N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2

Article abstract of DOI:10.1002/anie.201713075

The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

Full text of DOI:10.1002/anie.201713075

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: N-carboxyanhydride polymerization of glycopolypeptides that
activate antigen presenting cells through Dectin-1 and -2
Authors: Matthew Zhou, Corleone S Delaveris, Jessica R Kramer,
Justin A Kenkel, Edgar G Engleman, and Carolyn R. Bertozzi
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201713075
Angew. Chem. 10.1002/ange.201713075
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Angewandte Chemie International Edition
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COMMUNICATION
N-carboxyanhydride polymerization of glycopolypeptides that
activate antigen presenting cells through Dectin-1 and -2
+
+
Matthew N. Zhou , Corleone S. Delaveris , Jessica R. Kramer, Justin A. Kenkel, Edgar G. Engleman,
and Carolyn R. Bertozzi*
Abstract: The C-type lectins Dectin-1 and Dectin-2 contribute to
innate immunity against microbial pathogens by recognizing their
foreign glycan structures. These receptors are promising targets for
vaccine development and cancer immune therapy. However,
currently available agonists are heterogeneous glycoconjugates and
polysaccharides from natural sources. Here, we designed and
synthesized the first chemically-defined ligands for Dectin-1 and -2.
They comprised glycopolypeptides bearing mono-, di-, and
trisaccharides and were built via polymerization of glycosylated N-
carboxyanhydrides. Through this approach, we achieved
glycopolypeptides with high molecular weights and low dispersities.
We identified structures that elicit a proinflammatory response
through Dectin-1 or -2 in antigen presenting cells. With their native
proteinaceous backbones and natural glycosidic linkages, these
agonists are attractive for translational applications.
Dectin-1 (Dec-1) and Dectin-2 (Dec-2) are CLRs
expressed on antigen presenting cells such as dendritic cells
and macrophages that are best known for their roles in anti-
fungal and -bacterial immunity. Dec-1 binds to microbial β-1,3-
glucans, and is a key player in immunity against various
[
9]
pathogenic fungi in mouse models (Fig. 1a). Dec-1 signals
through phosphorylation of its immune-receptor tyrosine
activation motif (ITAM), leading to activation of NF-κB and AP-1.
This drives production of the proinflammatory cytokines,
[
10]
including TNFα and IL-6. Engagement of Dec-1 also promotes
[
10]
phagocytosis of the pathogenic cell.
Interestingly,
administration of particulate fungal β-1,3-glucans has been
shown to improve anti-tumor T cell responses in mice, and Dec-
1 was recently implicated in innate immune recognition of tumor-
[
11,12]
associated glycans and anti-tumor cytotoxicity.
Notably,
smaller soluble glucans bind the receptor but do not result in
immune cell activation, which is thought to require receptor
Microbial pathogens display a variety of glycans that
differ from human host structures. The innate immune system
employs glycan-binding proteins, including C-type lectin
receptors (CLRs), to recognize pathogen-associated motifs and
[13]
clustering. Dec-2 also plays an important role in anti-fungal
immunity, and might be similarly involved in anti-tumor innate
[14,15]
immunity.
It selectively binds to high-mannose glycans in a
[16]
microarray format
as well as fungal and mycobacterial
[
1]
trigger immune cell activation. Many CLRs are expressed on
antigen-presenting cells, and thus have been investigated as
[17]
structures that are rich in mannosides.
Like Dec-1, Dec-2
activates immune cells through NF-κB and AP-1 upon receptor
[
2]
targets for vaccine adjuvants and for antigen delivery. These
studies have primarily focused on dendritic cell-specific
intercellular adhesion molecule-3-grabbing non-integrin (DC-
clustering, but signals through the ITAM domain of an
[17c]
associated FcRγ subunit (Fig. 1a).
Previous work on Dec-1 and -2 has exclusively relied
on heterogeneous microbial extracts as sources of activating
ligands. However, the investigation of Dec-1 and Dec-2 as
translational targets for innate immune activation will require
chemically-defined agonists. Herein we report the development
of fully synthetic glycopolypeptide-based agonists of Dec-1 and -
[
3]
[4]
SIGN) and mannose receptor CD206 , although interest in
[
5]
other CLRs is growing as they become better characterized.
CLRs may also be attractive targets for provoking an anticancer
immune response in the typically immunosuppressive tumor
[
5]
microenvironment. This strategy is currently under clinical
[
6]
investigation with other classes of innate immune receptors ,
with the aim of complementing medicines that enhance adaptive
2
. Our design was inspired by a recent study of the fungal
pathogen Malassezia furfur that identified heavily Ser-O-α-1,2-
[
7]
immune mechanisms such as checkpoint inhibitors
and
mannobiosylated glycopeptides as potent agonists of Dec-2
[
8]
adoptive cell therapies.
[17]
signaling.
Accordingly, we synthesized densely glycosylated
polypeptides based on this motif (Figure 1b). Natural Dec-1
ligands are rich in β-1,3-glucan structures, which we also
integrated into glycopolypeptides.
[
+]
[+]
[*]
M. N. Zhou, C. S. Delaveris, Prof. C. R. Bertozzi
Department of Chemistry
Stanford University
Central to our approach was the use of N-
Stanford, Ca 94305
E-mail: bertozzi@stanford.edu
Prof. J. R. Kramer
Department of Bioengineering
University of Utah
Salt Lake City, UT 84112
Dr. J. A. Kenkel, Prof. E. G. Engleman
Department of Pathology and Medicine
Stanford University
Stanford, CA 94305
Prof. C. R. Bertozzi
Howard Hughes Medical Institute
Stanford University
carboxyanhydride (NCA) polymerization,
a
technique we
previously employed to synthesize monosaccharide-bearing
glycopolypeptides with low dispersities and tunable glycosylation
[18]
densities. The structural modularity of this method also allows
for conjugation of the glycopolypeptides to carriers and probes.
Some other glycopolymer platforms share these attributes,
including ring-opening metathesis polymerization (ROMP)-based
[
19,20]
materials
and reversible addition fragmentation chain
[21]
[22]
transfer (RAFT)-based glycopolymers that we
and others
have reported. However, NCA-derived glycopolymers possess a
native proteinaceous backbone and comprise all natural glycans,
glycosyl linkages, and amino acid building blocks. Thus, they
can be biodegraded to natural components, which we feel is
important for translational potential. Notably, NCA-derived
polypeptides are an established pharmaceutical treatment:
Stanford, CA 94305
[+]
These authors contributed equally to this work.
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Angewandte Chemie International Edition
10.1002/anie.201713075
COMMUNICATION
glatiramer acetate was approved by the FDA in 1996 as a
showed absorptions consistent with some helical character (Fig.
[
23]
[31]
multiple sclerosis therapy.
2b).
The larger steric bulk and hydration sphere of the
As building blocks for polymerization, we synthesized
O-glycosylated Ser NCAs bearing the β-1,3-glucan fragments β-
laminaribiose (Glc2) and β-laminaritriose (Glc3) to engage Dec-1,
as well as α-mannose (Man) and α-1,2-mannobiose (Man2) to
target Dec-2. We also prepared control glycopolymers adorned
with α-N-acetylgalactosamine-O-Ser (GalNAc) and β-lactose-O-
Ser (Lac), which should not bind either receptor. Glycosyl amino
acids 1a-f were prepared via the corresponding glycosyl
bromides, and the glycosylated amino acids were converted to
NCA monomers 2a-f via the Fuchs-Farthing method (Scheme 1,
disaccharide sidechains may inhibit the formation of organized
structures.
We next investigated immunostimulatory capabilities of
glucosylated and mannosylated glycopolypeptides through Dec-
1 and Dec-2, respectively. To create a particulate architecture
analogous to natural ligands on microbial cells, we covalently
attached Glc2- or Man2-glycopolypeptides to fluorescent, 0.8 µm
diameter polystyrene beads (Fig. 3a). Poly(ethylene glycol)
(PEG) and Lac-glycopolypeptide conjugated beads were used
as controls. Polymerizations were performed using Ni(II) catalyst
4, which installs a C-terminal azide group for post-polymerization
functionalization that is chemically orthogonal to the N-terminal
[
24]
S1). Glycosylated NCAs (glyco-NCAs) were isolated via cold
aqueous workup followed by anhydrous silica gel
chromatography to give highly pure monomers in 58-95%
[
18]
amino group (Scheme 1).
Amino- functionalized beads were
(BCN) N-
[
25,26]
yield.
treated with bicyclo[6,1,0]non-4-yn-9-yl
a
We next sought to polymerize the NCA monomers
using (PMe Co, which is known to induce controlled
hydroxysuccinimidyl ester and then conjugated to the azide-
terminated polypeptides or azido-PEG. Because steric crowding
could inhibit receptor binding to fully glycosylated homopolymers,
we used 50% and 65% glycosylated polypeptides for
3 4
)
[
27]
polymerization of other NCAs (Scheme S2). Although we had
previously polymerized α-GalNAc-O-Ser NCA, it was not clear
that higher-order glycans would be tolerated, as previous
attempts to polymerize disaccharide-conjugated NCAs had
Ser(Glc2/Glc3)
and
Ser(Man2/Lac),
respectively.
Glycopolypeptides were copolymerized with 1:1 Ala:Glu, all to
approximately 100 residues. Ala was chosen as an inert amino
acid spacer, while Glu was used to improve solubility.
Successful loading of glycopolypeptides onto beads was
measured via fluorescence intensity of polypeptides labeled with
fluorophores at the N-terminus (Fig S2).
[
28]
failed.
glycopolymers in the presence of (PMe
weights as high as 250 kDa and molar mass dispersities under
.15 (Tables S1-S6). Notably, this was the first demonstrated
To our delight, highly purified NCAs 2a-f formed
3 4
)
Co, with molecular
1
high-molecular weight polymerization of NCAs bearing di- and
trisaccharides.
We used a commercial reporter cell line, RAW-Blue, to
We
further
characterized
the
polymerization
study immune cell activation through Dec-1. Derived from the
RAW264.7 macrophage line, RAW-Blue cells possess an NF-
κB-dependent secreted alkaline phosphatase (SEAP), the
expression of which can be detected using colorimetric
substrates (Fig 3b). The parent RAW macrophages do not
express Dec-2, which is expected as expression is typically
parameters for various NCA monomers. Variation of
monomer:initiator (M:I) ratios for each glyco-NCA gave low-
dispersity glycopolypeptides whose lengths increased linearly
with stoichiometry (Fig. 2a, Tables S1,3-6). Polymer molecular
weights were higher than would be predicted assuming perfect
initiator efficiency, which was consistent with previous NCA
[
17]
associated with dendritic cells, not macrophages (Fig. S3).
[
29]
literature.
Remarkably, even disaccharide NCAs could be
Because we did not have access to a reporter line for Dec-2, we
used the Dec-2-expressing murine immature dendritic cell line
JAWS II (Fig. S3) and murine monocyte-derived dendritic cells
to probe this receptor. Proinflammatory cytokines TNFα and IL-6
were detected by ELISA, and microbead phagocytosis was
studied using flow cytometry and fluorescence microscopy (Fig
3b).
polymerized to well over 300 residues. We compared
polymerization kinetics of Ser(Man) NCA 2c and Ser(Man2)
NCA 2d to a non-glycosylated, protected Lys NCA and found
that rates were comparable, suggesting that our glyco-NCAs
would be incorporated stochastically during copolymerizations
(
Fig. S1).
Notably, Ser(Glc3) NCA 2b could only be fully
Treatment of RAW-Blue cells with Glc2- or Glc3-
polymerized at M:I ratios lower than ca. 30:1, likely due to trace
impurities that impeded polymerization at higher ratios.
glycopolypeptide-conjugated
beads
induced
increased
expression of the SEAP reporter protein in a Dec-1-dependent
manner, indicating activation of the NF-κB transcription factor
(Fig. 4a,b, S4). Promisingly, beads displaying Glc2- and Glc3-
glycopolymers elicited a comparable response to the bacterially-
3 4
Polymerization with (PMe ) Co in THF at an M:I ratio of 20:1
gave fully glycosylated chains over 100 residues in length,
indicating that steric hindrance is not a limitation in this reaction.
Promisingly, 2b could be efficiently copolymerized at higher M:I
ratios as a mixture of 20% Ser(Glc3) NCA with 80% Glu(OtBu)
NCA, yielding copolypeptides up to 365 residues (Table S5).
We previously reported a rigid extended conformation
[
32]
derived Dec-1 agonist curdlan.
Likewise, when JAWSII cells
were incubated with Man2-glycopolymer-coated beads, we
observed dose-dependent TNFα secretion (Fig. 4c). The cells
also responded to Man2- but not Lac-glycopolymer beads with a
modest increase in Dec-2-dependent phagocytosis (Figs. S5
and S6). However, the anti-Dec-2 blocking antibody has been
reported as a partial agonist, and indeed we observed low-level
TNFα expression in JAWSII cells, complicating analysis (Fig.
for α-GalNAc-O-Ser glycopolypeptides, which we and others
proposed is due to hydrogen bonding between the peptide
[
30]
backbone and the glycosyl acetamide. This was supported by
the unusual circular dichroism (CD) absorbances we observed.
In contrast, Glc2- and Man2-functionalized polypeptides 3a and
[
33]
S7a).
As an alternative model system, we assayed murine
3
d gave aqueous CD absorption spectra corresponding to
monocyte-derived dendritic cells in plates that had been coated
with Lac- or Man2-glycopolypeptides through passive adsorption.
disordered, random coil conformations, while Man-bearing 3c
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COMMUNICATION
3
92.
With these primary cells, we observed an exceptional cytokine
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a
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knockdown using the anti-Dec-2 blocking antibody when
compared Lac-glycopolymer controls (Fig. 4d, S7b). Remarkably,
although other mannose-binding CLRs are known, the cytokine
response was highly Dec-2 dependent; however, future studies
of how cell type and glycan structure affect the CLR activation
profile are of interest to us. Regardless, these results
demonstrate that the microbe-inspired glycopolypeptides
effectively mimic canonical agonists and induce expected
immunological outcomes.
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In summary, we developed a new class of synthetic
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Acknowledgements
[
This work was funded by grants from the National Institutes of
Health (GM59907; CA196657; CA209971) and by a Stanford
ChEM-H seed grant. C.S.D. was supported by a predoctoral
fellowship from the National Science Foundation (DGE-114747).
J.R.K. was supported by a postdoctoral research award from the
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Keywords: glycoconjugates • ring-opening polymerization •
glycopeptides • immunology • glycopolymers
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Figure 1. A) C-Type lectin receptors Dec-1 and Dec-2 recognize β-glucans and α-mannans, respectively, and induce expression of
cytokines such as TNFα and IL-6 through activation of NF-κB and AP-1. Dec-1 signals through a cytoplasmic signaling domain, while
Dec-2 requires an associated Fc receptor γ (FcRγ) subunit. B) Representative target glycopolypeptide structures based on natural β-
1
,3-glucan agonists of Dec-1 (left) and α-1,2-mannobiosylated agonists of Dec-2 (right).
Scheme 1. Synthesis of glycopolypeptides via metal-catalyzed ring-opening polymerization of N-carboxyanhydride (NCA) monomers
derived from O-glycosylated serine. Polymerization using (PMe Co affords glycopolypeptides with functionalizable N-termini,
3 4
)
whereas catalyst 4 additionally installs a C-terminal azide for dual end-functionalization.
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Figure 2. Polymerization parameters and circular dichroism analysis of glycopolypeptides. A) Representative polymerization data for
Man2 NCA 2d using (PMe Co, illustrating the linear relationship between monomer to initiator ratio [M]/[I] and number-average
molecular weight M , as well as molar-mass dispersities Đ . B) Circular dichroism spectra for glycopolypeptides.
3 4
)
n
M
Figure 3. Covalent attachment of glycopolypeptides to fluorescent polystyrene beads for cell culture evaluation of immune activation
and phagocytosis. A) Glycopolypeptides synthesized using azide-functionalized catalyst 4 were labeled with Alexa Fluor 488 NHS
ester and then conjugated to fluorescent BCN-functionalized beads. B) Glycopolypeptide-bead conjugates induce a variety of cellular
responses upon engagement with CLRs. Cytokine secretion was detected by ELISA, and phagocytosis could be observed by
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fluorescence microscopy and quantified by flow cytometry. Alternatively, NF-κB- and AP-1-induced expression of a secreted alkaline
phosphatase (SEAP) in the macrophage reporter cell line RAW Blue can be detected using a commercial chromogenic substrate.
Figure 4. Glucosylated and mannosylated glycopolypeptides induce receptor-specific inflammatory responses in cell culture. A)
Dose-response curve showing AP-1/NF-κB activation in RAW Blue cells when exposed to Glc2 glycopolypeptide-decorated beads.
Cells were incubated with beads for 16 h, after which supernatant aliquots were mixed with the chromogenic substrate, incubated for
four hours, and then absorbance measured at 620-655 nm. PEG-coated beads and the canonical agonist curdlan were used as
negative and positive controls, respectively. B) The same beads and cells were tested using the same general protocol in the
presence of an αDec-1 blocking antibody or an isotype control antibody to confirm that activation was Dec-1 dependent. C) JAWSII
cells were similarly incubated with Man2 glycopolypeptide-decorated beads, Dec-2 agonist furfurman, or PEG-beads for 16 h, and
dose-response relationships were measured via TNFα ELISA. D) Murine monocyte-derived dendritic cells were incubated for 20
hours with an aDec-2 blocking antibody or an isotype control antibody in plates that had been pretreated with Man2 or Lac polymers
to confirm that TNFα release was Dec-2-dependent. Data in panels A-C were measured in triplicate, those in panel D represent
duplicates; error bars indicate standard error of the mean.
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