A fully synthetic glycopeptide antitumor vaccine based on multiple antigen presentation on a hyperbranched polymer

Article abstract of DOI:10.1002/chem.201400256

For antitumor vaccines both the selected tumor-associated antigen, as well as the mode of its presentation, affect the immune response. According to the principle of multiple antigen presentation, a tumor-associated MUC1 glycopeptide combined with the immunostimulating T-cell epitope P2 from tetanus toxoid was coupled to a multi-functionalized hyperbranched polyglycerol by "click chemistry". This globular polymeric carrier has a flexible dendrimer-like structure, which allows optimal antigen presentation to the immune system. The resulting fully synthetic vaccine induced strong immune responses in mice and IgG antibodies recognizing human breast-cancer cells. Branching out: A tumor-associated MUC1 glycopeptide as a B-cell epitope combined with a tetanus toxoid T-cell epitope was coupled to hyperbranched polyglycerol (see figure). Due to the dendritic carrier structure, the antigen is presented in an optimal multiple display to the immune system. The fully synthetic and water-soluble vaccine induced antibodies that recognize human breast-tumor cells.

Full text of DOI:10.1002/chem.201400256

DOI: 10.1002/chem.201400256
Communication
&
Antitumor Agents
A Fully Synthetic Glycopeptide Antitumor Vaccine Based on
Multiple Antigen Presentation on a Hyperbranched Polymer
[
a]
[a]
[a]
[a]
[a]
Markus Glaffig, Bjçrn Palitzsch, Sebastian Hartmann, Christoph Schꢀll, Lutz Nuhn,
[b]
[b]
[a]
[a]
Bastian Gerlitzki, Edgar Schmitt, Holger Frey,* and Horst Kunz*
Dedicated to Professor Hans-Ulrich Reißig on the occasion of his 65th birthday
[1,4]
of the sequence PAHGVTSAPDTRPAPGSTAP.
As a conse-
quence, tumor-associated MUC1 exposes peptide backbone
epitopes in combination with the aberrant tumor-associated
carbohydrate antigens (TACAs), which should be qualified for
Abstract: For antitumor vaccines both the selected
tumor-associated antigen, as well as the mode of its pre-
sentation, affect the immune response. According to the
principle of multiple antigen presentation, a tumor-associ-
ated MUC1 glycopeptide combined with the immunosti-
mulating T-cell epitope P2 from tetanus toxoid was cou-
pled to a multi-functionalized hyperbranched polyglycerol
by “click chemistry”. This globular polymeric carrier has
a flexible dendrimer-like structure, which allows optimal
antigen presentation to the immune system. The resulting
fully synthetic vaccine induced strong immune responses
in mice and IgG antibodies recognizing human breast-
cancer cells.
[2]
the development of a cancer immunotherapy.
However, immunization using MUC1 sequences as sole vac-
cine component (B-cell epitope) failed to induce adequate
immune responses because these endogenous structures are
highly tolerated by the immune system. One approach to en-
hance the immunogenicity of these MUC1 antigens and there-
by overcome the immune self-tolerance consists of covalent
conjugation of synthetic MUC1 glycopeptides to immunosti-
[5]
mulating carrier proteins, for example, tetanus toxoid (TTox).
This highly immunogenic protein contains numerous T-cell epi-
topes. In addition, it provides multivalent presentation of anti-
[5]
gens promoting enhanced immunogenicity. Such protein-
based two-component vaccines can elicit strong humoral
immune responses in mice affording very high titres of anti-
It appears particularly attractive for a tumor therapy to induce
an immune response in a patient which is directed against his
[5]
bodies, which strongly bind to human cancer cells. Although
these protein-based vaccines are most promising candidates
for antitumor vaccination, they also induce immune reactions
against epitopes of the carrier protein. Unfortunately, this
might suppress the desired maximum immune response to-
[
1]
own tumor tissues. The selectivity and efficiency of an antitu-
mor vaccine and the quality of the induced immune response
do not only depend on the selected tumor-associated antigen
structure, but also on the particular presentation of the anti-
gen to the immune system, which should be as similar to the
situation on the cell surface as possible. From this point of
view terminal exposition of the antigens on carriers with den-
dritic architecture are considered particularly promising. The
tumor-associated mucin MUC1 has been identified as a promis-
ing target molecule for the development of antitumor vac-
[6]
wards the MUC1 glycopeptide. In order to circumvent the in-
duction of such undesired antibodies, fully synthetic two- and
three-component vaccines containing a MUC1 glycopeptide as
B-cell epitope and a single T-cell epitope were prepared with-
[7]
out the use of carrier proteins. Some of these fully synthetic
vaccines elicited significant immune reactions in mice, in par-
[
2]
cines. It is expressed on most epithelial tissues and over-ex-
ticular those containing Toll-like receptor 2 ligand structures as
[
3]
[7b–d,f]
pressed on the corresponding tumor tissues. Due to changed
activities of glycosyltransferases in tumor cells, tumor-associat-
ed MUC1 shows markedly altered glycosylation profiles of pre-
vailingly truncated glycans within the tandem repeat domains
additional immune-stimulating components.
For vaccines
consisting of a combination of the MUC1 glycopeptide antigen
with a single T-cell epitope peptide, the quality of the immune
response distinctly depends upon the properties of the T-cell
peptide. Of the T-cell epitope peptides P30 and P2, both de-
rived from tetanus toxoid and described as universal murine
[
a] M. Glaffig, B. Palitzsch, S. Hartmann, Dr. C. Schꢀll, L. Nuhn, Prof. Dr. H. Frey,
Prof. Dr. H. Kunz
Johannes Gutenberg University Mainz, Institute of Organic Chemistry
Duesbergweg 10-14, 55128 Mainz (Germany)
E-mail: hfrey@uni-mainz.de
[8]
and humane T-helper cell epitopes, the longer P30 epitope
[7e]
caused a strong immune reaction,
whereas the shorter
amino acid sequence P2 could not entail a sufficient immune
response. This may be due to the missing ability of P2 to self-
assembly and, thus, to present antigens in a multivalent
hokunz@uni-mainz.de
[
b] Dr. B. Gerlitzki, Prof. Dr. E. Schmitt
Johannes Gutenberg University Mainz
University Medical Center-Institute of Immunology
Langenbeckstrasse 1, Geb. 708, 55101 Mainz (Germany)
[7e]
form; this difference sheds light on the importance of the
above-mentioned presentation of the antigen.
Therefore, it is considered attractive to apply the strategy in-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201400256.
[9]
troduced by J. P. Tam for enhancing the immunogenicity of
Chem. Eur. J. 2014, 20, 1 – 6
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Communication
MUC1 glycopeptide antigens. In this strategy, peptide epitopes
were presented in a multiple form on a single branched oligo-
pling to hyperbranched polyglycerol as the carrier prevents an
entanglement of the vaccine. The nano-sized dendrimeric par-
ticles provide a presentation of the glycopeptide on the sur-
face and, thus, ensure a better accessibility of the bound anti-
gens to the immune system. Furthermore, the polydiversity of
the dendritic macromolecules reflects the multiple arrange-
ments on a cell surface. Consequently, these dendrimer-type
polymer vaccines appear more cell surface-like than all vac-
cines based on linear polymers. In addition, any mutually hin-
dering interactions of covalently bound antigens are reduced
by the flexibility of the hydrophilic polyether arms allowing for
an optimal exposure of each antigen to the immune system.
Due to the hydrophilic properties of hbPG, this core also en-
hances the solubility of synthetic vaccines in aqueous environ-
ment. The attachment of the antigens was conveniently realiz-
[
9]
lysine core. The thus achieved antigen multivalency provides
an opportunity for efficient clustering of B-cell Ig receptors and
[10]
stronger avidity between antigens and Ig receptors.
This
form of antigen presentation may facilitate vaccine uptake by
B-cells as antigen presenting cells (APCs) leading to enhanced
immune responses.
Taking into account both concepts (multiple antigen presen-
tation and defined T-helper cell epitopes), we recently de-
signed a fully synthetic polymer-based vaccine with separated
MUC1 and P2 T-cell epitopes orthogonally coupled to a multi-
step-synthesized
poly(N-(2-hydroxypropyl)methacrylamide)
[
11]
P(HPMA) as a linear non-immunogenic carrier.
In the present work, a new concept is described to combine
fully synthetic linear MUC1 glycopeptide–P2 T-cell epitopes in
a multivalent format by coupling to a globular, water-soluble
and readily available polymer. Multi-alkyne-functionalized hy-
I
ed by Cu-catalyzed Huisgen cycloaddition (“click chemistry”)
between the MUC1–P2 conjugate carrying an azido-terminated
[14]
triethyleneglycol acyl spacer
and the alkyne-functionalized
[
12]
perbranched polyglycerol (hbPG) was chosen, which is acces-
sible in a one-step copolymerization of glycidol 2 and its prop-
argyl ether 3 (ratio 28:5), in order to achieve, for the first time,
conjugation of glycopeptides to this polymer. Regarding the
structural similarity to the widely used and well-studied poly(-
ethylene glycol), polyglycerols are considered biocompatible
and non-immunogenic and, therefore, suitable as inert carriers
hbPG-polymers. The numbers of linked antigens can be adjust-
[12]
ed in this one-step reaction (Scheme 1).
A section of the tandem repeat domain from MUC1 includ-
ing the immunodominant motifs PDTRP and GSTA was chosen
as the B-cell epitope, as recent studies have revealed that anti-
[15,16]
MUC1 antibodies bind to these motifs preferentially.
Since
glycosylation influences the conformation of the MUC1 se-
quence and is important for the tumor selectivity of the in-
[
13]
for biomedical applications. The highly branched macromo-
lecule of globular and highly functional dendrimer-like struc-
ture provides ample space on its “surface” for multivalent pre-
sentation of relevant compounds, as for example, antigens. In
[16,17]
duced antibodies,
a Tn antigen was linked to threonine-18
within the GSTA region. There are three proline residues adja-
cent to this region of MUC1, which have significant conforma-
[
11]
[17b,18]
contrast to epitope coupling to the linear P(HPMA), the cou-
tional influence
and consequently, may affect the binding
Scheme 1. One-step synthesis of poly(glycerol-co-propargyl glycidyl ether) (hbP(G-GPE)) 4 and subsequent coupling of MUC1–P2 glycopeptide 5 by copper-
catalyzed azide–alkyne cycloaddition to the hyperbranched polyglycerol-based antitumor vaccine 6.
&
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Communication
affinity of the antibodies to the GSTA region. Hence, the 20
amino acid MUC1 tandem repeat sequence was extended by
two further amino acids (proline and alanine) resulting in the
[19]
22-mer peptide B-cell epitope PAHGVTSAPDTRPAPGSTAPPA.
As second component for the vaccine design, the T-cell epi-
[
8]
tope P2 (QYIKANSKFIGITEL) of tetanus toxoid was coupled to
the MUC1 B-cell epitope. In order to minimize mutual confor-
mational distortion, an immunogenically silent oligoethylene-
[
14]
glycol-spacer was inserted between the components.
The conjugation of the MUC1 glycopeptide with the defined
T-cell epitope can directly be integrated into antigen assembly
by solid-phase (glyco)peptide synthesis (SPPS). To this end, the
MUC1 glycopeptide, the T-cell epitope P2 and the two spacers
were prepared in a linear solid-phase synthesis according to
[5]
the established 9-fluorenylmethoxycarbonyl (Fmoc) protocol
starting from a TentaGel resin preloaded with a trityl-anch-
[
20]
ored Fmoc-protected alanine. Couplings of the spacers as
well as that of the Tn antigen threonine were carried out
under modified conditions (see the Supporting Information).
The completed MUC1–P2 peptide was detached from resin
with concomitant removal of all amino acid protecting groups
using trifluoroacetic acid (TFA), triisopropylsilane (TIS) and
water to afford the glycopeptide–peptide conjugate after pu-
rification by semipreparative HPLC. Subsequently, the O-acetyl
groups of the carbohydrate were removed using sodium meth-
oxide/methanol at pH<10 to avoid b-elimination. The com-
pletely deprotected glycopeptide 5 was isolated after addition-
al purification by HPLC in an overall yield of 11%.
Figure 1. a) SEC elugrams of hbP(G-GPE) 4, MUC1–P2 antigen 5 and vaccine
6 (eluent: HFIP). b) MALDI-TOF analysis of 6 (giving no indication of
amounts).
(Figure 1b). Signals with lower m/z ratio are probably caused
To obtain the vaccine, conjugation of MUC1–P2 5 with the
polymeric carrier hbPG 4 was performed by copper-catalyzed
by multiply charged conjugates (e.g., the peaks at about
À1
8000 gmol relate to doubly charged three and four bound
[
21]
azide–alkyne click-type cycloaddition (Scheme 1).
To this
antigens per polymer). The analytical SEC elugram also showed
end, an multi-alkyne functionalized hbPG with an average mo-
remaining glycopeptide–peptide conjugate 5, which does not
affect the immunological effects.
À1
[7e]
lecular weight of M =2770 gmol was synthesized. It carried
n
approximately five alkyne groups per polymer as calculated by
In order to evaluate the immunological properties of the
fully synthetic dendritic polymer vaccine, three wild-type
BALB/c mice were immunized subcutaneously together with
complete Freund’s adjuvant. Two further immunizations with
incomplete Freund’s adjuvant at intervals of 21 days were per-
formed by intraperitoneal applications. Five days after the
second boost, blood was drawn from tail veins of the mice,
and the three obtained sera were analyzed by enzyme-linked
immunosorbent assay (ELISA) in order to identify vaccine-in-
duced antibodies. The microtitre plates were coated with the
corresponding MUC1 glycopeptide 5 conjugate to bovine
serum albumin (BSA; see the Supporting Information). The
ELISA results of all three mice confirmed that the fully synthet-
ic multivalent vaccine induced a significant specific immune re-
sponse against the MUC1 B-cell epitope (Figure 2). Antibody
subtype analysis after the second immunization revealed pre-
vailing IgG1 isotype antibodies indicating MHCII-mediated
immune responses (Figure 2b). Interestingly, mouse 2 (showing
the highest titre) and mouse 3 additionally produced IgG2 anti-
bodies. The binding of the induced antibodies to human
1
[12]
H NMR spectroscopy (according to published procedure).
The alkyne moieties at the polymer were treated with the ter-
minal azido-functionalized spacer of the MUC1–P2 antigen 5 in
degassed water using copper(II) acetate and ascorbic acid at
408C for three days.
The coupling yielded the fully synthetic glycopeptide-hyper-
branched polygycerol vaccine 6, which was purified by semi-
preparative size exclusion chromatography (SEC). The copper-
free fraction collected at 2.8–3.9 min (see the Supporting Infor-
mation) was purified by dialysis. The structure of 6 was con-
firmed by matrix-assisted laser desorption and ionization mass
spectrometry (MALDI-TOF) and by analytical SEC with hexa-
fluoro-2-propanol (HFIP) as the eluent. Compared to the SEC
volumes of the starting compounds (MUC1–P2 peptide 5 and
alkyne-functional hbPG 4), the SEC elugram clearly showed
a shift to shorter elution times indicating covalent attachment
of the glycopeptides to the carrier (Figure 1a). MALDI-TOF
mass spectrometry (intensity is no measure of amounts of the
compounds) detected molecular masses of 16000–
À1
[22]
2
0000 gmol , which correspond to three and four bound
breast- tumor cells (MCF-7) was determined by flow cytome-
À1
MUC1–P2 antigens (M =4395.26 gmol ) per polymer. In addi-
try (FACS). All sera exhibited significant binding to MCF-7 cells
up to a recognition of 85% of the tumor cells in case of
mouse 3 (Figure 2c).
n
À1
tion, masses of approximately 11000 gmol were recorded,
which correspond to two (glyco)peptide antigens per polymer
Chem. Eur. J. 2014, 20, 1 – 6
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Communication
Figure 2. a) ELISA analysis of the antisera induced by vaccine 6; microtitre plates were coated with the corresponding MUC glycopeptide–BSA conjugate.
b) Antibody subtype analysis five days after the second immunization. c) FACS analysis of the binding of antisera to MCF-7 breast- tumor cells; negative con-
trol was obtained from mice treated with PBS buffer.
In conclusion, a novel efficient fully synthetic vaccine has
Keywords: antitumor agents · glycopeptides · hyperbranched
polymers · polyglycerol · tumor-associated MUC1
been prepared, which is based on biocompatible hyper-
branched polyglycerol as polymeric carrier. The globular struc-
ture of the macromolecular carrier ensures the presentation of
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Received: January 22, 2014
Published online on && &&, 0000
Chem. Eur. J. 2014, 20, 1 – 6
www.chemeurj.org
5
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Communication
COMMUNICATION
Branching out: A tumor-associated
MUC1 glycopeptide as B-cell epitope
combined with a tetanus toxoid T-cell
epitope was coupled to hyperbranched
polyglycerol (see figure). Due to the
dendritic carrier structure, the antigen is
presented in an optimal multiple display
to the immune system. The fully syn-
thetic and water-soluble vaccine in-
duced antibodies that recognize human
breast-tumor cells.
&
Antitumor Agents
M. Glaffig, B. Palitzsch, S. Hartmann,
C. Schꢀll, L. Nuhn, B. Gerlitzki, E. Schmitt,
H. Frey,* H. Kunz*
&
& – &&
A Fully Synthetic Glycopeptide
Antitumor Vaccine Based on Multiple
Antigen Presentation on
a Hyperbranched Polymer
&
&
Chem. Eur. J. 2014, 20, 1 – 6
www.chemeurj.org
6
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!