C-Mannosyl Lysine for Solid Phase Assembly of Mannosylated Peptide Conjugate Cancer Vaccines

Article abstract of DOI:10.1021/acschembio.9b00987

Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of DCs is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the induction of antigen-specific T cells. The natural O-mannosidic linkage is susceptible to enzymatic degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and streamline the synthesis of mannosylated peptide antigens, we here describe the development of an acid-stable C-mannosyl lysine, which allows for the inline introduction of mannosides during solid-phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization capacities and binding affinities as the O-mannosyl analogs. Moreover, the antigen conjugates were capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and providing enhanced gp100 presentation to CD8⁺ and CD4⁺ T cells, similar to their O-mannosyl counterparts. Our results demonstrate that the C-mannose lysine is a valuable building block for the generation of anticancer peptide-conjugate vaccine modalities.

Full text of DOI:10.1021/acschembio.9b00987

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Article
C-mannosyl lysine for solid phase assembly of
mannosylated peptide conjugate cancer vaccines
Tim P. Hogervorst, R.J. Eveline Li, Laura Marino, Sven C.M. Bruijns, Nico
J. Meeuwenoord, Dmitri V. Filippov, Herman S. Overkleeft, Gijsbert A. van
der Marel, Sandra J. van Vliet, Yvette van Kooyk, and Jeroen D. C. Codée
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00987 • Publication Date (Web): 11 Feb 2020
Downloaded from pubs.acs.org on February 15, 2020
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C-mannosyl lysine for solid phase assembly of mannosylated peptide
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conjugate cancer vaccines
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Tim P. Hogervorst †, R.J. Eveline Li †, Laura Marino , Sven C.M. Bruijns , Nico J. Meeuwenoord ,
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Dmitri V. Filippov , Herman S. Overkleeft , Gijsbert A. van der Marel , Sandra J. van Vliet , Yvette van
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Kooyk * and Jeroen D.C. Codée *
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Department of Bio-organic Synthesis, Faculty of Science, Leiden Institute of Chemistry, Leiden University, Leiden, The
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Netherlands, Amsterdam UMC- location Vrije Universiteit Amsterdam, dept. of Molecular Cell Biology and Immunology,
Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, Netherlands
† These authors have contributed equally to this work
* Correspondence:
Jeroen D.C. Codée
JCodee@chem.leidenuniv.nl
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Yvette van Kooyk
Y.vanKooyk@amsterdamumc.nl
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Graphical Abstract
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Abstract
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Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize
pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of DCs
is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a
frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the
induction of antigen-specific T cells. The natural O-mannosidic linkage is susceptible to enzymatic
degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this
reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring
orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and
streamline the synthesis of mannosylated peptide antigens, we here describe the development of an
acid-stable C-mannosyl lysine, which allows for the inline introduction of mannosides during solid-
phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly
of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-
associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization
capacities and binding affinities as the O-mannosyl analogs. Moreover, the antigen conjugates were
capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and
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providing enhanced gp100 presentation to CD8 and CD4 T cells, similar to their O-mannosyl
counterparts. Our results demonstrate that the C-mannose lysine is a valuable building block for the
generation of anti-cancer peptide-conjugate vaccine modalities.
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Keywords: glycopeptides, C-glycoside, peptide conjugate, vaccine model, DC-SIGN
INTRODUCTION
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Immunotherapy for cancer is gaining momentum. More and more therapies have reached the clinic or
are in advanced clinical trials, including immune checkpoint blocking (ICB) antibodies, chimeric antigen
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receptor T cells (CAR T cells), adoptive cell transfer (ACT), and dendritic cell (DC) vaccination. DC-
based strategies rely on the role of dendritic cells as key initiators of the adaptive immune system that
are crucial in the induction of memory responses. By antigen capture and processing, DCs can present
peptides to naïve T lymphocytes and skew their differentiation endpoints and by exposing DCs to
synthetic tumor (neo-)antigens, the immune response can be directed towards specific cancer-
associated antigens. Although animal models have demonstrated promising results for peptide-based
vaccine strategies, human trials often result in minimal tumor regression and only partial
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effectiveness . Vaccine efficacy may be improved by the incorporation of adjuvants that can target
Pattern Recognition Receptors (PRRs), which can induce DC maturation and improve antigen
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processing . Toll-Like Receptors (TLRs) are a family of PRRs, of which members have been successfully
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targeted with covalent adjuvant-antigen conjugates , to induce DC maturation and improve antigen
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processing and presentation . Another family of PRRs that has frequently been exploited as an
endocytic receptor to facilitate antigen cross-presentation are the C-type Lectin Receptors (CLRs). This
family of PRRs recognizes carbohydrate patterns, and are an essential determinant in discerning host
from foreign antigens. An often studied receptor is DC-SIGN (CD209), a CLR present on DCs that
internalizes carbohydrate modified antigens for cross-presentation to T cells. DC-SIGN recognizes
mannose and Lewis-type carbohydrate moieties on a wide variety of pathogens and is often targeted
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to activate specific signaling and tailor the immune response . DC-SIGN can also act as a scavenger
receptor that can induce receptor-mediated endocytosis. Due to its tetramic structure, a multivalent
presentation of its ligand enhances the avidity for DC-SIGN. Thus, while the affinity for a single
monomannoside ligand is low, targeting mannosylated constructs can be markedly increased by the
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multivalent presentation of the monosaccharides on a polyvalent core or carrier platform such as
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dendrimers, liposomes, or nanoparticles
. Vaccination with mannosylated antigens in mice has
demonstrated improved cytotoxic lymphocyte responses, stronger Th1 and Th2 responses and
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increased antibody responses . Addition of an adjuvant can further boost the generated immune
response of mannosylated conjugates. For example, the conjugation of multivalent mannosides and
TLR7 adjuvants to an antigen via a self-immolative linker resulted in a more robust and higher-quality
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humoral and cellular immune response in mice .
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In earlier work, we have also demonstrated the significance of antigen mannosylation . We
systematically increased the number of well-defined mono-, di-, and tri-mannosides on a peptide
backbone to evaluate the effect of multivalent presentation of DC-SIGN ligands on the peptide
antigens. We could also extend the conjugates with a secondary adjuvant. Using this strategy, we
generated precisely defined trifunctional conjugates (CLR-antigen-TLR), which effectively targeted DC-
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SIGN . This approach however, required the conjugation of O-mannosides via a Cu(I) catalyzed azide-
alkyne cycloaddition (CuAAC), which involved an additional purification step and limits the number of
available orthogonal handles that can be incorporated into the conjugates. Furthermore, O-
mannosides may be enzymatically cleaved rendering them less suitable for DC-SIGN targeting1
Therefore, we here report the design and synthesis of a C-mannose functionalized lysine building block
(1, Scheme 1). This C-mannoside lacks the exo-cyclic anomeric oxygen to render the glycosidic linkage
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resistant to the acidic conditions necessary for standard automated solid phase peptide synthesis
(SPPS). Besides, the C-glycoside is resistant to enzymatic hydrolysis. By attachment to an Fmoc-
protected amino acid building block, the mannoside can be incorporated via ‘inline’ synthetic
methodology, obviating post-synthesis conjugation steps and preventing the use of an azide-alkyne
click reaction. The C-mannoside building block has been used to generate peptide-antigen conjugates,
carrying one or six mannosides, in addition to a synthetic TLR7 ligand. The generated constructs have
been evaluated, in a side by side comparison to the corresponding O-mannoside clusters, for binding
affinity, uptake and antigen presentation capacity, revealing that C-mannosides can effectively be used
to replace their more labile O-counterparts in covalent antigen conjugates.
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RESULTS AND DISCUSSION
Synthesis of the C-mannoside lysine building block
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The synthesis of the key SPPS-ready mannosylated Fmoc amino acid is achieved in 11 steps and is
shown in Scheme 1. The crucial step in the synthesis of 1 is the introduction of the α-C-glycosidic bond.
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Based on previously reported work by Girard et al. , the anomeric allyl was introduced via a Hosomi-
Sakurai reaction using allyltrimethylsilane (allyl-TMS). The synthesis starts from methyl 2,3,4,6-tetra-
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O-benzyl-α-ᴅ-mannopyranoside 3, obtained by benzylation of methyl α-ᴅ-mannopyranoside 2 . The
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allyl group was introduced by treatment of the methyl mannoside with allyltrimethyl silane and
trimethylsilyl triflate in acetonitrile to provide C-mannoside 4. This reaction proceeded with high
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stereoselectivity and was complete within an hour when assisted by ultrasound irradiation . Selective
removal of the benzyl ethers in the presence of the allyl functionality was initially effected using either
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BCl
3
or a Birch reduction in liquid ammonia . However, both reactions proved difficult to scale up and
we therefore switched to the use of a single electron reduction using lithium naphthalenide in THF.
This reaction could be run on 80 mmol scale, to provide the desired tetra-ol, which was acetylated to
ease purification, delivering 5 in 54% yield. After the installation of four PMB ethers, the anomeric allyl
appendage was elongated through a cross-metathesis with methyl acrylate to afford α,β-unsaturated
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ester 7. The reduction of the double bond in this product with RuCl
MeOH was followed by saponification of the resulting ester 8 to obtain carboxylic acid 9. Fully
protected C-mannosyl lysine 12 was obtained by coupling of carboxylic acid 9 with the methyl ester of
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in the presence of NaBH and
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N -Fmoc protected lysine 11, using HCTU as condensation agent. Selective hydrolysis of the ester in
α
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the presence of the Fmoc group was achieved with LiOOH , which is more nucleophilic but less basic
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1
than LiOH , resulting in the isolation of key building block 1 in 79% yield. Altogether, SPPS-compatible
C-mannosyl 1 was synthesized in 20% yield over 11 steps.
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The use of per-acetylated mannosyl donors has previously shown to lead to anomeric mixtures, indicating that
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neighboring group participation falls short to effect stereoselective C-glycosylation reactions
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Scheme 1 Synthesis of C-mannoside lysine 1
OH
O
OBn
O
OBn
O
OAc
O
HO
HO
HO
BnO
BnO
BnO
BnO
AcO
BnO
AcO
a
b
c
BnO
AcO
2
OMe
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OMe
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d
OPMB
O
OPMB
O
OPMB
O
OPMB
O
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
g
f
e
PMBO
9
8
7
6
O
O
O
OH
OMe
OMe
h
-
+
Cl NH3
OPMB
O
OPMB
O
PMBO
PMBO
PMBO
PMBO
PMBO
PMBO
H
N
i
H
OMe
O
FmocHN
O
FmocHN
O
N
FmocHN
11
O
O
OMe
OH
10
1
1
11
1
1
1
1
12
13
14
15
Reagents and conditions: a) NaH, BnBr, TBAI, DMF, 78%; b) allylTMS, TMSOTf, ACN, 73%; c) either i. BCl
ii. Ac O, pyridine 95% or i. Li, naphthalene, THF, -20°C; ii. Ac O, pyridine, 54%; d) i. NaOMe, MeOH; ii. NaH, PMBCl,
TBAI, DMF, 69%; e) methyl acrylate, Grubbs 2nd gen. catalyst, DCM, 73%; f) RuCl
, NaBH , DCE/MeOH, 93%; g)
KOH, THF/H O, qnt.; h) 11, HCTU, DIPEA, DMF, 99% ; i) LiOH, H , THF/H O/t-BuOH, 79%.
3
, DCM;
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O
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Synthesis of the mannoside clusters
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1
Our previous work has described the synthesis of the O-mannoside clusters , and resulted in
monovalent- (24), bivalent- (25), trivalent- (26), and hexavalent (27) O-mannoside clusters.
Biotinylation of these compounds resulted in biotinylated O-mannoside clusters 28, 29, 30, and 31
respectively (Figure 1A). To enable the direct comparison to these clusters, we here generated clusters
containing 1, 2, 3 or 6 copies of the C-mannosyl through an SPPS approach (Scheme 2). Using Tentagel®
S RAM amide resin, Fmoc-Lys(Boc)-OH and Fmoc-Gly-OH were introduced successively, followed by
elongation with C-mannosyl 1 using a standard Fmoc protocol and HCTU as the condensation agent.
Building block 1 was introduced manually, using only a small excess of the amino acid and prolonged
coupling times (2 eq, overnight) to prevent the use of a large excess of 1. After completion of the
sequence, the N-termini were capped with acetyl groups resulting in immobilized peptides 12-15.
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122
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1
126
1
1
129
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24
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28
Subjecting resins 12-15 to a cleavage cocktail of TFA/TIS/H O (190/5/5, v/v/v) successfully removed
the Boc and PMB ethers and the peptide clusters were isolated after RP-HPLC purification to obtain
monovalent- (16), bivalent- (17), trivalent- (18) and hexavalent- (19) clusters in 7%, 14%, 6%, and 2%
2
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31
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yield respectively (Scheme 2, R = R ). Further functionalization via the introduction of a biotin handle
1
was achieved by reacting the primary amine of the C-terminal lysine with a biotin-N-
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hydroxysuccinimide (NHS) ester. This resulted in biotinylated mannoside clusters 20-23 (Scheme 2, R
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= R
2
).
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Scheme 2 Synthesis of C-mannoside clusters
OPMB
O
OH
O
PMBO
PMBO
PMBO
HO
HO
HO
=
NH2
R1
0
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2
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O
O
H
N
H
NH
NHBoc
NH
R
O
S
NH
H
a
b, c
O
O
O
O
H
N
H
N
NH2
N
H
N
H
N
H
N
H
=
HN
O
:
:
R2
R1 R2
O
O
O
O
n
n = 1 12
n = 2 13
n = 3 14
n
n = 1 16 20
n = 2 17 21
n = 3 18 22
Biotin
OPMB
O
OH
O
PMBO
PMBO
PMBO
HO
HO
HO
O
O
NH
NHBoc
NH
R
a
b, c
O
O
O
O
H
N
H
N
NH2
:
N
H
N
H
N
H
N
H
:
O
O
O
O
R1 R2
6
6
15
19 23
1
36
1
1
1
37
38
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Reagents and conditions: a) Fmoc-SPPS (1, HCTU, DIPEA, DMF); b) TFA, TIS, H O, (octanethiol, phenol):
(16: 7.0%; 17: 14%; 18: 6.0%; 19: 2.1%); c) BiotinOSu, DIPEA, DMSO: (20: 94%; 21: 72%; 22: 99%; 23:
80%).
2
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Binding of the C-mannoside clusters to moDCs
1
1
1
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43
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With the biotinylated constructs in hand, the effect of the O to CH
studying the binding, internalization and endosomal routing upon DC-SIGN engagement, of the C-
2
modification was evaluated by
2
1
mannoside clusters and their O-mannoside equivalents 28-31 (Figure 1A, 1B) . The binding of the
clusters to cellular membrane DC-SIGN was evaluated using DC-SIGN expressing monocyte-derived
DCs (moDCs)(Figure 1C, also see Supporting Information, Figure SI.1A). The assay was performed at a
temperature of 4°C to prevent the internalization of DC-SIGN from the cell membrane surface. The
cells were incubated with the biotinylated clusters for 30 minutes, after which unbound clusters were
washed away. Treatment of the cells with fluorophore-labeled streptavidin allowed quantification of
binding by flow cytometry. An increase in binding of bivalent C-mannoside 21, trivalent O-mannoside
30 and C-mannoside 22, hexavalent O-mannoside 31 and C-mannoside 23 was seen when compared
to the unstimulated control. Compared to a mannosylated polyacrylamide control, all compounds
showed better binding, with the exception of mono-O-mannoside 28. A clear valency-dependent
increase in binding was seen within each of the O- and C-mannoside sets. These results are in line with
our previous study, in which we compared the monomannoside clusters to clusters built up from more
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152
1
1
1
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complex di- and tri-mannosides . For all clusters higher valency led to higher binding affininty. Binding
of the hexavalent monomannoside cluster was comparable to the binding of the higher valent di- and
trimannosides. Blocking of the DC-SIGN receptor effectively diminished the binding, although low
residual binding with a similar valency-dependent increase remained (see SI, Figure SI.1B). The residual
binding suggests cluster recognition of other mannose-binding receptors on DCs, such as mannose
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receptor .
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We next selected the mono- and hexavalent O- and C-mannoside clusters to assess their
internalization. The assessment of cluster internalization was executed at 4°C, similar to the binding
assay. To this end, the cells were incubated for 60 minutes to saturate the immobilized DC-SIGN
receptors at the cell surface, followed by the removal of unbound clusters. Re-suspension of the
samples in warm (37°C) medium initiated internalization. At the indicated time points, samples were
taken and immediately put on ice. The signal loss of the membrane was measured through flow
cytometry upon treatment with a fluorophore-conjugated streptavidin. To exclude cluster-DC-SIGN
dissociation, we additionally gently fixed the cells with 1% paraformaldehyde for permanent
immobilization of DC-SIGN at the membrane. No dissociation of the mannosylated clusters from DC-
SIGN on fixed cells was measured (see SI, Figure SI.1C), indicating that the ligands are internalized upon
DC-SIGN binding. Internalization (>50%) of the hexavalent cluster 23 was seen after 5 minutes (Figure
1D). The complementary O-mannoside cluster (31) accomplished the same level of internalization after
15 minutes. Uptake of the monovalent clusters occurred at a slower rate, requiring 30 minutes for
internalization of approximately 50% of both the O- and C-mannose clusters (28, 20). The relative
internalization efficiency seems to mirror the binding profiles seen in Figure 1C.
163
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1
1
170
1
1
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Mono- and hexavalent cluster trafficking to the endosomes was further studied using pHrodo® Red.
This dye acts as a pH-sensitive sensor, as the fluorescence is considerably increased in acidic
environments, while it is quenched in the neutral extracellular environment. Prior to moDC
stimulation, the biotinylated mannoside clusters were treated with the avidin-conjugated fluorophore
(2:1). The pre-complexed clusters were subsequently added to the moDCs at 37°C, continued by
sample collection at each time point. The cells were washed, gently fixed, and fluorescence was
subsequently measured by flow cytometry. After 30 minutes, a 2-fold increase was visible in the
fluorescence of the hexavalent O- and C-mannoside clusters (Figure 1E). On the other hand, the
fluorescence of both monovalent clusters (28 and 20) was increased 2.5-folds at 30 minutes,
suggesting higher endosomal ligand concentrations. Although the C-mannosides resembled the O-
mannoside clusters, the deviation between mono- and hexavalent presentation is surprising. In the
previous binding and internalization assays, the hexavalent clusters 31 and 23 were superior to
monovalent mannoside presentation. The increased MFI of the monovalent over the hexavalent
clusters at 30 minutes could indicate faster endo-lysosomal trafficking of the smaller clusters, as the
1
1
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emitted fluorescence by the dye is higher with lower pH . Moreover, the mannosylated clusters
were pre-complexed with the pHrodo dye into a larger particle, possibly contributing to the altered
endocytosis . Altogether these results indicate that the C-mannoside clusters 20 and 23 were able to
convincingly resemble the DC-SIGN binding and internalization profiles of the O-mannoside
equivalents, encouraging the implementation of C-mannosylated antigen conjugates.
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Figure 1 Binding of the C-mannoside clusters to DC-SIGN
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(A) Structure of the mono-, di-, tri-, or hexavalent O-mannoside clusters, with and without biotin. (B)
Schematic representation of the binding, internalization, and endosomal routing assays. (C) Binding of
the biotinylated clusters to moDC DC-SIGN as measured by flow cytometry (left panel, eight donors),
and to moDC with blocked DC-SIGN (right panel). Polyacrylamide decorated with monomannosides or
200
2
2
2
2
2
2
01
02
03
04
05
06
Y
Le antigens were used as positive control. Paired Student’s t-test error: *, p < 0.05; **, p < 0.001. (D)
DC-SIGN-mediated internalization of the clusters was measured by flow cytometry. One donor is
depicted as a representative of four individuals. (E) The routing of the clusters to the
endosomes/lysosomes as measured by flow cytometry and normalized to a negative control. One
donor is depicted as representative of three individuals.
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Synthesis of mannosylated gp100 conjugates
2
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Melanoma derived from transformed pigment-carrying melanocytes is a highly lethal cancer, and this
3
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malignancy is considered one of the most immunogenic cancer types . We therefore selected the
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melanoma-associated antigen gp100 as our vaccine target, and we introduced C-terminal mannoside
3
6
clusters and a secondary TLR ligand to generate self-adjuvating peptide antigen vaccine conjugates .
Endosomal TLR7 was selected as PRR-target because we reasoned that the use of a cell surface PRR
targeting PAMP would lead to competition for binding with DC-SIGN on the outside of the DC surface
membrane. Furthermore, DC-SIGN-mediated endocytosis should deliver the conjugates to the
endosomes, where it will encounter the TLR7 receptor. The target gp100 peptide antigen combines
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+
the CD4 T cell binding epitope gp100280-288 and CD8 T cell binding epitope gp10044-59. Multiple
conjugates were synthesized bearing the TLR7 agonist either on the N- or C- terminus and carrying
either one or six mannosides, to study the effect of these modifications on antigen presentation.
19
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The SPPS of the conjugates started with the introduction of a monomethoxytrityl (Mmt) functionalized
Fmoc-Lys-OH on Tentagel® S RAM amide resin (Scheme 3). Both termini of the antigen sequence were
extended with four naturally occurring amino acids to act as spacers between the antigens, the
mannoside cluster, and the TLR7 ligand. The Cys60 in the N-terminal spacer was replaced with an
23
24
25
3
7
isosteric α-amino-butyric acid analog to prevent potential oxidation and peptide dimerization , a
2
1
modification we previously demonstrated not to affect antigen biology . The sequences were
elongated at the N-terminus with either one or six copies of the C-mannosyl 1, resulting in immobilized
peptides 33 and 34 (Scheme 3). Next, 33 and 34 were further extended at their N-termini with the
27
28
3
8
229
TLR7 ligand 2-butoxy-8-oxo-adenine, using the previously described Boc protected building block 32 ,
to give immobilized conjugates 35 and 36. Alternatively, the N-termini were acetylated, after which
the C-termini were further functionalized by selective removal of the C-terminal lysine Mmt-group,
2
2
2
233
2
2
30
31
32
3
9
followed by the introduction of the TLR7 ligand to provide 37 and 38 . It was observed that a cocktail
of TFA (1%) in DCM, led to partial removal of the PMB groups leading to the undesired esterification
of some of the carbohydrate alcohols with the TLR7 ligand. Therefore, milder conditions were explored
for the removal of the C-terminal Mmt-group. Eventually, the use of acetic acid in a mixture of
trifluoroethanol (TFE) and DCM (1/2/7, v/v/v), a cocktail first described to selectively cleave the Mmt
34
35
236
40
2
2
2
37
38
39
over methyltrityl and trityl-groups selectively , was found to be effective for the selective unmasking
of the MMT in the resin-bound protected conjugates. Using these conditions, the immobilized N-
terminal elongated conjugates 37 and 38 were successfully generated.
240
Initial attempts to deprotect and release the peptides from the resin with a cleavage cocktail of
2
2
2
244
2
2
41
42
43
TFA/TIS/H O (190/5/5, v/v/v) resulted in complex crude mixtures. Analysis of the mixtures indicated
2
that the poor quality of the crude material was due to side reactions of the cleavage and deprotection
step and not due to incomplete couplings. Reactive cationic species are liberated during the acidic
4
1
removal of the PMB ethers, which can react with functional groups in the unprotected peptide .
42
45
46
Howard et al. effectively scavenged PMB-cations using phenol as an electron-rich aromatic additive,
and when this additive was applied here, the quality of the crude mixture indeed improved. Further
optimization of the cleavage conditions was achieved by increasing the concentration of the
scavengers (up to 10% of the total volume) and increasing the volume of cleavage medium (effectively
diluting the concentration of reactive cationic species and reactive functional groups). Using this
optimized cleavage protocol all four peptides were successfully deprotected and released form the
resin. After RP-HPLC purification, the monovalent C-mannose conjugate 39 was obtained in 2.6% yield
after 36 couplings steps and the hexavalent C-mannose conjugate 40 in 1.3% yield over 41 couplings.
The conjugates 41 and 42 were isolated in 2.1% (after 36 steps) and 0.6% (after 41 couplings),
respectively (Scheme 3). Unlike the O-mannoside conjugates, we previously generated, these
constructs did not require additional conjugation and purification steps.
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To compare the activity of the C- vs. the O-mannoside conjugates, O-mannoside clusters 24 and 27
were functionalized with an alkyne handle (yielding 43 and 44) and conjugated through a CuAAC
ligation to the TLR7L- gp100 peptide 45 to obtain analogs 46 and 47.
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Scheme 3 Synthesis of C- and O- mannoside – gp100 – TLR7 agonist conjugates
:
-Val-Thr(tBu)-His(Trt)-Thr(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Pro-Gly-Pro-Val-Thr(tBu)-Ala-Asn(Trt)-Arg(Pbf)-Gln(Trt)-Leu-Tyr(tBu)-
[
AA]*
Pro-Glu(OtBu)-Trp(Boc)-Thr(tBu)-Glu(OtBu)-Ala-Gln(Trt)-Arg(Pbf)-Leu-Asp(OtBu)-
0
1
2
3
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9
0
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9
0
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9
0
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0
α
Abu-Trp(Boc)-Arg(Pbf)-Gly-
: -Val-Thr-His-Thr-Tyr-Leu-Glu-Pro-Gly-Pro-Val-Thr-Ala-Asn-Arg-Gln-Leu-Tyr-Pro-Glu-Trp-Thr-Glu-Ala-Gln-Arg-Leu-Asp-
[
AA]
α
Abu-Trp-Arg-Gly-
NH
2
NH
N
2
H
N
HO
HO
HO
OH
O
Boc
N
PMBO
PMBO
PMBO
OPMB
O
N
N
O
O
N
BuO
N
N
BuO
O
PMBO
PMBO
PMBO
OPMB
O
O
O
O
Mmt
HN
NH
2
2
HN
NH
HN
HN
O
O
O
Mmt
b
d
O
NH
NH
O
O
NH
H
N
[AA]
O
N
H
N
H
[
AA]*
N
H
N
H
O
O
n
O
O
n
H
N
n = 1 : 39
H
n = 1 : 35
n = 6 : 36
TLR7-L
C
gp100
[
AA]*
n = 6 :
N
H
N
H
40
n
O
O
n
n = 1 : 33
NH
N
2
NH
N
2
OPMB
O
HO
HO
HO
OH
O
n = 6 :
34
PMBO
PMBO
PMBO
Boc
N
H
N
N
N
O
O
N
N
BuO
BuO
a
O
O
O
O
c
d
O
O
O
O
HN
O
NH
HN
NH
NH
H
2
N
HN
HN
O
O
O
H
N
[
AA]*
[AA]
2
N
H
N
H
N
H
N
H
NH
N
2
O
O
O
O
n
n
Boc
N
n = 1 :
n = 6 :
37
38
n = 1 : 41
n = 6 : 42
N
C
gp100 TLR7-L
n
O
N
BuO
O
NH
N
2
NH
N
2
H
N
H
32
N
N
HO
HO
HO
OH
O
N
O
O
OH
N
N
BuO
BuO
O
O
O
N
O
N
HN
N
N
N
3
HN
O
N
HN
O
N
O
O
O
e
2
2
H
2
N
NH
NH
NH
2
O
HN
H
[
AA]
NH
2
g
[AA]
N
NH
2
N
O
AcHN
N
H
AcHN
N
H
O
OH
H
HO
HO
HO
O
O
O
45
O
O
n
OH
O
n = 1 : 46
n = 6 : 47
O
HO
HO
HO
O
gp100 TLR7-L
n
N
O
N
N
O
N
N
N
HN
:
R = H : 48 gp100
NHR
:
f
R = PEG-TLR7L : 49
gp100 TLR7-L
Ac
Gly-Lys-NH
2
O
O
N
H
H
N
n = 1 : 24
n = 6 : 27
NH
2
n = 1 : 43
n = 6 : 44
O
N
H
N
H
n
O
O
[AA]
O
NH
2
n
N
H
O
2
60
2
2
61
62
Reagents and conditions: a) Fmoc-SPPS (1, HCTU, DIPEA, DMF); b) Fmoc-SPPS (Fmoc-AEEA-OH or 32, HCTU,
DIPEA, DMF); c) i. Ac
2
O, DIPEA, DMF; ii. AcOH, TFE, DCM; iii. Fmoc-SPPS (Fmoc-AEEA-OH or 32, HCTU, DIPEA,
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DMF); d) TFA, TIS, H
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O, octanethiol, phenol: (39: 2.60% over 36 couplings; 40: 1.30 % over 41 couplings); 41:
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2.10% over 36 couplings); 42: 0.60% over 41 couplings; e) Fmoc-SPPS see reference ; f) Pent-4-ynoic acid
succinimidyl ester, DIPEA, DMSO: (43: 62%; 44: 66%); g) CuI, THPTA, DIPEA, H O, DMSO: (46: 34%; 47: 29%).
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Mannosylated gp100 conjugate efficacy as vaccination strategy
We analyzed the influence of the C-mannosylated antigens on dendritic cell maturation, cytokine
+
+
secretion, and antigen presentation to CD4 and CD8 T lymphocyte in relation to their O-
mannosylated peptide counterpart (Figure 2A). Furthermore, the effect of C- versus N- terminal
ligation of the TLR7 ligand was assessed.
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Dendritic cell maturation is considered an essential requirement for proper T lymphocyte activation
and polarization. As a measure of DC maturation, we quantified expression of the co-stimulatory
molecules CD86 (Figure 2B) and CD83 (see SI, Figure SI.1D) using the six variants of the trivalent
conjugates. The non-glycosylated gp100 48 and the bivalent gp100-TLR7L conjugate 49 were included
as controls. After overnight stimulation with the conjugates, we found that all conjugates induced
expression of CD86 and CD83. The C-mannoside conjugates did not hamper the DC maturation
processes and effectively elevated expression levels to the same extent as the O-mannosylated
conjugates. The position to which the TLR7 agonist was attached does not seem to affect the
maturation of the DCs in this assay.
74
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280
281
2
2
During maturation, DCs produce and secrete a tailored cytokine cocktail for subsequent T lymphocyte
skewing. The secreted cytokine profile depends on the triggered TLR, the signaling pathway of which
can be modified by DC-SIGN or other CLR engagement . To assess DC activation, we quantified four
key cytokines using a sandwich ELISA. IL-6 and IL-12 are primarily characterized as pro-inflammatory
cytokines, with functions aiding DC maturation and Th1 stimulation, respectively. Tumor necrosis
factor (TNF)-α is required for DC activation and proliferation, and reduces IL-10 mediated inhibition
82
83
4
3
284
2
2
2
288
2
2
85
86
87
4
4
during DC development . IL-10 is a cytokine that interferes with DC maturation and inhibits the
production of IL-12, and as such has been implied to play a role in skewing of naïve T lymphocytes to
45
46
89
90
Treg differentiation . Low levels of autocrine IL-10 prevent spontaneous maturation of DCs . Figure
2C shows that the stand-alone peptide (48) and gp100-TLR7L (49) minimally induced the production
of IL-6, while both the monovalent and hexavalent O-mannoside trifunctional conjugates 46 and 47
increased the secretion of this pro-inflammatory cytokine. The monovalent and hexavalent C-
mannoside conjugates 41 and 42 increased the IL-6 levels to a similar extent as their O-mannoside
counterparts. However, when moDCs were stimulated with conjugates 39 and 40, having both the C-
mannosides and the TLR7L attached to the N-terminus of the peptide sequence, cytokine production
was abrogated. A similar pattern observed for the IL-12 production profile: the O- and C-mannose
conjugates having the TLR7 ligand at the C-terminus of the conjugate were most active in stimulating
the production of this cytokine, while the gp100 peptide and gp100 peptide-TLR7L conjugate were less
active. The conjugates 39 and 40 induced low levels of the IL-12 cytokine. TNF-α expression levels were
minimally affected upon stimulation of the DCs with the various conjugates. Also here, the conjugates
carrying the TLR7 ligand and the mannosides at the same side of the peptide antigen showed least
activation. Finally, a low level of the anti-inflammatory IL-10 was detected in the ELISA with the non-
stimulated DCs, as well as for those treated with peptide 49. Figure 2C shows that while LPS effectively
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triggers the production of IL-10, the mannosylated conjugates do not induce the production of this
cytokine. Overall the cytokine production profiles of the O- and C-mannose conjugates appear to be
very similar for both the monovalent and hexavalent clusters. In addition, these experiments revealed
that the arrangement of the CLR and TLR ligands within the trifunctional conjugates has a great
influence on the activity of the conjugates. Possibly, the processing of these conjugates is different
from the conjugates bearing the CLR and TLR ligand on either side of the conjugate, due to differential
cleavage of the conjugates by proteases.
4
5
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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Finally, we studied the antigen presentation capacity of the DCs upon stimulation with all the
trifunctional conjugate variations. During DC maturation, the intrinsic focus of these cells shifts from
antigen endocytosis to antigen processing, major histocompatibility complex (MHC) molecule loading,
and presentation of the antigens for initiation of the T cell response. Upon recognition of the cross-
12
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+
3
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17
presented antigen in MHC-I, cytotoxic CD8 T cells induce programmed cell death of targeted cells. On
+
the other hand, CD4 T lymphocytes induce and support a cellular and humoral response upon antigen-
MHC-II binding. As both T-cell responses are needed for a robust immune response, we studied the
+
+
318
antigen (gp100) presenting capacity of the DCs to both CD8 and CD4 T cells after stimulation with the
47
3
3
19
20
trifunctional conjugates . To this end, DCs were stimulated for 30 minutes with the conjugates, before
+
+
washing and co-culturing with the CD8 HLA-A2.1 or CD4 HLA-DR4.1 restricted T cell clone for 24
hours. Activation of T cells was measured by quantification of the IFNγ cytokine produced. As shown
in Figure 2D, the conjugates carrying a TLR7 ligand are more active than the stand-alone peptide,
except for conjugate 40. The introduction of a CLR ligand also increases the antigen-presenting activity
of the conjugates, although the hexavalent C-mannoside conjugate appears to hamper antigen
presentation with respect to the monovalent conjugate partially. The presence of the TLR7 ligand and
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the hexavalent mannoside cluster on the same side of the conjugate blocks CD8 antigen presentation,
likely as the result of sub-optimal processing. In our previous study, in which we have investigated
gp100-conjugates bearing hexavalent clusters comprising di- and trimannosides we found that the
α1,2-dimannoside cluster gp100 conjugates, although being the best DC-SIGN binders, showed less
antigen presentation than a gp100-TLR7 conjugate lacking the carbohydrate cluster. Clusters
composed of α1,3- and α1,6-linked dimannosides or α1,3-α1,6-linked trimannosides showed slightly
enhanced antigen presentation. Taken together, these results show that optimal antigen presentation
requires not only DC-SIGN binding but also adequate processing of the incorporated antigens. These
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results were substantiated by the CD4 T cell activation assay, as similar gp100 antigen presentation
effects were seen (Figure 2E).Monovalent O- and C-mannosyl conjugates 46 and 41 improved antigen
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presentation to CD4 T cells most, and attachment of the TLR7 ligand to the same side as the CLR ligand
again nullified activity of the conjugates. Overall, also in these assays, the O- and C-mannosides
perform similarly. The combined results from the assays in Figure 2 show that the most attractive
vaccine conjugates require the antigenic peptide to be placed between an N-terminal CLR ligand and
a C-terminal TLR-ligand for antigen presentation and secretion cell activation.
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Figure 2 targeting efficacy of the mannoside-peptide conjugates
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(A) Schematic representation of the compounds and overview of the moDC maturation, cytokine
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secretion, CD4 and CD8 T lymphocyte antigen presentation. (B) Expression of co-stimulatory marker
CD86 as measured by flow cytometry. One out of four donors is depicted. LPS and 49 were included as
positive controls, as well as 48 as a negative control. (C) IL-6, IL-10, IL-12p70, and TNFα secretion of
four donors was measured using ELISA upon overnight stimulation with O- or C-mannoside conjugates.
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(D) Antigen presentation capacity of moDCs to CD8 T lymphocytes was quantified by the IFNγ
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production of activated T cells. The dashed lines represent the directionality between the donors when
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comparing mono and hexavalent mannosides analogs. (E) Antigen presentation capacity of moDCs to
CD4 T lymphocytes was quantified by the IFNγ production of activated T cells.
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CONCLUSION
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In conclusion, we have developed a C-mannosyl lysine that can be effectively used in solid-phase
peptide synthesis (SPPS) campaigns. The stability of the C-glycosidic linkage renders the mannoside
stable to both acidic reaction conditions employed during SPPS and enzymatic degradation. The
protecting groups on the building block were designed to be compatible with standard SPPS protocols
to allow the straightforward ‘in-line’ incorporation of the mannosylated residues in oligopeptides. This
allows for the generation of mannosylated conjugates without the necessity of a post-assembly
conjugation step requiring orthogonal click strategies. Not only does this streamline the synthesis of
these conjugates, it also ensures that bio-orthogonal handles, such as azides and alkynes, can be
incorporated into these multifunctional antigen-conjugates to allow these for the incorporation of
additional functionalities, such as other immune stimulating agents or fluorophores. We have applied
the mannosylated lysine in the assembly of a set of synthetic long peptide antigens to equip these with
either one or six mannosides to target the antigens to mannose-binding C-type lectins on professional
antigen-presenting cells to improve the antigenicity of the peptides. The conjugates were further
armed with a synthetic TLR7 ligand to further boost the response against the antigens. In comparing
the C- vs. the O-mannosylated conjugates for DC-SIGN mediated uptake, DC-maturation, and
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stimulation as well as CD4 and CD8 antigen presentation, the stabilized mannosides performed
virtually identical to their natural analogs. The conjugates bearing the mannosides and a TLR7 ligand
were shown to bind DC-SIGN and activate DCs, as indicated by pro-inflammatory cytokine release,
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upregulation of cell surface maturation markers and increased antigen presentation to both CD4 and
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CD8 lymphocytes. Notably, the relative position of the CLR and TLR ligands in the peptide antigen
conjugates played an important role in shaping the activity of the conjugates. The conjugates bearing
the mannose cluster and the TLR7 ligand on the same side of the conjugates proved to be poor
immune-stimulating agents, incapable of elucidating an effective pro-inflammatory response and
showing poor antigen presentation. These differences are likely due to differences in the processing
of the conjugates. As DC vaccination therapies hold great promise as an immunotherapeutic approach
to fight cancer, the development of more effective, tailor-made cancer vaccine conjugates, of which
the action is well understood and can be controlled, is of great importance. The conjugates described
here can be further equipped with biorthogonal visualization handles to allow tracking of the
conjugates during uptake and processing. Because the C-mannosyl lysine building block can be
incorporated in an ‘in-line’ manner and does not rely on a post-assembly conjugation step, often used
biorthogonal coupling partners, such as azides and alkynes, remain at one’s disposal for inclusion in
the conjugates.
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METHODS
Synthesis
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The synthesis of C-mannosyl 1 and all clusters and conjugates is described in the Supporting
Information.
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Cell isolation and culture
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Buffy coats of healthy donors were obtained from Sanquin Amsterdam (reference: S03.0023-XT).
Monocytes were isolated using a Ficoll (STEMCELL Technologies) and sequential Percoll (Sigma)
gradient centrifugation. The monocytes were differentiated to monocyte-derived dendritic cells
(moDCs) in RPMI 1640 (Invitrogen), supplemented with 10% FCS (Biowittaker), 1.000 U/mL penicillin
(Lonza), 1 U/mL streptomycin (Lonza), 262.5 U/mL IL-4 (Biosource), and 112.5 U/mL GM-CSF
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(Biosource), for five days. Flow cytometric monitoring of DC-SIGN (AZN-D1-Alexa488, in house ), CD83
and CD86 (both PE-conjugated, Becton Dickinson) expression was conducted for every donor.
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Binding of the mannose clusters to moDCs
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Day 5 moDCs (approximately 10 per condition) were washed and resuspended in ice-cold RPMI
medium (Invitrogen). The entire experiment was conducted at 4°C with pre-cooled reagents. DC-SIGN
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and mannose receptor were blocked with 20 µg/mL AZN-D1 (in house ) or purified mouse anti-human
CD206 antibody (Clone 19.2, BD Bioscience) respectively for 45 minutes. The biotinylated mannoside
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clusters (10 µM) or Lewis -conjugated polyacrylamide (1 µg/mL) as positive control was allowed to
bind for 30 minutes. The moDCs were washed with PBS and stained with an Alexa647-labelled
TM
streptavidin (Invitrogen ) in PBS supplemented with 0.5% BSA and 0.02% NaN (PBA) for 30 minutes.
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The moDCs were subsequently washed and fixed in PBS with 0.5% PFA, before the fluorescence was
measured by flow cytometry (CyAn™ ADP with Summit™ Software), and analyzed using FlowJo v10.
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Internalization of the mannoside clusters
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Day 5 moDCs (approximately 10 per condition) were washed and resuspended in ice-cold HBSS
medium (Thermo Fischer). The biotinylated mannoside clusters (20 µM) were added in ice-cold
medium to the moDCs for one hour, and washed away with ice-cold medium. Pre-warmed HBSS (37°C)
was added to the cells and were incubated at 37°C in a shaking heating block (800 RPM). Samples of
the cells were taken at the indicated time points (t = 0, 5, 10, 15, 30, 60 minutes), and immediately put
on ice. The moDCs were washed with ice-cold PBS supplemented with 0.5% BSA and 0.02% NaN3 (PBA)
and stained with Alexa647-labelled streptavidin (InvitrogenTM) for 30 minutes at 4°C. The
fluorescence was measured by flow cytometry (CyAn™ ADP with Summit™ Software), and analyzed
using FlowJo v10.
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Endosomal routing of the mannoside clusters
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Day 5 moDCs (approximately 10 per condition) were washed and resuspended in pre-warmed (37°C)
HBSS medium (Thermo Fischer). The biotinylated mannoside clusters (20 µM) were complexed with
pHrodo (2:1 ratio) for 15 minutes at RT. The pre-complexed pHrodo-labeled ligands were added to the
cells and were incubated at 37°C in a shaking heating block (800 RPM). Samples of the cells were taken
at the indicated time points (t = 0, 5, 10, 15, 30, 60, 120 minutes), and immediately put on ice. The
moDCs were washed with ice-cold PBS supplemented with 0.5% BSA and 0.02% NaN3 (PBA). The
fluorescence was measured by flow cytometry (BD LSRFortessa™ X-20 with FACSDiva Software), and
analyzed using FlowJo v10.
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moDC Cytokine secretion upon stimulation with the mannoside clusters
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Day 5 moDCs (approximately 50·10 per condition) were stimulated for 24 hours with the trifunctional
conjugates. Cytokines IL-6, IL-10, IL-12p40, and TNFα in the supernatant were measured by sandwich
ELISA according to manufacturer’s protocol (Biosource). The capture antibody was coated in NUNC
MaxiSorp plates (Nunc, Roskilde, Denmark) overnight at 4°C in PBA-0.05% BSA. The plates were
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blocked for 30 minutes at 37°C, using PBS supplemented with 1% BSA. Samples were added for 2 hours
at RT to allow binding, subsequently washed, and cytokine levels were detected using a peroxidase-
conjugated cytokine-specific detection antibody. After extensive washing, the binding was visualized
with 3,3’,5,5’-tetramethylbenzidine (Sigma Aldrich) and measured by spectrophotometry at 450 nm
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on the iMark Microplate Absorbance Reader (Bio-RAD).
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CD4 and CD8 Antigen presentation
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Day 5 moDCs of HLA-A2 and HLA-DR4 double positive donors (approximately 40·10 per condition)
were incubated with the different trifunctional conjugates (20 µM) for 30 minutes at 37°C. A short
gp100 peptide (gp100280-288) and a long gp100 peptide (gp100280-288, 40-59) were used as controls. The
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moDCs were washed and separated into two plates (30·10 for CD8 and 10·10 for CD4 T lymphocyte
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co-culture). Either a CD8 HLA-A2.1 restricted T cell clone transduced with the TCR specific for the
gp100280–288 peptide (approximately 10 cells per condition, E:T ratio 1:3) or a CD4 HLA-DR4.1
restricted T cell clone transduced with the TCR specific for the gp10044-59 peptide (approximately 105
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cells per condition, E:T ration 1:10) was added for overnight co-culture. The interferon γ cytokine
secretion was measured by sandwich ELISA, according to the manufacturer’s protocol (Biosource), and
measured by spectrophotometric analysis at 450 nm on the iMarkTM Microplate Absorbance Reader
(Bio-RAD).
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Statistics
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Unless otherwise stated, data are presented as the meanꢀ±ꢀSD of at least three independent
experiments or healthy donors. Statistical analyses were performed in GraphPad Prism v7.04.
Statistical significance was set at Pꢀ<ꢀ0.05 and it was evaluated by the Mann–Whitney U test.
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Supporting information
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All other synthetic procedures, supporting figures, NMR spectra and HPLC spectra are depicted in the
Supporting Information.
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Author information
6.1
Corresponding Author
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*E-mail: JCodee@chem.leidenuniv.nl
*E-mail: Y.vanKooyk@amsterdamumc.nl
6.2
ORCID
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Tim Hogervorst: https://orcid.org/0000-0002-4686-6251
R.J. Eveline Li: https://orcid.org/0000-0002-1928-925X
Yvette van Kooyk: https://orcid.org/0000-0001-5997-3665
Jeroen Codée: http://orcid.org/0000-0003-3531-2138
6.3
Author Contributions
TH and RL equally contributed to the work and both wrote the manuscript. The compounds were
synthesized by TH, assisted by LM, under the supervision of HO, DF, GM, and JC. NM aided in the
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peptide synthesis and purification of certain constructs. RL executed the binding, internalization,
endosomal tracking, maturation, and cytokine secretion experiments, and was assisted by SB in the
antigen presentation assay, supervised by SV and YK.
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Notes
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All authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
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Acknowledgements/Funding
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This work was funded by the NWO gravitation program 2013 granted to the Institute for Chemical
Immunology (ICI-024.002.009).
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References
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(1)
Khair, D. O.; Bax, H. J.; Mele, S.; Crescioli, S.; Pellizzari, G.; Khiabany, A.; Nakamura, M.; Harris,
R. J.; French, E.; Hoffmann, R. M.; Williams, I. P.; Cheung, A.; Thair, B.; Beales, C. T.; Touizer, E.;
Signell, A. W.; Tasnova, N. L.; Spicer, J. F.; Josephs, D. H.; Geh, J. L.; MacKenzie Ross, A.; Healy,
C.; Papa, S.; Lacy, K. E.; Karagiannis, S. N. Combining Immune Checkpoint Inhibitors:
Established and Emerging Targets and Strategies to Improve Outcomes in Melanoma. Front.
Immunol. 2019, 10, 453.
4
4
4
4
4
78
79
80
81
82
4
4
4
83
84
85
(2)
Boyiadzis, M. M.; Dhodapkar, M. V.; Brentjens, R. J.; Kochenderfer, J. N.; Neelapu, S. S.; Maus,
M. V.; Porter, D. L.; Maloney, D. G.; Grupp, S. A.; Mackall, C. L.; June, C. H.; Bishop, M. R.
Chimeric Antigen Receptor (CAR) T Therapies for the Treatment of Hematologic Malignancies:
Clinical Perspective and Significance. J. Immunother. Cancer 2018, 6, 137.
486
487
488
4
(3)
(4)
(5)
Rohaan, M. W.; van den Berg, J. H.; Kvistborg, P.; Haanen, J. B. A. G. Adoptive Transfer of
Tumor-Infiltrating Lymphocytes in Melanoma: A Viable Treatment Option. J. Immunother.
Cancer 2018, 6, 102.
89
490
4
4
Huber, A.; Dammeijer, F.; Aerts, J. G. J. V; Vroman, H. Current State of Dendritic Cell-Based
Immunotherapy: Opportunities for in Vitro Antigen Loading of Different DC Subsets? Front.
Immunol. 2018, 9, 2804.
91
92
4
4
4
4
4
498
499
93
94
95
96
97
Schreibelt, G.; Bol, K. F.; Westdorp, H.; Wimmers, F.; Aarntzen, E. H. J. G.; Duiveman-de Boer,
T.; van de Rakt, M. W. M. M.; Scharenborg, N. M.; de Boer, A. J.; Pots, J. M.; Olde Nordkamp,
M. A. M.; van Oorschot, T. G. M.; Tel, J.; Winkels, G.; Petry, K.; Blokx, W. A. M.; van Rossum,
M. M.; Welzen, M. E. B.; Mus, R. D. M.; Croockewit, S. A. J.; Koornstra, R. H. T.; Jacobs, J. F. M.;
Kelderman, S.; Blank, C. U.; Gerritsen, W. R.; Punt, C. J. A.; Figdor, C. G.; de Vries, I. J. M.
Effective Clinical Responses in Metastatic Melanoma Patients after Vaccination with Primary
Myeloid Dendritic Cells. Clin. Cancer Res. 2016, 22, 2155–2166.
500
501
(6)
(7)
Anguille, S.; Smits, E. L.; Lion, E.; van Tendeloo, V. F.; Berneman, Z. N. Clinical Use of Dendritic
Cells for Cancer Therapy. Lancet Oncol. 2014, 15, e257–e267.
502
5
5
Ho, N. I.; Huis in ’t Veld, L. G. M.; Raaijmakers, T. K.; Adema, G. J. Adjuvants Enhancing Cross-
Presentation by Dendritic Cells: The Key to More Effective Vaccines? Front. Immunol. 2018, 9,
2874.
03
04
5
5
05
06
(8)
Blander, J. M.; Medzhitov, R. Toll-Dependent Selection of Microbial Antigens for Presentation
by Dendritic Cells. Nature 2006, 440, 808–812.
ACS Paragon Plus Environment

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5
5
5
07
08
09
(9)
Cho, H. J.; Takabayashi, K.; Cheng, P.-M.; Nguyen, M.-D.; Corr, M.; Tuck, S.; Raz, E.
Immunostimulatory DNA-Based Vaccines Induce Cytotoxic Lymphocyte Activity by a T-Helper
Cell-Independent Mechanism. Nat. Biotechnol. 2000, 18, 509–514.
4
5
6
7
8
9
1
1
1
1
1
1
1
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(10) Deres, K.; Schild, H.; Wiesmüller, K.-H.; Jung, G.; Rammensee, H.-G. In Vivo Priming of Virus-
Specific Cytotoxic T Lymphocytes with Synthetic Lipopeptide Vaccine. Nature 1989, 342, 561–
564.
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9
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5
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13
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(11) Fujita, Y.; Taguchi, H. Overview and Outlook of Toll-like Receptor Ligand–Antigen Conjugate
Vaccines. Ther. Deliv. 2012, 3, 749–760.
5
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(12) Willems, M. M. J. H. P.; Zom, G. G.; Khan, S.; Meeuwenoord, N.; Melief, C. J. M.; Stelt, M. van
der; Overkleeft, H. S.; Codée, J. D. C.; Marel, G. A. van der; Ossendorp, F.; Filippov, D. V. N -
Tetradecylcarbamyl Lipopeptides as Novel Agonists for Toll-like Receptor 2. J. Med. Chem.
2014, 57, 6873–6878.
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(13) Ignacio, B. J.; Albin, T. J.; Esser-Kahn, A. P.; Verdoes, M. Toll-like Receptor Agonist Conjugation:
A Chemical Perspective. Bioconjug. Chem. 2018, 29, 587–603.
521
(14) Khan, S.; Bijker, M. S.; Weterings, J. J.; Tanke, H. J.; Adema, G. J.; van Hall, T.; Drijfhout, J. W.;
Melief, C. J. M.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V.; van der Burg, S. H.;
Ossendorp, F. Distinct Uptake Mechanisms but Similar Intracellular Processing of Two
Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells. J. Biol. Chem. 2007,
282, 21145–21159.
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5
5
5
26
27
28
29
(15) Gringhuis, S. I.; den Dunnen, J.; Litjens, M.; van der Vlist, M.; Geijtenbeek, T. B. H.
Carbohydrate-Specific Signaling through the DC-SIGN Signalosome Tailors Immunity to
Mycobacterium Tuberculosis, HIV-1 and Helicobacter Pylori. Nat. Immunol. 2009, 10, 1081–
1088.
5
531
532
30
(16) Bertolotti, B.; Sutkeviciute, I.; Ambrosini, M.; Ribeiro-Viana, R.; Rojo, J.; Fieschi, F.; Dvořáková,
H.; Kašáková, M.; Parkan, K.; Hlaváčková, M.; Nováková, K.; Moravcová, J. Polyvalent C-
Glycomimetics Based on l -Fucose or d -Mannose as Potent DC-SIGN Antagonists. Org. Biomol.
Chem. 2017, 15, 3995–4004.
5
33
534
5
5
(17) White, K. L.; Rades, T.; Furneaux, R. H.; Tyler, P. C.; Hook, S. Mannosylated Liposomes as
35
36
Antigen Delivery Vehicles for Targeting to Dendritic Cells. J. Pharm. Pharmacol. 2006, 58, 729–
737.
5
5
5
37
38
39
(18) Sangabathuni, S.; Vasudeva Murthy, R.; Chaudhary, P. M.; Surve, M.; Banerjee, A.; Kikkeri, R.
Glyco-Gold Nanoparticle Shapes Enhance Carbohydrate-Protein Interactions in Mammalian
Cells. Nanoscale 2016, 8, 12729–12735.
5
5
5
40
41
42
(19) Ahlén, G.; Strindelius, L.; Johansson, T.; Nilsson, A.; Chatzissavidou, N.; Sjöblom, M.; Rova, U.;
Holgersson, J. Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-
Specific Antibody and T Lymphocyte Responses. PLoS One 2012, 7, e46959.
5
5
545
546
43
44
(20) Wilson, D. S.; Hirosue, S.; Raczy, M. M.; Bonilla-Ramirez, L.; Jeanbart, L.; Wang, R.; Kwissa, M.;
Franetich, J.-F.; Broggi, M. A. S.; Diaceri, G.; Quaglia-Thermes, X.; Mazier, D.; Swartz, M. A.;
Hubbell, J. A. Antigens Reversibly Conjugated to a Polymeric Glyco-Adjuvant Induce Protective
Humoral and Cellular Immunity. Nat. Mater. 2019, 18, 175–185.
547
548
5
(21) Li, R.-J. E.; Hogervorst, T. P.; Achilli, S.; Bruijns, S. C.; Arnoldus, T.; Vivès, C.; Wong, C. C.;
Thépaut, M.; Meeuwenoord, N. J.; van den Elst, H.; Overkleeft, H. S.; van der Marel, G. A.;
Filippov, D. V.; van Vliet, S. J.; Fieschi, F.; Codée, J. D. C. C.; van Kooyk, Y. Systematic Dual
49
ACS Paragon Plus Environment

ACS Chemical Biology
Page 18 of 21
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
50
51
Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional
Mannosylated Antigen. Front. Chem. 2019, 7 . https://doi.org/10.3389/fchem.2019.00650.
5
5
52
53
(22) Tamburrini, A.; Colombo, C.; Bernardi, A. Design and Synthesis of Glycomimetics: Recent
Advances. Med. Res. Rev. 2019, No. April, 1–37.
5
5
54
55
(23) Zou, W. C-Glycosides and Aza-C-Glycosides as Potential Glycosidase and Glycosyltransferase
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibitors. Curr. Top. Med. Chem. 2005, 5, 1363–1391.
5
5
56
57
(24) Girard, C.; Miramon, M.; de Solminihac, T.; Herscovici, J. Synthesis of 3-C-(6-O-Acetyl-2,3,4-Tri-
O-Benzyl-α-d-Mannopyranosyl)-1-Propene: A Caveat. Carbohydr. Res. 2002, 337, 1769–1774.
5
5
58
59
(25) Jarikote, D. V.; O’Reilly, C.; Murphy, P. V. Ultrasound-Assisted Synthesis of C-Glycosides.
Tetrahedron Lett. 2010, 51, 6776–6778.
5
5
5
60
61
62
(26) Touaibia, M.; Krammer, E.-M.; Shiao, T.; Yamakawa, N.; Wang, Q.; Glinschert, A.;
Papadopoulos, A.; Mousavifar, L.; Maes, E.; Oscarson, S.; Vergoten, G.; Lensink, M.; Roy, R.;
Bouckaert, J. Sites for Dynamic Protein-Carbohydrate Interactions of O- and C-Linked
Mannosides on the E. Coli FimH Adhesin. Molecules 2017, 22, 1101.
563
5
565
64
(27) Sharma, P. K.; Kumar, S.; Kumar, P.; Nielsen, P. Selective Reduction of Mono- and
Disubstituted Olefins by NaBH4 and Catalytic RuCl3. Tetrahedron Lett. 2007, 48, 8704–8708.
566
567
5
5
(28) Newlander, K. A.; Callahan, J. F.; Moore, M. L.; Tomaszek, T. A.; Huffman, W. F. A Novel
Constrained Reduced-Amide Inhibitor of HIV-1 Protease Derived from the Sequential
Incorporation of .Gamma.-Turn Mimetics into a Model Substrate. J. Med. Chem. 1993, 36,
2321–2331.
68
69
5
5
5
70
71
72
(29) Boger, D. L.; Yohannes, D.; Zhou, J.; Patane, M. A. Total Synthesis of Cycloisodityrosine, RA-VII,
Deoxybouvardin, and N29-Desmethyl-RA-VII: Identification of the Pharmacophore and
Reversal of the Subunit Functional Roles. J. Am. Chem. Soc. 1993, 115, 3420–3430.
5
5
73
74
(30) Jencks, W. P.; Gilchrist, M. Nonlinear Structure-Reactivity Correlations. The Reactivity of
Nucleophilic Reagents toward Esters. J. Am. Chem. Soc. 1968, 90, 2622–2637.
5
5
5
5
75
76
77
78
(31) Raiber, E.-A.; Tulone, C.; Zhang, Y.; Martinez-Pomares, L.; Steed, E.; Sponaas, A. M.;
Langhorne, J.; Noursadeghi, M.; Chain, B. M.; Tabor, A. B. Targeted Delivery of Antigen
Processing Inhibitors to Antigen Presenting Cells via Mannose Receptors. ACS Chem. Biol.
2010, 5, 461–476.
5
580
581
79
(32) pHrodo Indicators for pH Determination - NL
https://www.thermofisher.com/nl/en/home/brands/molecular-probes/key-molecular-
probes-products/phrodo-indicators.html (accessed Sep 3, 2019).
582
5
5
(33) Hotta, C.; Fujimaki, H.; Yoshinari, M.; Nakazawa, M.; Minami, M. The Delivery of an Antigen
from the Endocytic Compartment into the Cytosol for Cross-Presentation Is Restricted to Early
Immature Dendritic Cells. Immunology 2006, 117, 97–107.
83
84
5
5
85
86
(34) Zhang, S.; Li, J.; Lykotrafitis, G.; Bao, G.; Suresh, S. Size-Dependent Endocytosis of
Nanoparticles. Adv. Mater. 2009, 21, 419–424.
5
5
5
87
88
89
(35) Vennepureddy, A.; Thumallapally, N.; Motilal Nehru, V.; Atallah, J.-P.; Terjanian, T. Novel
Drugs and Combination Therapies for the Treatment of Metastatic Melanoma. J. Clin. Med.
Res. 2016, 8, 63–75.
ACS Paragon Plus Environment

Page 19 of 21
ACS Chemical Biology
1
2
3
5
5
5
90
91
92
(36) Chan, M.; Hayashi, T.; Kuy, C. S.; Gray, C. S.; Wu, C. C. N.; Corr, M.; Wrasidlo, W.; Cottam, H.
B.; Carson, D. A. Synthesis and Immunological Characterization of Toll-Like Receptor 7
Agonistic Conjugates. Bioconjug. Chem. 2009, 20, 1194–1200.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
5
93
94
95
(37) Wlodawer, A.; Miller, M.; Jaskolski, M.; Sathyanarayana, B.; Baldwin, E.; Weber, I.; Selk, L.;
Clawson, L.; Schneider, J.; Kent, S. Conserved Folding in Retroviral Proteases: Crystal Structure
of a Synthetic HIV-1 Protease. Science (80-. ). 1989, 245, 616–621.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
5
5
96
97
98
(38) Gential, G. P. P.; Hogervorst, T. P.; Tondini, E.; van de Graaff, M. J.; Overkleeft, H. S.; Codée, J.
D. C.; van der Marel, G. A.; Ossendorp, F.; Filippov, D. V. Peptides Conjugated to 2-Alkoxy-8-
Oxo-Adenine as Potential Synthetic Vaccines Triggering TLR7. Bioorg. Med. Chem. Lett. 2019,
29, 1340–1344.
599
600
601
6
(39) Chan, M.; Hayashi, T.; Kuy, C. S.; Gray, C. S.; Wu, C. C. N.; Corr, M.; Wrasidlo, W.; Cottam, H.
B.; Carson, D. A. Synthesis and Immunological Characterization of Toll-Like Receptor 7
Agonistic Conjugates. Bioconjug. Chem. 2009, 20, 1194–1200.
02
603
6
6
(40) Matysiak, S.; Böldicke, T.; Tegge, W.; Frank, R. Evaluation of Monomethoxytrityl and
Dimethoxytrityl as Orthogonal Amino Protecting Groups in Fmoc Solid Phase Peptide
Synthesis. Tetrahedron Lett. 1998, 39, 1733–1734.
04
05
6
6
6
06
07
08
(41) Volbeda, A. G.; Kistemaker, H. A. V.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V.;
Codée, J. D. C. Chemoselective Cleavage of p -Methoxybenzyl and 2-Naphthylmethyl Ethers
Using a Catalytic Amount of HCl in Hexafluoro-2-Propanol. J. Org. Chem. 2015, 80, 8796–8806.
6
6
09
10
(42) Howard, K. T.; Chisholm, J. D. Preparation and Applications of 4-Methoxybenzyl Esters in
Organic Synthesis. Org. Prep. Proced. Int. 2016, 48, 1–36.
6
6
11
12
(43) Geijtenbeek, T. B. H.; Gringhuis, S. I. C-Type Lectin Receptors in the Control of T Helper Cell
Differentiation. Nat. Rev. Immunol. 2016, 16, 433–448.
6
6
6
13
14
15
(44) Brossart, P.; Zobywalski, A.; Grünebach, F.; Behnke, L.; Stuhler, G.; Reichardt, V. L.; Kanz, L.;
Brugger, W. Tumor Necrosis Factor Alpha and CD40 Ligand Antagonize the Inhibitory Effects
of Interleukin 10 on T-Cell Stimulatory Capacity of Dendritic Cells. Cancer Res. 2000, 60, 4485–
4492.
616
617
618
6
(45) de Smedt, T.; van Mechelen, M.; De Becker, G.; Urbain, J.; Leo, O.; Moser, M. Effect of
Interleukin-10 on Dendritic Cell Maturation and Function. Eur. J. Immunol. 1997, 27, 1229–
1235.
19
620
6
(46) Corinti, S.; Albanesi, C.; la Sala, A.; Pastore, S.; Girolomoni, G. Regulatory Activity of Autocrine
21
IL-10 on Dendritic Cell Functions. J. Immunol. 2001, 166, 4312–4318.
6
6
6
22
23
24
(47) Wieczorek, M.; Abualrous, E. T.; Sticht, J.; Álvaro-Benito, M.; Stolzenberg, S.; Noé, F.; Freund,
C. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational
Plasticity in Antigen Presentation. Front. Immunol. 2017, 8, 292.
6
6
6
6
25
26
27
28
(48) Engering, A.; Geijtenbeek, T. B. H.; van Vliet, S. J.; Wijers, M.; Liempt, E. van; Demaurex, N.;
Lanzavecchia, A.; Fransen, J.; Figdor, C. G.; Piguet, V.; van Kooyk, Y. The Dendritic Cell-Specific
Adhesion Receptor DC-SIGN Internalizes Antigen for Presentation to T Cells. J. Immunol. 2002,
168, 2118–2126.
6
6
631
29
30
(49) Schaft, N.; Willemsen, R. A.; de Vries, J.; Lankiewicz, B.; Essers, B. W. L.; Gratama, J.-W.;
Figdor, C. G.; Bolhuis, R. L. H.; Debets, R.; Adema, G. J. Peptide Fine Specificity of Anti-
Glycoprotein 100 CTL Is Preserved Following Transfer of Engineered TCR Genes Into Primary
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33
Human T Lymphocytes. J. Immunol. 2003, 170, 2186–2194.
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