Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles

Article abstract of DOI:10.1021/jacs.0c01928

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Full text of DOI:10.1021/jacs.0c01928

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Article
Prolonged innate immune activation by enzyme-sensitive
imidazoquinoline TLR7/8 agonist prodrug vesicles
Bi Wang, Simon Van Herck, Yong Chen, Xiangyang Bai, Zifu Zhong, Kim De Swarte,
Bart N. Lambrecht, Niek N. Sanders, Stefan Lienenklaus, Hans W. Scheeren, Sunil
A. David, Fabian Kiessling, Twan Lammers, Bruno G. De Geest, and Yang Shi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01928 • Publication Date (Web): 11 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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Prolonged innate immune activation by enzyme-sensitive imidazo-
quinoline TLR7/8 agonist prodrug vesicles
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Bi Wang†, Simon Van Herck#, Yong Chen#, Xiangyang Bai†, Zifu Zhong§, Kim De Swarte,^ Bart N.
Lambrecht,^± Niek N. Sanders§, Stefan Lienenklaus¶, Hans W. Scheeren†, Sunil A. David&, Fabian
Kiessling†, Twan Lammers†‡&*, Bruno G. De Geest#*, Yang Shi†*
† Institute for Experimental Molecular Imaging, Uniklinik RWTH Aachen and Helmholtz Institute for Biomedical Engineering, Faculty of
Medicine, RWTH Aachen University, 52074 Aachen, Germany
# Department of Pharmaceutics, Ghent University, Ghent, Belgium
§ Laboratory of Gene Therapy, Ghent University, Ghent, Belgium
^ Department of Internal Medicine and Pediatrics, Ghent University, VIB Center for Inflammation Research, Ghent, Belgium.
± Department of Pulmonary Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
¶ Institute for Laboratory Animal Science and Institute of Immunology, Hanover Medical School, Hannover, Germany.
& Virovax, Lawrance (KS), United States
‡ Department of Pharmaceutics, Utrecht University, 3584 CG Utrecht, The Netherlands
& Department of Targeted Therapeutics, University of Twente, 7500 AE Enschede, The Netherlands
Supporting Information Placeholder
ABSTRACT: Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent acti-
vators of antigen-presenting cells (APCs), but they also induce severe side effects due to leakage from the site of injection
into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of
an imidazoquinoline TLR7/8 agonist that in aqueous medium forms 200 nm vesicular structures. The conjugate contains
an endosomal enzyme-sensitive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native
form after endocytosis, which resulted in high in vitro TLR agonist activity. In vivo in mouse models, locally administered
vesicles provoked higher and long-lasting immune stimulation in terms of interferon expression in draining lymphoid
tissue compared to a non-amphiphilic control and the native TLR agonist. Moreover, the vesicles induced robust activa-
tion of dendritic cells in vivo.
strategies that mount tumor-specific immunity in patients
are highly promising to increase the response rate of the
treatment.4 Among several classes of immune-modula-
tory molecules, agonists of Toll like receptors (TLR) are
of major interest to induce activation of antigen present-
ing cells (APCs) and promote antigen presentation to T
cells in secondary lymphoid organs.5
INTRODUCTION
Immunotherapy has gained increasing importance in
clinical management of diseases with high mortality, in-
cluding cancer and infectious diseases. Especially for
cancer treatment, recent advances in immune therapy us-
ing adoptively transferred T cells and monoclonal anti-
bodies targeting the programmed death/ligand 1 (PD-
1/PD-L1) axis have achieved ground-breaking clinical
outcomes, including complete cure and long-term dis-
ease-free survival of late-stage cancer patients.1 Unfortu-
nately, the remarkable therapeutic benefit of PD-1/L1 an-
tibodies are only achieved in a fraction of patients.2
Hence, a huge unmet clinical need remains to render im-
mune therapy more efficiently in a broad population of
patients.
TLR7/8 agonists bind to receptors located on endoso-
mal membranes.6 Two small molecule TLR7/8 agonists
(imiquimod/Aldara and resiquimod/R-848) are on the
market as topical formulations, but are not suitable for
parenteral administrations due to severe systemic inflam-
mation caused by their rapid diffusion from the site of in-
jection when administered locally (i.e. subcutaneous or
intramuscular).7 Furthermore, the latter shortens the dura-
tion of such agonists, which is a downside for effective
immune-modulation.8 So far, various attempts have been
performed to enable site-specific immune-activation and
Evidence emerges that the response to immunotherapy
is highly dependent on pre-existing immunity in patients,
especially the status of cellular immunity.3 Therefore,
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Figure 1. Self-assembly of PEG-modified imidazoquinoline (IMDQ) amphiphile prodrug into vesicular nanoparticles. An
optimized amphiphile prodrug bearing two benzyl repeating units in the linker and PEG5k self-assembles into vesicles. Upon
endocytosis by antigen presenting cells, the vesicles degrade and native IMDQ is released, thereby binding to TLR7/8 receptors
and triggering immune-activation.
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to avoid systematic inflammation by TLR agonists, in-
cluding modification with lipid motifs9–11, chemical con-
jugation to bio/nanomaterials12–17 and physical entrap-
ment into nanoparticles via hydrophobic and electrostatic
interactions18–22. However, these strategies do not allow
for efficient release of the TLR agonist in native form in
cellular organelles where the corresponding TLR recep-
tors are located and hence might reduce the biological ac-
tivity.
For the design of the enzyme responsive linker, we
made use of benzyl carbamate residues capped by a β-
glucuronidase (β-GUS) sensitive glucuronide23,24, which
is conjugated to a PEG chain through an ester bond (Fig-
ure 1). Esterases as well as β-GUS are highly expressed
in endosomes, and upon cleavage of the ester bond and β-
GUS-mediated cleavage of the glucuronide, a self-immo-
lative elimination reaction takes place within the benzyl
carbamate residues, resulting in the release of IMDQ in
native form. Note that small molecule enzyme-responsive
prodrugs of imidazoquinolines have been reported be-
fore25, targeting metabolization by cancer cells that over-
express -galactosidase. This is an elegant approach to
induce innate immune activation in the tumor microenvi-
ronment in response to cancer cell metabolism-mediated
secretion of native imidazoquinoline. However, these
prodrugs have been designed to be hydrophilic which did
not alter the pharmacokinetic profile towards improved
lymphatic delivery.
Here, we report on conjugation of an imidazoquinoline
(IMDQ) TLR7/8 agonist to poly(ethylene glycol) (PEG)
through a hydrophobic enzyme-responsive and self-im-
molative linker (i.e. prodrug formation), which induces
self-assembly of the resulting amphiphiles into vesicular
nanoparticles (Figure 1). These structures traffic to
lymph nodes, disassemble under endosomal conditions
and provoke robust immune activation in vivo.
RESULTS AND DISCUSSION
Scheme 1. Synthesis route for PEG-GLn-IMDQ (n=1-3).
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Figure 2. (A) TEM image of self-assembled vesicles based on PEG5k-GL2-IMDQ (B) DLS plot of the PEG5k-GL2-IMDQ
vesicles with a mean diameter of 195 nm and low dispersity (0.2). (C) Stability of the PEG5k-GL2-IMDQ vesicles after dilution
o
in PBS 7.4 and incubated at 37 C. (n=3) (D) Enzymatic degradation scheme of PEG5k-GL2-IMDQ. (E) In vitro release of
IMDQ from PEG5k-GL2-IMDQ vesicles in the presence of an esterase and β-GUS at pH 5.0 and 37 oC (n=3).
The convergent synthesis route for the prodrug am-
phiphiles which we abbreviate as PEG-GLn-IMDQ (n:
number of benzyl carbamate residues in the β-GUS-
sensitive linker (GL)) is presented in Scheme 1. Firstly,
building blocks 4 and 8 were coupled via a carbamate
bond to yield compound 9 which contain a single benzyl
moiety. After deprotection, the obtained compound 10
was reacted with 8 yielding compound 11 which contains
two benzyl moieties. The same procedure was repeated to
obtain compound 13 which contains three benzyl moie-
ties. Deacetylation of 10, 12 and 14 yielded compounds
15-1,2,3 (“1,2,3” represents the number of benzyl repeat-
ing units in the linker). The methyl groups of 15-1,2,3
were then exchanged with propargyl alcohol to install an
alkyne moiety (compound 16-1,2,3). Next, the hydroxyl
group of 16-1,2,3 was activated and conjugated to the al-
iphatic amine of IMDQ. Finally, the IMDQ derivatives
with 1-3 benzyl repeating units (18-1,2,3) were conju-
gated with PEG (MW=5, 2 and 0.75 kDa) bearing an az-
ide group via copper(I)-catalyzed azide-alkyne cycload-
dition to yield the final compounds PEG-GLn-IMDQ
(n=1-3, number of benzyl moieties). As a control, 5kDa
PEG directly conjugated IMDQ (PEG5k-IMDQ) without
the β-GUS-sensitive linker was synthesized (synthesis
and characterization details in Supporting information).
PEG5k-GL1-IMDQ formed random aggregates in water
as shown by transmission electron microscopy (TEM)
and dynamic light scattering (DLS) (Figure S1). Interest-
ingly, PEG5k-GL2-IMDQ self-assembled into vesicular
nanoparticles (Figure 1) with a clearly depicted hollow
structure by TEM (Figure 2A) and a mean hydrodynamic
diameter of 195 nm (PDI=0.2) measured by DLS (Figure
2B). PEG5k-GL3-IMDQ formed larger particles (264
nm, PDI 0.2, Figure S1). In addition, also PEG2/0.75k-
GL2-IMDQ formed nanoparticles in aqueous medium,
however, these appeared to be unstable over time (cfr,
Figure S2 in Supporting Information) and TEM did not
indicate the formation of vesicular structures (Figure S1).
By contrast, PEG5k-GL2-IMDQ formed stable vesicles
(Figure 2C), which was selected for further investigation.
Vesicle-like polymeric nanostructures (e.g. poly-
mersomes) are considered particularly attractive drug de-
livery systems.26–35 Notably, vesicles based on synthetic
polymer-drug conjugates were very rarely reported (e.g.
conjugates of oligo(ethylene glycol)-camptothecin and its
analogue36,37).
Next, we investigated the enzyme-responsive prosperi-
ties of the PEG5k-GL2-IMDQ vesicles, under conditions
that mimic the endosomal milieu. The vesicles were incu-
o
bated at pH 5.0 (i.e. endosomal pH) and 37 C with an
The PEG-GLn-IMDQ with different number (1-3) of
the benzyl repeating units and MW of PEG (5, 2 and 0.75
kDa) were used to investigate the self-assembly behavior
in aqueous medium. Hereto, the conjugates were dis-
solved in tetrahydrofuran (THF) and added dropwise to
deionized water (v/v=1/1) in a low-energy sonication
bath. THF was evaporated under room temperature. As
shown in Figure S1, PEG5k-IMDQ was fully water-sol-
uble and did not form any ordered structures (e.g. PDI=1).
esterase to cleave the ester bond between the PEG chain
and GL2-IMDQ and β-GUS which subsequently hydro-
lyses the glucuronide motif. The residual linker was ex-
pected to be prone to a self-immolative reaction, thereby
releasing IMDQ in native form (Figure 2D). Of note, β-
GUS is predominantly present in endosomal compart-
ments,
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Figure 3. In vitro RAW Blue assay and in vivo immune activation. (A1) Time dependent activation curves. (A2) EC50 values
of the compounds. (n=6: Student t-test; ****:p<0.0001, **:p<0.01) (B1) IVIS imaging of IFNβ+/Δβ-luc reporter mice upon
subcutaneous injection of PEG5k-GL2-IMDQ vesicles and soluble PEG-IMDQ into the footpad. (B2) Fluorescence signal
detected at the local injection site and draining LN of the IFNβ+/Δβ-luc reporter mice. (n=3)
which is also the intracellular location of TLR7/8 recep-
tors. Hence, the PEG5k-GL2-IMDQ is optimal to selec-
tively release IMDQ upon cellular uptake to trigger the
TLR7/8 signaling pathway. To quantify the release kinet-
ics of native IMDQ from of the PEG5k-GL2-IMDQ ves-
icles, we measured native IMDQ by high-performance
liquid chromatography (HPLC). As sown in Figure 2E,
when the vesicles were incubated in the absence of the
enzymes, minimal release of IMDQ could be detected. By
contrast, in the presence of the enzymes, quantitative
IMDQ release reached a plateau after 4 days. This data
suggests efficient release of IMDQ from the vesicles un-
der endosome-mimicking.
small molecules by antigen presenting cells.11 To under-
score the amphiphilic nature of PEG5k-GL2-IMDQ and
their high cellular uptake, we loaded the vesicles with a
hydrophobic Cyanine3 derivative and assessed its cellular
uptake by confocal laser scanning microscopy. Whereas
the vesicles promoted substantial cellular internalization
of the dye (Figure S3), PEG5k-IMDQ did not have this
ability. Additionally, the fully water-soluble PEG5k-
IMDQ did not show any TLR agonistic activity (Figure
3A) in the Raw Blue reporter cell assay, thereby hinting a
poor cellular uptake of this construct and/or hindrance in
TLR7/8 triggering. Interestingly, the vesicles were even
more potent than native IMDQ after 72 hours of incuba-
tion (Figure 3A). Such behavior, showing increased po-
tency of a chemically modified IMDQ conjugate over na-
tive IMDQ has not been observed in any of our previous
Afterwards, we assessed the TLR agonistic activity of
the PEG5k-GL2-IMDQ vesicles in vitro using the RAW
Blue reporter cell assay, which is based on an engineered
mouse macrophage cell line that secretes embryonic alka-
line phosphatase (SEAP) in response to TLR triggering
and downstream nuclear factor kappa-light-chain-en-
hancer of activated B cells (NF-kB) signaling11. In these
experiments, PEG5k-GL2-IMDQ was compared to na-
tive IMDQ, PEG5k-IMDQ (i.e. IMDQ conjugated to a
PEG chain through an amide bond) and GL2-IMDQ
(IMDQ modified with the β-GUS-sensitive linker but
lacking the PEG chain) as controls. The dose-response
curves shown in Figure 3A indicate that PEG5k-GL2-
IMDQ vesicles and GL2-IMDQ showed increased activ-
ity in time, likely due to the release of IMDQ through en-
dosomal enzymatic activity. PEG5k-GL2-IMDQ vesicles
were more potent than the small molecule compound
GL2-IMDQ, which we speculate is due to the enhanced
dispersibility in aqueous medium of PEG5k-GL2-IMDQ
and/or improved cellular uptake of nanomaterials over
research on IMDQ-conjugated nanomaterials7,13,38,11
,
which highlights the role of the enzyme degradable linker
in conferring the PEG5k-GL2-IMDQ vesicles with high
biological activity.
To assess the in vivo biological behavior of the PEG5k-
GL2-IMDQ vesicles, we employed
a transgenic
IFNβ+/Δβ-luc mouse model39 engineered with a lucifer-
ase reporter gene linked to the expression of the type I
interferon, IFNβ. The vesicles were injected subcutane-
ously into the footpad of the mice, followed by non-inva-
sive luciferase bioluminescence imaging. At 24 hours
post-injection, potent innate immune activation occurred
in the draining popliteal lymph node (LN) and at the site
of injection, while PEG5k-IMDQ showed much less po-
tency (Figure 3B). Mice receiving soluble IMDQ devel-
oped a strong innate response throughout their whole
body immediately after injection (Figure S4), due to
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IMDQ rapidly decayed to baseline levels 72 hours post-
injection (Figure 3B and Figure S4).
Finally, we sought proof that PEG5k-GL2-IMDQ vesi-
cles are capable of activating dendritic cells (DCs) which
are the most potent class of professional APCs and a ma-
jor target cell population in immune therapy and vaccina-
tion. Flow cytometry analysis of the draining lymph node
24 hours post subcutaneous injection of the vesicles
showed a strong increase (~5-fold) in the number of DCs
(Figure 4A), indicating the capacity of the vesicles to in-
duce robust recruitment of DCs to secondary lymphoid
tissues. Furthermore, analysis of the expression of matu-
ration markers CD40, CD80 and CD86 on the surface of
DCs, indicated potent (over ~4-fold) induction of DC
maturation in mice by PEG5k-GL2-IMDQ vesicles com-
pared to untreated mice (Figure 4B). Taken together, our
in vitro reporter cell assay data and our in vivo data un-
derscore the ability of the PEG5k-GL2-IMDQ vesicles to
induce robust activation of DCs through TLR7/8 trigger-
ing.
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In addition, in both in vivo studies, neither signs of ad-
verse reactogenicity such as severe swelling, bruising and
redness at the site of injection, not any abnormal behavior
of mice treated with PEG5k-GL2-IMDQ vesicles was ob-
served, which gives a first indication regarding an ac-
ceptable safety profile of the vesicles.
CONCLUSIONS
In summary, we designed and synthesized PEG5k-GL2-
IMDQ, a novel amphiphilic polymeric prodrug of an im-
idazoquinoline TLR7/8 agonist that forms vesicles in
aqueous medium. These vesicles degraded specifically in
response to endosomal enzymes, thereby showing high in
vitro TLR agonistic potency. In vivo, in mouse models,
PEG5k-GL2-IMDQ vesicles provoked robust and long-
acting innate immune-stimulation in draining lymphoid
tissue and avoided systemic inflammation. Furthermore,
PEG5k-GL2-IMDQ vesicles efficiently promoted recruit-
ment of DCs to lymph nodes and induced robust DC mat-
uration.
In previous studies, IMDQ-conjugated nanoparticles
were shown to induce, upon intratumoral injection, potent
tumor-specific immune responses. When admixed with
protein antigens in a vaccination setting, robust antigen-
specific T cell and humoral response in mice were
mounted.7,42,38,43 Based on the highly efficient activation
of DCs in vivo by PEG5k-GL2-IMDQ vesicles, we expect
also these vesicles to be a highly potent immunotherapeu-
tic and vaccine adjuvant, which will be assessed in future
studies, including strategies to co-formulate antigen by ei-
ther entrapment into the vesicle core or conjugation to the
vesicle surface.
Figure 4. In vivo recruitment and activation of dendritic
cells (DCs) in draining lymphoid tissue. (A) Flow cytometry
analysis of the DC population in the draining lymph node in
response to subcutaneous injection of PEG5k-GL2-IMDQ
vesicles. (A1) contour plots and (A2) relative numbers of
DCs. (n=3: Student t-test; *:p<0.05) (B) Flow cytometry
analysis of the expression of maturation markers on DCs in
the draining lymph node in response to subcutaneous injec-
tion of PEG5k-GL2-IMDQ vesicles. (B1) histograms and
(B2) percentages of DCs expressing a specific maturation
marker. (n=3 Student t-test; ***:p<0.001, **:p<0.05)
the rapid leakage of IMDQ from the injection site and
subsequent entry into systemic circulation. It is important
to note that the signal in mice receiving PEG5k-GL2-
IMDQ vesicles was even detectable more than one week
post injection (Figure 3B), indicating prolonged immune
stimulation due to the sustained degradation and release
of IMDQ. Such long-acting immune stimulation has been
shown to enable less frequent administration and induce
more potent immune-modulation/vaccination ef-
fects.8,40,41 By contrast, the signal from PEG-IMDQ and
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website.
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Experimental details on chemical synthesis and characteri-
zation data, as well as methods for vesicle formulation and
in vitro/in vivo characterizations are presented.
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AUTHOR INFORMATION
Corresponding Authors
Yang Shi: yshi@ukaachen.de
Bruno G. De Geest: br.degeest@ugent.be
Twan Lammers: tlammers@ukaachen.de
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cica, B. J.; Decker, B.; Maciejewski, M.; Cheung, J.;
Yamane, H.; Smelkinson, M. G.; Francica, J. R.;
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G.; Jewell, C. M.; Lantz, O.; Piaggio, E.; Ishizuka, A.
S.; Seder, R. A. Peptide–TLR-7/8a Conjugate Vac-
cines Chemically Programmed for Nanoparticle Self-
Assembly Enhance CD8 T-Cell Immunity to Tumor
Antigens. Nat. Biotechnol. 2020, 38, 320-332.
(15) Wilson, D. S.; Hirosue, S.; Raczy, M. M.; Bonilla-
Ramirez, L.; Jeanbart, L.; Wang, R.; Kwissa, M.; Fra-
netich, 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
ACKNOWLEDGMENT
SVH acknowledges Ghent University of a postdoctoral
scholarship.
No competing financial interests exist. This work is sup-
ported by the European Research Council (ERC) under the
European Union’s Horizon 2020 research and innovation
programme (ImmunoBioSynth (817938), Picelles (813086),
Meta-Targeting (864121), the China Scholarship Council,
the European Union (European Fund for Regional Develop-
ment: TAKTIRA (EFRE-0801767), the German Research
Foundation (DFG: GRK/RTG 2375 Tumor-targeted Drug
Delivery (project number: 331065168) and SFB 1066), the
Aachen Interdisciplinary Center for Clinical Research
(IZKF; Project O3-2).
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