Download Antiviral applications of Toll-like receptor agonists

J Antimicrob Chemother 2012; 67: 789 – 801
doi:10.1093/jac/dkr588 Advance Access publication 18 January 2012
Antiviral applications of Toll-like receptor agonists
Nigel J. Horscroft*, David C. Pryde and Helen Bright
Pfizer Global R & D, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK
*Corresponding author. Tel: +41-788-84-95-60; E-mail: [email protected]
In the past, antiviral research has focused mainly on viral targets. As the search for effective and differentiated
antiviral therapies continues, cellular targets are becoming more common, bringing with them a variety of
challenges and concerns. Toll-like receptors (TLRs) provide a unique mechanism to induce an antiviral state
in the host. In this review we introduce TLRs as targets for the pharmaceutical industry, including how they
signal and thereby induce an antiviral state through the production of type I interferons. We examine how
TLRs are being therapeutically targeted and discuss several clinically precedented agents for which efficacy
and safety data are available. We describe some of the chemistries that have been applied to both small molecule and large molecule leads to tune agonist potency, and offer a differentiated safety profile through targeting certain compartments such as the gut or the lung, thereby limiting systemic drug exposure and affecting
systemic cytokine levels. The application of low-dose agonists of TLRs as vaccine adjuvants or immunoprotective agents is also presented. Some of the challenges presented by this approach are then discussed, including
viral evasion strategies and mechanism-linked inflammatory cytokine induction.
Keywords: TLR, immune modulation, therapy, virus
Introduction
In his 1908 Nobel lecture, Ilya Mechnikov commented on the use of
subcutaneously applied nucleic acids to protect patients from infection during and after surgery. He reported that ‘the results achieved
are so encouraging that it is possible to predict new progress in the
approach to the dressing of wounds’.1 One hundred years later we
have a better understanding of the role of pattern recognition receptors (PRRs) in sensing and responding to a variety of invading
pathogens.2 The detection of exogenous nucleic acids by Toll-like
receptors (TLRs) and RIG-I-like receptors (RLRs) is essential to
mounting an innate response to viral infection.3,4 A deeper understanding of the biochemical pathways of the innate immune
system has led to a number of innovative approaches to antiviral
therapy.5,6 Prosecution of these targets has proven challenging for
both biologists and chemists; nevertheless, this class of antiviral
therapy has grown rapidly in recent years.7
The innate immune system
PRRs are the sentinels of the innate immune system, recognizing
pathogen-associated molecular patterns (PAMPs), the components
of invading pathogens.8 PAMPs include various bacterial cell wall
components such as lipopolysaccharides (LPSs), peptidoglycans
(PGNs) and lipopeptides, as well as flagellin and viral nucleic acids.
TLRs
TLRs are the best-characterized PRRs and are evolutionarily conserved across a diverse range of species.9 They are homologues
of the Drosophila Toll gene first identified as being essential for
development and later for antifungal and antibacterial immunity.10,11 TLRs are type I transmembrane proteins featuring
an extracellular leucine-rich domain and a cytoplasmic tail
that contains a conserved Toll/IL-1 receptor (TIR) domain
(Figure 1a).12,13 The leucine-rich domain consists of 19 –25
leucine-rich repeats (LRRs) made up of an a-helix and a
b-strand connected by a loop. Each repeat contains the motif
XLXXLXLXX as well as conserved hydrophobic residues. The threedimensional structure of the human TLR3 ectodomain revealed
that this region forms a crescent shape, and, upon dimerization,
a groove is formed on the concave surface where the two
monomers meet.14,15 It is thought that negatively charged
double-stranded RNA binds in this groove.
TLRs are predominantly expressed in tissues involved in
immune function as well as those exposed to the external environment, such as lung and the skin. Most TLRs are located on the
plasma membrane, with the exception of TLR3, TLR7, TLR8 and
TLR9, which are expressed intracellularly, predominantly in endosomes.16 – 18 This intracellular expression is thought to minimize
the recognition of self RNA and DNA.19 To date, 11 human and
13 murine TLRs have been identified. However, only a subset of
these is thought to recognize viral components (Table 1). TLR3,
TLR7, TLR8 and TLR9 distinguish between double-stranded
RNA, single-stranded RNA and CpG-containing DNA, facilitating
the recognition of all viral species and inducing the expression
of type I interferons (IFNs). The signalling mechanisms leading
to the induction of type I IFNs differ depending on the TLR activated. Nevertheless, a wide variety of TLR agonists have been
shown to inhibit hepatitis B virus (HBV) and hepatitis C virus
# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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(a)
(b)
EXTRACELLULAR
5’-ppp-ssRNA
RIG-I
TLR7/8/9
Leucine-rich
repeats
TLR3
Mitochondrion
RIG-I
TRIF
MyD88
IRAK4
TRAF6
TRAF6
IRAK1
MAVS
TRAF3
FADD
TRAF6
1-2 cysteine-rich
regions
IRF3
NF-κB
IRF3
NF-κB IRF7
IRF7
NF-κB
Transmembrane
domain
Cytoplasmic domain
(highly conserved
TIR domain)
NF-κB
NF-κB
IRF7 IRF7
IRF7 IRF3
IRF3
IRF3
INTRACELLULAR
IFN beta, TNF
IFN alpha
IFN beta
IL-10, ISG15
Figure 1. (a) Schematic of a TLR type 1 transmembrane protein. (b) TLR signalling. Viral nucleic acids are primarily detected by either endosomal or
cytoplasmic PRRs. The resulting signalling cascades converge at the transcription factors NF-kB and IRFs 3 and 7. This leads to the production of a
number of cytokines, including the type 1 IFNs that initiate the innate response.
Table 1. PRRs involved in the recognition of viral components
PRR
Virus
Component recognized
haemagglutinin protein
Reference
TLR2
MV
124
TLR3
RNA and DNA viruses dsRNA
125–128
TLR4
RSV, VSV
envelope proteins,
glycoproteins
129–132
TLR7/8
RNA viruses: HCV,
FluV
ssRNA
3, 22,
133–136
TLR9
DNA viruses: HSV
CpG DNA
137–140
RIG-I
RNA viruses: NDV, SV, dsRNA, ssRNA with a
VSV, FluV, MV
5′ -triphosphate
4, 141– 143
MV, measles virus; RSV, respiratory syncytial virus; VSV, vesicular
stomatitis virus; HCV, hepatitis C virus; FluV, influenza virus; HSV, herpes
simplex virus; NDV, Newcastle disease virus; SV, Sendai virus; ds,
double-stranded; ss, single-stranded.
(HCV) (Table 2) and are discussed later.20 – 22 Based on a combination of cellular expression patterns and type I IFN signalling
pathways, TLR3, TLR7 and TLR9 are considered the best targets
for antiviral therapy.23
Signalling
TLR signalling consists of two distinct pathways (for review see
Brikos and O’Neill24). TLR7, TLR8 and TLR9 signal via the
myeloid differentiation primary response gene 88 (MyD88)-
790
dependent pathway, with the adaptor protein MyD88 playing a
pivotal role. Upon activation by single-stranded RNA or CpG
DNA these TLRs induce the recruitment of the adapter protein
MyD88 via its TIR domain. TIR domains initiate the signalling
cascade through TIR adapters, leading to downstream responses
tailored to specific pathogens. This leads to the activation of the
transcription factors nuclear factor-kB (NF-kB) and IFN regulatory factors (IRFs) 3 and 7 (Figure 1b). These in turn induce the
production of pro-inflammatory cytokines such as tumour necrosis factor-a (TNF-a), IL-1 and IL-6 (where IL stands for interleukin), and type 1 IFNs, respectively. Signalling via TLR3 is initiated
by double-stranded RNA. TLR3 activation is MyD88 independent
and relies on the adaptor molecule TIR-domain-containing
adaptor-inducing IFN-b (TRIF). However, the TLR3 signalling
cascade also results in the activation of NF-kB, IRF3 and IRF7,
and the production of pro-inflammatory cytokines and IFN.
RLR signalling, on the other hand, proceeds via the interaction
of the caspase activation and recruitment domain (CARD) of the
RLR with the CARD of the mitochondrial antiviral signalling
protein (MAVS), also known as IPS-1, VISA and Cardif.25 MAVS is
anchored to the mitochondrial outer membrane, a feature
essential for function (Figure 1b).26 In a similar fashion to TLR
signalling, a downstream cascade results in the activation of
NF-kB, IRF3 and IRF7.
From a therapeutic point of view, the key outcome of this
early signalling cascade is the production of type I IFNs. These
IFNs are then secreted and function in both an autocrine and
paracrine manner to produce more IFN, effectively amplifying
their own production. Interaction with the type I IFN receptor
leads to the induction of numerous genes that code for proteins
that are directly or indirectly antiviral.
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Table 2. Clinically precedented TLR agonists
Compound
Structure
Imiquimod
Target, intended viral disease
NH2
Company
Reference
TLR7, hepatitis C
3M
144
TLR7, hepatitis C
3M
38
TLR7, hepatitis C
Pfizer
43
TLR9, hepatitis C
Coley
55
TLR7, hepatitis C
Anadys
48
N
N
N
Resiquimod
NH2
N
N
N
O
HO
PF-4878691
NH2
N
N
N
NH
O
O
Actilon
S
oligonucleotide
Isatoribine
O
HO
HO
S
O
OH
N
OH
N
N
NH2
GS9620
structure not disclosed
TLR7, hepatitis B and hepatitis C
Gilead Sciences
65 –68
ANA-773
structure not disclosed
TLR7, hepatitis C
Anadys
50
IMO-2125
modified oligonucleotide
TLR9, hepatitis C
Idera Pharmaceuticals
73,74
Antiviral effector molecules
Imiquimod
Of the hundreds of genes up-regulated by IFN stimulation,
few have been thoroughly characterized. It is presumed
that many are involved in the establishment of an antiviral
state. Well-characterized examples of IFN-induced antiviral
proteins include protein kinase R (PKR), the myxovirus resistance (Mx) GTPases, the RNA-specific adenosine deaminase
(ADAR1), the ubiquitin-like modifier ISG15 and 2′ -5′ oligoadenylate synthetase (OAS) that activates endoribonuclease
L (RNase L).27 – 30
Imiquimod (Figure 2a) is an example of the imidazoquinoline
class of small molecule TLR7 agonists that have been the
subject of extensive research activity over the past three
decades, primarily by 3M Pharmaceuticals. Imiquimod,32 commercialized under the brand name Aldara, was initially launched
in 1997 for the topical treatment of genital and perianal warts
resulting from human papillomavirus (HPV) infection.33,34 In
2004 the product was commercialized in the USA for the
topical treatment of actinic keratosis and superficial basal cell
carcinoma. Phase II/III trials are currently under way for the
treatment of Bowen’s disease. At the time of launch, imiquimod
was shown to be a topical immune response modifier, capable of
inducing the synthesis of type I IFNs and other cytokines in
various cell types, although the precise mechanism of action of
the compound was only confirmed to be TLR7 agonism some
years later.35 A number of studies have shown imiquimod to
be poorly tolerated upon oral dosing, reportedly through a
Therapeutics
There are few TLR-targeted agents in current clinical development or launched for the treatment of viral infections
(Table 2).31 Those agents are described below, and on-going preclinical work with TLR ligands directed towards antiviral applications is detailed.
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(a)
(b)
N
N
(c)
NH2
NH2
NH2
N
N
N
N
N
N
O
S
HO
resiquimod
imiquimod
(d)
(f)
(e)
NH2
NH2
N
N
N
O
O
N
N
N
N
NH2
O
N
N
NH
O
OH
S
NH
O
3M-003
S
O
3M-011
852A (PF-4878691)
(g)
(h)
O
O
S
HO
O
S
HO
O
OH
N
N
O
HO
N
N
OH
O
NH2
O
NH2
ANA-245
N
N
O
ANA-975
(i)
NH2
N
N
H
(j)
NH2
H
N
N
O
N
N
O
H
N
O
N
N
N
MeO2C
SM-324405
SM-276001
(k)
(l)
NH2
N
MeO
O
H
N
O
N
N
NH2
N
F
F
H
N
O
N
F
SM-360320
PF-4171455
Figure 2. Small molecule TLR7, TLR8 and mixed TLR7/8 agonists. Examples of imidazoquinolines (a)–(f), nucleosides (g) and (h) and
8-hydroxyadenine derivatives (i)– (l).
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centrally mediated mechanism, based on studies carried out in
ferrets and possibly involving cytokine release triggered by imiquimod,36 although later studies with more potent non-emetic
agents would appear to call this hypothesis into question (see
the 8-Hydroxyadenine derivatives section below).
Other compounds from the imidazoquinoline series include
resiquimod (Figure 2b),37 a mixed TLR7/8 agonist that reached
Phase III for the topical treatment of genital herpes before
being discontinued due to a lack of adequate efficacy. Resiquimod has also been investigated as an oral treatment of HCV
infection in a Phase IIa study in which patients received a
0.01 mg/kg dose of resiquimod twice weekly for 4 weeks.38 At
this dose level the compound was well tolerated, but did not
result in meaningful effects on viral load or significant changes
in systemic levels of cytokines or biomarkers. Subsequent publications and patent applications have detailed further examples of
imidazoquinoline series variants in the peripheral functionality
around this core and covering a range of indications including
viral infection.39 – 41 Modest structural modifications have transformed these TLR7-specific agonists into TLR8-specific agonists
(Figure 2c) or mixed TLR7/8 agonists (Figure 2d and Figure 2e).
Using these tools, 3M has demonstrated TLR8 agonism correlates
well with higher levels of proinflammatory TNF-a induction than
IFN-a induction, while TLR7 agonism is biased towards greater induction of IFN-a.39 This indicates that a selective TLR7 agonist
could be preferable for antiviral indications. In 2007 3M sold the
majority of its TLR portfolio to the Coley Pharmaceutical Group,
which was itself later acquired by Pfizer.42 While Pfizer has continued to use oligonucleotides acquired from Coley as vaccine adjuvants, further development of the Coley small molecule TLR
agonist collection appears to have been limited to the most
advanced agent, which is discussed in the following section.
PF-4878691
852A (PF-4878691, Figure 2f) is an imidazoquinoline analogue
that was investigated by Coley for the treatment of cancers. It is
a potent TLR7 agonist modelled to dissociate its antiviral and inflammatory activities. Following the Pfizer acquisition, 852A was
repurposed for the potential treatment of HCV infection and progressed to a Phase I proof-of-mechanism study in healthy volunteers. 852A induced biomarkers of the immune and IFN responses
in a dose-dependent and dose-frequency-related manner.43
However, two subjects in the top (9 mg) dose group experienced
serious adverse events, characterized by flu-like symptoms, hypotension and lymphopenia, leading to termination of the study and
further clinical development of the compound.
Isatoribine
Isatoribine (ANA-245, Figure 2g),44 and its masked prodrug
ANA-975 (Figure 2h),45 is the most clinically studied oral TLR7
agonist reported to date, having entered Phase II studies for the
treatment of HCV infection. Similarly to imiquimod, this agent
was known to be an immune response modifier before its mechanism of action was found to be through activation of the TLR7
receptor.46 Anadys initially carried out a proof-of-concept study
in HCV patients with intravenous isatoribine administered once
or twice daily for 7 days or three times weekly for 14 days.47
The treatment was well tolerated, and in the highest once-daily
dose group, receiving an 800 mg dose, 8 of 12 patients showed
a statistically significant .0.5 log drop in viral load. This viral
load reduction was accompanied by a dose-dependent systemic
increase in the IFN-stimulated gene products 2,5-OAS and
ISG15. This study provided proof of principle that a systemically
dosed TLR7 agonist can produce viral load reductions in hepatitis
C patients. The observed decreases in viral load were modest, considering the relatively high dose used, and could reflect weak
primary pharmacology at the TLR7 receptor. Whether efficacy in
patients would increase with a more potent compound while
avoiding unacceptable increases in systemic cytokine levels, or
how this antiviral profile would perform as part of a combination
of therapies, remains to be seen.
Anadys partnered with Novartis to develop ANA-975 as an
oral agent, which was also found to be well tolerated when
administered to healthy volunteers, with efficient generation of
isatoribine in the systemic circulation through a sequence
of ring oxidation and ester hydrolysis. Since then, Anadys
suspended a 28 day Phase Ib study in HCV patients due to observations of dose-dependent ‘intense immune stimulation’ accompanied by polyclonal B-cell expansion in a concurrent 13 week
animal toxicity study.48 Development of a reformulated
ANA-975 was later discontinued following the results of a
further 13 week animal toxicity study. Analysis of this study indicated that daily dosing of the compound was unlikely to support
an adequate therapeutic index.
Interestingly, Anadys rationalized their prodrug approach primarily on the basis of avoiding exposure of gut immune tissue to
a TLR7 agonist,49 which they asserted would produce localized
gut-related side effects such as nausea, emesis and gastroenteritis, all symptoms of some of the known TLR7 agonists, which are
poorly tolerated by the oral route (see the Imiquimod section
above).47 ANA-975 also addressed the poor oral bioavailability
of isatoribine. Another prodrug of isatoribine, ANA-773, whose
structure has not been disclosed, is currently being developed
by Anadys for HCV. ANA-773 demonstrated an antiviral response
in HCV patients in a Phase I clinical trial that appeared to exceed
the efficacy seen previously with ANA-975. A Phase II trial was
expected to start in 2011, and it will be critical to observe if
the cytokine stimulation observed with ANA-975 is replicated
with ANA-773 in longer-term dosing, or if there is an inherent
design in the prodrug strategy of ANA-773 that limits systemic
exposure, and thus cytokine release.50 However, Roche recently
announced plans to acquire Anadys, and it is not currently
clear what projects will survive the acquisition.
CPG10101
Short sequences of synthetic oligodeoxynucleotides (ODNs) have
been shown to possess broad, potentially therapeutic applications as anti-infective, antitumour and antiallergic agents and
as Th1 vaccine adjuvants.51,52 ODNs containing sequencespecific unmethylated CpG motifs are abundantly present in
the DNA of several pathogens, but not in mammals, in which
their presence primes a host immune response through activation of the TLR9 receptor. This results in the activation and proliferation of immune cells to mount an immune response to
the pathogen challenge. Due to evolutionary divergence, the
precise sequence motifs that optimally stimulate immune cells
from one species may weakly stimulate the immune cells of
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another. For example, mouse TLR9 responds optimally to the
sequence GACGTT, and human TLR9 to GTCGTT. One major drawback of natural CpG ODNs is the susceptibility of the phosphodiester backbone to serum and cellular nucleases, which can
be addressed by using phosphorothioate backbones, which are
nuclease resistant.
Several CpG-containing phosphorothioate ODNs are in clinical
development, including several from the CpG platform of Coley
Pharmaceuticals. CpG-7909 (ProMune),53 a 24-mer B-class CpG
ODN, is the most advanced agent. Licensed to Pfizer, the compound entered Phase III trials for the treatment of non-small
cell lung cancer and breast cancer, but current development
has been halted, as it did not improve survival when added
to the current standard of care.54 Coley was also developing
CPG10101 (Actilon) for the treatment of chronic HCV.55
CPG10101 had progressed to a Phase IIa trial in combination
with pegylated IFN (PEG-IFN) and ribavirin in HCV patients who
had not previously responded to treatment, but it failed to
significantly reduce viral load. Coley decided to discontinue the
development of CPG10101 in order to focus its resources on
other product candidates in the company’s portfolio and to
seek a licensing partner for CPG10101.
8-Hydroxyadenine derivatives
Dainippon Sumitomo Pharmaceuticals Company Ltd has
partnered with AstraZeneca to investigate a series of
8-hydroxyadenine derivatives that strongly activate the TLR7
receptor and are structurally related to the base component of
isatoribine.56 SM-276001 (Figure 2i) is undergoing preclinical
investigation as a potential treatment for both HCV and HBV infection, and is one of a series of disclosed adenine derivatives that
demonstrate both high potency of IFN induction in vitro in
mouse splenocytes and in vivo in the mouse following oral
dosing. In cynomolgus monkeys, SM-276001 was shown to elicit
a superior systemic IFN induction response to resiquimod.57 – 59
Interestingly, Dainippon Sumitomo has shown that
SM-276001 is non-emetic in the ferret model at doses up to
30 mg/kg,57,60 while resiquimod is strongly emetic at doses
that achieve some 10-fold lower Cmax values. The researchers
at Dainippon Sumitomo postulate that the emetic behaviour of
resiquimod reflects a greater extent of CNS penetration.60 The
results of more detailed analysis of compound levels in the
brain or comparative cytokine profiles induced by different TLR7
ligands have not been released. Recent disclosures claim
extended ester prodrugs of this series61,62 for intratracheal
administration to inhibit allergen-induced airway inflammation,
but importantly did not induce any systemic IFNs. A key
feature of this class of compounds is the ‘antedrug’ concept, in
which an active ester (e.g. Figure 2j) is rapidly metabolized in
plasma to the much less active carboxylic acid equivalent. How
efficacious such an inhaled administration approach is for
respiratory tract infections remains unexplored. Analogous
functionality at the C2 position has also shown potent TLR7
agonism,63 such as the related SM-360320 (Figure 2k) that
inhibits HCV replication in hepatocytes both dependently and
independently of its ability to induce type I IFNs.22
The Pfizer group has disclosed a potent series of
8-oxo-deazapurines, such as PF-4171455 (Figure 2l), that are
being developed for the treatment of HCV.64
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GS9620
In a recent disclosure by Gilead Sciences,65 the selective TLR7
agonist GS9620 was shown to have a good pharmacology
profile in vitro and pharmacokinetic profile in animals. The compound has a reported in vitro PBMC stimulation EC50 of 291 nM
and a 30-fold selectivity over the TLR8 receptor. It shows good
solubility and has a modest pharmacokinetic half-life in preclinical species. Notably the hepatic extraction of the compound
is high, and the authors claimed that systemic IFN levels
appeared not to be driven by systemic compound exposure,
but primarily by engagement of gut-associated lymphoid tissue
(GALT). This was supported by orally administered GS9620
driving higher systemic IFN levels in cynomolgus monkeys than
an intravenous dose of the same compound, despite the latter
achieving a higher systemic exposure of the compound. The
compound induced cytokines and IFN-stimulated genes at low
doses in animals, and was well tolerated in monkeys when
dosed every other day at 1.5 mg/kg for 4 weeks. It induced a
protective antiviral response in woodchucks infected with woodchuck HBV and produced a reduction in viral load when administered to chimpanzees infected with HBV.66,67 Most notably,
GS9620 was safe and well tolerated when administered to
healthy human volunteers at single ascending doses of 0.3 –
12 mg.68 Treatment was accompanied by a dose-dependent
increase in cytokines and in IFN-stimulated genes, but IFN elevations were only detected with the 12 mg dose. The most
common adverse events seen with GS9620 were headaches,
chills and fever, but there were no serious adverse events or
discontinuations and GS9620 continues to be progressed for
the treatment of HBV and HCV. Its structure has not as yet
been disclosed. This study indicates that some of the toxicities
observed above with previous clinical studies using TLR7 agonists
may be surmountable. The observation that efficacy is preserved
in monkeys despite low systemic exposure may offer a means of
driving sufficient cytokine induction to be efficacious without
the intense immune system induction seen with some of the
previously investigated compounds. Further details of the
Gilead clinical programme are eagerly anticipated.
Immunomodulatory oligonucleotides
The next generation of oligonucleotide TLR agonists are exemplified by the immunomodulatory ODNs (IMOs) of Idera Pharmaceuticals (formerly Hybridon) that combine a novel synthetic
DNA structure, called an immunomer, and a synthetic YpR
motif, where Y and R are synthetic analogues of natural
bases.69,70 Naturally occurring CpG DNA (Figure 3a) forms
double-stranded helical structures; immunomers consist of two
3′ -3′ -linked identical DNA fragments, which are still able to
present two 5′ -termini to the TLR9 receptor. These secondgeneration ODNs have the advantage of greater metabolic
stability than their natural nucleotide predecessors, especially
when they incorporate a phosphorothioate backbone, and the
ability to induce a different cytokine profile depending on the
precise structure and sequence of the immunomer.
Early CpR IMOs (e.g. Figure 3b) in which the G base is replaced
by a synthetic analogue (R) demonstrated the same speciesspecific immunomodulatory activity seen with natural ODNs.
Interestingly, later YpG motifs (Figure 3c and d) in which the C
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(a)
(b)
O
S
–
P
O
O
O
N
S
S
N
O
P
O
P
O
N
N
S
O
N
P
O
O
O
NH
N
O
–
N
O
O
O
O
NH2
O
O
O
O
–
–
NH2
O
N
NH
N
O
NH2
NH2
CpG
CpR
(c)
(d)
O
S
–
P
O
O
O
S
P
N
–
P
O
NH2
O
N
O
O
O
–
S
N
OH
O
NH
O
O
O
O
O
O
N
S
O
N
N
–
P
N
O
O
O
NH
O
NH2
N
O
N
N
NH
NH2
Figure 3. Immunomodulatory oligonucleotides based on CpG sequences on a phosphorothioate backbone.
base is replaced with a synthetic analogue show speciesindependent activity, and different immunomers show a clear
structure–activity relationship depending on the exact structure
of the base used. For example, A-rich sequences at the 5′ -end of
the sequence confer TLR7 selectivity on the sequence, while
5-substituted C bases of YpG motifs can switch sequences to
being antagonistic.
IMOs have been shown to be well tolerated in mice at doses
up to 10 mg/kg following intraperitoneal, subcutaneous, intravenous or intratumoral administration, and have been optimized
further through modifications to linker structure and length.71
The most advanced molecule in the Idera portfolio is
IMO-2055,72 being developed for the treatment of cancer and
as a vaccine adjuvant. In a Phase I study in treatment-naive
HCV patients, IMO-2125 demonstrated similar efficacy to the
standard of care and was well tolerated.73
However, a Phase II study has been delayed due to atypical
lymphocytic proliferation observed in a 26 week non-clinical
rodent study.74 The company awaits the outcome of a
non-clinical non-human primate study. Oligoribonucleotide
analogues have also been developed as TLR7/8 agonists.75
Poly-ICLC
Poly-ICLC (Hiltonol) is a polyinosinic-polycytidylic acid stabilized
with poly-L-lysine and carboxymethylcellulose. It is a very
stable double-stranded RNA and potent TLR3 agonist with a
strong IFN-inducing ability. Preclinical studies in mice suggest
poly-ICLC and liposome-encapsulated poly-ICLC are safe and
offer broad-spectrum protection against influenza viruses as
well as other respiratory viruses, including respiratory syncytial
virus (RSV) and severe acute respiratory syndrome (SARS) virus
(reviewed in Wong et al.76). However, in a cotton rat model of
RSV or flu infection, efficacious doses of poly-ICLC were also
associated with augmented lung inflammation.77 Nevertheless,
Phase I/II clinical trials have shown poly-ICLC to be safe and efficacious when given to glioblastoma patients, improving their
survival time when added to standard care.78,79 Thus poly-ICLC
has a promising role to play as a safe, effective and broadspectrum antiviral and vaccine adjuvant; as such, a number of
Phase I studies in healthy volunteers are currently under way
(clinicaltrials.gov trial identifiers NCT00773097, NCT01127464,
NCT01299662 and NCT00646152).
Immunoprotection using TLR agonists
Prophylactic administration of a TLR agonist can also be beneficial by promoting an enhanced protective immune response. For
example, a single intranasal dose of an imidazoquinoline mixed
TLR7/8 agonist was shown to significantly reduce nasal viral
titres when given 72 h pre-challenge or 6 h post-challenge with
influenza virus in a rat model.80 Viral inhibition correlated with
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dose and the level of type I IFN induced. Interestingly, a second
pre-infection dose did not offer any additional viral suppression,
and prophylactic administration of the small molecule agonist
was found to be superior to using rat recombinant IFN-a.
The same group have shown that TLR8 agonism can enhance
immune responses in human neonatal antigen-presenting
cells.81 Corixa has published patent applications that detail
several members of their TLR4 receptor agonist platform as potential immunostimulants,82 at least some of which have been
shown to offer protection against viral challenge. For example,
a GlaxoSmithKline group has shown that pre-treatment of mice
with CRX-527 was found to enhance both CD4+ and CD8+ T
cell responses via induction of significant amounts of IL-12,
which offered protection against a challenge by influenza virus
and RSV.83
There have been similar reports of TLR3,84 TLR985 and TLR486
agonists also offering protection in murine models of influenza. A
group at the University of California has synthesized conjugates
of SM-360320 (Figure 2k) in which the parent compound is covalently attached to mouse serum albumin. The presence of the
conjugated group was found to enhance cytokine induction
in vitro, and was designed to keep the TLR7 agonist agent
within the lung when inhaled, thereby reducing the risk of systemic cytokine induction. Pre-treatment with the conjugate in
an infectious model of influenza virus produced significantly
delayed mortality.87
Vaccine adjuvant/immunostimulation using TLR agonists
Several of the agents described above are either being investigated or developed as vaccine adjuvants or general immune
system activators, in addition to several more that are described
briefly below.
Eisai has published on several lipid A analogues that are agonists of the TLR4 receptor. E-6020 is currently in preclinical development for use as a vaccine adjuvant.88,89 Corixa has taken a
non-toxic, truncated lipid A-based TLR4 agonist, monophosphoryl lipid A (MPL),90 into Phase III development as a vaccine
adjuvant.91 The TLR7 ligand, loxoribine, was being developed
by Johnson and Johnson as an immunostimulant,92 but its
development appears to have been discontinued. Idera’s
IMO-2055, known as Amplivax in adjuvant applications, is
being used by Immune Response Corporation alongside its investigational HIV vaccine technology in Phase II trials in HIV
patients.72 IC-31 is a TLR9 receptor agonist that is being used
as an adjuvant in several prophylactic vaccines (partnered with
Novartis). It is currently in Phase I trials.93,94 Dynavax has developed a vaccine technology based on a phosphorothioate
oligodeoxyribonucleotide vaccine containing a short DNA immunostimulatory sequence (ISS) that targets the TLR9 receptor.95
Their most advanced vaccine is Heplisav (ISS-1018), which recently completed a successful Phase III trial for the prevention
and immunotherapy of HBV.96 – 98 ISS-1018 also successfully
completed a Phase II clinical study for the treatment of B-cell
non-Hodgkin’s lymphoma.99 Following promising safety trials,
Dynavax has partnered with AstraZeneca to evaluate ISS-1018
in Phase II trials for asthma.100 The ISSs can be used alone or
linked with antigens. For example, Dynavax’s Phase III trials
are being conducted with ISS-1018 combined with either a
major ragweed allergen or a hepatitis B surface antigen.
796
Challenges
Viral evasion strategies
Viruses have evolved multiple mechanisms to evade the host
immune system. For example, the NS3/4A protease is a
key protein in HCV immune evasion.101 The protein blocks
TLR3-mediated signalling by cleavage of TRIF102 and
RIG-I-mediated signalling by cleavage of IPS-1.103,104 Since inhibition of NS3/4A proteolytic activity directly blocks viral replication
as well as restores immune function to infected cells, drugs
targeting NS3/4A would have dual pharmacodynamics, and
have indeed been shown to be highly efficacious in the
clinic.105 Other viruses evade the immune system in different
ways. Kaposi’s sarcoma-associated herpesvirus (KSHV), a
tumour-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by
incorporating viral homologues of the cellular IRFs.106 The vaccinia virus-encoded proteins A46R and A52R contain TIR
domains that interact with MyD88 and TRIF to inhibit immune
activation.107 Vaccinia virus also encodes N1L, a protein that
blocks TLR signalling by targeting kinases involved in the
cascade.108 The V proteins of paramyxovirus interact with
MDA5 to block immune activation,109,110 while influenza virus
uses its NS1 protein to block the production of IFN.111
While it is possible that these mechanisms could compromise
the efficacy of any therapy that seeks to activate the IFN
response, all of the evasion strategies characterized thus far
act within a virus-infected cell and have no effect on bystander
cells. It is likely that an exogenous immune activator could
mount an antiviral response in uninfected cells, preventing the
spread of the infection.
Safety
The immune system is a double-edged sword. Activating the
immune system can have unforeseen and catastrophic consequences, as witnessed during the TeGenero drug trial.112
Although TGN1412 did not target a PRR, there is some evidence
that might be of concern to companies developing drugs that
induce enhanced immune responses.
Phase I clinical studies with ANA-773 have demonstrated that
a TLR7 agonist can be efficacious and well tolerated; however, it
remains to be seen if this compound ultimately has sufficient
potency to significantly reduce viral load.
Clinical development of several TLR7 agonists has been
suspended due to safety concerns. Anadys suspended a 28 day
Phase Ib study of their TLR7 agonist ANA-975 in HCV patients
due to ‘intense immune stimulation’,48 and have since halted
all development. In mice, a single dose of resiquimod resulted
in a rapid and almost complete depletion of leucocytes from
the blood.113 This depletion lasted 24 h and was caused by the
retention of peripheral blood leucocytes in peripheral organs.
Leucopenia is a recognized feature of infections with viruses
that produce single-stranded RNA (e.g. influenza virus),114 as is
an increased susceptibility to bacterial superinfection in influenza
patients.115
Lymphopenia, together with severe hypotension and flu-like
symptoms, was reported by the Pfizer group in the recent Phase
I trial of PF-4878691.43 Pharmacokinetic –pharmacodynamic
Review
(PK –PD) modelling and preclinical toxicology data suggested that
daily dosing with TLR7 agonists was inappropriate due to exaggerated immune stimulation,48 thus the oral dosing regimen for
PF-4878691 was set at twice weekly. Nevertheless, antiviral activity (measured using an in vitro HCV replicon bioassay) was only
observed in the serum from volunteers who received doses
of PF-4878691 that were associated with mechanism-related
adverse events. The study concluded that increasing potency to
achieve greater antiviral efficacy would also induce more severe
side effects and that the therapeutic index for this mechanism,
based on systemic exposure, was not sufficient to progress at a
safe and effective dose.
CpG ODNs, acting via TLR9, have been implicated in triggering
autoimmune responses, including rheumatoid arthritis, systemic
lupus erythematosus and diabetes.116 – 119 TLR3 activation has
also been associated with lupus nephritis.120
Conclusions
The rationale for targeting TLRs as a means of treating viral
infections is based on the presence of naturally occurring
agonist molecules within invading viruses. Numerous other diseases can potentially be treated with PRR agonists and antagonists,121 – 123 and the field is set to expand. However, further basic
and applied research is required to develop our understanding of
TLRs and their downstream pathways before therapeutic application can become a reality. It is clear that Phase I safety
studies of any TLR agonists will be very closely examined for
both short- and long-term effects. Alternative dosing regimens
may need to be examined to determine whether dosing less frequently than once daily can maintain efficacy while increasing
safety. Targeting compounds to different immune system compartments could also be beneficial in limiting safety concerns.
In this regard, the emerging animal data with the Gilead
Sciences agent GS9620, in which systemic efficacy is observed
in monkeys despite low systemic drug exposure, is encouraging.
Also yet to be tested is whether low doses of a TLR7 agonist,
which does not show systemic pharmacology and is devoid
of significant side effects, can promote enough local TLR7
stimulation in internal organs such as the liver to mediate
antiviral activities in HCV patients.
Acknowledgements
We wish to thank Ben Sidders and Helena Büttner for assistance with the
graphics, and David J. Walsh for help in compiling the literature sources
used in the preparation of this review.
Transparency declarations
All authors were at the time of writing wholly employed by Pfizer Global
R & D.
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