Synthesis and vaccine evaluation of the tumor-associated carbohydrate antigen RM2 from prostate cancer

Article abstract of DOI:10.1021/ja403609x

We have successfully developed a [1+2+3] one-pot strategy to synthesize the RM2 antigen hexasaccharide that was proposed to be a prostate tumor antigen. The structure of the synthetic product was verified by NMR analysis and antibody binding assay using a glycan microarray. In addition, the synthetic antigen was conjugated to a mutated diphtheria toxin (DT, CRM197) with different copy numbers and adjuvant combinations to form the vaccine candidates. After vaccination in mice, we used glycan microarrays to monitor their immune response, and the results indicated that, when one molecule of DT was incorporated with 4.7 molecules of RM2 on average (DT-RM_4.7) and adjuvanted with the glycolipid C34, the combination exhibited the strongest anti-RM2 IgG titer. Moreover, the induced mouse antibodies mediated effective complement-dependent cytotoxicity (CDC) against the prostate cancer cell line LNCap.

Full text of DOI:10.1021/ja403609x

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Article
Synthesis and Vaccine Evaluation of the Tumor Associated
Carbohydrate Antigen RM2 from Prostate Cancer
Hong-Yang Chuang, Chien-Tai Ren, Chung-An Chao, Chung-Yi Wu, SACHIN
SHANKAR SHIVATARE, Ting-Jen R. Cheng, Chung-Yi Wu, and Chi-Huey Wong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja403609x • Publication Date (Web): 02 Jul 2013
Downloaded from http://pubs.acs.org on July 9, 2013
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Synthesis and Vaccine Evaluation of the Tumor Associated
Carbohydrate Antigen RM2 from Prostate Cancer
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†,
†
†
†
⊥
Hong-Yang Chuang , Chien-Tai Ren , Chung-An Chao , Chung-Yi Wu , Sachin S
Shivatare†,§,‡, Ting-Jen R. Cheng†, Chung-Yi Wu†,*, and Chi-Huey Wong †,⊥, ‡,*
†. Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2,
Nankang, Taipei 115, Taiwan.
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⊥. Department of Chemistry, National Taiwan University, 1 Roosevelt Road, Section
4, Taipei, 106, Taiwan.
§. Chemical Biology and Molecular Biophysics, Taiwan International Graduate
Program, Academia Sinica, Taiwan.
‡. Institute of Biochemical Sciences, National Taiwan University, 1 Roosevelt Road,
Section 4, Taipei, 106, Taiwan.
*To whom correspondence should be addressed: C-Y. Wu, Tel: +886-2-27871263,
Fax: +886-2-27898771, E-mail: cyiwu@gate.sinica.edu.tw or C.-H. Wong, Tel:
+886-2-27899400, Fax: +886-2-27853852, E-mail: chwong@gate.sinica.edu.tw (or
wong@scripps.edu).
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Abstract
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We have successfully developed a [1+2+3] one-pot strategy to synthesize the RM2
antigen hexasaccharide that was proposed to be a prostate tumor antigen. The
structure of the synthetic product was verified by NMR analysis and antibody binding
assay using a glycan microarray. In addition, the synthetic antigen was conjugated to a
mutated diphtheria toxin (DT, CRM197) with different copy numbers and adjuvant
combination to form the vaccine candidates. After vaccination in mice, we used
glycan microarrays to monitor their immune response, and the results indicated that
when one molecule of DT was incorporated with 4.7 molecules of RM2 in average
(DT-RM4.7) and adjuvanted with the glycolipid C34 exhibited the strongest anti-RM2
IgG titer. Moreover, the induced mouse antibodies mediated effective
complement-dependent cytotoxicity (CDC) against the prostate cancer cell line
LNCap.
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Introduction
An ideal target antigen for development of cancer vaccine should be specifically
expressed on cancer cells and accessible to the immune system. Tumor associated
carbohydrate antigens (TACAs) are not only most abundantly and sometimes
aberrantly expressed on the surface of cancer cells but also absent or rarely expressed
on normal cells.1 Many TACAs have been characterized for specific types of cancer2
and suggested for the development of tumor-specific vaccines.3,4 However, because
carbohydrates generally display poor immunogenicity, a glycan vaccine conjugated
with a carrier protein can stimulate the immune response. One example is the Globo
H-KLH vaccine, which was first synthesized by Danishefsky and co-workers via the
convergent glycal assembly strategy to Keyhole limpet hemocyanin (KLH)
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conjugate.5,6 In the phase I clinical trial of Globo H-KLH,7,8 ~700 (Globo H) GH units
conjugated to one KLH protein was administered together with QS21 adjuvant. The
treatment appeared to be fairly well-tolerated and was capable of inducing a high titer
of IgM antibodies that participate in complement-mediated tumor cell lysis. Currently,
this vaccine is in phase III clinical trials in Taiwan and in phase II clinical trials in
USA, Korea, Hong-Kong and India. Moreover the similar synthetic approaches have
been utilized to synthesize other vaccines (e.g., fucosyl-GM1-KLH9,10 and Ley-KLH11)
against other types of cancer.
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In this study, we focused on a novel TACA, the RM2 antigen (Fig. 1), a
glycosphingolipid (GSL) isolated and identified by Hakomari and colleagues from a
renal cell carcinoma cell line TOS-1 and prostate cancer cell lines LNCap and PC-3.12
It was immunocharacterized by the monoclonal antibody RM2 and, therefore, termed
the “RM2 antigen”. The RM2 antigen, β-1.4-GalNAc-disialyl-Lc, is highly expressed
on the prostate cancer cell surface as a glycolipid and possibly as a glycoprotein.13 In
addition, the RM2 antigen expression is closely associated with the prostate cancer
staging Gleason grading system, and is upregulated as the Gleason grade increases.14
Therefore, the RM2 antigen can also be developed as a new and novel marker for
prostate cancer. The structure of the RM2 antigen consists of a novel hybrid core with
the “ganglio-series” and the “disialyl lacto-series type 1 chain” groups (Fig. 1) and is
easily confused with the DSGG antigen, because (1) RM2 and DSGG are
indistinguishable on high performance thin-layer chromatography and (2) the renal
cell carcinoma expresses many types of disialogangliosides and other globo-series
monosialogangliosides, leading to varying proportions of globo-series and ganglio
/lacto-series antigens.15
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On the other hand, this internal hexasaccharide RM2 without the GlaNAc residue is
identical to α(2,3)/α(2,6)-disialyl lactotetraose (DSLc4), which was observed as a
colonic cancer-associated antigen by using a monoclonal antibody (FH9)16 for
siglec-7.17 Recently, Kiso and co-workers reported a systematic synthesis of DSLc4
via the α-stereoselective sialylation of O-6 and introduction of the sialyl-α-(2→
3)-D-galactose unit to the O-3 position of GlcNAc.18
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To further verify the proposed structure of the RM2 antigen, we chemically
synthesized the proposed hexasaccharide 1 and evaluated its recognition by
monoclonal antibody RM2. To our best knowledge, the RM2 carbohydrate antigen
has not been synthesized; the introduction of two sialic acid units to the compact and
rigid 3,4 dibranched galactoside unit is very challenging, due to (1) the presence of
the C1 carboxyl group of sialic acids reduces the reactivity of the anomeric position;
and (2) the lack of C-3 participating group fails to direct the stereoselectivity and, at
the same time, readily promotes the undesired 2,3-elimination. Many approaches have
been developed to address these problems, including: (1) the design of different
leaving groups at the anomeric center19-21; (2) the installation of TFA22 or Troc23 at the
N-5 position; (3) the 1,5- intramolecular lactamization24; (4) the use of acetonitrile as
solvent in glycosylation25; and (5) the installation of 5-N,4,O-carbonly group
(oxazolidinone ring) in sialic acid as protecting group.26 Recently, our group
incorporated
the
dibutyl
phosphate
as
leaving
group
into
the
N-acetyl-5-N,4-O-carbonyl-protected sialoside as a new sialyl donor to increase
α-selectivity and reaction yield.27
Besides the difficulty of sialylation, the development of a method for the
β-selective and efficient glycosylation of the galactosamine moiety at the 4-position
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of dibranched galactose is problematic owing to the steric hinderence of the adjacent
sialyl moiety. Here, we describe the use of our sialyl phosphate as donor to enable an
effective synthesis of the hexasaccharide of the proposed RM2 antigen using the
[1+2+3] one-pot sequential strategy28 with high yield and α-selectivity. The one-pot
oligosaccharide synthesis strategy has been recognized as a powerful method to
assemble complex oligosaccharides.29 There are three general one-pot strategies for
this purpose: (1) the chemoselective stategy,28,30,31 (2) the preactivation strategy,32 and
(3) the orthogonal strategy.33 Although the one-pot synthetic strategy is still needed to
build the individual differentially protected building blocks, the major advantage of
this method is that the purification of each coupling is not necessary. One just needs
to purify the final products after the one-pot reaction. We also synthesized some of the
truncated analogs of the proposed RM2 antigen in good yields and stereoselectivity.
With these synthetic carbohydrates in hand, we used the RM2 antibody for binding
analysis on a glycan array to confirm our synthetic structure. We then conjugated the
synthetic RM2 antigen to the carrier protein CRM197 to form the vaccine candidate
for vaccination in mice. Glycan arrays were used to monitor the titers of the induced
antibody and specificity. We further evaluated the efficacy of the vaccines by CDC
assay using the prostate cancer cell line LNCap.
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Fig. 1. The structure of a RM2 carbohydrate hapten 1. A: ganglio-series structure. B:
disialyl lacto-series type 1 structure.
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Results and discussion
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Retrosynthesis. Two main problems in the synthesis of the target compound 1 are
encountered: α-stereo- and regio-selective sialylation at O-6 of the GlcNAc residue
and O-3 of the Gal residue; and β-selective glycosylation of GalNAc at the O-4
position of the sialyl-α-(2,3)-D-galactose unit.
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After careful analysis of the structure of hexasaccharide 1, we decided to approach
the synthesis from a suitably protected hexasaccharide 2 (Fig. 2). The
hydroxylpentamine linker at the reducing end of the galactose residue was designed
for immobilizing hexasaccharide 1 onto the NHS-coated slides or conjugating to a
carrier protein to form a vaccine candidate. To use the step by step protocol for
oligosaccharide synthesis, orthogonal protecting groups were required to provide
potential acceptor sites for later glycosylation. Overall, the target hexasaccharide can
be divided into three parts, monosaccharide 3,34 disaccharide 4, and trisaccharide 5,
which are further divided into six monosaccharide building blocks 3, 7, 11, 12,27 15,
and 17.
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Fig. 2. Retrosynthetic analysis of the RM2 hapten.
Synthesis of GlcNAc building block 7. The synthesis of monosaccharide 7 can be
achieved from the known 4,6-O-benzylidene acetal 635 (Fig. 3). The
p-methoxybenzylation of 6 was carried out by p-methoxylbenzyl choride (PMBCl)
and sodium hydride (NaH) in DMF, followed by selective ring-opening of the
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4,6-O-benzylidene group with dibutylboranetriflate (Bu2BOTf) and borane-
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tetrahydrofuran complex (BH3 THF). After sequential removal of the phthalimido
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group and Troc formation, the desired product 7 was produced in four steps in 24%
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yield.
Fig. 3. Synthesis of GlcNAc building block 7. a. NaH, PMBCl, DMF, rt, 1 h; b.
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o
BH3 THF, Bu2BOTf, 0 C, 2 h; c. Ethylenediamine, ethanol, 80 C, 16 h; d. TrocCl,
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NaHCO3, THF, rt, 16 h, overall yield 24%.
Synthesis of galactose building block 11. The known compound 836 was acetylated
by the standard acetylation procedure (Fig. 4), and the acetylated compound was
glycosylated with the benzyl-5-hydroxypentylcarbamate in dichloromethane using
NIS/TfOH as a promoter to afford 9 in 60% yield. Compound 9 was de-acetylated
under the Zemplén condition; then, benzylation of the triol intermediate afforded 10
in 85% yield. The acetyl group was replaced by the benzyl group to increase the
reactivity of the galactose building block, followed by selective removal of the PMB
protecting group with DDQ to give 11 in 77% yield.
.
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Fig. 4. Synthesis of Galactose building block 11. a. Ac2O, pyridine, rt, 12 h; b. NIS,
o
cat. TfOH, benzyl 5-hydroxypentylcarbamate, CH2Cl2, -30 C, 3 h, 60% (over two
steps) ; c. NaOMe, MeOH, rt, 10 h; d. BnBr, NaH, rt, 2 h, 85% (over two steps); e.
DDQ, CH2Cl2/H2O, rt, 2 h, 91%.
Synthesis of trisaccharide 5. Compounds 7, 11, and 12 were used as starting
materials for the synthesis of trisaccharide building block 5 using an orthogonal
one-pot synthetic strategy (Fig. 5). The one-pot synthetic operation was performed by
sialyl phosphate donor 12 (1.5 eq.) and GlcNAc acceptor 7 (1.0 eq.) in the presence of
TMSOTf at -60 oC.
The second glycosylation between 7 and 11 was carried out by adding NIS (2.0
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eq.) to the reaction solution at higher temperature (-20 C). Finally, removal of the
PMB group by DDQ produced trisaccharide 5 as a single stereoisomer in 42% yield.
The configuration of the trisaccharide 5 was examined by NMR spectrometry, and the
3
new formed α-glycosidic bond was confirmed by coupling constant J(C1-H3ax) = 6.0
Hz.
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Figure 5. Orthogonal one-pot synthesis of trisaccharide 5. a. 7, 12, TMSOTf, MS 4Å,
CH2Cl2 : CH3CN = 1 : 2, -60 oC, 1 h; b. 11, NIS, -20 oC, 2 h; c. DDQ, CH2Cl2, H2O, rt,
12 h, overall yield 42%.
Synthesis of disaccharide 4. In order to synthesize the Neu5Ac α 2 → 3Gal
disaccharide with high α-stereoselectivity and yield, the thiophenyl group in the
anomeric center of 5-N,4-O-carbonyl-protected thiosialoside 1437 was replaced by the
dibutyl phosphate group at
0
oC under N-iodosuccinimide (NIS) and
trifluoromethansulfonic acid (TfOH) activation for 10 h to obtain 15 in 89% yield (α:β
= 13:1). In addition, compound 1636 was efficiently converted to the corresponding
6-O-benzyl-4-hydroxy derivative 17 using triethylsilane (Et3SiH) and
trifluorosulfonic acid (TfOH) in a high yield with excellent region-selectivity. With
the 3,4-dihydroxyl galactose acceptor 17 in hand, we first investigated the
stereoselectivity of several sialyl phosphate donors including 15 with galactose
acceptor 17 to produce Neu5Acα2→3Gal disaccharide (Table S1), and the results
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showed that the sialyl phosphate donor 15 had the excellent yields and
α-stereoselectivity. The glycosylation of 15 and 17 under the activation of TMSOTf in
CH2Cl2 at -78 oC for 2 h gave Neu5Acα2→3Gal disaccharide 4 as a single isomer in
87% yield (Fig 6). The configuration of the disaccharide 4 was examined by NMR
spectrometry, and the new formed α-glycosidic bond was confirmed by coupling
constant 3J(C1-H3ax) = 6.1 Hz.
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Fig. 6. Synthesis of disaccharide 4. a. NIS, TfOH, dibutylphosphate, MS 4Å, CH2Cl2,
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0 C, 10 h, 89%; b. Et3SiH, TfOH, MS 4Å, CH2Cl2, -78 C, 1 h, 85%; c. TMSOTf,
CH2Cl2, MS 4Å, -78 oC, 2 h, 87%.
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Fig. 7. Synthesis of hexasaccharide 2. a. TMSOTf, MS 4Å, CH2Cl2, -50 oC, 2 h, 73%;
b. NIS, TfOH, MS 4Å, CH2Cl2, 0 oC, 23 h, 62%.
Synthesis of compound 2. The preparation of the target hexasaccharide 2 was started
from the TMSOTf-promoted coupling of the GalNAc donor 334 with disaccharide 4.
After glycosylation, the fully protected trisaccharide compound 18 was isolated in
1
73% as a 6 to 1 mixture of rotamers (determined by H NMR spectroscopic analysis
of the crude reaction). Convergent synthesis of hexasaccharide 2 was achieved as a
single stereoisomer in good yield (62%) by glycosylation of trisaccharide acceptor 5
with trisaccharide donor 18 in the NIS/TfOH promoting system at 0 oC for 23 h (Fig.
7). We further conducted an orthogonal one-pot [1+2+3] glycosylation and
successfully synthesized the target hexasaccharide 2 (Fig. 8). Chemoselective
glycosylation of the phosphate donor 3 with the thioglycoside 4 under the
o
TBDMSOTf activation in CH2Cl2 at -50 C provided trisaccharide 18. Without
isolation, the acceptor 5 and NIS were subsequently added to the reaction vessel at 0
oC and the reaction was finished in 23 h to afford the protected hexasaccharide 2 as a
single stereoisomer in an overall yield of 32% based on 5.
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HO
O
OBn
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MeOOC
S
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OBz
BnO
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HN
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AcO
OAc
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COOMe
BnO
TBSO
(a)
OAc
O
AcO
AcN
O
O
O
BnO
OBn
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OBu
O
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BnO
HO
P
OBu
Bn
NHTroc
O
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O
O
NCbz
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(b)
OBn
NHTroc
5
32%
5
AcO
OAc
O
OBn
AcO
AcN
OAc
TBSO
COOMe
O
O
O
O
OBn
TrocHN
MeOOC
O
BnO
OBn
OBn
O
O
O
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Bn
NHTroc
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O
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OBn
NCbz
BnO
HN
O
O
Fig. 8. One-pot synthesis of hexasaccharide 2. a. 3, 4, TBDMSOTf, MS 4Å, CH2Cl2,
-50 oC, 2 h; b. 5, NIS, 0 oC, 22 h, 32% (over two steps) as a single stereoisomer.
Syntheses of truncated RM2 derivatives 20, 22, 24, and 26. Although RM2
antibody has been reported to recognize the RM2 antigen, its specificity for related
carbohydrates is not well studied. To understand the specificity of RM2 antibody, we
synthesized the RM2 antigen fragments 27, 28, 29, 30, and 31 to determine the
specificity of monoclonal anti-RM2 antibody. The syntheses of the RM2 fragments 20,
22, 24, and 26 followed the above method (Fig. 9). Donors 19,38 21,38 or 2327 were
coupled to acceptor 5 to give tetrasaccharide 20, pentasaccharide 22, or
pentasaccharide 24, respectively. In addition, treatment of the trisaccharide donor 18
and disaccharide acceptor 25 with NIS and a catalytic amount of TBDMSOTf at 0 oC
gave
the
protected
pentasaccharide
26
in
67%
yield.
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OAc
OAc
AcO
6
7
8
9
AcN
COOMe
O
O
O
O
O
O
BnO
OBn
O
O
BzO
S
Bn
O
BnO
O
OBz
(a)
O
O
NCbz
O
19
10
11
12
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18
19
20
21
22
23
24
25
26
27
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31
32
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60
O
O
OBn
NHTroc
20 (62%)
5
OBz
OAc
BzO
AcO
OAc
BnO
COOMe
AcO
AcN
OBn
OAc
O
O
O
OBn
AcO
AcO
TrocHN
BnO
OBn
O
O
O
O
Bn
O
BzO
O
S
BnO
O
O
O
NCbz
OBn
OBz
TrocHN
O
OBn
21
5
NHTroc
22 (68%)
O
O
(b)
5
OBz
BzO
OAc
OAc
AcO
AcN
O
O
CO2Me
O
O
O
O
O
S
O
O
BnO
AcN
AcO
OBn
O
O
O
O
OBz
Bn
CO2Me
BnO
O
O
O
O
OAc
O
NCbz
23
OAc
O
OBn
5
NHTroc
24 (60%)
(c)
OBz
O
O
O
CO2Me
O
N
Ac
AcO
OAc
OAc
Fig. 9. Synthesis of 20, 22, 24, and 26. a. NIS, TfOH, MS 4Å, CH2Cl2, 0 oC, 9 h, 62%;
b. NIS, TfOH, MS 4Å, CH2Cl2, 0 oC, 11 h, 68%; c. NIS, TfOH, MS 4Å, CH2Cl2, -20
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oC, 3 h, 60%; d. NIS, TBDMSOTf, MS 4Å, CH2Cl2, 0 oC, 8 h, 67%.
5
6
7
8
9
10
11
12
13
14
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18
19
20
21
22
23
24
25
26
27
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60
OH
HO
O
OH
O
OH
O
HO
HO
O
OH
O
AcHN
O
HO
O
26
O
NH2
O
HO
O
OH
NHAc
OH
AcHN
HO
CO2H
31 (13%)
b
OH
OH
OH
OH
CO2H
HO
AcHN
O
OH
HO
HO
HO
O
OH
O
O
O
HO
2
O
OH
AcHN
HO
O
O
O
NH2
O
HO
O
OH
NHAc
O
OH
AcHN
HO
CO2H
1 (16%)
OH
OH
Fig. 10. Global deprotection of 2, 5, 20, 22, 24, and 26. a. (i) LiOH‧H2O, Dioxane,
o
H2O, 90-95 C, 36 h; (ii) Ac2O, NaHCO3, H2O then LiOH, H2O, rt, 12 h; (iii)
o
Pd(OH)2, H2, CH3OH, H2O, rt, 12 h; b. (i) LiOH‧H2O, Dioxane, H2O, 90-95 C, 36
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h; (ii) Ac2O, pyridine, DMAP, 12 h; (iii) BF3.OEt2, CH3CN, 0 oC→rt, 18 h; (iv) LiOH,
5
6
H2O, rt, 12 h (v) Pd(OH)2, H2, CH3OH, H2O, rt, 12 h.
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8
9
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Global deprotection of the protected hexasaccharide 2 and pentasaccharide 26
was achieved using a four-step procedure: (i) hydrolysis of acyl protecting groups; (ii)
acetylation of the amine groups; (iii) removal of the acetonide groups; and (iv)
hydrogenolysis of the resulting benzyl ethers to provide the fully deprotected
hexasaccharide 1 and pentasaccharide 31. In addition, using a similar strategy,
deprotection of 5, 20, 22, and 24 gave the corresponding deprotected oligosaccharides
27-30, respectively, in good yields. These materials can be used for the preparation of
a glycan array for antibody binding study and for the preparation of glycoconjugates
for vaccine development.
To confirm that the synthesized hexasaccharide 1 was indeed the RM2 antigen and
to further understand the antibody binding specificity, we prepared an expanded
glycan array containing the synthesized hexasaccharide 1 and its analogs along with
the other ninety glycans prepared in our laboratory to form a glycan array for testing
(Fig. S4). All glycans were directly immobilized onto NHS-coated glass slides by
taking an aliquot from a stock solution of sugar at a fixed concentration (100μM).
The assay involved an initial treatment with RM2 (a mouse IgM anti-RM2
monoclonal antibody, a kind gift from Prof. Saito), followed by incubation with a
fluorescein-tagged anti mouse IgM secondary antibody against its primary antibody.
After one hour incubation, the slides were washed with ddH2O twice and scanned to
show the binding specificity of the antibody to printed oligosaccharides. The resulted
images showed that monoclonal RM2 antibody recognize hexasaccharide 1
specifically, indicating that the synthetic hexasaccharide 1 contains the same antigenic
epitope with which RM2 antibody reacts on prostate cancer cells (Fig. 11); therefore,
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we concluded that the synthesized hexasaccharide 1 was indeed the RM2 antigen.
Moreover, the structure assignment of the synthesized hexasaccharide 1 is confirmed
by both 1D and 2D-NMR (Fig. S1 and Table S2). Furthermore, to determine the
dissociation constants of RM2 and truncated analogs on the surface interacting with
the antibody in a multivalent manner, we followed the direct measurement method
developed in our laboratory,39 using different concentrations of antibodies and printed
sugars. The results showed that when the printing concentration is below 6.25 ꢀΜ, the
average distance between printed sugars is too far to have enough multivalent effect
with antibody RM2. At the printing concentrations from 100 ꢀΜ to 6.25 ꢀΜ, however,
we observed that the KD,surf values measured were narrowly distributed from the
individual curves (Fig. S2 and Table S3). Using the same method, the KD,surf values
were determined for the truncated RM2 analog 29 interacting with the antibody (Fig.
S3 and Table S4). Overall, the relative binding specificity of RM2 for the epitopes
was RM2 > pentasaccharide 29 (Table 1).
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60
anti-RM2 IgM(1:60)
4.0 10 7
×
3.0 10 7
×
2.0 10 7
×
1.0 10 7
×
0
Fig. 11. Binding specificity of monoclonal antibodies RM2 to RM2 antigen and its
fragments.
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Table 1. KD,surf (nM) values of different antibodies and
different RM2 analogs.
9
KD,surf (nM) SD(nM)
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Pentasaccharide,
Antibody
RM2
RM2 (1)
29
4.545 0.903
5.756 1.140
Synthesis of DT-RM4.7 as a vaccine candidate with glycolipid C34 as adjuvant.
To develop a general protocol40 for carbohydrate-carrier protein conjugation (Fig.
12 and Table S5), we adopted the thiol-maleimide coupling method for sialic
acid-rich compounds. The amine group of the hexasaccharide 1 was reacted with 2
equiv of 3,3´-Dithiobis(sulfo- succinimidylpropionate) (DTSSP) and an
amine-reactive N-hydroxysulfosuccinimide (sulfo-NHS) ester at each end of an
8-carbon spacer arm in pH 7.4 phosphate buffer at room temperature for 8 h to afford
the corresponding half ester. Next, the disulfide bond was cleaved in the presence of
o
dithiothreitol (DTT) at 40 C for 2 h to obtain the free thio product 32 as Michael
donors in 78% yield after purification on a size exclusion column LH-20. Furthermore,
in order to generate the thio active maleimide group on the protein, CRM197 was
reacted with an excess of N-(ε-Maleimidocaproyloxy) sulfosuccinimide ester
(Sulfo-EMCS) in pH 7.0 phosphate buffer for 2 h. The number of maleimide-linker on
the protein was determined by MALDI-TOF mass spectrometer. In average, 12.85
molecules of maleimide linkers were coupled on each molecule of diphtheria toxin
mutant CRM197. Finally, for protein conjugation, the purified thiolated
hexasaccharide 32 was incubated with the derivatized protein in pH 7.2 phosphate
buffer for 2 h at room temperature to obtain the RM2 antigen-CRM197
glycoconjugate which was shown to contain 4.7 molecules of RM2 antigen per
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molecule of CRM197 (DT-RM4.7) (Table S3). Our previous report showed that the
glyco-conjugate vaccine exhibited a better immunization result to induce a higher IgG
titer antibodies when combined with the α-galactosylceramide analogue C34 as an
adjuvant.30 To study the effect of adjuvant on Ab response, groups of BALB/c mice
were immunized intramuscularly with 2ꢀg of DT-RM4.7 in combination with 2ꢀg of
α-galactosylceramide C1, its analogue C34,40 or Alu (Fig. 13). Three vaccinations
were given at two-week intervals. Two weeks after the third injection, sera were
collected and subsequently tested with the previously mentioned glycan microarray
(96 glycans) (Fig. S5) to estimate the level and diversity of anti-RM2 related antibody.
The results showed that mouse anti-RM2 IgG titers increased as vaccination
proceeded and peaked after the third vaccination. Among the DT-RM4.7 vaccinated
groups, we found that DT-RM4.7/C34 induced higher levels of anti-RM2 IgG titers
than DT-RM4.7/C1 and DT-RM4.7/Alu after dilution to 12,000-fold (Fig. 13).
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Fig. 12. Scheme for producing synthetic candidate carbohydrate-based vaccine.
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Fig. 13. Female BALB/c mice were immunized with 2ꢀg of RM2 antigen of
DT-RM4.7 in combination with 2ꢀg of C1, C34, or Alu. Mouse serum was collected
two weeks after the final immunization, and the production of IgG against RM2
antigen and its truncated analogs after dilution to 12,000-fold. Data represent as total
fluorescence intensity of five mice the SEM.
Search for the best epitope ratio of DT-RM vaccine adjuvanted with C34.
After indentifying C34 as the most effective adjuvant for DT-RM4.7, we would like
to determine the best epitope ratio of vaccine by changing the amount of RM2 antigen
attached to each carrier protein DT (Fig. 13). Various equivalents of thiolated
hexasaccharide 32 and different protein concentrations generally would generate
diverse carbohydrate-protein ratios. The number of RM2 antigens on the protein was
determined by MALDI-TOF mass spectrometer. After dialysis and analysis, we were
able to conjugate, on average, 1.0, 3.0, 4.7, and 10 molecules of RM2 antigen to one
molecule DT to give DT-RM1.0, DT-RM3.0, DT-RM4.7, and DT-RM10.0 (Table S5).
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Using the same vaccination protocol mentioned previously, sera were collected 2
weeks after the third vaccination, and the elicited Abs were subsequently profiled by a
RM2-coated glycan microarray. In general, we observed that when immunized with
DT-RM3.0 alone without adjuvant, mice generated only low titers of anti-RM2 IgG
(Fig. S4 A). The glycan microarray results showed that, on average, 4.7 RM2 antigens
conjugated to one DT induced the most abundant IgG titers against RM2 (Fig. 14 and
Fig. S4). Alternatively, when immunized with DT-RM3.0 alone without adjuvant, mice
generated only low titers of anti-RM2 IgG (Fig. S4 A). When we fixed the signal to
noise (S/N) ratio ＞ 3, the induced IgG could be diluted to 12,000-fold before the
signal disappeared (Fig. S4 F). However, the induced IgM titer could only be diluted
to 60-fold (Fig. S4 E), and the IgM signals reached to the background after dilution to
200-fold.
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Interestingly, the specificity analysis of the induced IgG antibodies by
DT-RM4.7/C34 vaccine showed that the induced antibodies had a strong binding to the
RM2 antigen and weaker binding to its trisaccharide 27, tetrasaccharide 28,
pentasacharide 29, and pentasacharide 30 (Fig. 14). We observed that these
oligosaccharides all contain the same epitope trisaccharide 27 (NeuAc α 2 →
6GlcNAcβ1→3Galβ1→R). To further evaluate the vaccine specificity, we constructed
a glycan microarray created by STn, DSGG, RM2 antigen, RM2 antigen analogs, and
RM2 antigen fragments (Fig. 15). In general, specificity analysis of the induced
antibodies by DT-RM4.7/C34 vaccine showed that the induced antibodies mainly
bound to the RM2 antigen and to its trisaccharide epitope 27, a lesser extent to
disaccharide 32 (NeuAcα2→6GlcNAcβ1→R) and trisaccharide 33 (NeuAcα2→
6GlcNAcβ1→3Gluβ1→R). Besides, there is no detectable signal of binding to
NeuAc 2→6Gal, DSGG, or STn.
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It was reported that DT could induce antigen-specific T cell proliferation and
elevate splenocytes production of IL-2, IFN-γ, and IL-6, suggesting their role in the
Th1 driven pathway.41-43 Furthermore, the glycolipid C34 was able to induce higher
production of IFN-γ and IL-4, indicating a more Th1-skewed antigen.44 But, the
induced subtypes anti- DT-RM4.7/C34 antibodies were mainly IgG1 antibodies with a
trace amount of IgG2b and IgG2c antibodies and no detectable IgG2a (Fig. 16).
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Complement-dependent cytotoxicity (CDC) is one of the most potent cell killing
mechanisms mediating the immune response in which IgG or IgM antibodies bind to
antigens on the tumor or cancer cell surface. Complement activation, initiated through
complement protein C1q binding to the Fc region of the IgM or IgG isotype
antibodies, represents the important activity of antibodies against circulating tumor
cells and micrometastases. To evaluate the therapeutic potential of anti-RM2
antibodies, we tested the complement-dependent cytotoxicity with prostate cancer cell
line LNCap in the presence of new born rabbit complement. The ability of antibodies
to induce complement activation is strongly dependent on the antibody isotype and
epitope recognized. Thus, isotype IgG1 and IgG3 are able to activate the complement
cascade particularly well through C1q, in contrast to IgG2 and IgG445. As shown
above, our vaccine formulation resulted in a higher titer of subclass IgG1 antibodies
than other subclasses, and the immune serum showed a strong complement-mediated
cytotoxicity activity on the RM2-positive human prostate cancer cell line LNCap (Fig.
17). These data suggested that our carbohydrate-based vaccine DT-RM, based on the
chemically synthetic hexaccharide 1 and a mutated diphtheria toxin (DT) with
adjuvant C34 may create an efficient immune stimulation in human.
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DT-RM10
IgG (1:200)
C34
DT-RM4.7
DT-RM3.0
DT-RM1.0
27:
2.5×
107
2.0 107
28:
29:
×
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60
1.5×
107
30:
31:
1:
1.0 107
×
5.0×
106
0
Fig. 14. The induced IgG titers against RM2 antigen and glycan binding profile of
IgG collected from different epitope ratios of DT-RM/C34-immunized mice.
IgG (1:200)
DT-RM4.7 & C34
2.5 107
×
2.0×
107
1.5 107
×
1.0
×
×
107
106
0
5.0
Fig. 15. Specificity analysis of the induced antibodies by DT-RM4.7/C34 vaccine.
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IgG (1:200)
IgG1
5.0 107
4.0 107
3.0 107
2.0 107
1.0 107
0
IgG2a
IgG2b
IgG2c
IgG3
5
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60
Fig. 16. The glycan binding profiles of induced IgG subtypes titers collected from
DT-RM4.7/C34-immunized mice two weeks after the third injection.
CDC
DT-RM vaccination
ddH2O(control)
60
40
20
0
0
5
10
15
Serum dilution (%)
Fig. 17. CDC activity of the induced antibodies by serum from vaccination mice in
the presence of rabbit complement. Lysis of human prostate cancer cell line LNCap at
different concentrations.
Conclusion
In conclusion, we have successfully synthesized the prostate tumor carbohydrate
RM2 antigen and its analogs as single stereoisomers in every glycosylation step in
good yields. We used these synthetic glycans combined with other related glycans to
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form a 96-member glycan microarray to study the specificity of monoclonal RM2
antibody and confirmed the structure of our synthetic RM2 hexasaccharide antigen.
Moreover, the synthesized RM2 carbohydrate antigen was incorporated into the
carrier protein CRM197 in an average number of 1~10. After vaccination studies in
mice, we used the glycan microarray to monitor their immune response. Our results
indicated that when one molecule of DT was conjugated with 4.7 molecules of RM2
antigen in average (DT-RM4.7) and adjuvanted with the glycolipid C34 exhibited the
strongest anti-RM2 antigen titer. More importantly, this oligosaccharide-protein
conjugates are TD antigens, and the induced IgG antibody titers were much higher
than IgM antibody titers. In IgG subclasses analysis, IgG1 antibody was predominant
in the serum, while IgG3, a typical anti-carbohydrate antibody, also present in high
level. In addition, the induced mouse antibodies mediated effective
complement-dependent cytotoxicity (CDC) against the prostate cancer cell line
LNCap.
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Supporting Information Available
Supplementary schemes, synthetic methods, and experimental protocols. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
cyiwu@gate.sinica.edu.tw; chwong@gate.sinica.edu.tw
Notes
The authors declare no competing financial interest.
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Acknowledgement
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The authors thank Prof. Seiichi Saito in the University of the Ryukyus, Okinawa,
Japan for his kindly provided RM2 antibody, and Dr. Kun-Hsien Lin for providing the
adjuvant C34. This work was supported by Academia Sinica and National Science
Council, Taiwan. (Grant # NSC-100-2325-B-001-015 and NSC-101-2325-B-001-014
to CYW ).
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Table of Content
OAc
OBn
OAc
TBSO
OAc
OAc
Ac
N
Monosaccharide
O
COOM e
+
Disaccharide
O
Orthogonal TrocHN
O
OBn
O
O
BnO
O
O
MeOOC
O
one-pot
synthesis O
O
+
O
OBn
OBz
Trisaccharide
O
OBn
O
T rocHN
O
O
BnO
G lobal
D eprotection
HN
O
OR
BnO
DT
O
R M2
Vaccination
28
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