Please cite this article in press as: Duperret et al.
, Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010
Original Article
Synergy of Immune Checkpoint Blockade
with a Novel Synthetic Consensus
DNA Vaccine Targeting TERT
Elizabeth K. Duperret,1 Megan C. Wise,2,3 Aspen Trautz,1 Daniel O. Villarreal,3 Bernadette Ferraro,2 Jewell Walters,2
Jian Yan,2 Amir Khan,2 Emma Masteller,2 Laurent Humeau,2 and David B. Weiner1
1Vaccine Center, The Wistar Institute, Philadelphia, PA 19104, USA; 2Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA 19462, USA; 3University of Pennsylvania,
Philadelphia, PA 19104, USA
Immune checkpoint blockade antibodies are setting a new
standard of care for cancer patients. It is therefore important
to assess any new immune-based therapies in the context of
immune checkpoint blockade. Here, we evaluate the impact
of combining a synthetic consensus TERT DNA vaccine that
has improved capacity to break tolerance with immune check-
point inhibitors. We observed that blockade of CTLA-4 or, to
a lesser extent, PD-1 synergized with TERT vaccine, gener-
ating more robust anti-tumor activity compared to check-
point alone or vaccine alone. Despite this anti-tumor synergy,
none of these immune checkpoint therapies showed improve-
ment in TERT antigen-specific immune responses in tumor-
bearing mice. aCTLA-4 therapy enhanced the frequency of
T-bet+/CD44+ effector CD8+ T cells within the tumor and
decreased the frequency of regulatory T cells within the tu-
mor, but not in peripheral blood. CTLA-4 blockade syner-
gized more than Treg depletion with TERT DNA vaccine,
suggesting that the effect of CTLA-4 blockade is more likely
due to the expansion of effector T cells in the tumor rather
than a reduction in the frequency of Tregs. These results sug-
gest that immune checkpoint inhibitors function to alter the
immune regulatory environment to synergize with DNA vac-
cines, rather than boosting antigen-specific responses at the
site of vaccination.
INTRODUCTION
The magnitude of an adaptive immune response to a foreign antigen
is determined not only by the strength of interaction between the ma-
jor histocompatibility complex (MHC) and the T cell receptor (TCR)
but also by co-stimulation at the immunological synapse.1 Without
co-stimulation, T cells will fail to initiate an effective immune
response. Additional co-stimulatory molecules or immune check-
point molecules exist to control the amplitude of T cell activation
to prevent autoimmune responses. These immune checkpoints
(CTLA-4, PD-1, LAG3, and TIM3) are often necessary for initiation
of an immune response; however, as antigen persists, these check-
points ultimately serve to dampen the T cell effector function against
the foreign antigen.2 CTLA-4, PD-1, LAG3, and TIM3 are all ex-
pressed on T cells and limit T cell effector activity. Antibodies block-
Molecular Therapy Vol. 26 No 2 February 2018 ª 2017 The American Society of Gene and Cell Therapy.
ing these molecules have been shown to augment the effector activity
of tumor-specific T cells and additionally inhibit regulatory T cell
(Treg) activity and reduce tumor burden in preclinical models and/or
in clinical trials as mono-therapies.3–5
In particular, blockade of the immune inhibitory checkpoints PD-1
or CTLA-4 has shown promising results in the clinic for dozens of
tumor types, and PD-1 blockade has become a standard of care for
melanoma and non-small-cell lung cancer.6–8 However, response
rates to these mono-therapies are relatively low (33.7% response
for pembrolizumab [a-PD-1] and 11.9% response for ipilimumab
[a-CTLA-4] in melanoma patients), leaving room for improve-
ment.9 The lack of response for the majority of such patients may
be due to a lack of pre-existing tumor-associated T cell responses.
Strategies to combine PD-1 or CTLA-4 blockade with therapies
that prime T cells, such as radiation or irradiated cell-based vac-
cines, have shown improvements in pre-clinical models or in clin-
ical trials.5,10–17 These approaches, however, may rely on T cell
responses to neo-antigens within the tumor.18,19 Enhanced T cell
priming by vaccines may therefore be required to break tolerance
to self-antigens for patients with poor response to immune check-
point blockade. However, the mechanistic interactions between
immune checkpoint blockade antibodies and vaccines are poorly
understood.
Therapeutic peptide or DNA vaccination represents a more targeted
approach for directing T cells toward specific, less variable tumor-
associated antigens (TAAs). In mouse models, peptide vaccines
have been shown to synergize with immune checkpoint blockade.20,21
However, peptide vaccines are histocompatibility leukocyte antigen
(HLA) restricted and therefore cannot be used for all patients. Unlike
peptide vaccines, synthetic DNA (synDNA) vaccines are not HLA
restricted, are robustly presented on both MHC class I and MHC
Received 26 October 2017; accepted 14 November 2017;
https://doi.org/10.1016/j.ymthe.2017.11.010.
Correspondence: David B. Weiner, The Wistar Institute, 3601 Spruce St., Phila-
delphia, PA 19104, USA.
E-mail: dweiner@wistar.org
1
 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010

Molecular Therapy

Figure 1. Delivery of aCTLA-4 or aPD-1 Post-first Vaccination Synergizes with TERT DNA Vaccine above Checkpoint Alone in Generating Anti-tumor
Immune Response
(A) Experimental setup. Mice were implanted with TC-1 tumor cells on day 0 and then immunized four times at 1-week intervals starting 7 days after tumor implant. Mice were
given antibodies (200 mg per mouse) every 3 days starting 1 day after the first immunization. Antibody delivery was continued until 1 week after the final vaccination. (B, D, F,
and H) Tumor volume measurements over time for naive mice, mTERT vaccine-treated mice, or mice treated with mTERT vaccine plus aCTLA-4 (B), aPD-1 (D), or a
combination of aCTLA-4 and aPD-1 (F). (C, E, and G) Mouse survival over time for naive mice, mTERT vaccine-treated mice, or mice treated with mTERT vaccine plus
aCTLA-4 (C), aPD-1 (D), or a combination of aCTLA-4 and aPD-1 (G). For (B)–(G), n = 12–13 mice per group. (H) Tumor volume measurements over time for naive mice

(legend continued on next page)

2 Molecular Therapy Vol. 26 No 2 February 2018

 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010
www.moleculartherapy.org
class II, and can be designed using consensus sequences in order to
break tolerance.22
Our laboratory has pioneered efforts to develop therapeutic DNA
vaccines for patients with human papillomavirus (HPV)-associated
cervical intraepithelial neoplasia (CIN2/3).23,24 We incorporated
important improvements to the DNA vaccine platform, such as
plasmid optimization and use of in vivo electroporation, which has
directly led to enhanced CD4+ and CD8+ T cell responses.24–26 These
improvements led to the first DNA vaccine to demonstrate potent im-
mune responses and anti-neoplastic activity in a human clinical
trial.23,24 Specifically, a synthetic DNA vaccine targeting HPV-16
and HPV-18 E6 and E7 proteins (VGX-3100) showed clinical efficacy
for therapeutic treatment of cervical intraepithelial neoplasia 2/3
(CIN2/3) in women in a double-blind, placebo-controlled phase IIb
clinical trial.23
Next, we focused on developing a novel DNA vaccine platform to
help break tolerance to non-viral tumor-associated antigens, such
as TERT and WT-1.27,28 This novel platform is based on the concept
that the introduction of xenogeneic antigens into mice can induce
autoimmunity.29,30 We developed a synthetic consensus (SynCon)
approach in which gene sequences from various species are compared
to identify a consensus sequence (with approximately 95% homology
to the native gene sequence), an approach that has enhanced the ca-
pacity to break tolerance, yet maintains T cell killing against native
MHC class I-presented sequences. We reported that this synthetic
consensus vaccine design strategy is superior to native DNA-vac-
cine-encoded antigens for the WT-1 antigen.28
Here, we sought to further improve synthetic consensus DNA vac-
cine immune responses through combination therapy with im-
mune checkpoint blockade. We utilized a SynCon mouse TERT
DNA vaccine with enhanced capacity to break tolerance in combi-
nation with antibodies that block the immune checkpoint mole-
cules CTLA-4 or PD-1. We chose to test this combination therapy
in the TC-1 tumor model, which does not respond to immune
checkpoint blockade alone. We observe that blockade of
CTLA-4, PD-1, or a combination of the two effectively synergized
with SynCon TERT DNA vaccine to slow tumor growth in mice.
However, peripheral and systemic immune responses did not
necessarily correspond with anti-tumor activity of immune therapy
combinations. These studies highlight the potential application of
these DNA vaccine/immune checkpoint blockade combinations
in patients that respond poorly to immune checkpoint blockade
alone and allow for better rational design of combination thera-
pies. Furthermore, these results suggest that this synergistic effect
is due to the effect of immune checkpoint blockade on expanding
unvaccinated mice treated with aPD-1, aCTLA-4, or both aPD-1 and aCTLA-4. (I) Mouse survival over time for naive mice or unvaccinated mice treated with aPD-1,
aCTLA-4, or both aPD-1 and aCTLA-4. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5 cm. For (H) and (I), n = 9 mice per groiup. Each
experiment was repeated at least once to verify results. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey’s HSD test.
Significance for mouse survival was determined by the Gehan-Breslow-Wilcoxon test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars indicate ± SEM.
effector T cells in the tumor microenvironment rather than boost-
ing vaccine responses in the periphery.
RESULTS
CTLA-4 and PD-1 Blockade Synergizes with mTERT DNA
Vaccine in Generating Anti-tumor Immunity
For these studies, we utilized a synthetic consensus (SynCon) mouse
TERT DNA vaccine. This vaccine administered alone exhibits a
robust capacity to generate an immune response against matched,
SynCon peptides, while maintaining the capacity to break tolerance
to native mouse TERT peptides in C57BL/6 mice (Figures S1A and
S1B). For the remaining experiments, we focused on antigen-specific
immune responses targeting native mouse TERT peptides. In addi-
tion, this SynCon mouse TERT DNA vaccine is capable of slowing
tumor growth and prolonging survival in a proportion of the mice
in a TC-1 mouse tumor challenge model (Figures S2A and S2B).
We first examined whether aCTLA-4 or aPD-1 therapy would
improve anti-tumor activity in a therapeutic tumor challenge setting.
We implanted mice with TC-1 tumors and initiated immunizations
after palpable tumors formed 1 week following implantation (Fig-
ure 1A). Mice were immunized at 1-week intervals for a total of
four immunizations. Checkpoint treatment began 1 day following
the first immunization and continued until 1 week following the final
immunization (Figure 1A).
We observed that both a-CTLA-4 and a-PD-1 therapies synergized
with mTERT DNA immunization above vaccine therapy alone in a
therapeutic TC-1 tumor challenge model when immune checkpoints
were delivered 1 day post-first immunization (Figures 1B–1G). Both
a-CTLA-4 and a-PD-1 therapy alone had no initial impact in slowing
tumor growth and no significant impact on mouse survival (Figures
1H and 1I). SynCon mTERT immunization alone slightly slowed
tumor growth and improved survival compared to naive mice. How-
ever, a-CTLA-4 and a-PD-1 in combination with SynCon mTERT
robustly slowed tumor growth and significantly improved survival
compared to DNA alone or naive mice (Figures 1B–1E). Synergy
was greater for a-CTLA-4, in particular for survival, compared to
a-PD-1 (Figures 1B–1E).
Combination therapy of aCTLA-4 and a-PD-1 in both animal
models and in the clinic has shown improved anti-tumor responses
compared to treatment with each checkpoint alone.33–35 In particular,
combination therapy with aCTLA-4 and a-PD-1 was reported to
improve the anti-tumor immunity of cell-based vaccines.5,17 We
therefore examined whether a triple-combination therapy with
aCTLA-4, a-PD-1, and SynCon mTERT could further improve ther-
apeutic anti-tumor activity. We observed that combination therapy of
Molecular Therapy Vol. 26 No 2 February 2018 3
 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010

Molecular Therapy

Figure 2. Antigen-Specific Intracellular Cytokine Production upon TERT DNA Vaccination and Immune Checkpoint Blockade in Tumor-Bearing Mice
(A) Experimental setup. Mice were implanted with TC-1 tumor cells on day 0 and then immunized on days 7 and 14. Antibody delivery was started 1 day after the first
immunization and continued every 3 days. Mice were sacrificed on day 21 for immune cell analysis. (B–F) Intracellular cytokine staining of CD8 or CD4 T cells after stimulation
with native mouse TERT peptides for 5 hr. CD4 T cells were stained for IFN-g (B) or TNF-a (C). CD8 T cells were stained for IFN-g (D), TNF-a (E), or IFN-g/CD107a/T-bet (F).
Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group. A representative
of three independent experiments is shown. Error bars indicate ± SEM.

SynCon mTERT with both aCTLA-4 and a-PD-1 slightly improved
anti-tumor activity and survival of mice. This effect was greater than
the combined treatment of aCTLA-4 and SynCon mTERT (Figures
1F and 1G). The combination therapy alone without the DNA vaccine
exhibited little impact on tumor growth or on mouse survival (Figures
1H and 1I).
Surprisingly, there was no significant impact of any of the checkpoint
antibodies or combinations on systemic type 1 antigen-specific CD8+
or CD4+ immune responses, including expression of interferon-
gamma (IFNg) and tumor necrosis factor alpha (TNF-a) in CD4+
and CD8+ T cells or IFNg/CD107a/T-bet in CD8+ T cells compared
to mTERT DNA alone (Figures 2B–2F).

Impact of Immune Checkpoint Blockade on Systemic Immune
Responses in Tumor-Bearing Mice
Next, we explored immune responses in tumor-bearing mice treated
with combination vaccine and the aCTLA-4/aPD-1 immune check-
point blockade antibody therapy. We implanted mice with TC-1 tu-
mors, immunized mice on days 7 and 14, and sacrificed mice on day
21 (Figure 2A). Checkpoint inhibitor delivery was initiated one day
following the first immunization, and continued every 3 days until
day 21 (Figure 2A). We examined the antigen-specific immune re-
sponses in the spleen and the exhaustion marker PD-1 in the periph-
ery and in spleen.
Next, we examined PD-1 expression on CD4 and CD8 T cells in the
spleen and the periphery of tumor-bearing mice. In tumor-bearing
mice, aPD-1 had no impact on PD-1 expression in both CD4+ and
CD8+ T cells in spleen and in the periphery (Figures 3A–3D). How-
ever, both aCTLA-4 and a combination of aCTLA-4 and aPD-1
enhanced the frequency of PD-1+ CD4+ and CD8+ T cells in both
spleen and the periphery (Figures 3A–3D).
aCTLA-4 therapy is known to impact Tregs in a tissue-dependent
manner, with a more profound depletion of Tregs within the tumor
compared to other tissue sites.14,36 We therefore examined the

4 Molecular Therapy Vol. 26 No 2 February 2018

 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010

www.moleculartherapy.org

Figure 3. Phenotype of Peripheral and Spleen T Cells upon TERT DNA Vaccination and Immune Checkpoint Blockade in Tumor-Bearing Mice
(A–F) Staining of CD8 or CD4 T cells from spleen or peripheral blood of mice treated with the indicated therapies in accordance with the schedule in Figure 3A. CD8+
splenocytes (A) or PBMCs (B) were stained for PD-1. CD4+ splenocytes (C) or PBMCs (D) were stained for PD-1. CD4+ splenocytes (E) or PBMCs (F) were stained for CD25/
FoxP3. Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group.
A representative of three independent experiments is shown. Error bars indicate ± SEM.

frequency of CD4+/CD25+/FoxP3+ Tregs in spleen and in the periph-
ery upon immune checkpoint blockade therapy in combination with
SynCon mTERT DNA vaccine (Figures 3E and 3F). We observed that
all checkpoint therapies and combinations decreased the frequency of
Tregs in spleen, but slightly increased the frequency of Tregs in the
periphery (Figures 3E and 3F).
Impact of Immune Checkpoint Blockade on Tumor-Specific
Immune Responses in Tumor-Bearing Mice
Because of the surprising finding that immune checkpoint blockade
had no impact on TERT antigen-specific immune responses in
spleen, we next examined immune responses in the tumors of these
same mice. aCTLA-4 treatment resulted in significant depletion of
intra-tumoral Tregs (Figure 4A), whereas aPD-1 therapy did not
alter the frequency of intra-tumoral Tregs, and combination ther-
apy with both aCTLA-4 and aPD-1 resulted in only a modest
decrease in the frequency of intra-tumoral Tregs (Figure 4A).
Similar to the results observed in spleen and in the peripheral
T cells, the percentage of PD-1+ tumor-infiltrating CD8+ lympho-
cytes also increased upon aCTLA-4 treatment and, to a lesser
extent, combination therapy with aCTLA-4 and aPD-1 treatment
(Figure 4B).

Molecular Therapy Vol. 26 No 2 February 2018 5

 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010

Molecular Therapy

Figure 4. Phenotype of Tumor-Infiltrating Lymphocytes upon TERT DNA Vaccination and Immune Checkpoint Blockade
(A–F) Staining of CD8 or CD4 T cells isolated from the tumors of mice treated with the indicated therapies in accordance with the schedule in Figure 4A. mTERT-stimulated
CD3+ TILs were stained for CD4/CD25/FoxP3 (A), CD8/PD-1 (B), CD8/CD44 (C), CD8/T-bet (E), or CD8/CD44/Tbet (F). PMA-stimulated CD3+ T cells were stained for CD8/
CD44 (D). Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group.
A representative of three independent experiments is shown. Error bars indicate ± SEM.

Next, we examined the T cell activation markers CD44 and T-bet in
CD8+ tumor-infiltrating lymphocytes (TILs). aCTLA-4 treatment
alone enhanced the frequency of CD44+ CD8+ TILs (Figure 4C).
This effect was more pronounced after treatment of these TILs
with PMA (Figure 4D). Interestingly, aPD-1 and the combination
of aCTLA-4 and aPD-1 therapy did not significantly impact
CD44 expression in the TILs (Figures 4C and 4D). Both aCTLA-4
and the combination of aCTLA-4 and aPD-1 therapy enhanced the
frequency of T-bet+ CD8+ TILs (Figure 4E). This trend was similar
for CD8+ T cells that co-expressed T-bet and CD44 (Figure 4F). The
SynCon TERT DNA vaccine alone did not significantly impact
Tregs, PD-1, or CD44 expression in TILs (Figures 4A–4D).
Together, these results demonstrate that aCTLA-4 therapy expands
the effector T cell population within the tumor, likely resulting
in the strong anti-tumor effect when combined with TERT DNA
vaccination.
CTLA-4 Blockade Synergizes More Than Treg Depletion with
mTERT DNA Vaccine
It has been proposed that the primary mechanism for aCTLA-4 ther-
apy is depletion of Tregs in the tumor microenvironment. In order to
test the contribution of Tregs to the syngery between SynCon TERT
DNA vaccine and aCTLA-4 treatment, we compared the impact of
aCTLA-4 to aCD25, a depletion antibody that systemically depletes
Tregs.37 We verified that this aCD25 depletion antibody significantly
decreased the frequency of Tregs in the peripheral blood, in spleen,
and in the tumor (Figures 5A–5C). Importantly, the extent of Treg
depletion in the tumor was similar to that of CTLA-4 blockade (Fig-
ures 4A and 5A). In these experiments, while aCD25 in combination
with mTERT significantly improved anti-tumor responses, this effect
was not as strong as combination therapy of aCTLA-4 and mTERT in
terms of tumor growth or overall survival (Figures 5D and 5E).
Because CD25 can also be expressed by effector T cells, we examined

6 Molecular Therapy Vol. 26 No 2 February 2018

 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010

www.moleculartherapy.org

Figure 5. Delivery of aCTLA-4 in Combination with TERT DNA Vaccine Is Superior to Treg Depletion in Combination with TERT DNA Vaccine
(A–C) Staining of CD8 or CD4 T cells from the peripheral blood (A), spleen (B), or tumor (C) of mice treated with the indicated therapies in accordance with the schedule in
Figure 2. (D) Tumor volume measurements over time for mice with indicated treatment regimen. Mice were treated in accordance with the schedule shown in Figure 2A.
(E) Mouse survival over time for mice with indicated treatment regimen. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5 cm. Significance for
tumor volume measurements was determined by two-way ANOVA followed by Tukey’s HSD test. Significance for mouse survival was determined by the Gehan-Breslow-
Wilcoxon test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 10 mice per group. Error bars indicate ± SEM.

whether the aCD25 antibody treatment affected effector and central
memory T cells (Figures S3A–S3F). We found that, in the peripheral
blood, spleen and tumor, there was no depletion of effector CD8
T cells (CD44+/CD62Llo) or central memory CD8 T cells (CD44+/
CD62L+) with aCD25 antibody treatment (Figures S3A–S3F). These
data suggest that the mechanism of action of aCTLA-4 extends
beyond depletion of Tregs in the tumor and is likely due to the expan-
sion of effector T cells within the tumor (Figure 4).
DISCUSSION
Here, we utilized a synthetic consensus DNA vaccine targeting the
tumor antigen TERT in combination with immune checkpoint
blockade. We have previously shown strong immune responses for
our SynCon DNA vaccine platform in human clinical trials for viral
antigens such as HPV and Zika virus.23,38 We have now improved
this technology to help break tolerance to cancer-associated self-anti-
gens, such as WT-1 and TERT, which we have tested in both mice and
non-human primates.27,28 Because immune checkpoint blockade is
becoming the standard of care in cancer patients, it is important to
understand how these novel DNA vaccine therapies can work as com-
bination therapies.
In this study we observed robust synergy between SynCon mTERT
DNA vaccination and aCTLA-4 immune checkpoint blockade.

Molecular Therapy Vol. 26 No 2 February 2018 7

 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010
This combination exhibited long-term effects for prolonging mouse
survival in a therapeutic tumor challenge model. While aPD-1
showed some synergy with SynCon mTERT DNA immunization,
the effect was not as profound and did not result in significant prolon-
gation of mouse survival. Importantly, these mice did not exhibit anti-
tumor immunity after delivery of immune checkpoint antibodies
alone, suggesting that addition of a DNA vaccine to an immune
checkpoint blockade regimen may allow non-responders to become
responders. Interesting future studies may include testing the efficacy
of this combination therapy in tumor models that already show some
response to immune checkpoint blockade alone, to evaluate whether
increased immune potency could be achieved.
Only a handful of preclinical studies have examined the efficacy of
combining DNA vaccines with immune checkpoint blockade for
tumor therapy. Two studies showed modest improvement in anti-tu-
mor immunity by combining DNA vaccines targeting the MYB onco-
protein for colorectal cancer or the SSX2 cancer antigen for sarcoma
with aPD-1 antibody.39,40 However, neither of these studies used elec-
troporation in combination with the DNA vaccine, and therefore
both vaccines exhibited minimal anti-tumor effects on their own,
even when delivered only 1-2 days following tumor implantation.39,40
In the report, the DNA vaccine targeting MYB was completely inef-
fective if delivered 5 days or later after tumor implantation, and
was also ineffective at reducing tumor burden altogether in BALB/c
mice.39 Another study combined aCTLA-4 treatment with xenoge-
neic DNA vaccines targeting TRP-2, gp100 or PSMA in prophylactic
immunization studies.41 Interestingly, this study found that prophy-
lactic delivery of aCTLA-4 boosted anti-tumor immunity more when
given with the second immunization instead of the first immuniza-
tion.41 No previous studies have examined combination therapy
with DNA vaccines and aCTLA-4 blocking antibody in a therapeutic
tumor challenge model. In our studies we did not observe a significant
boosting in antigen-specific immune responses when aCTLA-4 was
delivered following the first or second immunization. The reasons
for this discrepancy are unclear, but may be antigen-dependent, or
masked by the immune potency of the electroporation enhanced syn-
thetic DNA vaccine itself.
While there are only a few studies using DNA vaccines in combina-
tion with aCTLA-4 or aPD-1 checkpoint antibodies, studies using
cell-based vaccines have explored these combinations more thor-
oughly. Irradiated tumor cells expressing GM-CSF or Flt-3 ligand
(GVAX or FVAX, respectively) have been frequently used as a
vaccine for combination therapy studies with immune checkpoint
blockade.5,14–17 Interestingly, the relative effects of aCTLA-4
compared to aPD-1 therapy appear to be dependent on both the
type of vaccine and the tumor model. A study that utilized a PLG
(poly[lactide-co-glycolide]) cancer vaccine, in which B16 tumor ly-
sates are incorporated into PLG scaffolds, showed that aCTLA-4
therapy synergized to a greater extent than aPD-1 therapy.15 How-
ever, a study comparing GVAX and FVAX with aCTLA-4 or
a-PD-1 therapy showed that for FVAX, aPD-1 therapy was superior
to aCTLA-4 therapy, and for GVAX, the two therapies were similar.17
8 Molecular Therapy Vol. 26 No 2 February 2018
Molecular Therapy
In an independent study GVAX synergized more with a-CTLA-4
therapy compared to aPD-1 therapy in CT26 tumors; however, re-
sponses to GVAX in combination with each checkpoint were similar
in ID8 tumors5. In each of these cell-based vaccine studies in which
triple-combination therapies were used (GVAX/FVAX, a-CTLA-4,
and a-PD-1), the triple-combination therapy was profoundly supe-
rior to any of the double- or mono-therapies.5,17 Our study showed
a clear bias toward DNA vaccine synergy with a-CTLA-4 blockade
over a-PD-1 blockade. Furthermore, the triple-therapy was only
slightly better than double-therapy with vaccine plus aCTLA-4, indi-
cating that patients receiving our vaccine may not benefit as much
from receiving both aCTLA-4 and aPD-1. This is useful information,
since the double-therapy with aCTLA-4 and aPD-1 has significantly
more toxicity compared to mono-therapy with either aCTLA-4 or
aPD-1 alone in humans.42
Furthermore, we did not observe a significant change in the T cell
exhaustion marker PD-1 expression on CD8 or CD4 T cells in the
spleen, periphery or tumor of both tumor-bearing mice after immu-
nization with SynCon TERT DNA vaccine alone. However, in other
studies upregulation of inhibitory T cell molecules, primarily PD-1,
has been observed on antigen-specific CD8 T cells in both mice and
humans after immunization with a variety of different vaccine plat-
forms, including an SSX DNA vaccine, HLA-A2-restricted NY-ESO
peptides formulated with Frend’s and CpG adjuvants, a recombinant
lentivector vaccine expressing melanoma associated antigen epitopes,
and a HPV E7 polypeptide vaccine.40,43–45 This lack of induction
of PD-1 after our potent DNA vaccine immunization may explain
why less synergy was observed between our vaccine and aPD-1
therapy.
We observed an interesting increase in PD-1 expression in T cells af-
ter aCTLA-4 treatment, but not aPD-1 treatment, in tumor-bearing
mice (Figure 3). Interestingly, other studies have also shown an in-
crease in the fraction of CD8+ T cells expressing PD-1 after mono-
therapy with aCTLA-4, aPD-1, or aPD-L1.17 Upregulation of
PD-1+ and PD-L1+ cells was also shown in aCTLA-4 therapy for hu-
man and murine bladder cancers.33 Blockade of the PD-1/PD-L1
signaling axis can only partially reverse an exhausted T cell pheno-
type; therefore, exploring methods of further improving immune acti-
vation in tumor-bearing mice remains important.46
Some differences between aCTLA-4 and aPD-1 blockade in the
context of DNA vaccines may be the result of the different effects
of aCTLA-4 and aPD-1 on Tregs. aCTLA-4 antibodies, unlike
aPD-1 antibodies, have demonstrated robust depletion of intra-
tumoral Tregs in mice.14,36 There is also some evidence for ipili-
mumab-dependent antibody-dependent cell-mediated cytotoxicity
(ADCC) ex vivo in patient samples; however, the extent to which clin-
ical aCTLA-4 antibodies can deplete Tregs in patient tumors remains
unclear.47 In the present study, aCTLA-4 antibody treatment was su-
perior to depletion of Tregs (with an aCD25 depletion antibody) in
combination with SynCon mTERT DNA vaccine (Figure 5). A sepa-
rate study found a similar result when comparing combination
 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010
www.moleculartherapy.org
therapy of GVAX or FVAX with aCD25 depletion, implying that
aCTLA-4 has additional effects beyond simple depletion of Tregs.16
This suggests that the synergy between TERT and aCTLA-4 is
most likely due to the expansion of effector CD8+ T cells within the
tumor, which express the markers CD44 and T-bet, which we
observed upon treatment with aCTLA-4 or aCTLA-4/aPD-1 combi-
nation therapy.
Importantly, we did not observe an increase in antigen-specific im-
mune responses in the spleen upon combination therapy with
TERT DNA vaccine and immune checkpoint blockade, despite the
synergy of these therapies in inducing anti-tumor immunity. These
data suggest that immune checkpoint blockade functions to alter
the immune microenvironment and expand effector T cells at the tu-
mor site rather than acting on antigen-specific cells in the periphery.
It is possible that addition of immune checkpoint blockade antibodies
may alter T cell trafficking and may drive antigen-specific immune
cells to peripheral tissues in the mouse.
Taken together, these studies support further rational combination
immune therapies with synthetic TERT DNA vaccination and im-
mune checkpoint blockade. In addition, antigen-specific immune re-
sponses in the periphery may not correlate with efficacy in patients.
MATERIALS AND METHODS
DNA Plasmids
The synthetic consensus TERT sequence was generated by using 6
TERT sequences collected from animals including mouse, rat, and
hamster. The consensus sequence was obtained after alignment of
these sequences using Clustal W. Two additional mutations were
added to abolish telomerase activity.31,32 All sequences were RNA
and codon optimized with an immunoglobulin E (IgE) leader
sequence and a Kozak sequence at the N terminus and cloned into
the modified pVAX vector (Genscript). The final SynCon mouse
TERT sequences shares 94.6% sequence identity with native mouse
TERT.
Peripheral Blood Mononuclear Cell and Splenocyte Isolation
For splenocyte isolation, spleens from mice were collected in com-
plete RPMI media containing 10% fetal bovine serum (FBS). Cells
were dissociated using a stomacher and then filtered through a
40-mm mesh filter. Red blood cells were then lysed using ACK Lysis
buffer (LifeTechnologies). Cells were then filtered again through a
40-mm mesh filter, counted, and plated for staining. For peripheral
blood mononuclear cell (PBMC) isolation, blood was collected in
4% sodium citrate to prevent clotting. Blood was then layered on
top of Histopaque 1083 (Sigma-Aldrich). Cells were spun for 1 hr,
and then cells from the buffy coat were separated and stained for
analysis.
Isolation of TILs
Tumors were dissociated using digestion mix, which consisted of
170 mg/L collagenase I, II, and IV (ThermoFisher), 12.5 mg/L DNase
I (Roche), and 25 mg/L Elastase (Worthington) in a 50/50 mixture of
Hyclone L-15 Leibowitz Media (ThermoFisher) and RPMI, supple-
mented with 10% FBS and 1% penicillin/streptomycin. Tumors
were minced and then transferred to a 50 mL conical filled with
10 mL of digestion mix. Cells were then filtered twice through a
40-mm mesh filter, counted, and stained.
ELISpot Assay
Splenocytes were stimulated in the presence of native mouse TERT
peptides (15-mer peptides spanning the entire native mouse protein,
overlapping by 9 amino acids, GenScript) for 24 hr in MABTECH
Mouse IFNg ELISpotPLUS plates. Spots were developed and counted
in accordance with the manufacturer’s protocol.
Intracellular Cytokine Staining and Flow Cytometry
2 million splenocytes per mouse were stimulated with native mouse
TERT peptides for 5 hr in the presence of GolgiStop GolgiPlug (BD
Bioscience). Phorbol myristate acetate (PMA, BD Bioscience) and
complete media were used as positive and negative controls, respec-
tively. Cells were incubated with FITC a-mouse CD107a (clone
1D4B, Biolegend) during the stimulation to stain for degranulation.
After stimulation, cells were washed and stained for viability using
LIVE/DEAD violet. Surface stain was then added, followed by fixation
and permeabilization using a FoxP3/transcription factor fixation/per-
meabilization kit (eBioscience). The following antibodies were used in
these studies: PECy5 aCD3 (clone 145-2C11, BD PharMingen),
BV510 aCD4 (clone RM4-5, Biolegend), BV605 aTNF-a (clone
MP6-XT22), PE aT-bet (clone 4B10, Biolegend), BV711 aPD-1
(clone 29F.1A12, Biolegend), APC aFoxP3 (clone FJK-16 s,
eBioscience), APCCy7 aCD8 (clone 53-6.7, Biolegend), AF700
aCD44 (clone IM7, Biolegend), and APC aIFNg (clone XMG1.2,
Biolegend). All data were collected on a modified LSRII flow cytom-
eter (BD Bioscience) and analyzed using FlowJo software (TreeStar).
Animal Immunization
Female 6- to 8-week-old C57BL/6 mice were purchased from Jackson
Laboratory. Animal care was in accordance with the guidelines of the
NIH and with the Wistar Institute Animal Care and Use Committee
(IACUC). Mice were immunized with 25 mg of each plasmid by intra-
muscular injection into the tibialis anterior (TA) muscle, followed by
electroporation (EP) using the CELLECTRA-3P adaptive constant
current device (Inovio Pharmaceuticals). Two 0.1-amp constant
current square-wave pulses were delivered through a triangular
three-electrode array. Each pulse was 52 ms in length with a 1-s delay
between pulses. For immunogenicity studies, mice were given a total
of three immunizations at 2-week intervals. For tumor challenge
studies, mice were given a total of four immunizations at 1-week
intervals.
Immune Checkpoint Blockade Antibodies
The following antibodies were used for animal studies (BioXCell):
a-mouse PD-1 (clone RMP1-14), a-mouse CTLA-4 (clone 9D9), or
aCD25 (clone PC-61.5.3). Mice were given 200 mg of each antibody,
delivered intraperitoneally, in accordance with the schedule in each
figure.
Molecular Therapy Vol. 26 No 2 February 2018 9
 Please cite this article in press as: Duperret et al., Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting
TERT, Molecular Therapy (2017), https://doi.org/10.1016/j.ymthe.2017.11.010
Tumor Challenge Studies
The TC-1 cell line was a gift from Dr. Yvonne Paterson from the Uni-
versity of Pennsylvania. This cell line was tested for Mycoplasma
contamination prior to freezing the cells, and the test results were
negative. Cells were thawed and cultured for fewer than 5 passages
prior to implantation into mice. Cells were cultured in DMEM
(Mediatech) supplemented with 10% fetal bovine serum (FBS). Cells
were subcutaneously implanted onto the right flank of C57BL/6 mice
(50,000 cells per mouse). 1 week following tumor implantation,
mouse tumors were measured, and mice were randomized to treat-
ment groups so that the average tumor volume and SD was equivalent
between groups. Tumors were monitored twice a week with electronic
calipers. Tumor volume was calculated using the following formula:
volume = (p/6)*(height)*(width2). Mice were euthanized upon any
sign of distress or sickness or when tumor diameters exceeded
1.5 cm, in accordance with IACUC guidelines.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism Software.
For each experiment, error bars represent the mean ± the SEM. For
all experiments, statistical significance was determined by one- or
two-way ANOVA, followed by Tukey’s post hoc honest significant
difference (HSD) test. For mouse survival studies, significance was
determined by Gehan-Breslow-Wilcoxon test.
SUPPLEMENTAL INFORMATION
Supplemental Information includes three figures and can be found
with this article online at https://doi.org/10.1016/j.ymthe.2017.11.010.
AUTHOR CONTRIBUTIONS
E.K.D, M.C.W., D.O.V., B.F., J.W., J.Y., A.K., E.M., L.H., and D.B.W.
were involved in study conception and design. E.K.D., M.C.W., A.T.,
D.O.V., B.F., J.W., J.Y., and A.K. were involved in the acquisition, the
analysis, and the interpretation of data. E.K.D. and D.B.W. drafted
the manuscript. All authors were involved in critically revising the
manuscript.
CONFLICTS OF INTEREST
M.C.W., B.F., J.W., J.Y., A.K., E.M., and L.H. are employees of Inovio
Pharmaceuticals and as such receive salary and benefits, including
ownership of stock and stock options. D.B.W. discloses grant funding,
industry collaborations, speaking honoraria, and fees for consulting.
His service includes serving on scientific review committees and advi-
sory boards. Remuneration includes direct payments, stock, or stock
options. He notes potential conflicts associated with his work with
Pfizer, Bristol Myers Squibb, Inovio Pharmaceuticals, Touchlight,
Oncosec, Merck, VGXI, and potentially others. Licensing of technol-
ogy from his laboratory has created more than 150 jobs in the private
sector of the biotechnology and pharmaceutical industry. The other
authors declare no competing financial interests.
ACKNOWLEDGMENTS
This work was supported by an NIH/NCI NRSA Individual Fellow-
ship (CA213795 to E.K.D.), a Penn/Wistar Institute NIH SPORE
10 Molecular Therapy Vol. 26 No 2 February 2018
Molecular Therapy
grant (CA174523 to D.B.W.), the Wistar National Cancer Institute
Cancer Center (P30 CA 010815), the W.W. Smith Family Trust (to
D.B.W.), funding from the Basser Foundation (to D.B.W.), and a
grant from Inovio Pharmaceuticals (to D.B.W.).
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