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Delivery of Stimulator of Interferon Genes (STING) Agonist
Using Polypeptide-Modified Dendrimer Nanoparticles in
the Treatment of Melanoma
Pere Dosta, Alexander M. Cryer, Michaela Prado, Michelle Z. Dion, Shiran Ferber,
Santhosh Kalash, and Natalie Artzi*
1. Introduction
Using the innate immune system to
instigate an antitumor response is an
increasingly attractive approach in cancer
immunotherapy. Engagement of pathogen-
associated molecular patterns (PAMPs)
with their respective pattern recognition
receptors (PRRs) was reported to elicit robust
downstream endogenous cytokine produc-
tion and immune cell activation,
[1]
which
is responsible for the potent immune
responses generated by vaccines and against
tumors. Cyclic dinucleotides (CDNs), such
as the second messenger 2 030-cyclic guano-
sine monophosphate-adenosine monophos-
phate (cGMP-AMP or cGAMP), are a class
of PAMPs that are generated upon sensing
cytosolic DNA.
[2]
The production of cGAMP
leads to agonism of stimulator of interferon
genes (STING),
[3]
enacting a type-I inter-
feron (IFN-I)-driven proinflammatory
program including the stimulation of
dendritic cells (DCs) and cross-presentation of tumor antigens
to T-cells, thereby priming them for antitumor effector functional-
ity.
[4]
This bridge between innate and adaptive antitumor immunity
positions STING as a critical regulator of immunosurveillance,
reinforced by studies in STING-deficient mice highlight-
ing increased susceptibility to tumorigenesis and diminished
responsiveness to immunotherapy such as immune checkpoint
inhibitors (ICIs).
[5]
The role of STING signaling in antitumor responses as well as
insufficient endogenous agonism has prompted investigations
into exogenous cGAMP and structural analogs as therapeutics
to promote antitumor immunity.
[6]
Intratumoral (IT) injection
of CDNs has reached phase I clinical trials;
[7]
however, CDNs
are anionic and highly hydrophilic, which restricts their entry into
the cytoplasm where STING resides.
[8]
Consequently, CDNs have
transient interactions with immune cells (e.g., DCs, macrophages)
in the tumor microenvironment (TME) and are rapidly eliminated
from the tumor site.
Biomaterial-based delivery strategies can be leveraged to
improve internalization into cells, therefore augmenting the
activity of adjuvants such as STING agonists.
[9]
Indeed, it has
previously been demonstrated that using nanoparticles (NPs)
of lipidic
[10]
or polymeric
[11]
origin to encapsulate CDNs can
Dr. P. Dosta, Dr. A. M. Cryer, M. Prado, M. Z. Dion, Dr. S. Ferber,
Dr. S. Kalash, Dr. N. Artzi
Institute for Medical Engineering and Science
Massachusetts Institute of Technology
Cambridge, MA 02139, USA
E-mail: nartzi@mit.edu, nartzi@bwh.harvard.edu
Dr. P. Dosta, Dr. A. M. Cryer, M. Prado, M. Z. Dion, Dr. S. Ferber,
Dr. S. Kalash, Dr. N. Artzi
Department of Medicine
Division of Engineering in Medicine
Brigham and Women’s Hospital
Harvard Medical School
Boston, MA 02115, USA
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/anbr.202100006.
© 2021 The Authors. Advanced NanoBiomed Research published by
Wiley-VCH GmbH. This is an open access article under the terms of
the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited.
DOI: 10.1002/anbr.202100006
The activation of stimulator of interferon genes (STING) in the cytosol by cyclic
dinucleotides (CDNs) enhances antitumor immunity through the induction of
proinflammatory cytokines, such as type-I interferons (IFN-I). However, the high
hydrophilicity and negative charge of CDNs hinders their delivery into cells. Here,
by developing a library of cationic polypeptide-modified dendrimers, it is shown
that CDNs can be efficiently delivered intracellularly in vitro and in vivo. With
respect to naked dendrimers, generation-5 polyamidoamine (G5-PAMAM) den-
drimers modified with arginine or with a mixture of arginine/lysine polypeptides
affords higher CDN-packaging capacity and leads to higher activation of IFN-I
and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
proinflammatory signaling pathway. In the B16-F10 murine model of melanoma,
the intratumoral administration of a synthetic CDN via arginine-modified G5-
PAMAM dendrimers at a low dose induces strong antitumor responses and
inhibits tumor growth. It is also shown that the combination of this therapy with
immune checkpoint blockade (ICB) further improves the therapeutic outcomes.
Cationic polypeptide dendrimers may be advantageous in the delivery of gene-
based immunomodulators for the treatment of solid tumors.
RESEARCH ARTICLE
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improve their antitumor activity. However, liposomes suffer
from poor packaging capacity and limited storage stability and
the encapsulation of small hydrophilic molecules within poly-
meric NPs remains challenging.
[12]
We reasoned that stable elec-
trostatic complexes could be formed between anionic CDNs and
cationic NPs, which would act to improve their stability, cytosolic
localization, and endosomal escape, resulting in greater antitu-
mor responses.
Dendrimers have an established history as drug delivery
vehicles
[13]
and have been used in immunotherapy as accessories
in antibody–drug conjugates or in vaccine formulations.
[14]
Polyamidoamine (PAMAM) dendrimers are hyperbranched
polymers consisting of tertiary amines throughout the den-
drimer core and terminal primary amines, the number of which
increases with each layer of branching, known as a generation.
[15]
This defined architecture yields cationic, low- polydispersity NPs
with many surface-reactive sites per NP. It has been noted that
highly localized cationicity is not well tolerated on a cellular
level,
[16]
with the external primary amines being the principal
drivers of this toxicity. Therefore, we leveraged the reactivity
of these amines to functionalize dendrimers with basic polypep-
tide motifs consisting of arginine, lysine, or histidine. By replac-
ing external amines with biocompatible amino acids, the
cytotoxicity is significantly attenuated, cellular internalization
and transfection efficiency are improved, and endosomal escape
can be better controlled.
[17]
In addition, it has been demonstrated
that these polypeptides can influence the nanoparticle composi-
tion, allowing the design of cell-specific nanoparticle formula-
tions.
[18]
Endowed with these properties, we hypothesized that
polypeptide-functionalized dendrimers could effectively complex
with CDNs and be internalized by immune cells, enhancing
CDN activity in vitro and in vivo. We investigated the physico-
chemical properties of the polypeptide-modified dendrimers,
their ability to deliver the synthetic CDN ADU-S100 to immune
cells, biocompatibility, cellular uptake, and antitumor efficacy
in vivo, as well as the associated immune responses.
2. Results
2.1. Synthesis of Polypeptide-Modified Dendrimers
We developed a library of dendrimers with diverse surface mod-
ifications using cationic polypeptides that can complex with CDNs
and form a wide range of NPs with distinct physicochemical
properties. Synthesis of polypeptide-conjugated dendrimers was
performed via a two-step reaction. First, the PAMAM G5 den-
drimer was modified with a bifunctional succinimidyl-[(N-malei-
midopropionamido)-diethyleneglycol]-ester linker [SM(PEG)
2
]by
conjugating the primary dendrimer amines to the succinimidyl
group. Then, the modified dendrimers were conjugated with
cationic polypeptide moieties through sulfhydryl–maleimide con-
jugation of the thiol group of cysteine-terminated polypeptides to
the maleimide group of the SM(PEG)
2
linker, obtaining around 79
peptides per dendrimer (Figure 1A and Figure S9, Supporting
Information). Polypeptide-modified dendrimers were purified by
dialysis and their molecular structure characterized by
1
H-NMR.
The chemical structure of the resultant dendrimerswas confirmed
by the presence of signals associated with the conjugated polypep-
tides (Figure S1–S8, Supporting Information).
To assess the effect of the polypeptides on our dendrimer for-
mulations, size (hydrodynamic diameter) and surface charge val-
ues were obtained using dynamic light scattering (DLS) and zeta
potential measurements, respectively (Figure 1). Whereas the
unmodified dendrimers were small (5.47 0.05 nm) and posi-
tively charged (14.77 6.32 mV), the end-modified dendrimers
were larger, ranging from 7 to 10 nm, and those modified with
arginine (D-CR3), lysine (D-CK3), and a mixture of lysine/histi-
dine (D-K/H) and arginine/lysine (D-R/K) showed an increase in
surface charge compared to nonmodified dendrimers (ranging
from þ24 mV to þ31 mV). In contrast, dendrimers modified
with histidine (D-CH3) or a mixture of histidine/arginine
(D-R/H) were less positive (þ10 mV) than nonmodified
dendrimers (þ15 mV). In addition, when the dendrimer
was only modified with a bifunctional SM(PEG)
2
linker (without
polypeptides), the zeta potential was markedly more negative
(7.07 0.34 mV). The addition of different polypeptides can
be used to tune the final nanoparticle surface charge, ranging
from neutral to positive.
2.2. Formulation and Biophysical Characterization of
Polypeptide-Modified Dendrimers
The CDN complexation efficacy of the newly synthesized
dendrimers was evaluated by agarose gel electrophoresis at dif-
ferent dendrimer/CDN ratios (w/w) (Figure 2 and Figure S10,
Supporting Information). A fluorescently labeled CDN [termed
CDN-F; c-(Ap-8-Fluo-AET-Gp)] was used to assess the complexa-
tion efficiency. Complexes prepared with unmodified dendrimer
and fluorescent CDN (CDN-F) revealed free CDN-F at ratios
below 64:1 dendrimer:CDN-F (w/w), while at a ratio of 64:1
complete CDN-F complexation was observed. D-CR3 showed
complete CDN-F retardation at 16:1 D-CR3/CDN-F ratios, sug-
gesting a more efficient complexation of CDN-F (0.5 CDN-F mol-
ecules per unmodified dendrimer vs 2 CDN-F molecules per
D-CR3). In contrast, D-CK3 required dendrimer-to-CDN-F ratios
similar to unmodified PAMAM dendrimer to achieve full CDN-F
complexation. Moreover, D-CH3 was not able to complex the
CDN-F at any of the tested ratios (Figure S10, Supporting
Information). These results suggest that, compared to unmodi-
fied PAMAM dendrimer, D-CR3 significantly increases the num-
ber of CDN molecules per dendrimer NP, whereas dendrimers
modified with lysine and histidine polypeptides contain a similar
or lower number of CDN molecules per dendrimer NP than the
unmodified dendrimer.
2.3. In Vitro Selection of Polypeptide-Modified Dendrimer for
Efficient ADU-S100 Delivery
Given the importance of interferon regulatory factor (IRF)3 and
nuclear factor (NF)-κBinefficient STING signaling, we assessed
the ability of functionalized dendrimers to deliver ADU-S100 and
stimulate IRF3 and NF-κB responses in human monocytic THP-
1 Dual cells (Figure 3). THP-1 Dual cells allow the simultaneous
study of the IRF3 pathway, by assessing the activity of a secreted
luciferase, and the NF-κB pathway, by monitoring
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Figure 1. Synthesis and characterization of polypeptide-modified dendrimers. A) Different polypeptides were used for the synthesis of a new family of
end-modified dendrimers (arginine-modified dendrimer, D-CR3; lysine-modified dendrimer, D-CK3; histidine-modified dendrimer, D-CH3; 50% arginine–
50% histidine modified dendrimer, D-R/H; 50% lysine–50% histidine modified dendrimer, D-K/H; and 50% arginine–50% lysine modified dendrimer,
D-R/K). B) Size (determined by DLS) and (C) Z-potential measurements of end-modified dendrimers (n¼3); data shown as mean SD.
Figure 2. Agarose retardation assay of polypeptide-modified dendrimer–CDN-F polyplexes. Polyplexes were formed using CDN-F and different
polypeptide-modified dendrimers at indicated w/w ratios and loaded onto an agarose gel to assess CDN-F mobility by electrophoresis for
A) nonmodified dendrimer and B) D-CR3 formulation.
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the activity of secreted embryonic alkaline phosphatase (SEAP).
D-CR3 and D-R/K achieved higher IRF3 activation than the
unmodified dendrimer. In contrast, D-CH3 or D-K/H showed
the lowest level of activation, similar to that of free ADU-S100.
D-CK3 or D-R/H showed high IRF3 activation at higher
ADU-S100 doses (500 n
M
). A similar trend was seen in terms
of NF-κB activation for all formulations (Figure 3B). A 16-fold
NF-κB increase was observed when ADU-S100 was delivered
using either the D-CR3 or D-R/K formulation at 250 n
M
. In con-
trast, no NF-κB activation was detected when D-CH3 or D-K/H
was used, and NF-κB expression was only detected at high
ADU-S100 doses when D-CK3 and D-R/H formulations were
used. In addition, we did not observe activation of IRF3 or
NF-κB in STING-deficient THP-1 Dual cells (Figure S12,
Supporting Information).
To determine if cellular internalization could account for
the differences in IRF3 and NF-κB, uptake of polypeptide-
modified dendrimers was assessed in THP-1 cells (Figure 3C).
Dendrimers were fluorescently labeled, and their uptake was
quantified by flow cytometry. We confirmed that formulations
showing high IRF3 activation exhibited higher cellular internali-
zation than those with low IRF3 activation. D-CR3, D-CK3, and
D-R/K were more efficiently internalized than the unmodified
dendrimer and D-CH3 and D-R/H showed the lowest internali-
zation. More than 90% of the cells showed nanoparticle uptake
for all of the dendrimer formulations, except those of the D-CH3
formulation. Interestingly, D-CK3 was efficiently internalized,
but less potent IRF3 activation was observed compared to D-
CR3 and D-R/K. This can stem from the fact that less CDN is
delivered per (Figure S10, Supporting Information). An increase
in the number of dendrimer surface amines in the case of D-CK3
can enhance its internalization. However, its lysine residues have
a lower pKa than arginine in D-CR3, which will impart weaker
electrostatic interactions between the D-CK3 and the CDN com-
pared to that with D-CR3.
To ensure the biocompatibility of functionalized dendrimers,
the viability of THP-1 cells was determined 24 h post ADU-S100
delivery using our library of dendrimer formulations
(Figure 3D). The cell viability of the polypeptide-modified den-
drimers was higher than that of unmodified dendrimers at a
CDN concentration of 500 n
M
, suggesting that the addition of
natural peptides can increase the biocompatibility of dendrimer
NPs. D-CR3 and D-R/K, which were highly efficient at delivering
ADU-S100, showed different cell viability profiles. While D-CR3
Figure 3. In vitro screening of polypeptide-modified dendrimers. A,B) Potent IRF3 and NF-κB activation following CDN delivery using polypeptide-
modified dendrimers. The polypeptide-modified dendrimers were screened for A) IRF3 activation and B) NF-κB activation using THP-1 Dual cells.
Cells were treated with different concentrations of ADU-S100 prior to measuring IRF3 and NF-κB 24 h posttreatment. Values were normalized to
untreated cells. C) Cell internalization efficiency of polypeptide-modified dendrimers in THP-1 Dual cells. Cells were treated with polypeptide-modified
dendrimers containing ADU-S100 at the final concentration of 125 n
M
, and fluorescence expression per cell was determined 2 h posttransfection by flow
cytometry. D) Cell viability of different concentrations of polypeptide-modified dendrimers was analyzed 24 h posttreatment. Samples were normalized to
untreated cells. Data are represented as mean SD (n¼3). Multiple comparisons among groups were determined using one-way ANOVA followed by a
Fisher’s LSD test. P-value: *p<0.05, **p<0.01, ***p<0.001.
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cell viability was greater than 80%, the D-R/K formulation
showed some toxicity at high CDN concentrations (500 n
M
).
Taking that into account, we selected the D-CR3 formulation
to deliver ADU-S100 in vivo.
2.4. IT Delivery of ADU-S100 with D-CR3 NPs Inhibits B16-10
Tumor Growth and Increases Survival
The recent approval of different checkpoint inhibitor therapies
for the treatment of immunogenic tumors has opened the door
to the treatment of different cancers; however, the development
of more efficacious therapies is still needed. Recently, it has been
demonstrated that ICIs antibodies, such as antiprogrammed cell
death protein 1 (anti-PD-1), have a synergistic effect with STING
agonist therapies.
[19]
Here, we assessed whether combination
therapy with D-CR3 NPs complexed with ADU-S100 (termed
D-CR3-ADU) and anti-PD-1 showed a significant benefit com-
pared to free ADU-S100 alone or D-CR3-ADU NPs alone in a
syngeneic subcutaneous murine melanoma model. ADU-S100
therapy (0.5 μg per dose) was initiated when the tumor volume
reached 100 mm
3
and was delivered intratumorally (IT) four
times, every 4 days, over the course of 12 days. Anti-PD-1 was
administered via intraperitoneal injection, in the combination
therapy groups, 24 h after ADU-S100 delivery (four injections,
every 4 days). Tumor size was monitored every other day
(Figure 4A). Control tumors treated with phosphate buffered
saline (PBS) injection grew rapidly, whereas tumors treated with
ADU-S100-containing formulations showed delayed tumor
growth (Figure 4B). D-CR3-ADU-treated mice showed slower
tumor growth and increased survival when compared to ADU-
S100. The combination of D-CR3-ADU and anti-PD-1 signifi-
cantly inhibited tumor growth compared to ADU-S100 alone.
A similar trend was observed when free ADU-S100 was com-
bined with anti-PD-1, where reduced tumor growth and
improved survival were observed. However, the NP-based deliv-
ery of ADU-S100, D-CR3 NPs, further inhibited tumor growth
and enhanced survival compared to the free drug form.
2.5. IT Delivery of CDN Modifies the Immune Profile
of the Tumor Microenvironment
To understand the mechanism by which the D-CR3 formulation
containing ADU-S100 resulted in enhanced antitumor activity,
we conducted immunohistochemical analysis of the TME 7 days
after delivery of the last IT dose (19 days after treatment
initiation) (Figure 5). We found that tumors treated with
D-CR3-ADU have less actively proliferating cells [determined by
hematoxylin and eosin (H&E) and Ki-67 staining] compared to
tumors treated with free ADU-S100, which is further reduced when
D-CR3-ADU is combined with anti-PD-1. In addition, infiltration of
CD8
þ
T-cells and upregulation of the immune checkpoint mole-
cule PD-L1 on tumor cells were observed when tumors were treated
with D-CR3-ADU compared to untreated tumors or those treated
with free ADU-S100. Similar results were obtained for the combi-
nation with anti-PD-1 compared to D-CR3-ADU alone.
Next, we conducted immunophenotyping of the TME and
tumor-draining lymph node (tdLN) 3 days after a single IT injec-
tion of ADU-S100, with or without D-CR3 complexation,
Figure 4. D-CR3-CDN polyplex therapy reduces tumor growth and increases animal survival, which is further enhanced when combined with anti-PD-1.
A) Study design of subcutaneous B16-F10 model. NP administration was initiated 8 days following tumor induction, when the tumor size reached
100 mm
3
. Intraperitoneal administration of anti-PD-1 was performed 24 h post NP administration. B) Tumor growth was monitored every other
day after the first administration. 0.5 μg of ADU-S100 delivered with D-CR3 NP, with and without anti-PD-1, resulted in statistically reduced tumor burden
compared to free ADU-S100. Data are represented as mean SD (n¼5). Multiple comparisons among groups were determined using uncorrected
Dunn’s test. C) Kaplan–Meier survival curves of mice treated with the indicated formulation using a 1000 mm
3
tumor volume or poor body condition as
the endpoint criterion. Statistical analysis (n¼5) was performed using a log-rank Mantel–Cox test.
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as well as with or without anti-PD-1 combination therapy. Flow
cytometry analysis of tumoral immune infiltrates (Figure 6A)
revealed an increase in the CD8
þ
/CD4
þ
T-cell ratio—a common
prognosticative biomarker of responsiveness to immunother-
apy
[20]
—in the D-CR3-ADU-treated groups, compared to
untreated and to free-ADU-S100-treated tumors. We also wit-
nessed a slight increase in the CD8
þ
/T
reg
(defined as CD4
þ
FOXP3
þ
) ratio in tumors treated with D-CR3-ADU and anti-
PD-1 and a trend towards an increased presence of tumor-
infiltrating T-cells (Figure 6B). Remarkably, the number of NK
cells was higher in all treated groups compared to untreated
tumors, but significantly more in the tumors treated with
D-CR3-ADU. A significant increase in activated granulocytic cells
(CD11b
þ
Gr-1
þ
, which includes monocytes, neutrophils, eosino-
phils, and myeloid-derived suppressor cells) was seen in all treat-
ment groups (Figure 6C). Moreover, increased expression of the
DCs activation marker CD86 was observed within the TME in all
the treated groups, compared to untreated tumors (Figure 6D).
Interestingly, DC infiltration in the tdLN was elevated in all the
treatment groups, with D-CR3-ADU in combination with aPD-1
having the highest DC expression (Figure 6E). Also, higher PD-1
expression was observed in CD8
þ
and CD4
þ
cells in the tdLN
when ADU-S100 was delivered with a D-CR3 NP (Figure 6F–G).
3. Discussion
The generation of endogenous CDNs in response to cytoplasmic
double-stranded DNA is an evolutionarily conserved innate
immune defense mechanism, resulting in the production of
IFN-I. Traditionally recognized as a response to viral infection,
this process has been increasingly implicated in antitumor
immunity.
[21]
Indeed, the potency of CDNs spurred efforts to
produce structural analogs of the endogenous adjuvant
Figure 5. D-CR3-ADU polyplex therapy reduces cancer cell proliferation, increases CD8
þ
cytotoxic T-cell infiltration and PD-L1 cancer cell expression.
Representative H&E staining (left, scale bar: 100 μm; right: scale bar: 50 μm), Ki-67 staining (scale bar: 100 μm), immunofluorescence staining of
CD8
þ
T-cells (red, CD8; blue, DAPI; scale bar: 100 μm), and PD-L1
þ
cancer cells (PD-L1þ, green; scale bar: 100 μm) in tumors harvested on day 19.
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cGAMP for use as antitumor therapeutics, such as ADU-S100.
[6a]
Here, we designed an NP system to overcome the delivery bar-
riers intrinsic to hydrophilic, anionic molecules such as CDNs
that require cytoplasmic delivery to enable their function.
Previous efforts for IT delivery of CDNs centralized around
encapsulation-based strategies whereby the CDN was housed
inside the NP. For instance, liposomes
[19a,22]
and polymeric
NPs such as polymersomes,
[11]
poly lactic-co-glycolic acid,
[23]
and acetalated dextran
[24]
have been used for the IT administra-
tion of CDNs. These formulations typically rely on intrinsic ele-
ments of the synthesized material (e.g., pH responsiveness,
endosomal disruption) for effective delivery. Depending on
the formulation and batch, the amount of CDN may vary, espe-
cially as hydrophilic molecules are difficult to encapsulate with
complete fidelity, and their storage stability can be compromised.
We present a two-component dendrimer system that is avidly
Figure 6. IT delivery of the D-CR3-CDN polyplex shifts the immune TME composition. A) Representative flow cytometry dot plot and quantification of
tumor-infiltrating CD4
þ
and CD8
þ
T cells’ratio. B) Ratio of CD8
þ
to Tregs cells in the TME. C) Flow cytometric quantification of the number of lym-
phocytes cells (CD3; CD3
þ
), natural killer cells (NK; NK1.1
þ
), macrophages (MΦ; CD11b
þ
F4/80
þ
), granulocytic cells (Gran; CD11b
þ
Gr-1
þ
), and den-
dritic cells (DC; CD11c
þ
MHCII
þ
) per milligram of tumor. D) Quantification of CD86 expression by IT DCs. E) Quantification of DCs in tdLN. F,G)
Quantification of PD-1
þ
expression by CD8
þ
and CD4
þ
cells in tumor-draining lymph node. Data are represented as mean SEM (n¼5).
Multiple comparisons among groups were determined using one-way ANOVA followed by a posthoc test. P-value: *p<0.05, **p<0.01, ***p<0.001.
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internalized by cells and requires only simple mixing that is
tuned to incorporate almost all CDN molecules, which has poten-
tial for scale-up. We,
[13b]
and others,
[13a]
have previously used
PAMAM dendrimers as nucleic acid delivery systems, which
is facilitated by electrostatic interactions between the cationic
dendrimer and anionic nucleic acid. We modified dendrimers
with polypeptide moieties with a dual-purpose function to
enhance the cationic nature of the dendrimer by virtue of addi-
tional amines while simultaneously attenuating the toxicity orig-
inating from unmodified PAMAM dendrimers. This was
achieved using a heterobifunctional linker that permits amide
bond formation with the dendrimer and Michael addition with
the thiol of polypeptide motifs of lysine, arginine, histidine, or
combinations thereof. Successful conjugation was confirmed
by
1
H-NMR and reflected by the increased size of all polypep-
tide-modified dendrimers and changes in surface charge
(Figure 1). Histidine-containing formulations were less posi-
tively charged than their lysine or arginine counterparts at phys-
iological pH due to the lower affinity for protons, resulting in
partial protonation. This was further reflected by the greater com-
plexation of ADU-S100 using D-CR3 compared to that of unmod-
ified dendrimer (Figure 2 and Figure S10, Supporting
Information). A combination of the increased amine groups
from polypeptide-modified dendrimer and cationicity was theo-
rized to be responsible for our observations, whereby D-CR3
could electrostatically entrap the most CDN-F as arginine has
the highest pKa values (pKa of side chain ¼12.48, 10.53, and
6.00, respectively, for arginine, lysine, and histidine). This is ben-
eficial as less dendrimer is required to deliver the same dose of
CDN-F, relative to other formulations.
Differential activation of IRF3 and NF-κB was observed
when we screened our formulations in vitro (Figure 3).
Greater activation of IRF3 was seen with NP formulations com-
pared to equimolar concentrations of free ADU-S100, particu-
larly beyond 250 n
M
. Dendrimer formulations containing
arginine were found to be the most potent inducers of IRF3
and NF-κB, implying that these particles were internalized effec-
tively and successfully delivered ADU-S100 to the cytoplasm. The
activation of IRF3, which is downstream of STING, leads to tran-
scription of IFN-Is and NF-κB signaling further elicits proinflam-
matory mediator activation.
[25]
Indeed, modification of
dendrimers with arginine has been described to improve the
transfection efficiency while maintaining biocompatibility,
[26]
matching our observations. NP formulations were differentially
internalized into immune cells in a modification-dependent
manner (Figure 3D). D-CH3 formulations were less efficiently
internalized than D-CR3 and D-CK3, which would explain the
lower IRF3 activation despite the potent endosomal escape prop-
erties of histidine.
[27]
Lysine-modified dendrimers were internal-
ized to a greater extent but were less potent activators of IRF3
compared to arginine-modified dendrimers. This may be due
to the lower number of CDN delivered per dendrimer molecule,
which may affect overall uptake and endosomal escape capaci-
ties. The mixture of arginine and lysine was well internalized
and potently activated IRF3 and NF-κB; however, this formula-
tion was more cytotoxic than the arginine alone.
Based on our in vitro data, we took forward the D-CR3 formu-
lation, featuring high CDN complexation and biocompatibility,
for in vivo evaluation. Previous reports demonstrated that high
doses of IT delivered CDN (up to 20 μg) induced tumor regres-
sion; however, the efficacy is severely diminished at lower
doses.
[28]
Here, we demonstrated that our NP formulation pre-
vented growth of a poorly immunogenic murine melanoma
model using 0.5 μg of ADU-S100, an effect that was further aug-
mented when used in combination with anti-PD-1 (Figure 4).
Interestingly, we observed that a low dose of free ADU-S100, par-
ticularly used in combination with anti-PD-1, was able to attenu-
ate tumor growth, although less effective than the D-CR3-ADU.
It is known that the intensity of STING signaling has a notable
effect on the type and magnitude of the immune response. Low-
dose IT administration of ADU-S100 was found to be more
immunogenic than ablative (over 100 μg) doses and was optimal
for combination with immune checkpoint blockade (ICB).
[19b]
Even by potentially dosing with immunogenic amounts of free
ADU-S100, our NPs were still able to improve antitumor
responses compared to ADU-S100. Notably, without anti-PD-1,
D-CR3-ADU was still able to attenuate tumor growth and to a
much greater extent than equivalent free ADU-S100 alone.
Indeed, the efficacy of our NP is comparable to that of other elec-
trostatic polymeric NP-CDN formulations
[29]
despite using lower
CDN dose.
These observations led us to dive deeper into the mechanism
underpinning antitumor efficacy. A decrease of proliferating
cells and an increase of CD8
þ
T-cell infiltration was observed
when ADU-S100 was delivered using D-CR3 compared to
tumors treated with free ADU-S100, which are hallmarks of anti-
tumor immune activation and induction of an immunogenic
TME (Figure 5). This was further corroborated by the upregula-
tion of the immune checkpoint molecule PD-L1 on tumor cells,
which is known to occur in response to IFN-β
[30]
—a direct down-
stream product of STING signaling—or IFN-γproduced by
tumor-infiltrating T-cells.
[31]
This suggests that increased
STING signaling within the TME, facilitated by the D-CR3,
resulted in greater production of IFNs such as IFN-β, leading
to greater CD8
þ
T-cell infiltration and PD-L1 expression.
Indeed, the improved therapeutic outcome seen with the inclu-
sion of anti-PD-1 may be in part due to the reduced immunosup-
pressive PD-1/PD-L1 interactions that would otherwise occur
upon STING-mediated upregulation of PD-L1.
In addition, flow cytometry analysis of the tumor immune
infiltrate (Figure 6) confirmed an increase in CD8
þ
/CD4
þ
T-cell
and CD8
þ
T-cell/T
reg
ratios in the D-CR3-ADU-treated groups
compared with ADU-S100-treated or untreated tumors.
Increased tumoral T
reg
presence is a negative clinical prognostic
indicator across a broad range of cancers,
[32]
and thus our
T-cell-related observations were encouraging. Interestingly, the
number of NK cells was higher in all treated groups compared
to untreated tumors, but significantly higher (twofold change) in
the tumors treated with D-CR3-ADU. It has been shown that
mobilization of NK cells in response to STING agonism can
induce tumor regression independently of CD8
þ
T-cells and is
a direct consequence of IFN-I production.
[33]
A significant
increase in activated granulocytic cells was evident in all treat-
ment groups, which is consistent with the notion that granulo-
cytes, particularly neutrophils, are among the first responders
that migrate to the site of inflammation—in this case induced
by STING activation. Interestingly, a trend of increase in the
number of macrophages (CD11b
þ
F4/80
þ
) and DCs (CD11c
þ
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MHC-II
þ
) compared to untreated tumors has only been observed
in groups treated with anti-PD-1, which may be responsible for
the improved efficacy witnessed when ADU-S100 was combined
with anti-PD-1, given the role of DCs and macrophages in
STING-mediated antitumor efficacy.
[34]
Moreover, increased
expression of the activation marker CD86 was evident on DCs
within the TME treated with free ADU-S100 or complexed using
D-CR3. Taken together, these immunological data show that
D-CR3-ADU administration can remodel the TME to a more
inflamed and immunoactive environment. Interestingly, we
observed an increase in the presence of DCs in the tdLN-treated
mice, which was most pronounced in D-CR3-ADU-treated mice.
This suggests that a higher level of antigen presentation may
have taken place as more DCs migrated from the TME to the
tdLN to then cross-present to and prime CD8
þ
T-cells.
4. Conclusion
In the current study, we describe a library of polypeptide-
modified dendrimers that can be used to effectively deliver
CDN in vitro and in vivo. We found that arginine-modified den-
drimers permit higher CDN complexation efficiency compared
to nonmodified dendrimers. Our data show that D-CR3-ADU
formulations are efficiently internalized into THP-1 cells, result-
ing in the activation of IRF3 and NF-κB, and present lower tox-
icity than nonmodified dendrimers. In vivo results demonstrated
that IT delivery of the D-CR3-ADU formulation in combination
with anti-PD-1 induced strong tumor regression in a murine
melanoma model. This polypeptide-modified dendrimer-based
system can serve as a platform for efficient delivery of STING
agonists in poorly immunogenic tumors, providing new oppor-
tunities for combination cancer therapy.
5. Experimental Section
Materials: All reagents and solvents were purchased from Sigma Aldrich
unless otherwise stated. Polypeptides (H─Cys─Arg─Arg─Arg─NH
2
,
H─Cys─Lys─Lys─Lys─NH
2
, and H─Cys─His─His─His─NH
2
) were
obtained from CPC Scientific with a purity of at least 90%. Generation
5 PAMAM dendrimer was purchased from Dendritech. Fluorescent
CDN (Ap-8-Fluo-AET-Gp) was obtained from Biolog, Inc.
Fluorescent Labeling of Dendrimer: G5 PAMAM dendrimer was fluores-
cently tagged with AlexaFluor 594 carboxylic acid, succinimidyl ester
(AF594) at 1:0.5 molar ratio (PAMAM dendrimer:AF594). Briefly, 14.22 μL
of AF594 at 10 mg mL
1
in dimethylformamide (DMF) was mixed with
10 mg PAMAM dendrimer in 0.1
M
bicarbonate buffer (pH 8.5) and
reacted for 1 h at room temperature in the dark. Fluorescent dendrimers
were washed with PBS and recovered by centrifugal filtration (10 kDa
MWCO Amicon Ultra-0.5 mL Centrifugal Filters, Millipore) at 14 000 g
for 10 min at 4 C.
Synthesis of Polypeptide-Modified Dendrimers: PAMAM dendrimer was
modified with bifunctional SM(PEG)
2
linker (Thermo Fisher), whereby
10 mg of dendrimer was dissolved in 500 μL of 0.1
M
phosphate buffer
at pH 7.5 and 500 μL PBS containing 19 mg of SM(PEG)
2
was added drop-
wise. The mixture was allowed to react for 30 min at room temperature.
The dendrimer was then mixed with equimolar SM(PEG)
2
to polypeptide
ratio and reacted at room temperature for 3 h. The polypeptide-modified
dendrimer was purified by dialysis against PBS for 2 days at 4 ºC. For struc-
tural analysis, modified dendrimers were freeze dried, dissolved in D
2
O,
and analyzed by
1
H-NMR, and recorded using a 400 MHz Varian NMR
spectrometer (NMR Instruments, Clarendon Hills, IL).
Quantification of the Ratio of Peptides per Dendrimer by UV–Vis
Spectroscopy: NanoDrop 2000c (Thermo Scientific, Tewksbury, MA) was
used to characterize the polypeptide-modified dendrimer. Absorbance
at 275 nm was used to determine the ratio of peptide to dendrimer.
Because the fluorescent dendrimer also absorbs at 275 nm, the equation
that follows was used to deconvolute the signals from each component
(dendrimer or peptide) and obtain the peptide-to-dendrimer ratio
(Equation (1)). The individual absorption coefficients were calculated from
the respective UV–vis standard curves.
ADendF &Peptide ¼ADendFþAPeptide (1)
εDendF &Peptide cDendF &Peptide ¼εDendFcDendFþεPeptide cPeptide
(2)
cDendF & Peptide ¼cDendF(3)
cpeptide
cDendF
¼εDendF &Peptide εDendF
εPeptide
(4)
Equation (1) is the peptide-to-dendrimer ratio calculation.
Biophysical Characterization of Polypeptide-Modified Dendrimers:To
assess CDN complexation, different fluorescent CDN (Ap-8-Fluo-AET-
Gp) to dendrimer ratios (w/w) between 0.5:1 and 64:1 were studied.
Dendrimer–CDN complexes were freshly prepared; for example, to form
100 μL of D-CR3 polyplexes, 50 μL of CDN at 0.05 mg mL
1
was mixed
with 50 μL of D-CR3 at 0.8 mg mL
1
. The dendrimer solution was added
to CDN solution, pipette mixed, and incubated at room temperature for
10 min. Dendrimer–CDN polyplexes were loaded in 4% E-Gel Precast
Agarose Gels (Thermo Fisher), run following the manufacturer’s instruc-
tions, and visualized in fluorescence mode. The size and surface charge
were determined by DLS and zeta potential measurements, respectively.
Polyplexes were prepared as previously described and after 10 min of incu-
bation at room temperature, 100 μL of dendrimer was diluted with 900 μL
of PBS and analyzed using a Zetasizer Nano ZS equipped with a He–Ne
laser (λ¼633 nm) at a scattering angle of 137(Malvern Instruments Ltd,
United Kingdom).
CDN Loading Efficiency:Dendrimer–CDN complexes were freshly pre-
pared as previously reported. To form 100 μL of D-CR3-CDN polyplexes,
50 μL of CDN at 0.05 mg mL
1
was mixed with 50 μLof
D-CR3 at 0.8 mg mL
1
.Toform100μL of dendrimer–CDN polyplexes,
50 μL of CDN at 0.05 mg mL
1
was mixed with 50μL of unmodified den-
drimer at 3.2 mg mL
1
. Noncomplexed CDN-F molecules were purified by
centrifugal filtration (10 kDa MWCO Amicon Ultra-0.5 mL Centrifugal
Filters, Millipore) at 14 000 gfor 10 min at 4 C and quantified by fluores-
cence (λ
ex
¼490; (λ
em
¼530) using a multimodal plate reader (TECAN).
Cell Lines:Mus musculus skin melanoma (B16-F10, from ATCC) was
maintained in Dulbecco’s minimum essential medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), 100 U mL
1
penicillin, and
100 μgmL
1
streptomycin, 2 m
ML
-glutamine. Human monocyte THP-1
Dual cells and THP-1 Dual KO-STING cells (InvivoGen) were maintained
in RPMI 1640 supplemented with 10% FBS, 2 m
ML
-glutamine,
25 m
M
HEPES, 100 μgmL
1
Normocin, Zeocin, 10 μgmL
1
Blasticidin
(InvivoGen), and 100 U mL
1
penicillin and 100 μgmL
1
streptomycin.
All cell lines were maintained in a humidified incubator at 37 C, 5% CO
2
.
In Vitro Evaluation of IRF and NF-κB Pathways: Human monocyte THP-1
Dual cells and THP-1 Dual KO-STING cells were seeded in 96-well plates at
110
5
cells per well and incubated with the different dendrimer NP for-
mulations or free CDN at CDN concentrations ranging from 0 to 500n
M
.
At 24 h post-treatment, IRF activity was examined using the QUANTI-Luc
reagent (InvivoGen) and NF-κB activity was determined using the QUANTI-
Blue reagent (InvivoGen) according to the manufacturer’s instructions.
Cell Viability: Cell viability was assessed using the MTS assay (Promega)
as instructed by the manufacturer. After 24 h treatment with varying
dendrimer–CDN NP formulations, MTS reagent was added to the cells
to achieve a final MTS concentration of 20% (v/v). Cells were incubated
at 37 C, 5% CO
2
up to 2 h and absorbance was measured at 490 nm using
a multimodal plate reader (TECAN).
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Cellular Uptake: THP-1 Dual cells were seeded in 12-well plates at
510
5
cells per well and treated with fluorescent polypeptide-modified
dendrimer at a concentration equivalent to 125 n
M
CDN. After 2 h at
37 C, 5% CO
2
, excess NPs were removed by washing with PBS and cells
were collected by centrifugation. Following fixation with 1% (w/v) parafor-
maldehyde, cells were analyzed by flow cytometry using a BD LSRFortessa
flow cytometer (BD Biosciences).
In Vivo Therapeutic Efficacy: Female C57BL/6 mice (6–8 weeks old) were
purchased from Charles River. All mouse procedures were conducted at
the Hale Building for Transformative Medicine and Koch Institute for
Integrative Cancer Research at the Massachusetts Institute of Technology
(MIT) under the protocol approved for this study by the Institutional
Animal Care and Use Committee (IACUC). To induce tumors, 5 10
5
B16-F10 cells in 100 μL of Hanks’balanced salt solution (HBSS) were
injected subcutaneously into the right flank of the mice. Upon reaching
50–100 mm
3
, tumors were intratumorally injected with 30 μL of PBS con-
taining D-CR3-CDN polyplexes or free CDN (0.5 μg). Mice were injected
four times with treatments spaced 4 days apart. Mice receiving ICB were
injected intraperitoneally with 100 μg of anti-PD-1 (clone RMP1.14, Bio X
Cell) 24 h post IT injection. The tumor size was measured every other day
via caliper measurements, and the tumor volume was calculated using the
equation V¼(LWH)/π÷6. Body weight was measured contempora-
neously with tumor volume. Mice were euthanized when tumors reached a
volume of 1000 mm
3
or for otherwise poor body condition.
Analysis of Immune Infiltrate: B16-F10 tumors were harvested,
chopped, and digested in a solution of HBSS supplemented with collage-
nase I, II, and IV (100 ng mL
1
), and DNase I (1 μgmL
1
) for 2 h at 37 C.
TdLNs were harvested and mechanically dissociated. Single-cell suspen-
sions of tumors and tdLNs were filtered through a 40 μm nylon cell
strainer. Tumor cells were further treated with ACK Lysing Buffer (Gibco).
Cells were counted and stained with fluorescent antibodies at a concen-
tration of 1 10
6
cells mL
1
in 100 μL cell-staining buffer (BioLegend).
Intracellular staining was performed using a FoxP3 Staining Buffer Set
(Miltenyi) according to the manufacturer’s protocol. The following anti-
mouse antibodies were used for flow cytometry were purchased from
BioLegend: CD45 APC-Cy7 (clone 30-F11), NK-1.1 BV710 (clone
PK136), FOXP3 PE (clone MF-14) IFN-γBV421 (clone XMG1.2),
CD279 (PD-1) FITC (clone 29 F.1A12), CD45 BV785 (clone 30-F11),
CD11b BV421 (clone M1/70), Gr-1 APC-Cy7 (clone RB6-8C5), CD8a
BV421 (clone 53-6.7), CD86 BV510 (clone GL-1), CD206 PE (clone
C068C2), and MHCII BV605 (clone M5/114.15.2) CD11c APC (clone
N418). The following antimouse antibodies were purchased from BD
Biosciences: CD3 BB700 (clone 17A2), CD4 BUV395 (clone GK1.5),
CD8a BUV737 (clone 53-6.7), F4/80 BUV395 (clone T45-2342), CD103
BUV395 (clone M290), and CD80 BUV737 (clone 16-10A1). Live cells
were gated using LIVE/DEAD (Thermo Fisher) aqua (cat. no. L34966),
green (cat. no. L34970), or NIR (cat. no. L34976). Stained cells were
analyzed by flow cytometry using a BD LSRFortessa flow cytometer
(BD Biosciences) and all data were analyzed using FlowJo software
(Flowjo LLC).
Immunohistochemistry, Immunofluorescence, and Imaging: B16-F10
tumors were resected kept in 10% (v/v) formalin for a minimum of
24 h and then in 70% (v/v) ethanol until processing. H&E as well as
expression of Ki-67, CD8, and PD-L1 protein was assessed by immunohis-
tochemistry (IHC) from histological sections (5 μm) of tumors. H&E stain-
ing was performed using standard protocols. For protein expression,
parrafin-embedded sections were deparrafinized, rehydrated, and washed
in distilled water. Antigen retrieval was then performed in citrate buffer
(pH 6.0) at 125 C for 5 min. To quench endogenous peroxidase activity,
samples were incubated with 3% (v/v) H
2
O
2
for 5 min at room tempera-
ture and washed with PBS-Tween (PBS-T). The samples were then blocked
with blocking buffer consisting of 10% (v/v) donkey serum, 1% (w/v) BSA
in PBS-T for 60 min at room temperature. Sections were then incubated
with antimouse CD8a (Thermo Fisher, cat. no. PA5-81344) or anti-mouse
PD-L1 (Proteintech, cat. no. 66248-1-Ig) overnight at 4 C in a humidified
chamber. After rinsing with PBS-T, samples were incubated with either
goat antirabbit AF594 (abcam, cat. no. ab150080) or goat antimouse
AF488 (Jackson ImmunoResearch, cat. no. 115-543003) for 2 h at room
temperature in the dark. The slides were washed with PBS-T, stained with
Hoescht 33342 (Thermo Fisher), washed again with PBS-T, and mounted
using ProLong Diamond antifade mountant (Thermo Fisher). For Ki-67
expression, sections were run using a LabVision Autostainer 360
(Thermo Fisher) following an automated protocol using a polymer-based
antibody detection system (Vector Laboratories) and visualization with
3,30-diaminobenzidine. H&E and Ki-67 images were obtained using an
Aperio AT2 slide scanner (Leica Biosystems) and fluorescence images
were captured using a Nikon Ti-E microscope and were processed with
ImageJ software.
Statistical Analysis: Statistical analyses were conducted using Graph-Pad
Prism 8 (GraphPad Software). All data are reported as mean SEMs. For
in vitro experiments, a minimum of n¼3 biological replicates were used
per condition in each experiment. Pairwise comparisons were performed
using Student t-tests. Multiple comparisons among groups were deter-
mined using one-way ANOVA followed by a posthoc test. For in vivo
experiments, a minimum of n¼5 biological replicates were used per con-
dition in each experiment. Multiple comparisons among groups were
determined using Kruskal–Wallis test with uncorrected Dunn’s test.
Kaplan–Meier survival curve statistical analysis was determined using
the two-tailed Mantel–Cox test. No specific preprocessing of data was per-
formed prior to statistical analyses. Differences between groups were con-
sidered significant at p-values below 0.05 (*p<0.05, **p<0.01,
***p<0.001).
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors thank the Hale Building for Transformative Medicine for the
assistance with animal housing. The authors thank Swanson
Biotechnology Center at the Koch Institute for Integrative Cancer
Research at the Massachusetts Institute of Technology (MIT) for assis-
tance with animal experiments and facilities, especially the microscopy,
flow cytometry, and histology cores. The authors thank the Department
of Comparative Medicine at MIT. The authors thank G. Paradis for
FACS assistance with Cancer Center Support (FACS core). The authors
thank Kathleen S. Cormier and Charlene Condon for histology and immu-
nohistochemistry assistance. The authors thank Takeda Pharmaceuticals
for provision of ADU-S100.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
Research data are not shared.
Keywords
dendrimer nanoparticles, immunotherapy, intratumoral delivery,
melanoma, stimulator of interferon genes agonist
Received: January 4, 2021
Revised: March 10, 2021
Published online: April 9, 2021
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