Published OnlineFirst May 4, 2011; DOI: 10.1158/1078-0432.CCR-10-3262
Clinical
Cancer
Research
Cancer Therapy: Preclinical
Intratumoral Administration of TLR4 Agonist Absorbed into
a Cellular Vector Improves Antitumor Responses
Meghan B. Davis1, David Vasquez-Dunddel1, Juan Fu1, Emilia Albesiano2,
Drew Pardoll2, and Young J. Kim1,2
Abstract
Purpose: Because toll-like receptor (TLR) agonists have been well characterized as dendritic cell (DC)
activators, we hypothesized that the admixture of TLR4 agonist into a cellular vector could improve the
antitumor response in vivo.
Experimental Design: Granulocyte macrophage colony stimulating factor secreting whole cell tumor
cell vector (GVAX) was formulated with lipopolysaccharide (LPS), a TLR4 agonist, and its intratumoral
therapeutic efficacy was tested in three different murine models. We utilized immunohistochemistry,
fluorescence-activated cell sorting, enzyme-linked immunosorbent spot (ELISPOT), and in vivo CTL
analysis to assess both local innate immune responses within the tumor tissue as well as the downstream
generation of antitumor T-cell responses.
Results: Intratumoral treatment of LPS-absorbed GVAX showed efficacy in improving an antitumor
response in vivo in comparison with GVAX alone. Improved antitumor efficacy of this novel admixture was
not present in TLR4 signaling impaired mice. In the CT26 model, 40% to 60% of the mice showed
regression of the transplanted tumor. When rechallenged with CT26 tumor cells, these mice proved to be
immunized against the tumor. Tumors treated with TLR4 agonist–absorbed GVAX showed increased
infiltrating CD4 and CD8 T cells as well as increased numbers of CD86þ cells in the tumor tissue. Draining
lymph nodes from the treated mice had enhanced number of activated CD86þ, MHCIIþ, and CD80þ DCs
in comparison with GVAX alone and mock-treated groups. ELISPOT assay and in vivo CTL assay showed
increased numbers of CTLs specific for the AH1 tumor antigen in mice treated with LPS-absorbed GVAX.
Conclusion: TLR4 on antigen-presenting cells in the tumor microenvironment may be targeted by using
cell-based vectors for improved antitumor response in vivo. Clin Cancer Res; 17(12); 3984–92. 2011 AACR.
Introduction
The role of toll-like receptor (TLR)-mediated inflammation in the tumor microenvironment is a complex process
whose role in carcinogenesis is still unclear. Various tumor
models dissecting the downstream MyD88 and NF-kB
signaling pathways initially showed procarcinogenic role
of TLR signaling (1–3). However, Salcedo and colleagues
have shown antitumor role of MyD88 signaling, whereas
Garrett and colleagues have noted no carcinogenic effect of
MyD88 signaling in Rag2/ mice (4, 5). TLR signaling in
Authors' Affiliations: Departments of 1Otolaryngology–Head and Neck
Surgery, 2Oncology and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
M.B. Davis and D. Vasquez-Dunddel contributed equally to this work.
Corresponding Author: Young J. Kim, Johns Hopkins University School
of Medicine, 1650 Orleans Street, CRB1 Building, Suite 4M61,
Baltimore, MD 21231. Phone: 410-550-0460; Fax: 410-550-2870; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-10-3262
2011 American Association for Cancer Research.
3984
the hematopoietic compartment, however, has been
shown to elicit antitumor responses, which have translated
into multiple clinical trials (6, 7). In the context of infection, TLR4 agonists have been shown to render dendritic
cell (DC) activation immunogenic whereas lack of TLR4
signaling can lead to tolerance (8). Medzhitov showed
phagocytosed microbial antigens can be more efficiently
presented in the presence of lipopolysaccharide (LPS; ref.
9). Cumulative, one valid strategy is to increase TLR stimulation directed toward increasing the number of activated antigen-presenting cells (APC) within the tumor
microenvironment in vivo (10–12).
This study focuses on intratumoral injection as a direct
means of modifying the tumor microenvironment to
enhance both innate and adaptive antitumor immunity.
To minimize potential TLR4 signaling on the tumor cells
and to target the APCs in the tumor microenvironment, we
evaluated intratumoral delivery of cellular vectors loaded
with TLR4 agonist. The implication from studies of Medzhitov is that colocalization of TLR4 agonist and antigen can
potentially enhance antitumor response when given as part
of a combinatorial cellular vector with tumor antigens (9).
An excellent candidate as a cellular vector for localized
TLR agonist delivery is GVAX. The paracrine granulocyte
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Published OnlineFirst May 4, 2011; DOI: 10.1158/1078-0432.CCR-10-3262
TLR4 Agonist in Cellular Vectors Can Improve Immunotherapy
Translational Relevance
Use of toll-like receptor (TLR) agonists as adjuvants
for advanced malignancies in patients is still controversial. One limitation is that TLR signaling can have mixed
results in the tumor microenvironment. To target TLR4
signaling in antigen-presenting cells within the tumor
microenvironment, we have formulated TLR4 agonist
with a safe cellular vaccine, GVAX, which is currently
undergoing clinical trials for multiple solid tumors. The
following preclinical studies, therefore, provide scientific rationale for potential modification of a well-studied tumor vaccine platform to improve its clinical
response in solid tumors amenable for intratumoral
injections such as head and neck tumors.
macrophage colony stimulating factor (GM-CSF) delivery
afforded by GVAX cells dramatically increases the numbers
of myeloid cells, including DCs, macrophages, and granulocytes, at the site of injection (13–15). GVAX has been
found to be safe from numerous phase I trials but was
recently found to have uncertain efficacy in a phase III trial
for advanced hormone refractory prostate cancer (16). As a
cellular vector, however, GVAX may offer a starting platform to immunize the patients with multiple tumor antigens and deliver apoptotic and cellular debris loaded with
TLR4 agonists to activate APCs in the tumor microenvironment as part of a combinatorial therapy.
To activate the locoregional APCs, LPS was formulated
with GVAX cells, and this novel combinatorial regimen was
injected intratumorally and studied for antitumor efficacy
in several murine models.
Methods
Murine tumor cell lines
The SCCFVII/SF head and neck squamous carcinoma,
B16-F0 melanoma, and B16-F0 transduced to secrete GMCSF cell lines were cultured in RPMI 1640 with 10% fetal
calf serum, penicillin (100 U/mL) and streptomycin (100
U/mL). CT26 was cultured similarly with MEM nonessential amino acids (Sigma), 1 mmol/L sodium pyruvate, and
2 mmol/L of L-glutamine. The bystander B78HI cells transduced with GM-CSF were cultured in the same media as
CT26 with the addition of Hygromycin B (1 g/L; Roche).
Mice
Adult (>50 days of age) female C57BL/6, BALB/C, C3H/
HeOuJ, and C3H/HeJ mice were purchased from Jackson
Laboratory and housed according to the JHU Animal Care
and Use Committee. C57BL/6 MyD88/ and C57BL/6
MyD88/TRIF/ mice were obtained from Dr. Franck
Housseau (Johns Hopkins University).
TLR4 agonist formulation with GVAX
Lipofectamine (120 mg/mL), LPS from Escherichia coli
026:B6 (10–50 mg/mL), and LPS-BODIPY (Molecular
www.aacrjournals.org
Probes) were combined into liposome complex. Cells for
LPS formulation were seeded at a density of 5 105 cells
were incubated with the Lipofectamine-LPS complex for 6
hours, washed 5 times prior to injection. To quantitate LPS
incorporated into cells, Limulus amebocyte lysate (LAL)
assay (Cambrex) was done as directed by the manufacturer.
LPS (23 EU/ng) concentrations ranging from 0 to 3.0 EU/
mL were used as standards. Prior to lethal irradiation, LPSabsorbed GVAX was cultured to ensure cell growth.
Annexin staining verified no evidence of apoptosis after
formulation (data not shown).
In vivo vaccine treatment assay
C57BL/6 mice were injected s.c in the right flank with 5 104 B16-F0 cells. Three to 5 days later, 106 lethally irradiated (150 Gy) B16 GM-CSF (GVAX), 106 lethally irradiated (150 Gy) LPS-formulated GVAX or LPS-absorbed
293T cells were injected intratumorally. In some cases, the
vaccines were injected in the contralateral limb from the
tumor-inoculated limb. C3H/HeOuJ mice and BALB/c
mice were used with SCCFVII/SF cells and CT26 cells,
respectively with comparable methods.
Immunohistochemistry
Frozen CT26 tumor were cut in 10 mm thickness and
blocked with 1% bovine serum albumin 30 minutes at
RT. Anti-mouse CD45 (eBioscience) and anti-mouse
CD4–fluorescein isothiocyanate (FITC), CD8–FITC,
and CD86–FITC antibodies (BD Pharmingen) incubated
at 4 C for 1 hour were used. Anti-CD45–Cy3 (Invitrogen) was used as secondary in some cases. 40 ,6-diamidino-2-phenylindole was used as counterstain for 10
minutes. Cells in 10 randomly selected fields at 40
magnification were counted by using Nikon Eclipse F800
microscope and developed with Nikon DS-Qi1mc camera. NIS-Element AR 3.0 was the software used for these
experiments.
DC activation assay
Spleens and draining lymph nodes (DLN) from tumorchallenged mice were harvested 5 days post-GVAX and
TEGVAX treatment. Crushed spleens were digested in
media containing DNAse I (Roche) and Liberase Blendzyme 2 (20,000 Mandl U/mL; Roche). DC-enriched populations were obtained by depleting CD3þ and CD19þ, and
gated for CD11cþ and B220þ. These were evaluated by a
multicolored fluorescence-activated cell sorting analysis
by using CD80, CD86, and MHCII antibodies from BD
Biosciences.
ELISPOT assay
Enzyme-linked immunosorbent spot (ELISPOT) plates
(MultiScreenHTS filter plate; Millipore) were coated with a
mouse IFN-g Ab (MabTech) for 24 hours and 4T1 breast
cancer cells were pulsed with 10 mg/mL of AH1 peptide
overnight. A total of 106 CD8 cells from spleen and lymph
nodes were plated in triplicates to be cocultured with
pulsed or unpulsed 105 4T1 cells or stimulated with 1
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Davis et al.
1 h incubation
10 µg/mL of LPS
6 h incubation
25 µg/mL of LPS
50 µg/mL of LPS
25 µg/mL of LPS
100
100
100
100
80
80
80
80
60
60
44.8
60
71.6
60
74.2
40
40
40
40
20
20
20
20
0
0
0
100
101
102
103
104
100
101
102
103
104
99.4
0
100
101
102
103
104
100
101
102
103
104
LPS-BODIPY
Figure 1. Formulation of TLR4 agonist into GVAX. To optimize the absorption of LPS, the concentrations of Lipofectamine and LPS-BODIPY fluorophore
conjugate were varied as noted. Cells were washed 5 times, and LPS was undetectable in the final wash by using the LAL assay. Optimal absorption
was at 6 hours incubation with 40 mg/mL of Lipofectamine and 25 mg/mL of LPS per 1 105 cells. These conditions were used for quantitation of LPS on a per
cell basis.
mmol/L of phorbol 12-myristate 13-acetate and 10 ng/mL
of Ionomycin as positive controls. On day 3, biotinylated
anti-mouse IFN-g Ab (MabTech) and Strepavidin–horseradish peroxidase for ELISPOT (BD) were added. AEC
Substrate Reagent Set for ELISPOT (BD) was used to
develop spots and analyzed by using an ELISPOT Plate
Reader (Immunospot).
In vivo CTL assay
A CellTrace CFSE Cell Proliferation Kit (Molecular
Probes) was used to test the cytolytic activity of CTLs in
CT26 tumor–challenged mice treated with or without
TEGVAX. Splenocytes were processed and pulsed with
either b-gal or AH1 peptide at a concentration of 10 ug/
mL for 90 minutes. The b-gal population was carboxyfluoroscein succinimidyl ester (CFSE)-labeled low (0.5 mmol/L)
and the AH1 population was CFSE-labeled high (5 mmol/
L). After 10 minutes, both CFSE-labeled cells were injected
into the mice at 107 cells/mouse. Twenty-four hours postinjection, splenic cells were harvested and analyzed for
detection of ratios of CFSE-labeled cells (17). Peptidespecific killing was calculated by using the formula (1%
of CFSE peptide/% of CFSE no peptide) 100.
Statistical analysis
We used paired t test to calculate 2-tailed P value to
estimate statistical significance of differences between 2
treatment groups by using Excel software. Kaplan–Meier
curves were generated by using GraftPad Prism software
and analyzed with log-rank test. Statistically, significant
P values are labeled in the figures and the legends with
asterisks.
Results
TLR4 agonist formulation with GVAX
To enhance locoregional innate immune cell activation
and minimize systemic toxicity of TLR4 stimulation
3986
Clin Cancer Res; 17(12) June 15, 2011
and minimize TLR4 stimulation on tumor cells, LPS was
formulated into GVAX (TEGVAX–TLR agonist–enhanced
GVAX). We used a commercially available vector, Lipofectamine, to optimize absorption of LPS into GVAX cells
prior to lethal irradiation. LPS-BODIPY flurophore was
used to show that 25 mg/mL of LPS for 6 hours resulted in
99.4% of the cells to be labeled (Fig. 1). LAL assay
(Cambrex) was then used to show that 4.73 0.2 ng
of LPS was absorbed into 5 105 cells by using the
Lipofectamine method that optimized LPS formulation
(data not shown). For each of the in vivo murine tumor
experiments, aliquots of TEGVAX were tested by using
LAL assay and murine GM-CSF ELISA assay to ensure a
comparable amount of LPS and GM-CSF in the TEGVAX
formulation. The typical GM-CSF secreted ranged from 50
to 200 ng/mL/106 cells/24 h.
TEGVAX can induce in vivo antitumor response in
multiple murine models
We initially tested the efficacy of intratumoral TEGVAX
injection in a B16 murine model, whereby TEGVAX was
delivered intratumorally 3 to 5 days after tumor inoculation in a therapeutic model. One rationale for intratumoral injection was to ensure that locoregional APCs
that circulate between the tumor and the DLNs were
targeted. These timepoints of treatment relative to initial
tumor implantation were selected because we previously
showed that anergic and tolerant tumor-specific T cells
were present as early as 3 days after B16 injection (18). As
shown in Figure 2, intratumoral TEGVAX administration
decreased the growth rate of the B16 tumor in comparison with intratumoral injection of GVAX alone, which
translated to survival curve differences (Fig. 2A). However, all the mice treated with TEGVAX eventually developed large tumors. We injected equimolar amount of LPS
intratumorally as in the TEGVAX treatment group and
these mice were no different than mice treated with
GVAX or PBS (Supplementary Fig. S1). Equimolar LPS
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TLR4 Agonist in Cellular Vectors Can Improve Immunotherapy
1.00
3.50
3.00
∗
2.50
2.00
1.50
1.00
GVAX
0.50
TEGVAX
0.00
16
14
18
20
GVAX
TEGVAX
50
∗∗
25
0
24
22
75
5
0
10
Time (d)
15
20
25
Days
3.50
100
GVAX
3.00
Percent survival
Relative volume – cm3
B
TEGVAX
2.50
2.00
∗
1.50
1.00
0.50
0.00
14
16
18
20
22
24
26
28
75
GVAX
TEGVAX
∗∗
50
25
0
30
0
5
10
Time (d)
15
Days
20
25
30
C
100
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
CT26
CT26+TEGVAX
∗
CT26+GVAX
Percent survival
Rechallenged
CT26+293LPS
75
50
∗∗
GVAX
TEGVAX
25
0
7
9
11
Time (d)
injected with GVAX separately without formulation also
did not show an antitumor response, suggesting that the
specific formulation of LPS with a cellular vector is
critical for its antitumor response (Supplementary
Fig. S1).
To test whether TEGVAX can also show an antitumor
response in other murine models, SCCFVII/SF cells were s.c
injected into the flanks of syngeneic C3H/HeOuJ mice with
a wild-type TLR4 (Fig. 2B) with comparable results as the
B16 model.
Finally, intratumoral administration of TEGVAX was
also tested in the CT26 colon carcinoma model, in which
it also showed an antitumor response (Fig. 2C). For
CT26, 40% to 60% of the mice treated with TEGVAX
actually showed regression of tumor. Mice whose tumor
regressed completely after intratumoral TEGVAX administration rejected subsequent challenges with CT26, indicating that the initial intratumoral treatment resulted in
systemic immunization. For the CT26 model, we also
tested LPS formulated with lethally irradiated 293 cells in
equimolar amounts as TEGVAX. This experiment was
done to determine whether colocalization of LPS and
www.aacrjournals.org
Percent survival
Relative volume – cm3
A
Relative volume – cm3
Figure 2. TEGVAX can induce an
antitumor response in vivo. A, B16
inoculated C57BL/6; B, SCCFVII/
SF inoculated C3H/HeOuJ; and C,
CT26 inoculated BALB/c mice
were treated with appropriate
PBS, GVAX, or TEGVAX
intratumorally typically from 3 to
5 days after the tumor injection. In
vivo tumor progression in the
TEGVAX group in all the murine
models studied was statistically
slower than those in the GVAX
group (*, P < 0.05; left). A total of 10
to 20 mice per group were used,
and all the experiments replicated
3 times. For the B16 and the
SCCFVII/SF models, there were
no differences between GVAXtreated group and the PBStreated group (Supplementary
Fig. S1 and data not shown). In the
CT26 model, 40% to 60% of the
mice in the TEGVAX group
experienced a regression of their
palpable tumor to undetectable
levels by 3 weeks. Equimolar LPS
formulated into 293T cells also
showed no difference in growth
rate. Mice showing regression of
tumor were rechallenged with
CT26 tumor (2 106) and no
tumor growth were noted. In all 3
murine models, the reduced tumor
growth rate correlated with
enhanced survival for mice in the
TEGVAX group compared with
those in the GVAX group
(**, P < 0.01; right).
13
15
0
10
20
30
40 50
Days
60
70
80
tumor antigen in TEGVAX was important for the antitumor response. LPS formulated with cells not expressing
an identical set of tumor antigens as the treated tumor
did not elicit an antitumor response in the CT26 model
(Fig. 2).
Antitumor response to TEGVAX is MyD88-TRIF and
TLR4 dependent
To verify that the in vivo antitumor response noted above
were because of the TLR4 signaling, B16 tumor was treated
with TEGVAX and GVAX in mice with MyD88/ and
MyD88/TRIF/ genotypes (Fig. 3A). Small differences
noted early between the GVAX group and PBS groups
eventually disappeared at later timepoints in MyD88/
mice. MyD88 and TRIF are essential intracellular mediators
of TLR4 signaling, and the complete absence of these
downstream TLR4 signaling mediators also abrogated
the enhanced in vivo antitumor response noted in wildtype mice. For the SCCFVII model, the experiment was
carried out in C3H/HeJ mice that lack functional TLR4,
and, once again, the antitumor effect of TEGVAX was
abrogated (Fig. 3B).
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Davis et al.
MyD88 -/4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
MyD88 -/- TRIF -/Relative volume - cm3
Relative volume - cm3
A
Wt
GVAX
TEGVAX
8
10
12
14
18
16
20
22
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Wt
GVAX
TEGVAX
10
24
12
14
16
Time (d)
B
TLR4 deficient C3H/HeJ
Relative volume - cm3
18
Time (d)
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
C3H/HEOUJ+GVAX
C3H/HEOUJ+TEGVAX
C3H/HEJ+SCCFVII+GVAX
C3H/HEJ+SCCFVII+TEGVAX
∗
10
12
14
16
18
20
22
20
22
24
Figure 3. Improved in vivo
antitumor response noted for
TEGVAX is MyD88 dependent and
TLR4 dependent. A, using the B16
model in the MyD88/ mice and
MyD88/TRIF/ mice, the in
vivo tumor growth rate was not
delayed by the vaccine treatment
conducted intratumorally 3 to
5 days after tumor inoculation.
B, for the SCCFVII model, the
experiment was done in C3H/HeJ
that lack TLR4, and for these mice,
TEGVAX treatment did not show
the antitumor response.
Experiments by using C3H/HeOuJ
mice replicated results from Figure
2 (*, P < 0.05). All these
experiments were replicated 3
times with 10 mice per group.
24
Time (d)
TEGVAX induces enhanced T-cell infiltration and APC
maturation in the tumor
To examine the potential mechanism of the in vivo
antitumor responses from TEGVAX treatment, the tumor
tissue was harvested and analyzed for lymphocytic infiltrate. As shown in Figure 4, tumor treated with TEGVAX
had quantitatively increased CD4 and CD8 infiltration in
comparison with the control and the GVAX-treated
tumors. Moreover, given our hypothesis that TLR4 agonist stimulates the locoregional APCs to mature, we
probed the tissue with aCD86 and noted quantitative
enhancement of CD86þ cells in the tumor treated with
TEGVAX. We found no significant differences in the
infiltration of F4þ macrophages between the treatment
groups (data not shown).
TEGVAX augments DC activation in the
locally DLNs
The immunostaining data from Figure 4 were consistent
with the hypothesis that TLR4 agonist–absorbed cell vaccines can increase the number of activated locoregional
DCs. Given that the LPS absorbed into GVAX is probably
phagocytosed into the infiltrating DCs as cellular debris
and micelles with tumor antigens, we predicted that there
would be increased number of activated locoregional DC
population with TEGVAX treatment. To test this, we purified DCs from the DLNs from tumor-bearing mice treated
with either PBS, GVAX, or TEGVAX and gated for the
conventional DC population with B220 and CD11c. Multi-
3988
Clin Cancer Res; 17(12) June 15, 2011
parametric staining of DC activation marker, CD86, CD80,
and MHCII from these gated cells, shows that DCs from the
TEGVAX-treated group has greater population of activated
phenotype (Fig. 5).
Tumor-specific cytotoxic T cells are expanded in mice
treated with TEGVAX
CT26 model is associated with a well-characterized
immunodominant CT26 antigen, AH1 that can facilitate
quantitation of tumor-specific cytotoxic T cells. To test
whether the in vivo antitumor responses for TEGVAX that
activates locoregional DCs can increase the downstream
population of tumor-specific cytotoxic effector cells, ELISPOT assays were conducted (19). T cells from the DLN
and spleen were harvested and IFN-g producing cytotoxic T
cells screened in the presence of MHC class I Ld restricted
AH1 peptides pulsed with APC in the ELISPOT assay.
Although minimal AH1-specific T cells were detected on
days 3 to 5 after treatment with TEGVAX (data not shown),
by day 8, there were statistically significant AH1-specific T
cells in the TEGVAX group in both the DLNs and the spleen
as shown in Figure 6A. In vivo CTL assays also showed
enhanced cytotoxic T-cell priming for the AH1 peptide in
the TEGVAX-treated group in comparison with the control
groups (Fig. 6B). The "average killing" value calculated
between TEGVAX and GVAX only reached statistical significance at P < 0.07, but each of the experiments showed
consistent trend for enhanced number of AH1-specific T
cells in the TEGVAX-treated groups. In vivo CTL assays with
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TLR4 Agonist in Cellular Vectors Can Improve Immunotherapy
A
CD4+CD45
CD8+CD45
CD86
Untreated
GVAX
TEGVAX
B
*P < 0.01
Positive cells numbers
Figure 4. TEGVAX treatment
increases the lymphocytic and
APC infiltration into the tumor
microenvironment.
Immunohistochemical staining
was used to detect the expression
of CD4þ, CD8þ, CD86þ, and
CD45þ cells in CT26 tumor treated
with control, GVAX, or TEGVAX.
The yellow cells represent the
colocalized staining of CD4 or
CD8 with CD45 in the first 2
columns. The arrow points to the
nonaggregating, diffusely
infiltrating lymphocytes in the
tumor tissue. The third column
represents CD86 conjugate
staining alone. These cells were
formally quantified in 10 randomly
selected fields per slide at 40
magnification. Statistical
differences were obtained in the
CD4, CD8, CD86, and CD45 cells
between the GVAX and TEGVAX
groups (P < 0.01). These
experiments were replicated at
least 3 times.
200
150
CD8
50
CD45
p15E-specific T cell from the B16 model also showed
enhanced p15E-specific T cells in the TEGVAX group (data
not shown).
Discussion
In this article, we tested the efficacy of intratumoral
injection of TEGVAX, a novel TLR4 agonist–formulated
GVAX, whereby we were able to absorb TLR4 agonist into
GVAX cells. We showed that TLR4 agonist–absorbed
GVAX has significantly improved antitumor response in
3 different therapeutic murine models, including SCCFVII/
SF, Bl6, and CT26. For the CT26 model, TEGVAX injected
intratumorally was able to prevent tumor growth in 40% to
60% of the mice, whereas GVAX or LPS injection alone had
no such effect.
By adding a potent TLR4 agonist into the tumor microenvironment via a cellular vector with our TEGVAX reagent,
we showed that the growth rate of the inoculated tumor is
significantly blunted. We believe that it is not only the
addition of TLR4 agonist, but also its novel delivery
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CD4
100
0
CD86
Untreated
GVAX
TEGVAX
absorbed into lethally irradiated vaccine cells with tumor
antigens as vectors. Equimolar LPS injection alone did not
have an in vivo response. Injection of equimolar LPS
directly into the tumor separately from GVAX and control
experiments by using LPS absorbed into 293T cells also did
not produce the antitumor response in our mice models
(Fig. 2 and Supplementary Fig. S1). The effect of TLR4
agonist in the tumor microenvironment is a complex
process that has produced conflicting in vivo responses.
Recent reports showed that TLR4 expressed on HNSCC
cell lines could promote carcinogenesis (3). However,
expression of TLR4 in HNSCC primary tumor is heterogeneous and it is unclear in whether TLR4 signaling is a
critical carcinogenic signaling in vivo. Others have shown
that TLR4 stimulation can break CD8þ T-cell tolerance and
eradicate established tumors (17, 20). MyD88 signaling,
which is downstream to TLR4, has been shown to be
procarcinogenic in some animal models (5). However,
our results are consistent with a recently presented hypothesis developed by Medzhitov and others that TLR4 agonists
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Davis et al.
TEGVAX
MHC II
GVAX
52.1
41
3.42
3.42
5.22
3.48
89.6
1.74
MHC II
CD86
16.4
73.6
6.14
85.1
8.18
1.82
5.26
3.51
CD80
Figure 5. TEGVAX treatment increases activated DCs in the DLNs. A total
of 10 to 20 CT26 tumor–bearing BALB/c mice were treated with PBS
(not shown), GVAX, or TEGVAX peritumorally 3 days after the tumor
injection. DCs were isolated from the DLN 5 to 8 days after treatment by
enzymatic digestion. B220-CD11cþ conventional DCs were gated and
CD86, MHCII, and CD80 staining were analyzed as shown. TEGVAX had
an increased number of CD86þMHCIIþ and CD80þMHCIIþ DCs in the
DLN in comparison with GVAX-treated group. These experiments were
replicated more than 3 times. The results from these other experiments are
depicted in Supplementary Fig. S4.
absorbed into micelles can induce an upregulation of
antigen presenting machinery in the phagolysosome by
the APCs (9).
With TEGVAX, our strategy was to dress syngeneic cells as
"foreign" cells with strong danger signals that can activate
APCs in the tumor microenvironment. LPS formulated into
cellular vaccines may be preferentially targeted toward the
locoregional APCs such as DCs. We hypothesized that
apoptotic cellular debris with TLR4 agonist from the
injected TEGVAX cells would be phagocytosed by the local
APCs such that TLR4 signaling on the tumor cells would be
minimized. Hence, this would explain the consistent lack
of in vivo antitumor response in the mouse groups treated
with LPS alone or with LPS and GVAX without the absorption formulation. When we examined the draining LN,
TEGVAX treatment was associated with an increased number of activated DCs as defined by high expression of
CD86þ, CD80þ, and MHC class IIþ cells in comparison
with GVAX-treated groups as shown in Figure 5. GVAXtreated groups also had increased number of activated DCs
but less than TEGVAX-treated DCs, which is consistent with
the immunohistochemical data from Figure 4. GM-CSF
from GVAX can also activate DC cells that explain the
increased number of activated DC in GVAX group in
comparison with the control group treated with PBS alone.
3990
Clin Cancer Res; 17(12) June 15, 2011
The lack of antitumor response in GVAX treatment group
may stem from possible upregulation of myeloid derived
suppressor cells that has been associated with GM-CSF
signaling in the tumor microenvironment (21). Regardless,
TEGVAX-treated groups had greater number of activated
DCs in comparison with the GVAX or the PBS-treated
groups to potentially induce tumor-specific CTL response
in our experimental models.
Activated APCs can prime tumor-specific T cells as downstream effectors, so we examined the tumor tissue from
each of the treated and the control groups for CD4 and
CD8 T-cell infiltration by using immunohistochemistry.
Quantitatively, TEGVAX-treated tumors showed greater
infiltration of both CD4 and CD8 T cells in comparison
with GVAX- or PBS-treated tumors. Whether the increase in
CD4þ cells is also associated with any changes in Treg or
Th17 cells are still being investigated. Regardless of this,
there appeared to be a positive correlation between T-cell
infiltration and the overall tumor response in vivo. We
stained for natural killer cells in the tumor tissue but did
not see any qualitative differences (data not shown).
We quantitated the tumor-specific, Ld-restricted, AH1specific, IFN-g–secreting CTLs from each of the control
and treated groups in the CT26 model, and both ELISPOT
and in vivo CTL assay showed that TEGVAX-treated mice
had increased the number of AH1-specific CTLs. These
results strongly suggest that one downstream consequence
of TEGVAX treatment is the upregulation of antitumor
cytotoxic IFN-g–producing T cells in both the DLNs and
the spleen that can slow the rate of the tumor growth.
Consistent with this mechanistic picture is an experiment,
whereby TEGVAX injected on the contralateral limb from
the tumor growth site had comparable in vivo antitumor
response as intratumoral injection as shown in Supplementary Fig. S2. Both intratumoral and systemic treatments
were lymphocyte dependent as shown by the abrogation of
these effects in Rag2/ mice. Although intratumoral injection can elicit both innate and adaptive immune response,
injection of TEGVAX on the contralateral limb predominantly increases the priming of naive T cells into vaccinespecific T cells.
In terms of the architecture of the tumor microenvironment, TEGVAX-treated tumor had broad infiltration of
CD86þ cells. Although both PBS and GVAX-treated tumor
had localized aggregates of lymphocytes and APCs, TEGVAX-treated tumors had diffuse infiltration of CD4þ,
CD8þ, and CD86þ cells (see arrow in Fig. 4). These ectopic
lymphoid aggregates have been described in pancreatic
cancer specimens from patients treated with GVAX (ref.
22; Jaffee and Zhang, personal communication). The significance of these aggregates is unclear, but there seems to
be a negative correlation between these aggregates and the
tumor response in vivo.
Given that one limitation of GVAX is limited number of
activated APCs, development of TEGVAX is a step toward
an efficacious combinatorial immunotherapeutic reagent
for advanced cancer patients. GVAX has been found to be
safe in multiple phase I trials, and preclinical in vivo efficacy
Clinical Cancer Research
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Published OnlineFirst May 4, 2011; DOI: 10.1158/1078-0432.CCR-10-3262
TLR4 Agonist in Cellular Vectors Can Improve Immunotherapy
Number of spots per 5 × 10E5 cells
A
800
700
TEGVAX(spleen)
600
GVAX(spleen)
Untreated(spleen)
500
*P < 0.01
400
TEGVAX(LN)
GVAX(LN)
300
*P < 0.01
Untreated(LN)
200
100
0
CD8a + PMA
+ lonomycin
CD8a + APC
+ AH1
B
CD8a + APC
*P < 0.07
10
9
Average killing
8
7
6
- TEGVAX
- GVAX
- PBS
5
4
3
2
1
0
Groups
Figure 6. TEGVAX treatment increases the number of tumor-specific
CD8þ T cells. A, CT26 tumor–bearing BALB/c mice were treated with PBS,
GVAX, or TEGVAX, and 5 days later CD8þ cells were isolated and
purified from the spleen and lymph nodes. ELISPOT assays were done by
using 4T1 cells as APCs and AH1 peptide, and IFN-g levels were
measured. The number of AH1-specific IFN-g producing T cells was
statistically greater in the TEGVAX group compared with GVAX group
in both the spleen and the draining LN (P < 0.01). B, in vivo CTL assays
were used to measure cell killing by AH1-specific CTLs in CT26
tumor–bearing mice treated with or without TEGVAX (see Methods section
for calculation of average cell killing). The average cell killing, which
measure AH1-specific CTLs, was higher in the TEGVAX group compared
with the untreated groups (P < 0.07). These experiments were replicated
3 times.
are currently formulating TEGVAX with a lower amount of
LPS on a per cell basis to titrate the minimal amount of LPS
required for in vivo response in murine models. We are also
currently developing TEGVAX with nontoxic lipid A
mimetics.
Much like the vaccines for infectious agents, the importance of TLR4 adjuvants has been acknowledged by the
fact that the multiple clinical cancer vaccines have been
typically mixed with TLR4 agonists. The MAGE-A3 ASCI
lung cancer vaccine for NSC lung cancer and Cervarix for
cervical cancer contains monophosphoryl lipid A, a TLR4
agonist (7). Bacillus Calmette Guerin, another TLR4 agonist, was found to be important for low-volume melanoma disease (24). Our article, however, points out the
importance of TLR4 agonist formulation in terms of
in vivo efficacy. Simple mixing of cellular vaccine and
adjuvants may be a suboptimal method to integrate the
power of danger signals into the combinatorial vaccine.
Future studies will also compare different TLR4 agonists
that have been tested in patients with comparable
formulations.
In conclusion, we report a novel formulation of GVAX
tumor vaccines with improved antitumor efficacy with
intratumoral injection in several murine models. Intratumoral injection of vaccine is potentially feasible in select
cases of accessible tumors such as head and neck and skin
malignancies. Moreover, intratumoral injection has important translational implications for neoadjuvant therapy
prior to surgery in patients with a high risk of relapse.
Intratumoral injection prior to resection could generate
systemic antitumor immune responses that could eliminate the micrometastasis undetectable at the time of surgery, which are the sources of tumor relapse.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
of TEGVAX, as shown in this article, warrants further
investigation. Of greatest concern is the use of TLR4 agonist
therapy in cancer patients. Goto and colleagues had previously injected 10 mg of LPS into cancer patients with
tolerable side effects that were not greater than WHO grade
3 (23). Extrapolating from previous trials with GVAX in
cancer patients in which 108–9 GVAX cells were used, this
would amount to micrograms of LPS injected submucosally or subdermally in human subjects as currently formulated in TEGVAX. To minimize the exposure of LPS, we
Grant Support
This work was supported by NIH K23-DE018464–02 and Triological
Society/American College of Surgeon Career Developmental Award (Y.J.
Kim).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received December 8, 2010; revised April 14, 2011; accepted April 21,
2011; published OnlineFirst May 4, 2011.
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Published OnlineFirst May 4, 2011; DOI: 10.1158/1078-0432.CCR-10-3262
Intratumoral Administration of TLR4 Agonist Absorbed into a
Cellular Vector Improves Antitumor Responses
Meghan B. Davis, David Vasquez-Dunddel, Juan Fu, et al.
Clin Cancer Res 2011;17:3984-3992. Published OnlineFirst May 4, 2011.
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