Conjugation Chemistry-Dependent T-Cell Activation with Spherical Nucleic Acids

Article abstract of DOI:10.1021/jacs.7b12579

Spherical nucleic acids (SNAs) can be potent sequence-specific stimulators of antigen presenting cells (APCs). When loaded with peptide antigens, they can be used to activate the immune system to train T-cells to specifically kill cancer cells. Herein, th

Full text of DOI:10.1021/jacs.7b12579

Communication
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Conjugation Chemistry-Dependent T‑Cell Activation with Spherical
Nucleic Acids
Kacper Skakuj,⊥,† Shuya Wang,⊥,‡ Lei Qin,§ Andrew Lee,*,∥ Bin Zhang,*,§ and Chad A. Mirkin*,†
†Department of Chemistry and the International Institute for Nanotechnology, Northwestern University, Evanston, Illinois 60208,
United States
‡Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, Illinois 60208, United States
§Division of Hematology-Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States
∥Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States
S
* Supporting Information
to APCs but also the processing and chemical structure of the
ABSTRACT: Spherical nucleic acids (SNAs) can be
potent sequence-specific stimulators of antigen presenting
cells (APCs). When loaded with peptide antigens, they can
be used to activate the immune system to train T-cells to
specifically kill cancer cells. Herein, the role of peptide
chemical conjugation to the DNA, which is used to load
SNAs with antigens via hybridization, is explored in the
context of APC activation. Importantly, though the antigen
chemistry does not impede TLR-9 regulated APC
activation, it significantly augments the downstream T-
cell response in terms of both activation and proliferation.
A comparison of three linker types, (1) noncleavable, (2)
cleavable but nontraceless, and (3) traceless, reveals up to
an 8-fold improvement in T-cell proliferation when the
traceless linker is used. This work underscores the critical
importance of the choice of conjugation chemistry in
vaccine development.
antigen. Indeed, for small molecule and peptide delivery,
activity can be highly dependent on the type of conjugation
chemistry employed.14−16 When designing the next generation
of vaccines, such as immunostimulatory SNAs, it is imperative
to understand the impact of the conjugation chemistry used to
attach the antigen to the DNA that loads the antigen on the
SNA construct. Specifically, because chemical modifications can
influence peptide antigenicity, it may be important to devise
general strategies that can be used with a wide array of peptides,
to deliver pristine antigens with no chemical appendages.
Herein we report the use of three linkage types, a disulfide
reduction-activated traceless linker, a disulfide reduction-
activated cleavable linker, and a noncleavable linker (Figure
1A,B), for attaching a human melanoma-specific antigenic
peptide, gp100 (KVPRNQDWL), to SNAs. The study is
designed to probe the importance, or lack thereof, of generating
pristine antigens for immune activation. The gp100 antigen was
chosen as a model system because of its clinical relevance to
human diseases and high potential for translation.17
ubtle changes in the chemical architecture of nanoparticle
Sconstructs can significantly influence biological function,
including biodistribution properties,1−3 drug release,4−6 and
cellular internalization.7−10 To rationally design nanoparticles
with desired properties, researchers should focus on character-
izing the attributes that can be systematically changed and
where structure−function relationships can begin to be defined.
For example, SNA architectures, synthesized by arranging linear
oligonucleotides on the surfaces of nanoparticle templates, have
shown promise as probes in diagnostics11 and as therapeutic
lead compounds in medicine.12 In the latter category, their
ability to enter cells via endosomal pathways and agonize or
antagonize toll-like receptors makes them highly promising
immunomodulatory agents.13 In the case of cancer vaccines,
SNAs can also be used to carry antigens that provide selective
training of the immune system through T-cell activation and
proliferation. From a chemistry perspective, this creates both a
challenge and an opportunity. Out of the many ways of
combining components required for T-cell activation and
proliferation, which ones are best, and do they result in
significant differences in efficacy?
Immunostimulatory SNAs were synthesized using a lip-
osomal core with TLR9-stimulatory CpG B oligonucleotides
(see Table S1 for sequences), tagged with a Cy5 dye, and
immobilized on the core surface through intercalation by using
a cholesterol anchor on the 3′ end.18,19 Antigens were attached
to the SNA as one of three gp100−DNA conjugate types, 1−3,
made with DNA complementary to the CpG adjuvant. CpG
anchor stands were all hybridized to the conjugates prior to
their addition to liposomes, and these duplexes were added at a
75:1 ratio to liposomes. All design parameters, such as the 1:1
ratio of antigen to adjuvant, DNA and gp100 concentrations
were kept constant across the SNA structures investigated, only
the identity of the linker differed.
Conjugates 1−3 were synthesized by first attaching one end
of the linker to a peptide amine, followed by attachment of
thiolated DNA to the other. The amine residue of the antigen
was used as a chemical point for conjugation because this
strategy can be adapted to other antigens, all of which have at
least one primary amine at their N-terminus. The three distinct
linker chemistries were chosen to test general considerations
The way antigen molecules are incorporated in synthetic
Received: November 28, 2017
vaccines could impact not only quantities of antigen delivered
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b12579
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Journal of the American Chemical Society
Communication
liposomes (Figure 1D). The ζ-potentials of the SNAs were on
average −26.7 1.7 mV, a decrease of approximately 20 mV
compared to the bare liposomes, which can be attributed to the
added negative charge carried by the DNA backbone (Figure
1E).
Codelivery of both adjuvant and antigen is crucial for
efficient T-cell activation.24 In order to characterize the
codelivery of these components, we used bone marrow-derived
dendritic cells (DCs) as a model system, because they are the
most effective professional APCs of the immune system.25
Confocal microscopy images show that both the AlexaFluor488
(AF488)-labeled gp100 antigen (green) and Cy5-labeled CpG
adjuvant (red) have been internalized by DCs after incubation
with 1-SNAs for 15 min (Figure 2A). We further quantified the
Figure 1. (A) Schematic design of the immunostimulatory SNA. (B)
Three distinct linker chemistries were used to make the antigen-DNA
conjugates 1−3: NDEC (traceless), SPDP (cleavable), and BMPS
(noncleavable), respectively. (C) Cholesterol-modified cyanine-5
(Cy5)-tagged anchor DNA, conjugate and anchor duplex, and SNA
were characterized using 1% agarose gel, imaged by Cy5 fluorescence.
(D,E) DLS shows an increase in diameter along with a decrease in zeta
potential, measured at pH 7, between the bare liposome and the SNAs.
Samples for DLS were prepared without the Cy5 modification.
for antigen attachment (Figure 1B). A commercially available
noncleavable linker (N-(β-maleimidopropyloxy) succinimide
ester, BMPS) was used to create conjugate 3, which has no
readily cleavable bonds. A commercially available cleavable
linker (succinimidyl 3-(2-pyridyldithio)propionate, SPDP) was
used to prepare conjugate 2, which cleaves in the reducing
environment of the cell but leaves a molecular pendant group
(3-mercaptopropionamide) attached to the antigen. Finally, a
traceless linker (4-nitrophenyl 2-(2-pyridyldithio)ethyl carbo-
nate, NDEC)15,16,20−22 was incorporated to create conjugate 1
(see Figures S1−S2 for synthesis). The traceless linker
incorporates a disulfide, which upon reduction, results in an
intramolecular cyclization that releases the antigen in an
unmodified form.
We incubated conjugates 1−3 at pH 7.4 with 10 mM
glutathione and characterized the decomposition products
using PAGE and MALDI-MS. These experiments confirm that,
under cell-mimicking reduction conditions,23 the BMPS
conjugate 3 does not release an antigen, the SPDP conjugate
2 releases an antigen that is modified with a chemical pendant,
and the NDEC conjugate 1 regenerates an unmodified gp100
peptide (see Figures S3−S5). We also characterized the rate of
conjugate cleavage by synthesizing 1−3 using a fluorescein
labeled gp100 peptide and a quencher-containing oligonucleo-
tide to form a FRET reporter. The fluorescence of this reporter
increases upon cleavage of the linkage between the peptide and
DNA. We find that conjugates 1 and 2 have similar cleavage
half-lives of approximately 31 and 54 min in 20 mM GSH,
respectively. Conjugate 3 did not show an increase in
fluorescence (Figure S6).
Figure 2. (A) Confocal microscopy images show gp100 antigen
(AF488, green) and the CpG adjuvant (Cy5, red) inside mouse
dendritic cells. (B,C) Flow cytometry measurements after 15 min
incubation. Values are an average of three replicates (see Figure S8 for
additional replicates).
codelivery of these components using flow cytometry (Figure
2B). The SNA architecture formulation resulted in a doubling
of codelivery efficiency (double positive of AF488 and Cy5)
compared to the linear mixture, as measured over background
fluorescence control (medium only) (Figure 2C). In addition,
we observed no significant effect of 1-SNA on cell viability at
concentrations below 1 μM using an MTT assay (Figure S7).
T-cell receptor transgenic CD8+ T-cells (from pmel-1 mice)
specifically recognizing gp100 were used to study the efficacy of
the immunostimulatory SNAs at eliciting gp100-specific CD8+
T-cell responses.26 The splenocytes from pmel-1 mice were
treated with each SNA individually at different concentrations
for 72 h to determine a dose−response curve (Figure
3A,B).27,28 We observed that CD8+ T-cell proliferation
(measured by eFluor 450 dilution) was dependent upon
linkage type, the only parameter that differs across the three
SNAs. The extent of proliferation was similar across the three
structures when splenocytes were treated at the highest
concentration range (1−10 nM in gp100); however, at lower
concentrations, the T-cell proliferation differed significantly
among the three treatment groups (1−100 pM in gp100).
Notably, 1-SNAs even produced detectable T-cell proliferation
at 100 fM treatment whereas the two other SNAs failed to
show any effect. The calculated EC50 values indicate that 1-SNA
(EC50 = 2.3 pM) is approximately three times more potent than
2-SNA (EC50 = 6.4 pM), which itself is approximately three
times more efficacious than 3-SNA (EC50 = 18 pM). This
observation reveals the significance of antigen conjugation
chemistry on the ability of SNAs to induce antigen-specific T-
cell proliferation.
SNAs synthesized with the three conjugates were charac-
terized by agarose gel electrophoresis. A shift in electrophoretic
mobility was observed between the single stranded CpG DNA,
the duplex with the gp100−DNA conjugate, and the SNA
(Figure 1C). Additionally, the SNAs all have indistinguishable
z-average hydrodynamic diameter, of 83.7 0.4 nm (PDI 0.075
0.012). An increase of approximately 13 nm over the bare
B
DOI: 10.1021/jacs.7b12579
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Journal of the American Chemical Society
Communication
simply on synthetic convenience, but instead the choice should
be made by considering its impact on the immunogenicity of
the delivered antigen. This knowledge underscores the impact
of conjugation chemistry on immunostimulatory nanother-
apeutic constructs and will inform the design of future vaccines,
beyond those based upon the SNA architecture.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b12579.
■
S
Experimental details, oligonucleotide sequences, and
characterization data (PDF)
AUTHOR INFORMATION
Corresponding Authors
*chadnano@northwestern.edu
*bin.zhang@northwestern.edu
*andrew.lee3@northwestern.edu
■
ORCID
Figure 3. (A) Flow cytometry data showing CD8+ T-cell proliferation
following incubation of pmel-1 splenocytes with the three types of
SNAs at 10 pM concentration. (B) Dose−response curve of SNA
treatment on T-cell proliferation. Average and standard deviation for
three replicates are shown for each point (see Figure S9 for additional
replicates). The curves are three-parameter dose−response fits with a
shaded 95% confidence interval of the fit. (C) Secreted TNF-α, IFN-γ,
Granzyme B, and IL-6 were quantified by ELISA at a 10 pM treatment
concentration, **** p < 0.0001.
Chad A. Mirkin: 0000-0002-6634-7627
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research reported in this publication was supported by the
National Cancer Institute of the National Institutes of Health
under Award U54CA199091. S.W. was supported by a
fellowship associated with the Chemistry of Life Processes
Predoctoral Training Program T32GM105538 at Northwestern
University. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health. The authors also acknowl-
edge support from the Vannevar Bush Faculty Fellowship
program sponsored by the Basic Research Office of the
Assistant Secretary of Defense for Research and Engineering
and funded by the Office of Naval Research grant N00014-15-
1-0043. The project described was also supported by the IDP
Sherman Fairchild Foundation through the Robert H. Lurie
Comprehensive Cancer Center, and the Prostate Cancer
Foundation and Movember Foundation under award
17CHAL08
To further evaluate the impact of conjugation chemistry on
T-cell activation, we quantified the release of IFN-γ, TNF-α,
granzyme-B, and IL-6 for all three SNAs using ELISA at a 10
pM SNA treatment concentration (Figure 3C). Consistent with
results of T-cell proliferation, we show that T-cells treated with
the traceless 1-SNAs secrete higher levels of the cytokine
activation markers IFN-γ and IL-6, compared to the 2-SNA and
3-SNA groups. This shows that traceless NDEC conjugation
chemistry leads to higher T-cell activation. Granzyme B and
TNF-α secretion, which results from 1-SNA treatment, is also
higher than all other groups, indicating the increased potential
of T-cell-mediated killing of tumor cells.
Optimum T-cell activation and proliferation depend on
MHC-antigen-TCR binding as well as the activation state of the
APCs. The observed differences in SNA efficacy could be due
to different levels of APC activation. Therefore, we compared
the activation levels of DCs across the SNA types by
quantifying the expression of the costimulatory markers,
CD40 and CD80 (Figure S10). All SNA types caused
upregulation in the expression of the two receptors compared
to a medium only control. No difference in APC activation
among the three SNA types was observed, indicating that the
activation of DCs, caused by the interaction of CpG
oligonucleotides with TLR receptors in the endosomes, is
likely independent of the linkage chemistry used to form the
gp100−DNA conjugates.
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DOI: 10.1021/jacs.7b12579
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