Fully synthetic vaccines consisting of tumor-associated MUC1 glycopeptides and a lipopeptide ligand of the toll-like receptor 2

Article abstract of DOI:10.1002/anie.201000462

(Figure Presented) Fragment condensation of a protected Pam₃Cys lipopeptide with unprotected tumor-associated MUCI glycopeptides opens the way to vaccine candidates such as A. These vaccines elicit selective immune responses in mice although they lack an immunodominant carrier protein. In principle, such vaccines are applicable to humans.

Full text of DOI:10.1002/anie.201000462

Communications
DOI: 10.1002/anie.201000462
Vaccine Synthesis
Fully Synthetic Vaccines Consisting of Tumor-Associated MUC1
Glycopeptides and a Lipopeptide Ligand of the Toll-like Receptor 2**
Anton Kaiser, Nikola Gaidzik, Torsten Becker, Clara Menge, Kai Groh, Hui Cai, Yan-Mei Li,
Bastian Gerlitzki, Edgar Schmitt, and Horst Kunz*
Dedicated to Professor Horst Kessler on the occasion of his 70th birthday
Mucin glycoprotein structures on epithelial tumor cells are
characteristically different from the mucin structures on
healthy cells.[1] However, the immunogenicity of these
tumor-associated glycoproteins is too low to overwrite the
endogenous tolerance of the immune system. Therefore, they
can not be used directly as antitumor vaccines. Recently, it
was demonstrated that glycopeptides from the tandem repeat
region of tumor-associated mucin MUC1 conjugated to a T-
cell epitope peptide from ovalbulmin furnish fully synthetic
vaccines which elicit a strong, highly specific immune
response in transgenic mice.[2] An even stronger and highly
specific immune response was induced by immunization of
wild-type balb/c mice with a vaccine containing the tumor-
associated MUC1 glycopeptide bound to tetanus toxoid as the
carrier protein.[3] This type of vaccines has the advantage of
being applicable to humans. Of course, such MUC1 glyco-
peptide/tetanus toxoid vaccines also elicit immune reactions
against tetanus toxoid. To suppress the generation of an
anticarrier immune reaction, for example in booster immu-
nizations, an alternative form of a synthetic vaccine must be
developed in which the tumor-associated MUC1 glycopeptide
is covalently bound to a general immunostimulating structure.
Toll-like receptor ligands, for example tripalmitoyl-S-glycer-
ylcysteine peptides like Pam3CysSer(Lys)4 described by
Bessler, Jung et al.,[4] represent such immunostimulating
structures. Recently, Boons et al.[5] reported vaccines consist-
ing of a glycoundecapeptide of the tandem repeat unit of
MUC1 containing the monosaccharide TN-antigen side chain
directly coupled to a T-cell epitope from polio virus[6] and the
aforementioned TLR2 agonist. These constructs induced
selective immune reactions in mice. During the synthesis of
these vaccines the O-deacetylation of the N-acetylgalactos-
amine part was achieved by transesterification with hydrazine
in methanol.[7] This procedure is not applicable to the
synthesis of glycopeptides bearing neuraminic acid.
To benefit from the immunostimulating effects of
Pam3Cys Toll-like receptor ligands in synthetic MUC1
glycopeptide vaccines supplementing the tetanus toxoid
conjugates, we developed a fragment condensation to attach
the Pam3CSKKKK lipopeptide to tumor-associated MUC1
glycopeptides to give fully synthetic vaccines A.
To minimize the influence of the lipopeptide and its basic
side chains on the conformation of the MUC1 glycopeptide
antigen, an oligoethylene glycol spacer was placed between
the TLR2 ligand and the B-cell epitope. Provided the
activated carboxylic group of the lipopetide bears only acid-
labile protecting groups and the saccharide part of the
glycopeptide already is deprotected, the final acidolytic
deprotection should not affect the palmitic esters and
should afford a pure fully synthetic vaccine.
The N-terminally and side-chain-protected lipopeptide
was synthesized on a resin functionalized with the 2-phenyl-2-
trimethylsilylethylester (PMTSEL) anchor.[9] This anchor
molecule is cleavable under neutral conditions by use of
tetrabutylammonium fluoride trihydrate in dichloromethane.
Fmoc-Lys(Boc)-OH was treated with 4-(2-hydroxy-1-trime-
thylsilylethyl)phenoxyacetic acid allyl ester[9,11] (1) according
to the procedure reported by Steglich and Neises[10] to give the
anchor ester molecule 2 (Scheme 1). The allyl ester 2 was
cleaved selectively using catalytic amounts of tetrakis(triphe-
nylphosphine)palladium(0) and N-methylaniline[12] as the
allyl scavenger. The obtained anchor carboxylic acid 3 was
coupled to amino-functionalized Tentagel[13] resin using
TBTU/HOBt to yield the resin 4 preloaded with Fmoc-
Lys(Boc) (Scheme 1). The lipopetide 5 was assembled on
resin 4 following the Fmoc strategy. After cleavage of the
PMTSEL anchor with fluoride,[9] the side-chain-protected
TLR2 ligand hexapeptide 5 was isolated in 81% yield
(Scheme 2).[15]
[*] A. Kaiser, N. Gaidzik, T. Becker, C. Menge, K. Groh, Prof. Dr. H. Kunz
Johannes Gutenberg-Universitꢀt Mainz
Institut fꢁr Organische Chemie
Duesbergweg 10–14, 55128 Mainz (Germany)
Fax: (+49)6131-392-4786
E-mail: hokunz@uni-mainz.de
H. Cai, Prof. Dr. Y.-M. Li
Tsinghua University Beijing (China)
B. Gerlitzki, Prof. Dr. E. Schmitt
Johannes Gutenberg-Universitꢀt Mainz
Institut fꢁr Immunologie,Mainz (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft,
the Jꢁrgen Knop-Stiftung, and the Chinesisch-Deutsches Zentrum
fꢁr Wissenschaftsfꢂrderung, Beijing, program number GZ561. Toll-
like receptors are receptors that resemble the protein coded by the
Toll gene; they are also known as pattern recognition receptors.
Lipopeptide 5 selectively deprotected at the terminal
carboxylic group can now be used for fragment condensa-
tions. For coupling reactions, the fully deprotected spacer-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201000462.
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tion). After completion of
the reaction, acidolytic
deprotection, and workup,
the lipoglycopeptide vac-
cines 9, 10, and 11 were
isolated in yields of 20–
25%. They were character-
ized by MALDI-TOF and
high-resolution ESI mass
spectrometry and by two-
dimensional 1H NMR spec-
troscopy.[18] The synthesis
of the Pam3Cys MUC1 gly-
copeptide vaccine 11 gives
evidence that the described
fragment
condensation
between the activated pro-
tected lipopetide and the
Scheme 1. Synthesis of the preloaded PMTSEL resin: Fmoc=fluorenyl-9-methoxycarbonyl, DIC=diisopropyl-
carbodiimide, DMAP=4-dimethylaminopyridine, NMA=N-methylaniline, TBTU=O-benzotriazol-1-yl-
N,N,N’,N’-tetramethyluronium tetrafluoroborate,[14] HOBt=1-hydroxybenzotriazole, DIPEA=diisopropylethyl-
amine (Hꢁnig’s base).
completely
deprotected
glycopeptide antigen is
useful for the conversion
of important tumor-associ-
ated glycopeptide antigens
containing neuraminic acid
into
vaccines.
To evaluate the immu-
TLR2-ligand-based
nogenicity of the TLR2
ligand glycopeptide vac-
cines, balb/c-J mice were
immunized with Pam3Cys-
icosaglycopeptide conju-
gate 10 in combination
with complete Freundꢀs
adjuvant (CFA). After
every 20 days, booster
immunizations with 10 and
incomplete Freundꢀs adju-
vant (IFA) were per-
formed. Five days after the
second boost, the induced
antibodies were deter-
mined regarding their bind-
ing to the immobilized con-
jugate 12 of the MUC1
antigen glycopeptide with
bovine serum albumin[2a,3]
(Scheme 4) by means of
an ELISA based on
double-antibody technique
(Figure 1).
Scheme 2. Synthesis of the Pam3Cys lipopeptide 5. HBTU=O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium
hexafluorophosphate, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate,
HOAt=7-aza-1-hydroxybenzotriazole,[16] NMM=N-methylmorpholine, NMP=N-methylpyrrolidone.
functionalized MUC1 glycopeptides with TN- (6), T- (7), and
2,6-sialyl-T-antigen side chains (8) were synthesized on a resin
equipped with a trityl anchor according to an already
described procedure[17] (Scheme 3). To accomplish the frag-
ment condensation, lipopeptide 5 was converted to its active
ester by reaction with HATU/HOAt.[16] Subsequently, a
solution of the amino-functionalized MUC1 glycopeptide 6,
7, or 8 (0.5 equiv) was added (see the Supporting Informa-
The results of the ELISA analysis indicate that a specific
humoral immune response had been elicited in all three mice.
The antiserum titers were not as high as those for the
corresponding MUC1 tetanus toxoid vaccine,[3] but the effect
is reproducible and shows that the general mechanism of the
immunological activation by TLR2 agonists can be applied to
antitumor vaccines based on MUC1 glycopeptides.
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Communications
Scheme 3. Synthesis of MUC1 glycopeptide TLR2 agonist vaccines 9–11 by fragment condensation. TIS=triisopropylsilane.
glycosylated in the same position but with different tumor-
associated carbohydrate antigens such as TN-antigen (13c) or
sialyl-TN-antigen (13d) do bind to the induced antibodies.
However, the antibodies do not recognize MUC1 glycopep-
tides of the same sequence that are glycosylated in a different
position, as for example 14 and 15,[2a] and also not the peptide
sequence 16 from mucin MUC4[19] (Figure 2).
The behavior of the antibodies induced by the T-antigen
MUC1 TLR2 agonist vaccine 10 suggests that the recognition
process is more dominated by the peptide sequence of the
antigen and its conformation than was previously observed
for vaccines containing neuraminic acid and a T-cell epitope[2]
or the tetanus toxoid.[3] The incomplete neutralization of the
antibodies by the T-antigen MUC1 structure 13a contained in
the vaccine 10 could indicate an influence of the Pam3Cys
lipopeptide in 10 on the B-cell epitope.
Figure 1. Determination of the induced immune reaction against 10 in
balb/c-J mice.
To determine the specificity of the induced immune
reaction, the antibodies were subjected to incubation with the
already described MUC1 glycopeptides 13–15[2a] and a MUC4
peptide[19] 16 (Scheme 4). These neutralization experiments
with the antisera induced by vaccine 10 showed that the T-
antigen MUC1 glycopeptide 13a as well as the unglycosylated
MUC1 peptide 13b and MUC1 glycopeptides that are
In conclusion, the combination of tumor-associated mucin
glycopeptide antigens with lipopeptide Toll-like receptor 2
ligands furnished efficient fully synthetic vaccines, which can
be generally synthesized through fragment condensation with
unprotected amino-functionalized glycopeptides. These vac-
cines can be combined advantageously with vaccines[3] based
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Scheme 4. The MUC1 glycopeptide BSA conjugate 12 and MUC1 and MUC4 peptides and glycopeptides 13–16 used for the neutralization of the
antibodies induced by vaccine 10 (Figure 2). BSA=bovine serum albumine.
on tetanus toxoid and principally are applicable to humans.
They also offer the possibility to prevent the application of
the complete Freundꢀs adjuvant.
Received: January 26, 2010
Published online: April 1, 2010
Keywords: glycopeptides · lipopeptides · synthetic vaccines ·
.
Toll-like receptor 2 · tumor-associated antigens
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Figure 2. Neutralization of the antibodies induced by vaccine 10 using
MUC1 and MUC4 peptide and glycopeptides 13–16 (Scheme 4).
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Communications
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24
[15] 5: ½aꢀD ¼ꢁ5.6 degcm3 gꢁ1 dmꢁ1 (c = 1 gcmꢁ3, CHCl3); HR-ESI-
MS (positive): m/z: 1554.19 ([M+Hꢁ4 ꢃ Boc]+, calcd 1553.62).
1H NMR data are shown in the Supporting Information.
[16] L. A. Carpino, A. El-Faham, C. A. Minor, F. Albericio, J. Chem.
Soc. Chem. Commun. 1994, 2001.
[17] See Schemes 1–3 in Ref. [3] and Scheme 2 in Ref. [2a].
[18] Details concerning the fragment condensation, deprotection,
and characterization are given in the Supporting Information. 9:
23
8 mg (from 20 mg of 6); ½aꢀD ¼ꢁ66.5 degcm3 gꢁ1 dmꢁ1 (c =
0.37 gcmꢁ3, H2O); MALDI-TOF (dhb, positive): m/z: 3786.36
[M+H]+, calcd 3785.29. 10: 6.7 mg (from 20 mg of 7);
22
½aꢀD ¼ꢁ48.4 degcm3 gꢁ1 dmꢁ1
(c = 0.3 gcmꢁ3
,
H2O);
tR =
39.1 min; MALDI-TOF (dhb, positive): m/z: 3947.64 [M+H]+,
calcd
3964.32.
11:
6.1 mg
(from
20 mg
, H2O); HR-ESI-
of
8);
22
½aꢀD ¼ꢁ37.4 degcm3 gꢁ1 dmꢁ1 (c = 0.5 gcmꢁ3
MS (positive): m/z: 1413.14 [M+3H]3+; calcd 1413.14.
[19] C. Brocke, H. Kunz, Synthesis 2004, 525.
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