-Rifai, M. Faure, A. Claudy, J. Clin. Pathol. 1998, 51,
588–592; d) V. V. Glinsky, G. V. Glinsky, K. Rittenhouse-Olson,
M. E. Huflejt, O. V. Glinskii, S. L. Deutscher, T. P. Quinn, Cancer
Res. 2001, 61, 4851–4857.
[4] S. Adluri, F. Helling, S. Ogata, S. Zhang, S. H. Itzkowitz, K. O.
Lloyd, P. O. Livingston, Cancer Immunol. Immunother. 1995, 41,
185–192.
[5] a) S. D. Kuduk, J. B. Schwarz, X.-T. Chen, P. W. Glunz, D. Sames, G.
Ragupathi, P. O. Livingston, S. J. Danishefsky, J. Am. Chem. Soc.
1998, 120, 12474–12485; b) G. Ragupathi, R. R. Koganty, D. Qiu,
K. O. Lloyd, P. O. Livingston, Glycoconjugate J. 1998, 15, 217–221;
c) S. F. Slovin, G. Ragupathi, C. Musselli, C. Fernandez, M. Diani,
D. Verbel, S. Danishefsky, P. Livingston, H. I. Scher, Cancer Immu-
nol. Immunother. 2005, 54, 694–702.
[6] a) J. W. Woollen, P. G. Walker, Clin. Chim. Acta 1965, 12, 647–658;
b) H. S. Singer, I. A. Schafer, N. Engl. J. Med. 1970, 282, 571;
c) G. F. Springer, P. R. Desai, Carbohydr. Res. 1975, 40, 183–192.
[7] G. Ragupathi, P. Damani, K. Deng, M. M. Adams, J. Hang, C.
George, P. O. Livingston, D. Y. Gin, Vaccine 2010, 28, 4260–4267.
[8] a) D. Urban, T. Skrydstrup, J.-M. Beau, Chem. Commun. 1998, 955–
956; b) B. Kuberan, S. A. Sikkander, H. Tomiyama, R. J. Linhardi,
Angew. Chem. 2003, 115, 2119–2121; Angew. Chem. Int. Ed. 2003,
42, 2073–2075; c) L. Cipolla, M. Rescigno, A. Leone, F. Peri, B. La
Ferla, F. Nicotra, Bioorg. Med. Chem. 2002, 10, 1639–1646.
[9] a) D. R. Bundle, J. R. Rich, S. Jacques, H. N. Yu, M. Nitz, C.-C.
Ling, Angew. Chem. 2005, 117, 7903–7907; Angew. Chem. Int. Ed.
2005, 44, 7725–7729; b) J. R. Rich, W. W. Wakarchuk, D. R. Bundle,
Chem. Eur. J. 2006, 12, 845–858.
To conclude, the vaccine candidate b-V is immunogenic in
mice and induces specific IgG titers against triglycopeptide
L18.
This work demonstrates for the first time that a C-disac-
charide analogue of an O-disaccharide sugar epitope can be
used to construct vaccine candidates. This opens the possi-
bility to obtain antigens, which are not metabolized too
quickly in the body and thus should lead to vaccines capable
to induce a desired immune response with a single or limit-
ed number of injections. Very interesting is the finding that
the b-C-disaccharide analogue of the TF disaccharide sugar
epitope
(b-d-Galp-(1-3)-a-d-GalNAcp-O-l-serine)
has
a much stronger immune response than its a-C-disaccharide
stereoisomer. We believe that the use of different linkers,
different N-terminus capping of the conjugates are not caus-
ing difference in immunogenicity.[21]
It is thus possible that the efficiency and high stereoselec-
tivity of the immune response observed with our vaccine
candidate b-V corresponds to a stimulation of the natural
immune response that mice normally have toward the genu-
ine TF epitope (auto-antigen) of naturally formed cancer
cells. This leaves the possibility to construct therapeutic vac-
cines based on C-disaccharide analogues of natural O-disac-
charide epitopes.
Acknowledgements
[10] a) C. Mersch, S. Wagner, A. Hoffmann-Roeder, Synlett 2009, 2167–
2171; b) F. Yang, X.-J. Zheng, C.-X. Huo, Y. Wang, Y. Zhang, X.-S.
Ye, ACS Chem. Biol. 2011, 6, 252–259.
[11] F. Karch, A. Hoffmann-Rçder, Beilstein J. Org. Chem. 2010, 6, No.
47, DOI: 10.3762/bjoc.6.47.
[12] a) B. Lꢄpez-Mꢀndez, C. Jia, Y. Zhang, L.-H. Zhang, P. Sinay, J. Ji-
menez-Barbero, M. Sollogoub, Chem. Asian J. 2008, 3, 51–58; b) L.
Drouin, E. C. Stanca-Kaposta, P. Saundh, A. J. Fairbanks, S.
Kemper, T. D. W. Claridge, J. P. Simons, Chem. Eur. J. 2009, 15,
4057–4069.
We thank the Swiss National Science Foundation (Bern) and the SER
(Bern, European COST D13/0001/99 and FP6-TRIoH projects) for sup-
port. We also thank the University of Lausanne (Mrs. A. Charon) for
a Peace Grant in favor of L.A. We are grateful also to Dr. R. Scopelliti
(X-ray analysis), M. Marial Rey (NMR spectroscopy), and Francisco Se-
pulveda (MS) for their technical help. The biological assays would not
have been possible without the help of Dr. Wolfgang Froestl (AC
Immune).
[13] V. Garcꢅa-Aparicio, M. Sollogoub, Y. Bleriot, V. Colliou, S. Andre,
J. L. Asensio, F. J. Canada, H.-J. Gabius, P. Sinay, J. Jimenez-Bar-
bero, Carbohydr. Res. 2007, 342, 1918–1928.
Keywords: antibodies · antigens · cancer · glycosides ·
[14] a) A. S. Bhat, J. Gervay-Hague, Org. Lett. 2001, 3, 2081–2084; b) T.
Uchiyama, O. Hindsgaul, ChemInform 1996, 27, DOI: 10.1002/
chin.199642226.
immunoassays
[15] a) A. Itoh, S. Ozawa, K. Oshima, H. Nozaki, Bull. Chem. Soc. Japan
1981, 54, 274–278; b) A. Itoh, S. Ozawa, K. Oshima, H. Nozaki, Tet-
rahedron Lett. 1980, 21, 361–364.
[16] D. Horton, J. P. Roski, P. Norris, J. Org. Chem. 1996, 61, 3783–3793.
[17] a) P. B. Alper, S. C. Hung, C. H. Wong, Tetrahedron Lett. 1996, 37,
6029–6032; b) R.-B. Yan, F. Yang, Y. Wu, L.-H. Zhang, X.-S. Ye,
Tetrahedron Lett. 2005, 46, 8993–8995.
[18] G. J. P. H. Boons, P. Hoogerhout, J. T. Poolman, G. A. Van der Mar-
el, J. H. Van Boom, Bioorg. Med. Chem. Lett. 1991, 1, 303–308.
[19] L. J. Williams, C. R. Harris, P. W. Glunz, S. J. Danishefsky, Tetrahe-
dron Lett. 2000, 41, 9505–9508.
[1] a) G. Springer, Science 1984, 224, 1198–1206; b) T. Feizi, Cancer
Surv. 1985, 4, 245–269; c) S. Hakomori, Cancer Res. 1985, 45, 2405–
2414; d) K. O. Lloyd, Semin. Cancer Biol. 1991, 2, 421–431.
[2] a) C.-Y. Wu, P.-H. Liang, C.-H. Wong, Org. Biomol. Chem. 2009, 7,
2247–2254; b) W. Dullenkopf, G. Ritter, S. R. Fortunato, L. J. Old,
R. R. Schmidt, Chem. Eur. J. 1999, 5, 2432–2438; c) K. M. Koeller,
C.-H. Wong, Glycoscience 2001, 3, 2305–2352; d) A. Kaiser, N.
Gaidzik, U. Westerlind, D. Kowalczyk, A. Hobel, E. Schmitt, H.
Kunz, Angew. Chem. 2009, 121, 7688–7692; Angew. Chem. Int. Ed.
2009, 48, 7551–7555; e) A. Hoffmann-Rçder, A. Kaiser, S. Wagner,
N. Gaidzik, D. Kowalczyk, U. Westerlind, B. Gerlitzki, E. Schmitt,
H. Kunz, Angew. Chem. 2010, 122, 8676–8681; Angew. Chem. Int.
Ed. 2010, 49, 8498–8503; f) T. Buskas, S. Ingale, G.-J. Boons, Angew.
Chem. 2005, 117, 6139–6142; Angew. Chem. Int. Ed. 2005, 44, 5985–
5988; g) A. Kaiser, N. Gaidzik, T. Becker, C. Menge, K. Groh, H.
Cai, Y.-M. Li, B. Gerlitzki, E. Schmitt, H. Kunz, Angew. Chem.
2010, 122, 3772–3776; Angew. Chem. Int. Ed. 2010, 49, 3688–3692;
h) U. Westerlind, H. Kunz, Chimia 2011, 65, 30–34.
[20] D. Udy, J. Am. Oil Chem. Soc. 1971, 48, A29A33.
[21] S. von Mensdorff-Pouilly, Expert Rev. Vaccines 2010, 9, 579–594.
Received: February 3, 2012
Published online: && &&, 0000
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L. Awad, P. Vogel et al.
Immunoassays
L. Awad,* R. Madani, A. Gillig,
M. Kolympadi, M. Philgren, A. Muhs,
C. Gꢀrard, P. Vogel* ......... &&&& — &&&&
A C-Linked Disaccharide Analogue of
Thomsen–Friedenreich Epitope
Induces a Strong Immune Response in
Mice
Anticancer vaccine: Antigen 2 made
out of a tripeptide of b-d-Galp-(1!3)-
CH2-a-d-GalNAc-O-Ser and conju-
gated with Keyhole limpet hemocyanin
(KLH) protein induced early and
strong immune response (IgG antibod-
ies) in mice. Antigen 3 made out of a-
C-galactoside analogue did not induce
such immune response (see scheme).
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