'Multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines

Article abstract of DOI:10.1021/ja4046857

Mucin-related carbohydrates are overexpressed on the surface of cancer cells, providing a disease-specific target for cancer immunotherapy. Here, we describe the design and construction of peptide-free multivalent glycosylated nanoscale constructs as potential synthetic cancer vaccines that generate significant titers of antibodies selective for aberrant mucin glycans. A polymerizable version of the Tn-antigen glycan was prepared and converted into well-defined glycopolymers by Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization. The polymers were then conjugated to gold nanoparticles, yielding 'multicopy-multivalent' nanoscale glycoconjugates. Immunological studies indicated that these nanomaterials generated strong and long-lasting production of antibodies that are selective to the Tn-antigen glycan and cross-reactive toward mucin proteins displaying Tn. The results demonstrate proof-of-concept of a simple and modular approach toward synthetic anticancer vaccines based on multivalent glycosylated nanomaterials without the need for a typical vaccine protein component.

Full text of DOI:10.1021/ja4046857

Communication
pubs.acs.org/JACS
‘Multicopy Multivalent’ Glycopolymer-Stabilized Gold Nanoparticles
as Potential Synthetic Cancer Vaccines
Alison L. Parry,†,§ Natasha A. Clemson,† James Ellis,† Stefan S. R. Bernhard,† Benjamin G. Davis,*,§
and Neil R. Cameron*,†
†Department of Chemistry and Biophysical Sciences Institute, Durham University, South Road, Durham, DH1 3LE, U.K.
§Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, U.K.
S
* Supporting Information
ABSTRACT: Mucin-related carbohydrates are overex-
pressed on the surface of cancer cells, providing a
disease-specific target for cancer immunotherapy. Here,
we describe the design and construction of peptide-free
multivalent glycosylated nanoscale constructs as potential
synthetic cancer vaccines that generate significant titers of
antibodies selective for aberrant mucin glycans. A
polymerizable version of the Tn-antigen glycan was
prepared and converted into well-defined glycopolymers
by Reversible Addition−Fragmentation chain Transfer
(RAFT) polymerization. The polymers were then
conjugated to gold nanoparticles, yielding ‘multicopy-
Figure 1. Overview of approach to develop gold nanoparticle-based
synthetic anticancer vaccines. Breast cancer cells express aberrant
multivalent’ nanoscale glycoconjugates. Immunological
studies indicated that these nanomaterials generated strong
and long-lasting production of antibodies that are selective
to the Tn-antigen glycan and cross-reactive toward mucin
proteins displaying Tn. The results demonstrate proof-of-
concept of a simple and modular approach toward
synthetic anticancer vaccines based on multivalent
glycosylated nanomaterials without the need for a typical
vaccine protein component.
mucins displaying Core 1 glycans such as the Tn-antigen glycan. This
‘multicopy-multivalent’ presentation is mimicked by displaying Tn-
antigen glycan glycopolymers on the surface of nanoparticles.
feature a dense presentation of glycans attached to a protein
backbone. We hypothesized that presenting core 1 glycans such
as αGalNAc in a ‘multicopy-multivalent’ manner14 might
produce a nanoparticle with a surface that mimics much
more closely the surface of cancer cells which engage the
surface receptors of cells of the immune system, and thus
produce an effective synthetic vaccine (Figure 1).
Novel Tn glycan monomer 2 was synthesized using a
nonparticipatory glycosyl donor sugar reactant to create the
desired α-anomeric stereochemistry (Figure 2).15 α-Glycosyla-
tion of the linker moiety with glycosyl donor 1 in diethyl ether/
DCM gave the azido-glycoside in 87%. Following successful
attachment of the sugar precursor, the azide functionality was
converted to acetamide using a one-pot Staudinger reduction16-
acetylation procedure. The unwanted β-anomer was readily
removed to give linked pure α-glycoside. Selective methanolysis
followed by careful neutralization allowed removal of the
acetate protecting groups to yield the pure Tn α-anomer 2. In
this way, gram quantities of the polymerizable antigen building
block were readily generated.
Controlled glycopolymers were prepared by Reversible
Addition−Fragmentation chain Transfer (RAFT) polymer-
ization.17 Polymers of varied length and composition could be
prepared (Table 1) by varying feedstock composition, ratios
and conditions. First, a Tn-antigen glycan monocomponent
homopolymer was created in an optimum yield of 65% in a
ealthy cells of the mammary gland are characterized by
H
the surface presentation of branched, O-linked core 2
glycans containing high levels of N-acetyl-D-glucosamine
(GlcNAc). However, proteins on the surface of breast cancer
cells instead present mainly linear, truncated core 1 mucin-type
glycans such as α-N-acetyl-D-galactosamine (αGalNAc, the Tn-
antigen glycan) (Figure 1), with complete or near-complete
absence of core 2 residues.1 These differences have been
targeted as a strategy for cancer immunotherapy.2 Accordingly,
multivalent glycoconjugates have been prepared in which
mucin glycans are presented on a variety of scaffolds, including
peptides,3 lipopeptides,4 dendrimers5 and proteins.6 Some of
these approaches have developed as far as clinical trials.7
Nanomaterials represent an alternative platform for the
presentation of glycans8 that allow greater synthetic control and
higher density than on current protein scaffolds. Carbohydrate-
presenting gold nanoparticles (AuNPs)9 decorated with small
molecule thiolated glycans have been used as tools to study
carbohydrate−carbohydrate interactions,10 in antiadhesive
therapy,11 and as anti-HIV12 and cancer vaccine candidates.13
However, these typically monomolecular sugar coatings do not
represent well the structure of mucin glycoproteins, which
Received: May 19, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja4046857 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(TEM) (Figures 2, S1 and S2). All samples contained a narrow
size distribution of particles of diameter between 5 and 20 nm,
except for PEG40Tn10 which resulted in a polydisperse sample
(Figures S1 and S2). Determination of antigen and polymer
loading using thermogravimetric analysis (TGA) indicated that
not only could loading be tuned, but also that the use of longer
polymer chains allowed the creation of smaller nanoparticles
(Table 1, entries 4 and 5) with a lower mass fraction of gold
core.
The Tn-antigen glycan presenting nanoparticles were
analyzed for their efficacy and ability to induce an immune
response in vivo. New Zealand White rabbits (n = 3) were
immunized at days 0, 14, 28, and 56 with either polyTn-NP or
free polymer solution. Serum samples were taken at day 0
(preimmune bleed), 42 and 70 and antibodies present were
quantified using an ELISA assay against the synthetic antigens.
The results are presented in Figure 3a. The free polymers gave
low or negligible response, whereas all glyconanoparticles
generated a higher response that increased over time as judged
by antibody (IgG) titers. Examination of the data in Figure 3a
reveals a strong influence of nanoparticle composition on
immunological properties. The highest titers were observed for
AuNPs prepared with the polymers PEG25Tn25 and PEG80Tn20,
with weaker responses observed for PEG40Tn10 and PEG50Tn50
(subscript denotes number average block length). The
polydispersity of PEG40Tn10 may reduce its stability in vivo
and hence produce lower titers than the other particles.
The relationship observed between carbohydrate density and
immune response is notable. It appears that the optimum Tn-
antigen glycan density is 20−25 units per polymer chain,
regardless of chain length. Antigen induced cross-linking of B
cell receptors leads to B cell activation and antibody
production, whereas cross-linking with coreceptors can either
increase or suppress B cell response.19 It is likely that the
glycoconjugate carbohydrate density has a strong influence on
the subtle interplay between these factors and therefore on the
production of antibodies.
Figure 2. Preparation and characterization of Tn-antigen gold
nanoparticles (for abbreviations see Supporting Information).
Reagents, conditions and yields: (i) NaN3, CAN, CH3CN, −20 °C,
30 h, 80%; (ii) PhSH, DiPEA, CH3CN, 1 h, 72%; (iii) K2CO3,
CCl3CN, dichloromethane (DCM), 8 h, 62%; (iv) HEMAm,
TMSOTf, Et3N, Et2O/DCM (2:1), −20 °C, 30 min, 80%; (v)
DPPE, DCM, 1 h then Ac2O, DMAP, Et3N, H+ resin, 55% α-anomer;
(vi) K2CO3, MeOH, 65%; (vii) PEGMA, CPADB, ACVA, 70 °C, 48 h,
51−75%. Insets show representative dynamic light scattering data
(top) and TEM image (bottom) for glyconanoparticles (scale bar = 20
nm).
solvent mixture of DMF:H2O (7:3). The resulting polymer was
analyzed by SEC and possessed a number-average molecular
weight (Mn) of 14.2 kDa and a polydispersity index (PDI) of
1.16 (see Table 1). Next, the Tn-antigen glycan monomer 2
and poly(ethylene glycol) methyl ether methacrylate (PEGMA;
Mn = 300 Da) were copolymerized at varying comonomer
feedstock ratios, with [total monomer]0/[CTA]0 ratios of 100/
1 and 50/1, and after 48 h all proceeded with excellent overall
conversion (see Table 1). The number- and weight-average
molecular weights (Mn and Mw) determined by SEC were in
good to excellent agreement with those predicted and the PDIs
ranged from 1.12 to 1.23.
Sodium borohydride was then used to reduce simultaneously
HAuCl4 to Au0 and the dithioester end groups of the RAFT
polymers to thiol,18 thereby forming a range of Tn-antigen
glycan nanoparticles (polyTn-NPs) in situ (Figure 2 and Table
1). The polymer coronas of these particles varied in Tn glycan
density (20, 50, and 100 mol %) and polymer DPn (50 or 100
units). The size of the nanoparticles was confirmed by dynamic
light scattering (DLS) and transmission electron microscopy
Barchi et al. have prepared glycosylated gold nanoparticles
bearing the Thomson-Friedenreich (TF) antigen and demon-
strated moderate antibody responses.13b Direct comparison
with their data is difficult since optical density values rather
than serial dilution titers were reported; nonetheless, it seems
that the maximum response of our nanoparticles is of the same
order of magnitude.
To probe the ability of the nanoparticle-generated antibodies
to recognize naturally occurring antigens, cross-reactivity with
Table 1. Data for the Synthesis and Characterization of Glyconanoparticles
a
d
polymer
conv
yield
(%)
Mn, Th
Mn
PDI
Dh
FAu
[Pol]
[Tn]
b
c
d
e
f
g
h
(%)
(kDa)
(kDa)
(nm)
(%)
(mmol)
(mmol)
Tn50
65
52
73
59
68
75
11.1
15.3
12.9
29.0
27.7
14.2
15.0
16.9
40.4
30.4
1.16
1.12
1.18
1.15
1.18
16
25
13
9
34
61
45
6
4.7 × 10−5
4.1 × 10−5
3.3 × 10−5
2.3 × 10−5
2.6 × 10−5
2.3 × 10−3
2.0 × 10−4
2.0 × 10−4
3.0 × 10−4
1.0 × 10−3
i
i
i
i
PEG40Tn10
PEG25Tn25
PEG80Tn20
PEG50Tn50
99, 95
90, 70
99, 75
99, 75
7
21
a
PEG = poly(ethyleneglycol) methyl ether methacrylate, Tn = Tn-antigen glycan monomer 2, subscript = target degree of polymerization.
b
Determined by 1H NMR spectroscopy by comparison of the integrals of the monomer alkene peaks to a selected polymer peak in the spectrum of
c
d
e
the crude polymer. Theoretical Mn, at observed conversion, determined from [monomer]0:[CTA]0;. Determined by SEC. Mean hydrodynamic
diameter determined by dynamic light scattering. Mass fraction of Au per mg of nanoparticle, determined by thermogravimetric analysis. Quantity
of polymer per mg of nanoparticle, determined by thermogravimetric analysis. Quantity of Tn-antigen glycan per mg of nanoparticle, determined by
thermogravimetric analysis. Values refer to copolymer first and second block, respectively.
f
g
h
i
B
dx.doi.org/10.1021/ja4046857 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
n.r.cameron@durham.ac.uk; ben.davis@chem.ox.ac.uk
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the MRC and EPSRC (joint grant reference
G0700080) for funding. N.R.C. acknowledges the P2M RNP
programme of the European Science Foundation. B.G.D. is a
recipient of a Royal Society Wolfson Merit Award. Professor
Quentin Sattentau (Sir William Dunn School of Pathology,
University of Oxford) is thanked for helpful discussions.
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