Translational Cancer Mechanisms and Therapy Clinical

Cancer
Research
Tumor Microenvironment Remodeling by
Intratumoral Oncolytic Vaccinia Virus Enhances
the Efficacy of Immune-Checkpoint Blockade
Hong Jae Chon1,2,3, Won Suk Lee1,2, Hannah Yang1,2, So Jung Kong1,2, Na Keum Lee1,2,
Eun Sang Moon4, Jiwon Choi4, Eun Chun Han2, Joo Hoon Kim1,2, Joong Bae Ahn3,
Joo Hang Kim1, and Chan Kim1,2

Abstract
Purpose: Cancer immunotherapy is a potent treatment conversion of a noninflamed tumor into an inflamed tumor.

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modality, but its clinical benefit depends on the tumor's JX virotherapy led to enhanced abscopal effects in distant
immune profile. Here, we used mJX-594 (JX), a targeted and tumors, with increased intratumoral infiltration of CD8þ
GM-CSF–armed oncolytic vaccinia virus, as a strategy to re- T cells. A depletion study revealed that GM-CSF is an
model the tumor microenvironment (TME) and subsequently indispensable regulator of anticancer efficacy of JX. Dual-
increase sensitivity to aPD-1 and/or aCTLA-4 immunotherapy. combination therapy with intratumoral JX and systemic
Experimental Design: The remodeling of the TME was aPD-1 or aCTLA-4 further enhanced the anticancer immune
determined using histologic, flow-cytometric, and NanoString response, regardless of various treatment schedules. Of note,
immune profiling analyses. JX was intratumorally injected triple combination immunotherapy with JX, aPD-1, and
into implanted Renca kidney tumors or MMTV-PyMT trans- aCTLA-4 elicited the most potent anticancer immunity and
genic mouse breast cancers with or without aPD-1 and/or induced complete tumor regression and long-term overall
aCTLA-4. Various combination regimens were used to evalu- survival.
ate immunotherapeutic anticancer responses. Conclusions: Our results show that intratumoral JX treat-
Results: Intratumoral injection of JX remodeled the TME ment induces dramatic remodeling of the TME and
through dynamic changes in the immune system, as shown more potently suppresses cancer progression with immune-
by increased tumor-infiltrating T cells and upregulation of checkpoint blockades by overcoming resistance to
immune-related gene signatures. This remodeling induced immunotherapy.

Introduction effector functions on tumor cells (11–13). Therefore, additional

Cancer immunotherapy with immune-checkpoint inhibitors therapeutic interventions are required for these non–T cell–
(ICI) targeting PD-1 or CTLA-4 has demonstrated a potent and inflamed tumors to appropriately remodel the TME to render
durable therapeutic efficacy and emerged as a new weapon in the these tumors more sensitive to ICI treatments (8, 14).
war on cancer (1–6). However, the clinical efficacy of ICIs is Oncolytic viruses have been proposed as a novel class of
confined to tumors with a T cell–inflamed tumor microenviron- anticancer therapy, and viruses with different backbones and
ment (TME; refs. 7, 8). In poorly immunogenic tumors with few transgenes are currently being evaluated in clinical trials (15–
tumor-infiltrating lymphocytes (TILs), TME lacks the type I inter- 17). Although the success of oncolytic viruses was initially pre-
feron signature and chemokines for T-cell recruitment (9, 10). dicted during the past decade based on their faster replication and
Moreover, tumor vasculatures and stromal components may pose enhanced oncolytic capability, they are now beginning to be
a barrier against intratumoral trafficking of T cells and their recognized as an immunotherapeutic because the strongest and
most durable responses after oncolytic virotherapy are coupled
with successful induction of antitumor immunity with increased
1
tumor-specific effector and memory T cells (16, 18–21). None-
Medical Oncology, CHA Bundang Medical Center, CHA University, Seongnam,
theless, because the therapeutic efficacy of oncolytic viruses was
Republic of Korea. 2Laboratory of Translational Immuno-Oncology, Seongnam,
Republic of Korea. 3Yonsei Graduate School, Yonsei University College of
greatly hindered by the immunosuppressive TME, releasing the
Medicine, Seoul, Republic of Korea. 4SillaJen, Inc., Seoul, Republic of Korea. brakes of the immune system is critical to maximize the immu-
notherapeutic efficacy of oncolytic viruses (22–25). Therefore, the
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/). combination of oncolytic viruses and ICIs is a rational and
appealing strategy to overcome the poorly immunogenic and
H.J. Chon and W.S. Lee contributed equally to this article.
immunosuppressive TME.
Corresponding Author: Chan Kim, CHA Bundang Medical Center, 59 Yatap-ro, JX-594 (pexastimogene devacirepvec, Pexa-vec) is an oncolytic
Bundang-gu, Seongnam 13496, Republic of Korea. Phone: 82-31-780-7590; vaccinia virus that is engineered to express an immune-activating
Fax: 82-31-780-3929; E-mail: chan@cha.ac.kr
transgene, GM-CSF, and that has the viral thymidine kinase gene
doi: 10.1158/1078-0432.CCR-18-1932 disrupted (26, 27). JX-594 showed impressive anticancer activity
2018 American Association for Cancer Research. with low toxicity in preclinical and clinical studies. It has become

1612 Clin Cancer Res; 25(5) March 1, 2019

 Potentiation of Immunotherapy by Oncolytic Vaccinia Virus

tor. All cell lines were used within 10 passages and confirmed to
Translational Relevance be Mycoplasma free using the MycoAlert Mycoplasma Detection Kit.
Cancer immunotherapies, such as immune-checkpoint
inhibitors (ICI), have demonstrated potent therapeutic effica- Generation and quantification of virus
cy. However, many cancer patients have immunosuppressive mJX-594 (JX), provided by SillaJen, Inc., is a Western Reserve
tumors, leading to resistance against immunotherapy and strain of vaccinia virus encoding murine GM-CSF in the vaccinia
consequently limited therapeutic response. JX-594 (Pexa-vec) thymidine kinase gene locus under the control of the p7.5
is one of the most promising oncolytic virus platforms in promoter (35, 36). This virus was amplified in HeLa S3 cells
clinical development as one of the few oncolytic viruses in prior to purification. In brief, HeLa S3 cells were infected and
phase III clinical trials. Here, we show that a murine version of incubated with recombinant vaccinia virus for 3 days, collected by
JX-594 (JX) remodels the tumor microenvironment by facil- centrifugation, then homogenized and centrifuged once more.
itating the accumulation of T cells. As a result, poorly immu- The virus-containing supernatant was layered onto a 36% sucrose
nogenic tumors become sensitive to ICIs, augmenting the cushion and centrifuged at 32,900  g, and the purified viral pellet
immunotherapeutic efficacy. Of note, the triple combination was resuspended in 1 mmol/L Tris, pH 9.0. To determine the viral
therapy of JX, aPD-1, and aCTLA-4 maximizes anticancer titer, serially diluted virus in serum-free DMEM was applied onto a
immunity and induces durable regression with improved monolayer of U-2 OS cells for 2 hours, and then 1.5% carboxy-
overall survival. These findings demonstrate the potential of methylcellulose in DMEM supplemented with 2% FBS was added.

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JX in combination with ICIs for improving anticancer immune After 72 hours, cells were stained with 0.1% crystal violet and
responses. plaques were counted.

Tumor models and treatment regimens

Tumors were implanted by subcutaneous injection of 2  105
Renca cells into the right flank of wild-type BALB/c mice. When
one of the most feasible and promising oncolytic virus plat-
tumors reached >50 mm3, mice were treated with either PBS or
forms in clinical development as one of the few oncolytic
1  107 plaque-forming units (pfu) of JX by intratumoral injec-
viruses in phase III clinical trials (27–30). In addition to its
tion every 3 days. For the bilateral tumor model, 2  105 Renca
oncolytic and vascular disrupting activity, JX-594 is proposed to
cells were implanted subcutaneously into the right flank, and 1 
exert an in situ cancer vaccination effect because it can elicit the
105 Renca or CT26 cells were implanted subcutaneously into the
adaptive immune response against tumor antigens for selective
left flank 4 days later. For the cell depletion study, antibodies
tumor disruption and subsequent additional tumor antigen
against CD4 (200 mg, clone GK1.5; Bio X Cell), CD8 (200 mg,
release (31, 32). Although JX-594 is now in a phase III ran-
clone 53-6.72; Bio X Cell), or GM-CSF (200 mg, clone MP1-22E9;
domized clinical trial (NCT02562755) in advanced hepatocel-
Bio X Cell) were intraperitoneally injected along with intratu-
lular carcinoma (33), few studies have characterized its
moral JX treatment. For immune-checkpoint blockade, anti–PD-1
immune modulatory functions in primary TME as well as
(10 mg/kg, clone J43; Bio X Cell) and/or anti–CTLA-4 (4 mg/kg,
distant lesions after JX-594 treatment (34). Moreover, the
clone 9D9; Bio X Cell) antibodies were injected intraperitoneally,
optimal combination of JX-594 with immunotherapeutics such
every 3 days depending on the dosing schedule. Tumors were
as ICIs has not yet been pursued and verified.
measured every 2 or 3 days using a digital caliper, and tumor
Here, we comprehensively dissected the dynamic remodeling
volumes were calculated using the modified ellipsoid formula
of the TME with a mouse variant of JX-594 (mJX-594, WR.
[1/2  (length  width2)]. On day 50, the surviving mice with
TKmGM-CSF, hereafter referred to as JX) and investigated its
complete tumor regression were rechallenged with 2  105 Renca
immunotherapeutic potential to provide a rational combinatorial
or CT26 cells in the left flank and monitored for tumor growth and
strategy with ICIs in poorly immunogenic tumor models.
survival. Mice were euthanized when tumors reached 1.5 cm in
diameter or when mice became moribund. Female MMTV-PyMT
Materials and Methods transgenic mice were purchased from The Jackson Laboratory.
Starting at 9 weeks after birth, the volume of every palpable tumor
Mice and cell lines
nodule (>20 mm3) was measured, and the total volume of all tu-
Male BALB/c mice between 6 and 8 weeks of age were purchased
mors combined was used to calculate the tumor burden per
from Orient Bio Inc., and female MMTV-PyMT transgenic mice
mouse. MMTV-PyMT mice were randomized according to their
(FVB/N) were purchased from The Jackson Laboratory
initial tumor burden, and were treated with 4  107 pfu of JX with
(#002374). Mice were housed in a specific pathogen-free animal
or without anti–PD-1 (10 mg/kg) or anti–CTLA-4 (4 mg/kg)
facility at CHA University (Seongnam, Korea). All animal experi-
antibodies at the indicated time points. In particular, JX was
ments were approved by the Institutional Animal Care and Use
injected intratumorally (1  107 pfu per tumor) in 4 randomly
Committee (IACUC, #170025) of CHA University and were
selected palpable tumors. After 4 weeks of treatment, mice were
carried out in accordance with the approved protocols. The Renca
anesthetized, and tissues were harvested for further analyses.
murine renal cancer cell line and the CT26 murine colon cancer
Analyses for MMTV-PyMT were performed as previously
cell line were obtained from the ATCC (#CRL-2947) and Korean
described (37, 38).
Cell Line Bank (#80009). The human cancer cell lines HeLa S3 and
U-2 OS were also originally obtained from ATCC (#CCL-2.2 and Statistical analysis
#HTB-96). These cells were maintained in RPMI-1640 medium or Statistical analyses were performed using GraphPad Prism 7.0
Dulbecco's modified Eagle medium (DMEM), each supplemen- software (GraphPad Software) and PASW statistics 18 (SPSS).
ted with 10% fetal bovine serum (FBS) and 1% penicillin/ Values are represented as mean standard error of the mean
streptomycin, and were incubated at 37 C, 5% CO2 in an incuba- unless otherwise indicated. Statistical differences between means

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 Chon et al.

were tested using unpaired Student t tests. Survival curves were T-cell infiltration in both peritumoral and intratumoral regions
generated using the Kaplan–Meier method, and statistical differ- were also dose-dependent (Supplementary Fig. S2B). Indeed,
ences between curves were analyzed using the log-rank test. The flow-cytometric subset analysis of the lymphoid cell compart-
level of statistical significance was set at P < 0.05. ment revealed that the JX-induced increase in absolute numbers
of intratumoral CD8þ and CD4þ T cells was dose-dependent
(Supplementary Fig. S2C and S2D). Although the number of
Results CD4þFoxp3þCD25þ regulatory T cells increased following the
JX converts immunosuppressive noninflamed tumors into triple administration of JX (Supplementary Fig. S2E), the ratio of
inflamed tumors CD8þ T cells to regulatory T cells was 5.3-fold higher compared
To determine the immunomodulatory potential of the onco- with that of control treatment (Supplementary Fig. S2E), implying
lytic virus JX, we extensively examined temporal changes in TME an overall increase in T-cell effector function in the TME by JX
after single intratumoral injections of JX into the Renca tumors, treatment. Additionally, the expression of ICOS and granzyme B
which are resistant to ICIs (39). The tumoral level of JX was (GzB), which are costimulatory and T-cell activation markers,
already high at day 1, peaking at day 3, but was barely detectable at was increased in CD8þ T cells following JX treatment (Supple-
day 7 after the injection (Fig. 1A and B). Conversely, tumor vessel mentary Fig. S2F). To confirm the presence of tumor-specific T
density was markedly reduced between days 1 and 3 but was cells that were recruited into the tumor after JX treatment, IFNg
recovered at day 7 and thereafter after the injection (Fig. 1A and B; ELISPOT assays were performed with isolated TILs and spleno-

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Supplementary Fig. S1B and S1C), indicating that JX is a potent cytes. The result was a marked increase in IFNg-secreting T cells
but transient tumor vessel disruptor. Of note, the population of against Renca tumor cells within tumors and spleens of JX-treated
CD8þ cytotoxic T cells within the tumor, which comprise the most mice compared with control mice. This finding indicated the
critical aspect of anticancer immunity, began to increase strikingly presence of tumor-specific CD8þ T cells in tumors as well as
at day 5, peaking at day 7, and remaining at a high density at 2 in the lymphoid organ (Supplementary Fig. S2G). Further
weeks after injection (Fig. 1A and B), demonstrating distinct and subset analysis of the myeloid cell compartment revealed no
long-lasting conversion of the noninflamed tumor into a T cell– significant change in CD11bþGr1þ myeloid cell fraction in
inflamed tumor by JX. By comparison, CD11cþ dendritic cells tumors treated with JX (Supplementary Fig. S2H). However, the
(DC) transiently emerged at day 3 and then decreased in tumors. CD11bþLy6GLy6Cþ monocytic myeloid cell fraction was in-
However, DCs accumulated in draining lymph nodes from day 5 creased, whereas the CD11bþLy6GþLy6Cint granulocytic myeloid
where they interacted with CD8þ T cells (Fig. 1A; Supplementary cell fraction was reduced, indicating polarization of myeloid
Fig. S1D). In addition, the level of PD-L1 was low at day 0 and cells following JX treatment (Supplementary Fig. S2I). These
upregulated after JX treatment (Fig. 1A and B). Intriguingly, the findings demonstrate that repeated JX administration enhances
PD-L1 upregulation followed just after a massive influx of CD8þ anticancer immunity, leading to increased infiltration of activated
TILs (Fig. 1C), indicating activation of the PD-1/PD-L1 axis in an T cells and repolarization of myeloid cells.
attempt to negatively regulate T cell–mediated immunity. Most
PD-L1þ cells were cytokeratinþ tumor cells, and some were Intratumoral injection of JX leads to systemic and cancer-
CD11bþ myeloid but were not T cells (Fig. 1D). Thus, JX is not specific immune responses
only a transient tumor vessel disruptor but also a potent and To determine whether local injection of JX could induce a
durable anticancer immunity enhancer. In contrast to JX-treated systemic immune response for regulating noninjected distant
tumors, control tumors showed no significant changes in CD31þ tumors, we administered JX into the right-side tumor after
blood vessels, CD8þ cytotoxic T cells, CD11cþ DCs, or PD-L1þ implantation of Renca tumor cells into both side flanks. This
cells (Supplementary Fig. S1A). treatment suppressed the growth of both right and left (opposite,
To elucidate the cancer immune pathways modulated by JX, we not injected side) Renca tumors (Fig. 2A). In line with tumor
further analyzed changes in expression of 750 immune-related growth inhibition on both sides, infiltrations of CD8þ T cells at
genes in the Renca tumor following JX monotherapy, using a intratumoral regions were increased by 7.9- and 5.5-fold in both
PanCancer Immune Profiling panel. Of note, expression levels of right and left Renca tumors (Fig. 2B), suggesting that local JX
the genes (100 genes) related to immune modulation, including virotherapy can activate systemic anticancer immunity.
activation of type I IFN signaling, DC maturation, and T-cell Next, to exclude the possibility of direct viral spread to the
activation, were significantly different between control- and JX- distant tumors through systemic circulation after the local vir-
treated tumors (Fig. 1E and F). In particular, the genes related to otherapy, we examined the presence of JX in the left, noninjected
inhibitory immune checkpoints (Pd-1, Pd-l1, Ctla-4, and Lag-3) Renca tumors and found no immune-detective JX in the left
and agonistic immune checkpoints (Icos, Gitr, and Cd27), Th1 and tumors (Supplementary Fig. S3). This finding indicated that the
Th2 responses, and M1 macrophage polarization (Nos2 and anticancer activity of JX in distant tumors was systemically
Cd86) were upregulated in JX-treated tumors compared with immune-mediated and not a result of systemic viral spread.
control-treated tumors (Fig. 1G). These results indicate that JX To evaluate whether the observed systemic immune response
elicits long-term immune activation through dynamic changes in was tumor-specific, we performed a similar experiment using mice
the TME to remodel noninflamed tumors into T cell–inflamed implanted with Renca tumors on the right flank and CT26 tumors
tumors that can respond to ICIs. on the left flank. Intratumoral treatment of the right, Renca tumor
with JX markedly decreased the growth of the injected tumor,
JX augments intratumoral infiltration of CD8þ T cells and whereas the growth of the left, untreated CT26 tumor was unaf-
induces myeloid cell repolarization fected (Fig. 2C). Moreover, the number of CD8þ T cells was not
JX-induced delay of tumor growth was dose-dependent (Sup- changed in CT26 tumors but was highly increased in Renca
plementary Fig. S2A). In parallel, JX-induced increases in CD8þ tumors (Fig. 2D), indicating that JX virotherapy induces a

1614 Clin Cancer Res; 25(5) March 1, 2019 Clinical Cancer Research
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Figure 1.
JX converts immunosuppressive noninflamed tumors into inflamed tumors. Renca tumors were implanted subcutaneously (s.c.) into BALB/c mice and treated
with a single intratumoral injection of 1  107 plaque-forming units (pfu) of mJX-594 (JX) when tumors reached >50 mm3. A, Representative images of Renca
tumors treated with JX. Tumor sections were stained for JX, CD31, CD8, CD11c, and PD-L1. B, Quantifications of the JXþ area, CD31þ blood vessels, CD8þ cytotoxic
T cells, CD11cþ dendritic cells, and PD-L1þ cells.  , P < 0.05 versus day 0. C, Temporal changes in JX, CD8, and PD-L1 in the TME after JX treatment. D, Images
showing upregulated PD-L1 expression (red) in various cell types (green) within the TME after JX treatment. Note that the expression of PD-L1 was observed
mainly in Pan-CKþ tumor cells (arrowheads), and some CD11bþ myeloid cells (arrow) also occasionally expressed PD-L1, whereas CD3þ T cells did not. E,
NanoString immune-related gene-expression heat map. Red and green colors represent upregulated and downregulated genes, respectively. F, Volcano plot
showing changes in immune-related gene expressions in JX-treated tumors. Red line, P < 0.05. G, Comparisons of gene expressions related to inhibitory immune
checkpoints (IC), agonistic ICs, Th1 response, Th2 response, TME, and myeloid cell. Pooled data from two experiments with 5 animals per group. Values are mean
 SEM.  , P < 0.05 versus control. Two-tailed Student t test was used. Scale bars, 50 mm.

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 Chon et al.

Anticancer immunity plays a critical role in the overall

therapeutic efficacy of JX
To determine which components of the immune system are
responsible for the therapeutic efficacy of JX, we examined its
effect on tumors in mice treated with neutralizing antibodies
against CD8, CD4, or GM-CSF (Supplementary Fig. S4A). Of
special note, depletion of either CD8þ or CD4þ T cells abro-
gated the effective tumor growth inhibition by JX (Supple-
mentary Fig. S4B and S4C), emphasizing the importance of an
immune-mediated mechanism rather than direct oncolysis in
JX-induced tumor growth inhibition. Intriguingly, depletion of
CD4þ T cells at the time of JX injection reduced intratumoral
infiltration of CD8þ T cell (Supplementary Fig. S4D), indicating
that CD4þ T cells are involved in the activation and recruitment
of CD8þ T cells in the TME. However, depletion of CD8þ T cells
did not significantly alter infiltration of CD4þ T cells (Supple-
mentary Fig. S4D), indicating that CD8þ T cells did not affect

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CD4þ T cells in the TME. These findings indicate that intratumoral
JX treatment induces priming of CD8þ and CD4þ T cells, which
may interact with each other to mediate anticancer immunity.
Previous virotherapy based on herpes and vaccinia viruses used
GM-CSF as an immune-activating transgene, which recruits and
activates antigen-presenting cells that subsequently trigger T-cell
response (40). However, the use of GM-CSF is still controversial
because of its potential immunosuppressive roles in tumor pro-
gression, such as inducing proliferation of myeloid-derived sup-
pressor cells (18). Therefore, we explored whether GM-CSF is
required for the therapeutic effect of JX. Interestingly, depletion of
GM-CSF negated the antitumor effect of JX and reduced both
CD8þ and CD4þ T cell levels, suggesting that GM-CSF is critical
for the immunotherapeutic efficacy of JX (Supplementary Fig. S4C
and S4D). Thus, both CD8þ and CD4þ T cells are indispensable
mediators of the anticancer effect of JX, and GM-CSF is an essential
regulator of T-cell activation for the JX treatment.

Combination of JX with immune-checkpoint blockade elicits

an enhanced anticancer effect with augmented infiltration of
T lymphocytes into the tumor
As shown earlier, whereas JX inflames the TME by enhancing
the recruitment of CD8þ T cells, it concomitantly increases the
expression of PD-L1, which hinders the anticancer effects of
cytotoxic T cells. On the other hand, ICI monotherapy is ineffec-
tive in noninflamed, T cell–insufficient tumors (8). Therefore, we
sought to combine the two modalities to compensate for their
respective weaknesses.
The combination of anti–PD-1 antibody (aPD-1) and JX
reduced tumor growth by 70%, whereas aPD-1 and JX mono-
Figure 2. therapy delayed tumor growth by 23% and 44%, respectively
Intratumoral injection of JX leads to systemic and cancer-specific immune
(Fig. 3A). In support of these findings, CD8þ T cells were more
responses. Mice were s.c. injected with Renca tumor cells in the right flank
and with Renca or CT26 tumors in the left flank. Arrows indicated highly infiltrated in both peritumoral (2.5-fold) and intratumoral
intratumoral JX treatment. A, Growth curves of JX-injected Renca tumor and (2.4-fold) regions of the tumors treated with combination therapy
noninjected Renca tumor. B, Representative images and comparisons of than those treated with JX (Fig. 3B and C). Furthermore, CD31þ
CD8þ T cells in the JX-injected and noninjected tumors. C, Growth curve of tumor blood vessels were decreased by combination therapy
JX-injected Renca tumor and noninjected CT26 tumor. D, Representative compared with control (peritumoral and intratumoral regions,
images and comparisons of CD8þ T cells in the JX-injected and noninjected
1.8-fold and 2.6-fold, respectively; Fig. 3B and C; Supplementary
tumors. Unless otherwise denoted, n ¼ 5 for each group. Values are
mean  SD.  , P < 0.05 versus control. ns, not significant. Two-tailed Student Fig. S5A), and tumor apoptosis was most severely induced in
t test was used. Scale bars, 50 mm. tumors treated with combination therapy compared with all other
groups (Fig. 3B and C; Supplementary Fig. S5B). Similarly to our
tumor-specific CD8þ T-cell response. Thus, local JX treatment can initial findings (Fig. 1A–C), although the PD-L1 expression was
elicit systemic and tumor-specific anticancer immunity with lym- minimal in control tumors, it was upregulated by 2.1- to 3.7-fold
phocyte infiltration to distant tumors. in both peritumoral and intratumoral regions of JX-treated

1616 Clin Cancer Res; 25(5) March 1, 2019 Clinical Cancer Research
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Figure 3.
Combination of JX with aPD-1 elicits an enhanced anticancer effect with augmented infiltration of T lymphocytes into the tumor. Renca tumor–bearing mice
were treated with or without JX and aPD-1 on the indicated days (arrows). A, Comparisons of tumor growth. Mean and individual tumor growth curves over time.
B and C, Representative images (B) and comparisons (C) of CD8þ T cells, CD31þ blood vessels, activated caspase-3 (casp-3)þ apoptotic cells, and PD-L1þ cells in
the peritumoral and intratumoral regions. D, Diagram depicting the mechanism by which the immunosuppressive TME is overcome by a combination therapy of
JX and ICI. Pooled data from two experiments with 7 animals per group. Values are mean  SEM.  , P < 0.05 versus control; #, P < 0.05 versus JX; $, P < 0.05
versus aPD-1. ns, not significant. Two-tailed Student t test was used. Scale bars, 100 mm.

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 Chon et al.

tumors (Fig. 3B and C; Supplementary Fig. S5B), implying that The therapeutic efficacy of concurrent combination therapy
PD-L1 involves an adaptive negative feedback mechanism that with ICIs and oncolytic viruses varies depending on the virus
dampens anticancer immunity after oncolytic virotherapy. administration route because viral clearance by adaptive immu-
Next, to determine whether combination therapy is effective nity may differ when the oncolytic virus is injected either system-
against distant untreated tumors as well as injected tumors, we ically (intravenous) or locally (intratumoral; refs. 22, 41). There-
treated mice carrying bilateral Renca tumors with JX and/or aPD-1 fore, we hypothesized that the administration route could affect
(Supplementary Fig. S5C). The combination therapy more the efficacy of concurrent combination therapy. To test this
potently suppressed the growth of distant untreated tumors hypothesis, we compared intravenous versus intratumoral injec-
compared with JX or aPD-1 monotherapy. tion of JX concurrently with aPD-1 (Supplementary Fig. S8A and
Therefore, our findings indicate that combining JX and ICI not S8B). Intriguingly, in tumors treated with intravenous JX, JX
only potentiates the systemic immunotherapeutic effect of JX tumoral levels were remarkably reduced with concurrent aPD-
virotherapy but also overcomes resistance against ICI monother- 1 treatment. In contrast, in tumors treated with intratumoral JX,
apy through enhanced anticancer immunity by increasing CD8þ concurrent aPD-1 treatment had almost no effect on tumoral
T-cell infiltration (Fig. 3D). levels of JX. Therefore, concurrent aPD-1 treatment seems less
We further validated our hypothesis by testing the efficacy of likely to affect JX if JX is administered via intratumoral injection.
combination treatment with anti–CTLA-4 antibody (aCTLA-4) Collectively, combination therapy with intratumoral JX injec-
and JX. Although tumor growth was modestly inhibited by either tion and systemic ICI led to an effective anticancer immunity

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JX (42.0%) or aCTLA-4 (20.0%) monotherapy, combination regardless of treatment schedule, suggesting that intratumoral
therapy displayed the most potent inhibitory effect (57.6%; administration of JX could minimize the potential antagonism
Supplementary Fig. S6A). In addition, CD8þ T cells were more with systemic ICI treatment.
highly accumulated in both peritumoral (1.9-fold increase) and
intratumoral (1.9-fold increase) regions of tumors treated with Triple combination of JX, aPD-1, and aCTLA-4 induces
combination therapy compared with JX (Supplementary Fig. S6B profound tumor regression and provides a long-term survival
and S6C). CD31þ tumor blood vessels were also disrupted in both benefit in implanted kidney cancer
peritumoral and intratumoral regions of combination therapy– As dual combination of JX and ICIs did not induce complete
treated tumors compared with control (2.1-fold and 3.8-fold tumor regression, we explored the effect of triple combination
reductions, respectively; Supplementary Fig. S6B and S6C). Fur- therapy using JX, aPD-1, and aCTLA-4. Although the dual com-
thermore, flow cytometry revealed that intratumoral infiltration bination of aPD-1 and aCTLA-4 delayed tumor growth by 14.5%
of CD8þ and CD4þ T cells was also increased by JX and aCTLA-4 and JX monotherapy inhibited tumor growth by 36.9%, the triple
combination therapy (Supplementary Fig. S6D). Taken together, combination inhibited tumor growth by 76.5% (Fig. 5A). Of note,
these results indicate that combination therapy using JX and ICIs a few mice (40%) of this triple combination group exhibited
can overcome the resistance against immunotherapy in immu- complete tumor regression, which was not observed in any other
nosuppressive TMEs, resulting in enhanced anticancer effects. groups (Fig. 5B). Furthermore, mice with complete tumor regres-
sion were tumor-free for more than 14 weeks after treatment
The efficacy of combination immunotherapy with intratumoral cessation. They were also fully protected against rechallenge with
JX and ICIs is not largely affected by treatment schedule Renca tumor cells but were not immune to CT26 tumor cells,
Because ICIs can negatively affect viral replication and lead to suggesting the establishment of an effective, long-term, and
premature clearance of the oncolytic virus, previous studies tumor-specific immune memory (Fig. 5C).
explored the optimal schedules of treatment using combinations To establish that the potent anticancer effects induced by triple
of systemic oncolytic virotherapy and ICIs and reported that some combination therapy could translate into a long-term survival
combination schedules could antagonize the therapeutic effica- benefit, we performed survival analyses of tumor-bearing mice
cy (22, 41). However, the dependency of local oncolytic virother- (Fig. 5D). Mice treated with triple combination immunotherapy
apy on the treatment schedule of ICIs has not been reported. To showed a remarkably better overall survival compared with results
establish the optimal combination schedule for intratumoral JX with monotherapy or dual-combination therapy. Intriguingly, the
and ICIs, we compared the following: (i) simultaneous admin- difference between dual and triple combination therapy was not
istration of JX and ICI (schedule I); (ii) initiation of ICI 3 days after remarkable early in the treatment period, but it increased over
administration of JX (schedule II); and (iii) administration of JX 3 time and was maintained for a long time. Therefore, triple
days after initiation of ICI (schedule III; Fig. 4A). All combination combination therapy is needed to induce durable immunother-
schedules delayed tumor growth by 40% (Fig. 4B). Likewise, apeutic effects and longer survival. In conclusion, these findings
levels of tumor-infiltrating CD8þ and CD4þ T cells were increased demonstrate that triple combination immunotherapy has the
by >8.0-fold and >4.0-fold, respectively, and the expression of potential to induce complete tumor regression and long-term
ICOS and GzB in CD8þ T cells was remarkably increased com- survival.
pared with control regardless of the treatment schedule (Fig. 4C
and D). The triple combination therapy enhances anticancer immune
Similar to combination therapy with JX and aPD-1, the com- responses in a spontaneous breast cancer model
bination of JX and aCTLA-4 inhibited tumor growth by 40% To validate the long-term immunotherapeutic efficacy of the
regardless of the treatment schedule (Supplementary Fig. S7A). triple combination therapy in immune-resistant tumors, we used
Furthermore, intratumoral infiltration of CD8þ and CD4þ the MMTV-PyMT transgenic mouse model, which is a spontane-
T lymphocytes (>7-fold and >7-fold increases, respectively) and ous breast cancer model with intrinsic resistance to immune-
GzB and ICOS expression in CD8þ T cells were greater regardless checkpoint blockade (42). After 4 weeks of treatment, mice
of the treatment schedule (Supplementary Fig. S7B and S7C). treated with the triple combination of JX, aPD-1, and aCTLA-4

1618 Clin Cancer Res; 25(5) March 1, 2019 Clinical Cancer Research
 Potentiation of Immunotherapy by Oncolytic Vaccinia Virus

Figure 4.
The efficacy of combination
immunotherapy with intratumoral
JX and systemic ICIs is not largely
affected by treatment schedule.
Mice were s.c. implanted with Renca
tumor cells and treated with JX plus
ICIs on various schedules. A,
Diagram depicting various
treatment schedules. Arrows

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indicate treatment with either
intratumoral delivery of JX (red) or
systemic delivery of ICIs (blue). B,
Comparison of tumor growth in
mice treated with JX and aPD-1
using different treatment schedules.
Mean and individual tumor growth
curves over time. C, Representative
flow-cytometric plot showing
tumor-infiltrating CD8þ and CD4þ
T-cell fractions. D, Comparisons of
absolute numbers of CD8þ, CD4þ,
CD8þICOSþ, and CD8þGzBþ cells
per gram of tumors. Pooled data
from two experiments with 7
animals per group. Values are mean
 SEM.  , P < 0.05 versus control.
ns, not significant. Two-tailed
Student t test was used.

exhibited a significant reduction in overall tumor burden by triple combination immunotherapy with JX and ICIs can elicit a
48.1% and a delay in the development of palpable tumor nodules robust anticancer immune response even in a poorly immuno-
compared with control mice (Fig. 6A–D). Furthermore, triple genic spontaneous breast cancer model.
combination therapy led to a 48.1% reduction in average tumor
nodule size and better overall survival compared with other
treatments (Fig. 6E and F). Histologic analyses revealed less- Discussion
invasive carcinoma with well-preserved tumor margins in the Here, we demonstrate that combination therapy with JX and
triple combination group, indicating that triple combination ICIs is an effective therapeutic strategy for immune-resistant
effectively delays tumor progression and invasion (Fig. 6G). tumors. The combination therapy leads to an immunologic
Moreover, intratumoral recruitment of CD8þ T cells was further "boiling point" in which a cold, noninflamed tumor is sufficiently
increased by 2.0-fold in tumors treated with triple combination inflamed to enable the host immune system to eradicate tumor
therapy compared with those treated with JX monotherapy cells. The most profound effect was observed with triple immu-
(Fig. 6H). However, tumor vascular density was similar among notherapy with JX, aPD-1, and aCTLA4, which induced complete
the treatment groups (Fig. 6H), indicating that the vascular dis- regression in 40% of Renca tumors, one of the most resistant
rupting effect is not long-lasting after repeated JX injections. syngeneic tumors to immunotherapy. This strong efficacy can be
Finally, the number of hematogenous lung metastases was sig- explained by the mutually complementary cooperation of onco-
nificantly reduced in the triple combination group (Fig. 6I), lytic virus and ICIs.
indicating an effective antimetastatic action by the triple combi- JX-594 is an oncolytic virus in the most advanced stage of
nation therapy. Taken together, these results demonstrated that clinical trials and acts through various mechanisms (27, 32, 33).

www.aacrjournals.org Clin Cancer Res; 25(5) March 1, 2019 1619

 Chon et al.

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Figure 5.
The triple combination of JX and aPD-1 and aCTLA-4 induces profound tumor regression and provides a long-term survival benefit in kidney cancer. Mice were
s.c. implanted with Renca tumors and treated with or without JX and immune-checkpoint blockades for PD-1 and CTLA-4 on the indicated days (arrows). A,
Comparisons of tumor growth. Mean and individual tumor growth curves over time. Pooled data from two experiments with 8 animals per group.  , P < 0.05
versus control; #, P < 0.05 versus JX; $, P < 0.05 versus aPD-1þ aCTLA-4. ns, not significant. Two-tailed Student t test was used. B, Waterfall plot showing the
maximal percent changes from baseline in tumor size. C, Comparison of tumor size after injection of Renca or CT26 tumor cells into mice with complete tumor
regression or into na€ve mice. D, Kaplan–Meier plot for overall survival. n ¼ 8–11 for each group. Log-rank test was used.

Although it can rapidly induce direct oncolysis and vascular virus to prevent early shutdown of oncolytic virotherapy–
disruption in tumors, these effects are transient and mostly induced anticancer immunity (18).
diminish within 1 week of injection. Thereafter, CD8þ T cells Although ICI monotherapy revolutionized the treatment land-
extensively infiltrate the tumor to initiate anticancer immune scape of cancer, its dramatic therapeutic response is confined to a
responses. However, at the same time, tumors begin to evolve subset of patients (1, 43). This outcome gave rise to the concept of
to avoid immune-mediated elimination by upregulating immunologically "hot" or "cold" tumors: hot tumors respond
immune inhibitory checkpoint molecules such as PD-1, PD- well to ICIs because they are immunologically inflamed with TILs
L1, or CTLA-4 in the TME. Because the most potent and durable and show high expression of PD-L1, whereas cold tumors are
anticancer effects of an oncolytic virus are achieved when it is refractory to ICIs because of the paucity of CD8þ TILs and
coupled with successful induction and maintenance of antitu- immunosuppressive TME (9, 24). Therefore, current efforts are
mor immunity, it is reasonable to combine ICIs with oncolytic focused on overcoming resistance to ICIs by converting

1620 Clin Cancer Res; 25(5) March 1, 2019 Clinical Cancer Research
 Potentiation of Immunotherapy by Oncolytic Vaccinia Virus

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Figure 6.
The triple combination therapy delays tumor growth and metastasis in a spontaneous breast cancer model. Growth of spontaneous mammary tumors of MMTV-
PyMT mice was analyzed starting from 9 weeks after birth. Samples were harvested 13 weeks after birth. A, Diagram depicting the treatment schedule. Arrows
indicate treatment with or without intratumoral delivery of JX and systemic delivery of aPD-1 (P) and aCTLA-4 (C). B, Representative image showing gross
appearance of tumors. Dotted-line circles demarcate palpable mammary tumor nodules. C, Comparison of total tumor burden. Tumor burden was calculated by
summating the volume of every tumor nodule per mouse. D, Comparison of the number of palpable tumor nodules. E, Comparison of volume of each tumor
nodule. Each tumor nodule in MMTV-PyMT mice is plotted as an individual dot. F, Kaplan–Meier curves for overall survival. Log-rank test was used. G, H&E-stained
tumor sections showing intratumoral regions. Acinar structures of JX and JX þ P þ C groups are early, less-invasive lesions (Ea) showing the distinct boundary
with the surrounding mammary adipose tissue (Adi). Invasive ductal carcinoma regions (Ca) of Cont and P þ C have massively invaded into the surrounding
tissue and formed solid sheets of tumor cells with no remaining acinar structure. H, Representative images and comparisons of CD8þ T cells and CD31þ tumor
blood vessels in tumor. I, Representative lung sections stained with H&E and comparison of the number of metastatic colonies per lung section. Arrows indicated
metastatic foci. Unless otherwise denoted, n ¼ 8–9 for each group. Values are mean  SD.  , P < 0.05 versus control; #, P < 0.05 versus JX; $, P < 0.05 versus
aPD-1þ aCTLA-4. ns, not significant. Two-tailed Student t test was used in C–E, H, and I. Scale bars, 200 mm.

www.aacrjournals.org Clin Cancer Res; 25(5) March 1, 2019 1621

 Chon et al.

immunologically cold tumor to hot tumors. In this respect, our In this study, we were not able to exclude the possibility that the
result identifies JX as an ideal combination partner for ICIs. It can immunogenicity of mouse model was affected by a tumor
selectively replicate in tumor cells, destroy them, and release implantation–induced inflammatory reaction (50). Although we
tumor antigens to stimulate the host immune system. Moreover, performed every treatment 10 or 12 days after tumor implanta-
our study shows that JX can dramatically convert the TME from a tion to minimize inflammatory reaction, the level of the response
cold to hot state by inducing intratumoral inflammatory to treatment that we observed in this study may not fully reflect the
responses: induction of Th1 responses along with activation and immune reaction in human cancer. Therefore, the findings of this
recruitment of T cells, upregulation of PD-L1, and polarization of preclinical study should be confirmed in clinical trials.
myeloid cells toward M1. Intriguingly, the replication and spread Several clinical trials are ongoing to investigate the efficacy of
of oncolytic viruses are more active in cold tumors where there are JX-594 in combination with aPD-1, aCTLA-4, or aPD-L1 to
few immune cells to eliminate the virus, whereas hot tumors with target various solid cancers, including liver, kidney, and colon
ample resident TILs can induce premature clearance of virus and cancers (ClinicalTrials.gov: NCT03071094, NCT02977156,
attenuate its therapeutic effects (24). Therefore, together with the NCT03294083, and NCT03206073). Thus, we will be able to
results of this study, JX emerges as an optimal combination verify the findings of this study in a clinical setting in the near
partner for ICIs, especially for noninflamed cold tumors with future.
intrinsic resistance to immunotherapy. In conclusion, these results indicate that intratumoral injection
GM-CSF is the most commonly used therapeutic genetic pay- of JX induces a profound remodeling of the TME from cold to

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load of oncolytic viruses (18, 44, 45). Two oncolytic viruses in the hot state and elicits robust anticancer immunity in combination
most advanced phases of clinical trials, T-Vec and Pexa-Vec with ICIs, overcoming immunotherapy resistance.
(JX-594), are both armed with GM-CSF (46). Although GM-CSF
is generally known to induce proliferation of various immune Disclosure of Potential Conflicts of Interest
cells such as DCs, there is a concern regarding unwanted prolif- No potential conflicts of interest were disclosed.
eration of immunosuppressive cells such as myeloid-derived
suppressor cells (23, 47). In the present study, we revealed that Authors' Contributions
JX did not significantly alter the fraction of intratumoral Conception and design: H.J. Chon, W.S. Lee, E.S. Moon, C. Kim
CD11bþGr1þ cells. In addition, neutralization of GM-CSF ablat- Development of methodology: H.J. Chon, W.S. Lee, E.S. Moon, C. Kim
Acquisition of data (provided animals, acquired and managed patients,
ed the therapeutic efficacy of JX, which was partly because of the provided facilities, etc.): H.J. Chon, W.S. Lee, H. Yang, S.J. Kong, E.S. Moon,
reduction in CD8þ and CD4þ TILs, indicating that GM-CSF has an Joo Hang Kim, C. Kim
indispensable role in anticancer immunity elicited by JX. Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
Previous studies have reported that although the combination computational analysis): H.J. Chon, W.S. Lee, H. Yang, E.S. Moon, C. Kim
of an oncolytic virus and ICIs elicits an impressive immune Writing, review, and/or revision of the manuscript: H.J. Chon, W.S. Lee,
E.S. Moon, J. Choi, Joo Hang Kim, C. Kim
response, the therapeutic efficacy can be affected by administra-
Administrative, technical, or material support (i.e., reporting or organizing
tion route and treatment schedule (22, 41, 48). In particular, data, constructing databases): H.J. Chon, E.S. Moon, J. Choi, E.C. Han,
when both the oncolytic virus and ICIs are systemically admin- Joo Hoon Kim, C. Kim
istered simultaneously, the combination could be antagonistic Study supervision: E.S. Moon, J.B. Ahn, C. Kim
because of the ICI-induced antiviral immunity that can facilitate Other (conduct in vivo experiment): N.K. Lee
premature viral clearance, indicating the importance of an ade-
quate time gap in between treatments for the oncolytic virus to Acknowledgments
induce a successful anticancer immunity (41, 49). In the present This study was supported by the National Research Foundation of Korea
(NRF-2016R1C1B2014671 to C. Kim) grant funded by the Ministry of Science,
study, local injection of JX consistently induced anticancer immu-
ICT and Future Planning, and by the Bio and Medical Technology Development
nity without being significantly affected by administration Program of the National Research Foundation (NRF-2016M3A9E8941664 to
sequences. We presume that this result is attributable to the H. Chon). This study was also cosupported by the Global High-Tech Biomed-
intratumoral injection having provided the oncolytic virus a icine Technology Development Program of the National Research Foundation
sufficient time lag to inflame the TME before being detected and (NRF) and Korea Health Industry Development Institute (KHIDI) funded by the
eliminated by systemic antiviral immunity. Indeed, in tumors Korean government (MSIP and MOHW; No. 2015M3D6A1065644 and
HI15C3517).
treated with intratumoral JX, concurrent aPD-1 treatment had
almost no effect on the tumoral level of JX, in contrast to the
The costs of publication of this article were defrayed in part by the payment of
markedly decreased level of JX in tumors treated with intravenous page charges. This article must therefore be hereby marked advertisement in
JX. Therefore, intratumoral virotherapy may be more suitable for accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
designing clinical trials with the ICI and oncolytic virus combi-
nation compared with systemic virotherapy in terms of admin- Received June 18, 2018; revised October 22, 2018; accepted December 6,
istration schedule. 2018; published first December 11, 2018.

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