A pH-sensitive Macromolecular Prodrug as TLR7/8 Targeting Immune Response Modifier

Article abstract of DOI:10.1002/chem.201702942

The chemical synthesis and biological activity of novel functionalized imidazoquinoline derivatives (ImQ) to generate Toll-like receptor (TLR) 7/8 specific prodrugs are presented. In vivo activity of ImQs to induce inflammation was confirmed in zebrafish

Full text of DOI:10.1002/chem.201702942

A Journal of
Accepted Article
Title: A pH-sensitive macromolecular prodrug as TLR7/8 targeting
immune response modifier
Authors: Stefan Aichhorn, Anne Linhardt, Angela Halfmann, Markus
Nadlinger, Stefanie Kirchberger, Manuela Stadler, Barbara
Dillinger, Martin Distel, Alexander Dohnal, Ian Teasdale, and
Wolfgang Schoefberger
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To be cited as: Chem. Eur. J. 10.1002/chem.201702942
Link to VoR: http://dx.doi.org/10.1002/chem.201702942
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A pH-sensitive macromolecular prodrug as TLR7/8 targeting
immune response modifier
Stefan Aichhorn[a,†], Anne Linhardt[b,†], Angela Halfmann[c], Markus Nadlinger[a], Stefanie Kirchberger[d], Manuela
Stadler[d], Barbara Dillinger[d], Martin Distel*[d], Alexander Dohnal*[c], Ian Teasdale*[b], and Wolfgang Schöfberger*[a]
Abstract: The chemical synthesis and biological activity of novel
functionalized imidazoquinoline derivatives (ImQ) to generate Toll-like
receptor (TLR) 7/8 specific prodrugs are presented. In vivo activity of
ImQs to induce inflammation was confirmed in zebrafish larvae. After
covalent ligation to fully biodegradable polyphosphazenes (ImQ-
polymer), the macromolecular prodrugs were designed to undergo
intracellular pH-sensitive release of ImQs to induce inflammation
through binding to endosomal TLR7/8 (danger signal). We showed
ImQ dissociation from prodrugs at a pH 5 pointing towards endosomal
prodrug degradability. ImQ-polymers strongly activated ovalbumin-
specific T cells in murine splenocytes as shown by increased
proliferation and expression of the IL-2 receptor (CD25) on CD8+ T
cells accompanied by strong IFN-γ release. ImQ prodrugs presented
here are suggested to form the basis of novel nanovaccines, e.g. for
intravenous or intratumoral cancer immunotherapeutic applications to
trigger physiological antitumor immune responses.
even via intratumoral delivery, and hence the risk of systemic
immune response represents a severe limitation of the applicable
dose to levels below that required for activation. One of the most
widely investigated groups of TLR agonists are imidazoquinolines,
a family of synthetic small molecules, including imiquimod (R837),
resiquimod (R848), gardiquimod, and other variants. Herein we
present
chemically modified as such to enable reversible covalent ligation
to synthetic and/or biological macromolecules, i.e.
a
novel imidazo[4,5-c]quinolin-4-amine agonist,
macromolecular prodrugs. The rational for a macromolecular
prodrug approach, alongside potential gains in therapeutic
efficacy[6,7] is the location of the TLR7/8 receptors in the
endosome, well understood to be the major intracellular gateway
for macromolecules.[8] During the last decade, polymer-based
nanomedicines or polymer therapeutics[9,10] have become an
important tool for drug delivery[6] and theranostics[11]. Although
there can be clincial issues with heterogeneity and reproducibility
the use of macromolecular prodrugs has been shown in a wide
number of cases to increase the efficacy and positively influencing
the biodistribution of therapeutics.[6,9] Poly(organo)phosphazenes
are a versatile class of polymers with immense potential for
application in nanomedicine.[12,13] Recent advances in
polyphosphazene synthesis allow simple access to controlled
molecular weights, controlled hydrodynamic volumes and high
Agonists of toll-like receptors (TLRs) are being actively pursued
for their ability to activate the immune system for a host of
therapeutic applications, including cancer immunotherapy.[1]
Murine TLR7[2] and human TLRs 7 and 8[3] are known to sense
guanosine-based drugs that induce an antiviral response in vivo,
stimulating the development of tolerogenic antigen-presenting
dendritic cells (DCs), producing selective cytokines that drive T
cell responses. To date, most DC-based tumor immune-
therapeutic strategies involve ex vivo loading of DCs with tumor-
associated antigens and immune-stimulatory agents (adjuvants)
and subsequent re-injection into the patient for in vivo T cell
activation.[4] This represents a highly promising but labor and cost
intensive methodology. Thus, a more direct, pharmaceutical
approach could be of significant value to the field.[5] However, low
water solubility.[14] One of relatively few fully degradable[13,15]
,
water soluble polymers available, essential design features in
avoiding the deleterious effects associated with post-drug-release
accumulation of high molecular weight macromolecules in the
organism[7]. Moreover, certain poly(organo)phosphazenes
themselves appear as potent immunoadjuvants in vaccine
delivery in terms of their complexes with protein antigens leading
to activation and antigen presentation of DCs[16]. Recently, a pH
releasing nanogel approach was successfully applied to induce
superior antibody and T-cell responses against a tuberculosis
molecular weight molecular adjuvants share
a
poor
pharmaceutical profile and rapid diffusion from the local tissue,
antigen.[17]
Herein, we demonstrate the design of
macromolecular prodrugs, capable of undergoing a controlled
intracellular release of the low-molecular weight TLR agonist as
a so-called “danger signal” to induce inflammation through
binding to endosomal TLR7/8. However, since known
imidazoquinoline based agonists lack appropriate sites for
conjugation to suitable pH release systems, the implementation
of novel synthetic imidazoquinoline analogues is necessary. We
report the synthesis of novel a imidazo[4,5-c]quinoline derivative
with an aliphatic ketone moiety attached to the benzylic group,
which was suitable for coupling to macromolecules equipped with
hydrazide linkers to give pH cleavable hydrazone linkage[18], well-
proven to provide a clean rapid release in the acidic endosomal
environment, thus to potentially produce a controlled, intracellular
presentation of the TLR agonist. The group of David et al. have
[a]
Prof. Dr. Wolfgang Schöfberger, Dr. Stefan Aichhorn, DI Markus
Nadlinger, Institute of Organic Chemistry, Johannes Kepler University
Altenberger Straße 69, A-4040 Linz (Austria)
E-mail: wolfgang.schoefberger@jku.at)
[b]
Prof. Dr. Ian Teasdale, Anne Linhardt MSc.
Institute of Polymer Chemistry, Johannes Kepler University Linz (JKU)
Altenberger Straße 69, A-4040 Linz (Austria)
E-mail: ian.teasdale@jku.at
[c]
Dr. Alexander Dohnal, DI Angela Halfmann, Dr. Martin Distel, Dr. Stefanie
Kirchberger, Manuela Stadler MSc, Barbara Dillinger MSc
Tumorimmunology and Innovative Cancer Models, St. Anna
Kinderkrebsforschung e.V. CCRI – Children’s Cancer Research Institute
Zimmermannplatz 10, 1090 Vienna (Austria)
E-mail: alexander.dohnal@ccri.at; martin.distel@ccri.at
† These authors contributed equally to this work.
investigated
the
structure-activity
relationships
of
imidazoquinoline analogues.[19]
Supporting information for this article is given via a link at the end of the
document
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Scheme 1. Reagents and conditions: (i) 2-bromobenzylamine, NEt3, THF, r.t.; (ii) iron powder, NH4Cl, valeraldehyde, 2-BuOH, air, reflux; (iii) but-3-en-2-ol, Pd(OAc)2,
NEt3, DMA, 130°C; (iv) mCPBA, CH2Cl2, reflux; (v) I.) benzoyl isocyanate, CH2Cl2, reflux; II.) NaOMe, MeOH, reflux.
It was demonstrated that maintaining high agonistic activity of
imidazoquinolines narrows the possibilities to introduce functional
groups down to a few certain positions of the 1H-imidazo[4,5-
c]quinoline active core. Most notably, position C4 is required to
hold a primary amine and C2 a short aliphatic chain. In short,
position N1 would appear to be the preferred site for
functionalization for subsequent conjugation, since a wide variety
of substitution patterns are tolerated at this position. Our initial
concept was to synthesize halogenated 1-benzyl-2-butyl-1H-
imidazo[4,5-c]quinolin-4-amines, which then act as substrates for
cross-coupling reactions to install various keto-functionalities. To
ensure rapid pH-driven dissociation, an aliphatic ketone was
introduced. Considering the circumstances mentioned above, a
synthesis route was designed starting with 4-chloro-3-
nitroquinoline, a common precursor for fused quinoline-type
heterocyclic frameworks (Scheme 1).[20] A functionalized benzylic
group can be installed at this stage by substitution of the C4-
the highest-yielding reaction was shown to be preparation of 3
from 2 with but-3-en-2-ol (81 %, Scheme 1). The N5-oxide 4 could
be accessed in very good yield by employing meta-
chloroperoxybenzoic acid as an oxidizing agent, without the
requirement for hitherto unsuccessful ketal protection of the
ketones. These formations proceeded slowly, taking 60 h. The
same was true for the final preparation of C4-amines, where the
reaction conditions also involved refluxing in dichloromethane for
two to three days. For this purpose, benzoyl isocyanate was
added as the nitrogen source. The concluding step consisted of
refluxing the intermediate with sodium methoxide in methanolic
solution to release the primary amine. The synthesis of 5 resulted
in good yield (53 %). In order to assess the effect of chemical
modification on the activity of the novel TLR agonist, a series of
tests were carried out in living zebrafish larvae as shown in Figure
1. Stimulation with 5 resulted in NF-kB signaling activation in
zebrafish macrophages visualized in Tg(mpeg1:mCherry)gl23 [23]
/
chlorine with
a
benzylamine. Treatment of 4-chloro-3-
Tg(6xHsa. NFKB: EGFP)nc1 [24] double transgenic animals, which
have macrophages demarcated by mCherry expression and
report NF-κB activity by EGFP expression (Fig.1A).
nitroquinoline with 2-bromo benzylamine using triethylamine in
THF solution gave quinolin-4-amine 1 in excellent yield. By
combining and adjusting two published one-pot procedures for
the formation of benzimidazoles starting from ortho-nitroaniline,[21]
5 showed stronger induction of NF-κB than the known TLR7/8
agonist R848 as quantified by flow cytometry (Fig.1B). QPCR
further confirmed induction of inflammatory cytokines IL1b, IL6
and Tnf-α upon 5 stimulation for 6h (Fig.1C). In addition, IL1b
induction upon stimulation with 5 was reduced in MyD88 mutant
zebrafish, indicating a MyD88 dependent signal pathway as
expected for specific TLR7/8 activation (Fig.1D).[25] In the next
step, water soluble polyphosphazenes decorated with hydrazide
linkers (43 wt%) were prepared as previously reported (see ref.[26]
and SI-2). The novel TLR agonist 5 could be successfully bound
to the polymer due to hydrazone formation with the carbonyl
groups (Scheme 2). After purification by dialysis, UV-Vis, GPC
and DLS measurements were used to prove covalent drug
loading. The percentage of loading was calculated using the UV-
Vis absorbance at 324 nm (SI-3). Conjugation between 3 and 6
weight percent of the macromolecular conjugate was achieved
1-benzyl-2-butyl-1H-imidazo[4,5-c]quinoline
2
could
be
synthesized in 83 % yield. This transformation consisted of the
reduction of the nitro group with iron powder aided by ammonium
chloride in iso-butanol combined with the annulation of a 1H-2-
butyl-imidazole motif employing valeraldehyde. The established
route to carry out amino-functionalizations with quinoline-
derivatives is to firstly subject compounds such as 3 to N-
oxidization (compare Scheme 1, 4), before conversion with
nitrogen-bearing reagents. Aliphatic ketones could be
successfully installed in a single step via a Heck-type reaction
using an allyl alcohol along with triethylamine,[22] to give a 3-oxo-
butyl-chain attached to the benzyl-group. By subjecting various
halobenzyl-imidazoquinoline derivatives to this palladium acetate-
catalyzed coupling, suitable conversions were observed. By far
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representing a loading yield of between 10 and 25 percent of the
linkers (SI-3).
carriers is associated with particle formation and enhanced
cytokine production.[28] Double internalization of the agonist, via
its chemical binding location close to the backbone of the brush
type polymers is further pronounced by the agglomeration of the
hydrophobic units in aqueous solutions, thus offering the potential
for a so-called ‘stealth-effect’[29], reducing the probability of
agonist presentation prior to intracellular release. The release of
5 from the conjugates was analyzed by HPLC. Within a period of
24 h at 37°C, 100% release from the polymer-drug conjugates
could be observed for the samples exposed to pH 5 and only 50%
for the conjugates stored at pH 7.4 (Figure 2b).
Figure 1. In vivo studies of the immune- activating functions of 5. (A) & (B)
NFκB:eGFP induction by 5 in mpeg:mCherry positive macrophages in live
zebrafish larvae. Zebrafish larvae (4 dpf) were stimulated with DMSO (control),
R848 (positive control) or 5 for 6h. A) Representative confocal images of
macrophages in live NFκB:eGFP/mpeg:mCherry transgenic larvae. Upper
panel: DMSO control (arrow demarcates macrophage). Lower panel: 5 (B) Flow
cytometry analysis of NF-κB induction in single-cell suspensions of zebrafish
larvae. Cells were pre-gated on mpeg:mcherry macrophages and analysed for
NFκB:eGFP expression. (C) Quantitative real- time PCR for RNA expression of
the inflammatory cytokines il1b, il6, tnfa in wildtype zebrafish larvae after 6 h
activation with 5. DMSO was used as negative control. (n= 3) *P<0.05; ** P<0.01
(D) Il1b RNA expression in MyD88 mutant and wildtype zebrafish larvae after 6
h of stimulation with TLR ligands R848 and 5. DMSO was used as negative
control. (n= 4-5) Scale bars in A are 10 µm.
Scheme 2. Chemical conjugation of TLR agonist 5 to the poly(organo)-
phosphazene (simplified structure) via a hydrazone linkage.
The release rate is comparable to published data using similar
hydrazide based polymer systems.[26,30] At pH 5, approximately
50 % release of compound 5 was observed after 2.5 hours and
full release was observed after 30 hours, whereas at pH 7.4 only
approx. 50% release of 5 was observed during the whole period
measured. As complete clearance of the macromolecular carrier
is a stringent requirement for subsequent in vivo applications, the
degradation profiles of the polymers were also investigated. The
degradation studies of the conjugates at 37°C, pH 5 and 7
measured by size exclusion chromatography, showed that the
polymers are stable over a short period of time in an aqueous
environment but degrade significantly to small molecules under
these simulated physiological conditions within 10 weeks (SI-5).
These results are comparable to previous degradation studies of
amino substituted poly(organo)phosphazenes but could be easily
accelerated or decelerated as and when required[11]. Compound
5, upon release from polymer-ImQ conjugate is expected to
stimulate T cells through internalization and activation of APCs
such as DCs. Thus, in order to analyse the immunological activity
of polymeric prodrugs we started investigating cellular uptake and
localization of ImQ-conjugated within murine DCs. Compound 5-
conjugated ImQ polymer accumulated in generated DCs and co-
localized with intracellular endosomal and lysosomal vesicles
(Figure 3 A-D). Overall, a total of 87 % of all DCs internalized ImQ-
based macromolecular prodrug.
The GPC curves of the polymer conjugates after drug loading,
measured in DMF, are shifted to lower retention volumes (lower
hydrodynamic volume), presumably due to the hydrophobicity of
the conjugated drugs, whilst still possessing a relatively narrow Đ
of 1.3 (SI-4). The hydrodynamic volume is a crucial parameter in
terms of bio-distribution and cell uptake. Dynamic light scattering
studies showed that whilst the hydrophilic polymers show
hydrodynamic diameters in the 10 nm range, they form
supramolecular associates in the 100 nm range upon conjugation
of the hydrophobic drug as a result of hydrophobic interactions
(Figure 2). This phenomenon which has been previously reported
with similar phosphazene based systems[27]is thought to be
assisted by the high backbone flexibility relative to carbon based
polymers. Furthermore, in a work with different polymer-TLR7/8
agonist conjugates, Lynn et al. recently showed that increased
densities of Toll-like receptor agonists arrayed on polymer
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The capacity of ImQ-conjugate activated DCs to stimulate T cells
via DCs was further analysed in T cell / DC co-cultures.
vesicles. The capacity of ImQ-conjugate activated DCs to
stimulate T cells via DCs was further analysed in T cell/DC co-
cultures.
Figure 2. a) Schematic representation of the intra- and intermolecular
agglomeration and self-assembly of the polymers upon conjugation of the
hydrophobic drug in aqueous solvents. b) Molecular size distribution by intensity
as detected by dynamic light scattering for polymers 2–5 in phosphate buffer at
pH 7.4 (polymer concentration 1 mg/mL, dh - hydrodynamic diameter). c)
Release of 5 from the conjugate at 37°C in acidic environment (acetate buffer,
pH 5), and a neutral solution (pH 7.4, tris buffer). The amount of the released
drug was estimated using a calibration curve for the free drug.
We used splenocytes from OT-I mice, which among other immune
cells comprise APCs and predominantly CD8+ T cells. Such T
cells entirely express a transgenic T cell receptor, which is specific
for a short ovalbumin related peptide sequence (SIINFEKL)
primarily presented by APCs. Thus strong APC-mediated T cell
responses can be raised in an OT-I splenocyte culture upon
TLR7/8 ligation together with SIINFEKL-peptide. In order to
investigate the effect on T cells, we measured the degree of T cell
activation, as well as the secretion of IFN-γ, a cytokine that is
associated with activation of CD8+ effector T cells (Figure 3 E-F).
The activation marker CD25 was significantly increased on CD8+
T cells in stimulation cultures using conjugates, R837 or R848
compared to the pristine polymer alone. The dose response curve
using the conjugate was comparable to the course of proliferation
induced by R837, between 0.1 and 5 μM. The amount of IFN-γ
released into the culture was between 200 and 600 ng/ml after
the stimulation with 0.1 to 5 µM using conjugate, R837 or R848
compared to 200 ng/ml for the polymer backbone or SIINFEKL-
peptide alone. The conjugates induced the highest IFN-γ release
at 5 µM, which strongly declined with decreasing conjugate
concentrations in the splenocytes cultures down to levels of the
negative controls as shown by polymer or SIINFEKL peptide
alone. Based on our observation the conjugate strongly
stimulates murine CD8+ T cells presumably by DC-mediated T
cell priming involving TLR7/8 signaling in DCs.
Figure 3. A-D: Confocal microscopy images of dendritic cells incubated with
polymer- ImQ conjugates. Murine BMDCs (A) were stained with LysoTracker®
Green (504 nm/511 nm) for 1 hour (B) and with polymer-ImQ (390 nm/415 nm).
LysoTracker® Green accumulates in cellular compartments with low internal pH
and stains lysosomes (green, B). Polymer-ImQ accumulates in lysosomes and
in endosomes (C and D, purple regions indicated by arrows). E-F) Conjugates
activate T cells and IFN-γ release. Splenocytes were isolated from transgenic
OT-I mice, labeled with CFSE and stimulated for 4 days with R837, R848 or
conjugate, together with the ovalbumin derived SIINFEKL-peptide, which
stimulates ovalbumin specific CD8+ T cells from OT-I mice. On day 4, CD8+ T
cells were analyzed for CD25 expression and CFSE diluted cells, considered
as proliferating T cells by flow cytometry. E) CD25 expression depicted as mean
fluorescence intensity (MFI) is shown for splenocytes cultures containing 1 µM
conjugate. F) Absolute cell numbers of proliferating CD8+ T cells per splenocyte
culture were calculated in CFSE diluted dividing T cells using a dosage ranging
from 0.1 to 5 µM. G) Supernatants from splenocyte cultures were analyzed for
IFN-γ secretion. Mean  standard deviation from triplicate analysis is shown for
CD25 expression, *) p < 0.05, **) p < 0.01. Polymer alone and SIINFEKL peptide
alone dissolved in DMSO served as negative controls.
In conclusion, the chemical synthesis and biological activity of a
novel functionalized imidazoquinoline (ImQ) derivative bearing a
3-(3-oxobutyl)-benzyl moiety at the N1-position of the ImQ was
presented. The keto-group was introduced to provide a site for
reversible binding to hydrazide groups attached to modified, water
soluble and biodegradable polyphosphazenes. The resulting
hydrazone functionality served as an acid labile linkage between
the polymeric carrier system and the immune response modifier.
Compound 5-conjugated ImQ polymer accumulated in generated
DCs and co-localized with endosomal and intracellular lysosomal
In splenocytes from OT-I mice stimulated with polymer-ImQ
conjugates in the presence of SIINFEKL-antigen, CD8+ T cells
proliferated and strongly released IFN-γ into the supernatant
when compared to known immune response modifiers resiquimod
(R848) and imiquimod (R837), used as positive controls. In
addition, CD8+ T cells showed high expression of the IL-2
receptor (CD25), considered as an activation marker for T cells
after polymer-ImQ conjugate stimulation. Ongoing in vivo studies
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We acknowledge the financial support of the Austrian Research
Promotion
Agency
(FFG-Project
836532
“Cancer-
immunotherapy”) and of the Austrian Science Fund (FWF-
P28167-N34). The NMR spectrometers were acquired in
collaboration with the University of South Bohemia (CZ) with
financial support from the European Union through the EFRE
INTERREG IV ETC-AT-CZ program (project M00146, "RERI-
uasb"). We would like to thank Dr. Markus Himmelsbach for the
measurement of ESI-MS spectra, Dr. Susana Pascoal for fish
husbandry and Dr. Michiel van der Vaart and Dr. Graham
Lieschke for kindly providing zebrafish strains A.L and I.T thank
Prof. Oliver Brüggemann for his support of the project.
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Keywords: Imidazoquinoline • TLR7/8 agonists • immune
response modifiers • pH-sensitive polymer • nanovaccines
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10.1002/chem.201702942
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COMMUNICATION
A novel pH-sensitive
Stefan Aichhorn, Anne Linhardt, Angela
Halfmann, Markus Nadlinger, Stefanie
Kirchberger, Manuela Stadler, Barbara
Dillinger, Martin Distel*, Alexander
Dohnal*, Ian Teasdale*, and Wolfgang
Schöfberger*
polyphosphazene-imidazoquinoline
conjugate serves as a TLR7/8
targeting macromolecular prodrug for
T cell activation.
Page No. – Page No.
A pH-sensitive macromolecular
prodrug as TLR7/8 targeting immune
response modifier
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