Mucin architecture behind the immune response: Design, evaluation and conformational analysis of an antitumor vaccine derived from an unnatural MUC1 fragment

Article abstract of DOI:10.1039/c5sc04039f

A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines.

Full text of DOI:10.1039/c5sc04039f

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Mucin architecture behind the immune response:
design, evaluation and conformational analysis of
an antitumor vaccine derived from an unnatural
MUC1 fragment†
Cite this: Chem. Sci., 2016, 7, 2294
a
b
b
a
Nuria Martınez-Saez, Nitin T. Supekar, Margreet A. Wolfert, Iris A. Bermejo,
´
´
cd
e
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Ramon Hurtado-Guerrero, Juan L. Asensio, Jesus Jimenez-Barbero,
Jesus H. Busto, Alberto Avenoza, Geert-Jan Boons,* Jesus M. Peregrina*
and Francisco Corzana*
´
´
´
a
a
b
a
´
´
a
A tripartite cancer vaccine candidate, containing a quaternary amino acid (a-methylserine) in the most
immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in
transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was
capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated
tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide
backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to
enzymatic degradation than the natural counter part. In spite of these features, the immune response
elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural
threonine. These observations were rationalized by conformational studies, indicating that the
presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in
immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids
have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc
and the threonine residues. The results described here will be helpful to the rational design of efficacious
cancer vaccines.
Received 23rd October 2015
Accepted 6th December 2015
DOI: 10.1039/c5sc04039f
www.rsc.org/chemicalscience
shows simple and truncated carbohydrates because of the
incomplete glycosylation. As a result, different tumour-associ-
Introduction
Mucin MUC1 is an O-glycoprotein strongly over-expressed in ated carbohydrate antigens (TACAs), such as the Tn determi-
1
–5
6
various tumours. In healthy tissues, the MUC1 backbone nant (a-O-GalNAc-Ser/Thr), are exposed to the immune system
7
presents complex oligosaccharides, while in cancer cells it and can be recognized by different antibodies. Due to this
unique characteristic, MUC1 derivatives are attracting great
interest as a potential tool in developing therapeutic vaccines
a
3,8–10
Departamento de Qu ´ı mica, Universidad de La Rioja, Centro de Investigaci ´o n en
for the treatment of cancer.
have succeeded in clinical trials due to the fact that most
natural TACAs are tolerated by the immune system and glyco-
sidases can reduce their in vivo bioavailability. One way to
overcome this issue is through the use of chemical modica-
tions of the antigens to generate non-natural determinants. A
However, to date, none of them
S ´ı ntesis Qu ´ı mica, Madre de Dios 53, 26006 Logro n˜ o, Spain. E-mail: francisco.
corzana@unirioja.es; jesusmanuel.peregrina@unirioja.es
11
b
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road,
12
Athens, Georgia 30602, USA. E-mail: gjboons@ccrc.uga.edu
c
BIFI, University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n,
13
Campus Rio Ebro, Edicio I+D, Zaragoza, Spain
d
14–16
Fundaci ´o n ARAID, 50018, Zaragoza, Spain
number of TACA mimics, comprising C-glycosides
and
have been incorporated into carbohydrate-
based vaccines. Additionally, the use of homoserine and
e
17–19
Instituto de Qu ´ı mica Org a´ nica General, IQOG-CSIC, Juan de la Cierva 3, 28006
S-glycosides,
Madrid, Spain
f
Structural Biology Unit, CIC bioGUNE, Parque Tecnol ´o gico de Bizkaia Building 801A,
3
20,21
b -homothreonine conjugates
to construct mucin-like
4
8160 Derio, Spain
g
glycopeptides and derivatives that incorporate uorine
IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain
22,23
h
atoms
have been proposed.
Department of Chemical and Physical Biology, Centro de Investigaciones Biol ´o gicas,
CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Nevertheless, it is important to mention that the structural
†
Electronic supplementary information (ESI) available: Characterization of the basis for the design of these vaccines remain unclear. From the
new compounds, biological assays and MD simulations. See DOI:
view of molecular recognition, the Pro-Asp-Thr-Arg (PDTR)
1
0.1039/c5sc04039f
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sequence comprises the minimal epitope for most of anti- routes that are summarized in Scheme 1. The rst one uses
7
24
MUC1 antibodies, such as SM3, which has a potential use in derivative 5 as a glycosyl donor, which was easily prepared from
25
the early diagnosis and treatment of breast cancer. Although compound 4 and following the methodology described in the
29
the crystal structure of this antibody in complex with a small literature. Derivative 4 was obtained from commercially
2
5
peptide was reported some years ago, we have recently re- available 3,4,6-tri-O-acetyl-D-galactal
3 (see ref. 30). The
31
ported the X-ray structures of short glycopeptides bound to adequately protected a-methylserine 9, as N-Fmoc and tert-
SM3. The analysis of these structures reveals that the threo- butyl ester protecting groups, was synthesized starting from
nine (Thr) residue of this epitope adopts a helix-like confor- a-methylserine 7, obtained by methodology described by us.
mation and that its b-methyl is engaged in a hydrophobic Initially, to carry out the glycosylation of protected amino acid 9
contact with the surface of the antibody.
26
32
33
34
with derivative 5, we examined the method of Liebe and Kunz
With these considerations in mind, we designed a novel Tn using AgCO3 and AgClO4 in a mixture of toluene and
antigen mimic based on the quaternary amino acid a-methyl- dichloromethane (DCM), obtaining a mixture of a/b anomers in
27
28
serine (MeSer). This amino acid favors helix-like structures
and has a methyl group at Ca that can establish the hydro- by column chromatography and its azide group was trans-
phobic contact commented above with the antibody. formed into an acetamido group by reduction with Zn in the
In the present work, we have incorporated this unnatural Tn presence of CuSO to give compound 12 in an excellent yield.
a ratio of 3 : 2 in a moderate yield. The a-anomer 11 was puried
4
into the most immunogenic domain of a MUC1 fragment, The tert-butyl ester group of this compound was removed to
designing a three-component cancer vaccine (Fig. 1), similar to afford the required building block 2. In order to improve the
8
that previously reported by Boons and co-workers. We have yield of the glycosylation reaction, as well as the anomeric ratio,
35
demonstrated that the unnatural glycosylated epitope exhibits we used a modication of the trichloroacetimidate method.
better stability in human serum when compared to the natural Therefore, the imidate-derivative 6 was prepared by hydrolysis
derivative. The novel vaccine is able to elicit a potent immune of the nitro group of compound 4 (ref. 36), followed by treat-
response in transgenic mice, recognizing both glycosylated and ment with 2,2,2-triuoro-N-phenylacetimidoyl chloride in the
unglycosylated tumour-associated MUC1 derivatives and native presence of DBU. The use of the acid glycosylation method
MUC1 antigen present on cancer cells. The effectiveness of this forced us to prepare a different and convenient protected
vaccine is comparable (but not better) to that observed for its quaternary amino acid. N-Fmoc-a-methylserine 8 was converted
homologue derivative with threonine. To explain the experimental into benzyl ester derivative 10 using benzyl bromide in the
data, we have performed an extensive conformational analysis on presence of cesium carbonate. The glycosylation of this
the vaccine in the free state in water as well as bound to phos- compound with imidate 6 was carried out with triic acid
ꢁ
pholipid-based liposomes. The analysis involves the use of NMR in diethyl ether at ꢀ40 C for 15 min, giving a 70% yield of
and Molecular Dynamics (MD) simulations. The conformational a/b anomers in a ratio 9 : 1. a-Anomer 13 was puried by silica
studies point out that the extra exibility of the side chain and the gel column chromatography and its azide group was reduced to
glycosidic linkage of the unnatural GalNAc-MeSer fragment have acetamido group to give compound 14.
an unfavorable impact on the molecular recognition by the
The benzyl ester of the latter compound was removed by
immune system. This information reinforces the idea that the hydrogenolysis, giving building block 2 in a good overall yield
design of more efficient vaccines based on MUC1 has to involve (Scheme 1).
the use of glycopeptides that can imitate the conformational
The synthesis of vaccine candidate 1 was carried out by
preferences of the aberrantly glycosylated natural MUC1 epitope. microwave-assisted solid phase peptide synthesis (MW-SPPS)
employing a Rink amide AM LL resin, Fmoc-protected amino
35
acids and derivative 2 (Scheme 2). The incorporation of
building block 2 and the assembly of Fmoc-Pam -Cys-OH were
Results and discussion
2
manually performed under microwave-assisted coupling
conditions.
Prior to the preparation of vaccine candidate 1, we optimized
the synthesis of the unnatural building block 2 (Scheme 1) in
a large scale. To this end, we developed two divergent synthetic
Fig. 1 Novel vaccine incorporating the unnatural amino acid a-methylserine at the MUC1 epitope.
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Scheme 1 Synthesis of building block 2.
We examined then the stability of the unnatural MUC1
Next, groups of MUC1.Tg mice (C57BL/6; H-2b) that express
derivative in human plasma. To this end, we synthesized human MUC1 (ref. 37) were immunized four times at biweekly
glycopeptides APDThr(a-D-GalNAc)RP and APDMeSer(a-D-Gal- intervals with liposomal preparations of compound 1 and
NAc)RP (ESI†). The degradation of the glycopeptide containing empty liposomes as control. One week aer the last immuni-
threonine was faster (around 1.8-fold) compared to the unnat- zation, the mice were sacriced and the serum harvested. Mice
ural compound. We hypothesized that this resistance may immunized with 1 elicited a specic anti-MUC1 IgG antibody
translate to an increase in in vivo stability and bioavailability response (Table 1 and Fig. 2), which was signicantly different
and hence lead to stronger and longer-lasting antigenic from the control group immunized with empty liposomes (EL).
responses.
To examine the immunity of lipoglycopeptide 1, it was IgG2a,b responses, indicating a mixed Th1/Th2 response.
incorporated into phospholipid-based small vesicles (ESI†). The The observed high IgG3 titer is typical of an anti-carbohy-
Sub-typing of the IgG antibodies shows signicant IgG1 and
aliphatic chain of the palmitic acid in the TLR2 agonist favours drate response. IgM antibody titers were low and not signi-
the incorporation of the vaccine into liposomes, which may cantly different from the control. Low amounts of IgG
enhance the circulation time of the vaccine and allows for the antibodies raised against the Thelper epitope were measured;
presentation of the glycopeptide epitope in a multivalent indicating that candidate vaccine 1 does not suffer from
8
manner.
signicant immune suppression.
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Scheme 2 Synthesis of vaccine 1. The incorporation of building block 2 and the assembly of Fmoc-Pam
2
-Cys-OH were manually performed.
0
0
HBTU ¼ N,N,N ,N -tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate. HATU ¼ 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate. DIPEA ¼ N,N-diisopropylethylamine. TFA ¼ trifluoroacetic acid. TIS ¼ triisopropylsilane.
DMF ¼ N,N-dimethylformamide.
a
Table 1 ELISA anti-MUC1 and anti-Thelper antibody titers after 3rd &
We found that the antibodies produced by vaccine 1 could
recognize glycosylated and unglycosylated MUC1 epitopes with
similar titers (Fig. 3), indicating that antibodies recognize the
peptide and not the glycan.
4
th immunization
b
d
Immunization
1
EL
In addition, the ability of the mouse antisera to recognize the
1500 MUC1 antigen expressed on cancer cells was investigated.
600 Serum samples were added to MUC1 expressing MCF7 human
breast cancer and C57mg.MUC1 mammary gland tumor cells,
and recognition was established using a FITC-labeled anti-
mouse IgG antibody. As shown in Fig. 4, antisera obtained from
100
IgG total, MUC1, 3rd imm
IgG total, MUC1, EP
18 400
19 000
4900
1900
9900
800
c
IgG1, MUC1, EP
IgG2a, MUC1, EP
IgG2b, MUC1, EP
IgG3, MUC1, EP
IgM, MUC1, EP
0
1000
400
15 000
9600
IgG total, Thelper, EP
1000
100 immunizations with vaccine 1 displayed recognition of MUC1
tumour cells, whereas no binding was observed for the controls.
a
Anti-MUC1 and anti-Thelper antibody titers are presented as median
values for groups of mice. ELISA plates were coated with BSA-MI-
Of note, vaccine 1 did not improve the effectiveness of
8
CTSAPDT(a-D-GalNAc)RPAP conjugate for anti-MUC1 antibody titers or a previously reported natural threonine-containing derivative
NeutrAvidin-biotin-Thelper for anti-Thelper antibody titers. Titers were
determined by linear regression analysis, with plotting of dilution
versus absorbance. Titers are dened as the highest dilution yielding
(ESI†). Prompted by these results, we investigated whether the
unnatural scaffold inuences the presentation of the epitope.
an optical density of 0.1 or greater relative to normal control mouse To this end, we ran 100 ns MD simulations on phospholipid-
b
c
sera.
Liposomal preparations were employed. EP stands for
based liposomes loaded with only a lipoglycopeptide 1 molecule
d
endpoint (serum samples aer 4 immunizations). EL stands for
to simplify the system. For comparative purposes, we performed
empty liposomes. MI stands for maleimide. Individual anti-MUC1
titers for IgG total, IgG1, IgG2a, IgG2b, IgG3 and IgM, and anti-Thelper also the MD simulation on the threonine analogue.
epitope for IgG total are reported in Fig. 2.
According to these simulations, which start with an extended
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Fig. 3 Anti-MUC1 (glycosylated and unglycosylated) antibody titers
after 4 immunizations with either empty liposomes (n ¼ 3) or vaccine 1
in liposomes (n ¼ 5) as indicated. ELISA plates were coated with (a)
BSA-MI-CTSAPDT(a-D-GalNAc)RPAP conjugate for anti-MUC1(Tn)
titers or (b) BSA-MI-CTSAPDTRPAP conjugate for anti-unglycosylated
MUC1 titers. EL stands for empty liposomes. MI stands for maleimide.
Each data point represents the titer for an individual mouse and the
horizontal lines indicate the mean for the group of mice. Asterisks
indicate statistically significant difference (** P < 0.01, * P < 0.05).
Fig. 4 Cell recognition analysis for specific anti-MUC1 antibodies.
Serum samples (1 : 50 diluted) were incubated with (a) MCF7 or (b)
C57mg.MUC1 cells. After incubation with FITC-labeled anti-mouse
IgG antibody, the fluorescence intensity was assessed in cell lysates.
AU indicates arbitrary fluorescence units; PB stands for pre-bleed; EL
stands empty liposomes. Each data point represents an individual
mouse and the horizontal lines indicate the mean for the group of
mice. Asterisks indicate statistically significant difference (** P < 0.01, *
P < 0.05).
conformation of the peptidic backbone, both lipoglycopeptides
have a clear tendency to adopt folded conformations, allowing
the sugar moiety to be close to the liposome surface at the end
of the MD simulations. This feature is more marked in the
analogue with threonine.
Fig. 2 Anti-MUC1 and anti-Thelper antibody titers after (a) 3 or (b–h) 4
immunizations with either empty liposomes (n ¼ 3) or vaccine 1 in
liposomes (n ¼ 5) as indicated. ELISA plates were coated with (a–g)
BSA-MI-CTSAPDT(a-D-GalNAc)RPAP conjugate or (h) NeutrAvidin-
The solvent-accessible surface area (SASA) calculated for the
biotin-Thelper and titers were determined by linear regression analysis, GalNAc residue, which gives an idea of its accessibility, is ca.
plotting dilution vs. absorbance. Titers were defined as the highest 1.8-fold higher for the unnatural vaccine (Fig. 5), indicating that
dilution yielding an optical density of 0.1 or greater over that of normal
control mouse sera. EL stands for empty liposomes. MI stands for
maleimide. Each data point represents the titer for an individual mouse
and the horizontal lines indicate the mean for the group of mice.
the unnatural scaffold does not hinder the Tn-mimicry to
interact with the immune system.
Attending to our simulations, this result does not explain the
Asterisks indicate statistically significant difference (*** P < 0.001, ** P experimental evidence. Accordingly, we focused our conforma-
<
0.01, * P < 0.05) and NS indicates no significant difference.
tional analysis on the epitope fragments. Thus, glycopeptides g1
and g2 (Fig. 6a) were synthesized and subjected to detailed
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backbone, in agreement with previous structural studies on
26,40–43
MUC1-derived glycopeptides modied by GalNAc.
Con-
cerning the glycosidic linkage, it adopts the ‘alternate’ confor-
mation in the unnatural glycopeptide g1, with f torsional angle
ꢁ
around 80 , in agreement with the exo-anomeric effect, and
ꢁ
with the j dihedral close to 180 (Fig. 7). This geometry of the
44
glycosidic linkage is, as expected, observed in the analogue
glycopeptide with serine also studied in this work (ESI†). In
ꢁ
contrast, the ‘eclipsed’ conformation, with f around 120 , is
preferred in glycopeptide g2 (ref. 45). This particular confor-
mation is characterized by a NOE cross-peak between the amide
groups of GalNAc and Thr (ESI†). Consequently, the carbohy-
drate moiety adopts different orientations with respect to the
peptide backbone in the investigated compounds. Regarding
the side chain of the glycosylated residue, while in g2 it is
ꢁ
1
locked, showing a value close to 60 , in glycopeptide g1 the c
torsional angle is rather exible, showing two similar populated
ꢁ
ꢁ
conformers, with values around 60 [g(+)] and 180 [anti] (Fig. 7).
These conformational differences were also observed when
peptide fragments, g1 (amino acids in blue in Fig. 1) and g2
(natural epitope), are part of the 10-mer MUC1 derivatives,
indicating the generality of the different conformational
behavior. These ndings were deduced from the 100 ns MD
simulations carried out on the liposomes (Fig. 5 and ESI†).
Therefore, although the unnatural glycopeptide exhibits
a higher population of the bioactive backbone conformation
with respect to the natural epitope, it displays a higher exibility
at the sugar moiety. Presumably, the extra exibility of the
unnatural epitope will have a negative impact on the molecular
Fig. 5 Ensembles obtained from 100 ns MD simulations on liposomes
consisting of 65 dodecylphosphocholine lipids loaded with the thre- recognition by the immune system. This hypothesis might be
onine-containing lipoglycopeptide (upper panel) or derivative
1
validated by the excellent results recently reported by Nativi and
co-workers with a non-natural vaccine based on a conforma-
(
bottom panel). The peptide backbone is shown in green, the GalNAc
unit in purple and the liposomes in brown. The solvent-accessible
surface area (SASA) calculated for the GalNAc residues is shown.
13
tional restricted Tn antigen. In summary, our data reinforces
the idea that the design of more efficient cancer vaccines has to
involve the use of glycopeptides that can emulate the peculiar
conformational preferences of the a-GalNAc-Thr glycosidic
conformational analysis by combining NMR and MD simula-
tions. The proton–proton distances obtained from 2D-NOESY
experiments (Fig. 6a) were used as restraints in MD simulations
45
linkage, found in aberrantly glycosylated natural MUC1
epitope.
38,39
(MD-tar).
This type of calculation provides a distribution of
low energy conformers able to reproduce the experimental data. Conclusions
The good agreement obtained in our simulations between the
experimental and calculated distances validates the theoretical
study (see ESI†).
A MUC1-based vaccine containing the quaternary amino acid
a-methylserine at the most immunogenic domain has been
synthesized and the immunogenicity examined in transgenic
mice. The vaccine elicits robust antibody titers recognizing
glycosylated and unglycosylated tumour-associated MUC1
derivatives and native MUC1 antigen present on cancer cells. In
spite of the peptide backbone of the novel vaccine presenting
the bioactive conformation in solution and it being more
resistant to enzymatic degradation than the natural one, the
immune response elicited by this unnatural vaccine is not
superior to the threonine analogue. To rationalize these
observations, we have performed conformational studies on the
unnatural vaccine and on its natural counterpart. These studies
indicate that the presentation and dynamics of the sugar moiety
displayed by the MUC1 derivative may be critical for a strong
immune response. The present work offers a new way to
Fig. 6b and c show the ensemble obtained from 20 ns MD-tar
simulations in explicit water. The unnatural residue in
compound g1 adopts a helix-like conformation in water, which
is validated by the NOE cross-peaks between the methyl group
of MeSer and NH protons of MeSer and Arg residues (Fig. 6a).
The folded conformation adopted by the unnatural residue is in
agreement with previous studies on other glycopeptides
27
carrying this quaternary amino acid. However, the presence of
a-methyl group provides exibility to the peptide backbone. The
most populated conformer (30%) is similar to the bioactive
26
conformation found in the crystal structures. This conforma-
tion is barely populated in the free state for glycopeptide g2,
which exhibits an extended conformation (around 78%) for the
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ꢁ
Fig. 6 (a) Glycopeptides g1 and g2 and sections of the 500 ms 2D NOESY spectrum (400 MHz) in H
2
O/D
2
O (9 : 1) at 25 C and pH ¼ 5.6 showing
the amide cross-peaks. Diagonal peaks and exchange cross-peaks connecting NH protons and water are negative (green color). The NOE
contacts are represented as positive cross-peaks (blue color). Ensembles obtained for glycopeptides g1 (b) and g2 (c) through the 20 ns MD-tar
simulations in water, together with the main conformation found in solution for the backbone of the glycopeptides (lower panels). The backbone
of g1 and g2 are superimposed onto the geometry of the APDThr(a-O-GalNAc)RP glycopeptide in complex with SM3 antibody (PDB entry: 5A2K).
rmsd ¼ root-mean-square deviation.
Asociaci ´o n Espa n˜ ola Contra el C ´a ncer en La Rioja for a grant.
We also thank CESGA for computer support. G.-J. B. acknowl-
edges the National Cancer Institute of the US National Institutes
of Health for support (R01CA88986). Animal studies have been
approved by the Institutional Animal Care and Use Committee
(IACUC) of the University of Georgia.
Notes and references
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Fig. 7 Differences obtained from the MD-tar simulations between the
glycosidic linkage and the side chain of a-methylserine and threonine
residues in compounds g1 and g2, respectively.
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behavior of their components. The designed unnatural amino
acids have to be able to emulate the particularities of the
glycosidic linkage between the GalNAc and the threonine resi-
dues. Our results provide foundational information for the
rational design of carbohydrate-based vaccines against cancer.
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Acknowledgements
We thank the Ministerio de Econom ´ı a y Competitividad
(projects CTQ2012-36365/FEDER, CTQ2012-32025/FEDER,
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