Combina$on of PV-­‐10 Immuno-­‐chemoabla$on and Systemic an$-­‐CTLA-­‐4 An$body Therapy in Murine Models of Melanoma Eric A. Wachter , Savannah O. Blair, Jamie M. Singer and H. Craig Dees Abstract No. 4755 Provectus Pharmaceu$cals, Inc., Knoxville, TN USA American Associa$on for Cancer Research Annual Mee$ng – Washington, DC 6 -­‐ 10 April 2013 Lung Metastasis + Flank Tumor Model (B16-­‐F10) PV-­‐10 Immuno-­‐Chemoabla$on [1],[2] Background: Elsas et al. reported CTLA-­‐4 blockade in combina$on with F10/GM vaccine reduced lung metastases (no flank tumor). Both treatments commenced 1 day ajer tail vein injec$ons of 5 x 104 B16-­‐F10 cells (9H10 IP dose: 100 μg, 50 μg, 50 μg, days 1, 4 and 7). CTLA-­‐4 blockade alone resulted in > 200 lung mets in each animal, but hamster IgG control showed some lung met reduc$on. Since hamster IgG was not reported to have an effect in any of the other literature reviewed, we did not contemplate using that control in these studies. We first wanted to ensure that 9H10 or some IgG component therein did not reduce lung metastases in our model. Murine Model: C57BL/6 (6 females per group) Tumor InducEon: Solitary SQ tumors were induced in the flank by injec$on of B16-­‐F10 tumor cell line (2 x 105 cells). Lung metastases were produced by synchronous injec$on of tumor cells into the tail vein (2.5 x 105 B16-­‐
F10 cells). Treatment: 8 days post tumor induc$on, a single IL injec$on of 50-­‐100 μL of PV-­‐10 (dose based on tumor size) or a control injec$on of 100 μL IL saline was administered into the flank lesion. 100 μg of 9H10 (BioXcell) was administered intraperitoneally (IP) on day 0, and 50 μg each on days 3 and 6. Follow up: 19 days post tumor induc$on, lungs were removed and metastases were counted on the anterior and posterior sides using a 1 mm2 grid. Each 1 mm2 of tumor $ssue was counted as 1 lung metastasis. Lung Metastasis Results Intralesional
Injection
10-20 min
PV-10 accumulation
in tumor lysosomes
Systemic
Tumor-specific
immune response
detectable in 7 days
30-60 min
Lysosomal
rupture
Tumor cell
rupture
Treatment: IP 9H10 (x3) IP 9H10 (x3) + Bilateral Flank Model: High Dose 9H10 (B16-­‐F10) Discussion Bilateral Flank Model: In order to discriminate the systemic effect of PV-­‐10 alone from that in combina$on with CTLA-­‐4 blockade, 3 dose levels of 9H10 were assessed in an aggressive melanoma model with four treatment groups (C57BL/6, 10 per group) randomized by tumor burden immediately prior to treatment. Tumors were induced in each flank by subcutaneous injec$on of B16-­‐F10 tumor cells (2 x 105 cells per tumor). Mice were required to have palpable tumor burden in both flanks prior to randomiza$on on treatment day 0. Only the larger of the flank tumors was infiltrated with saline (control) or PV-­‐10 in each animal. The untreated flank tumor was followed as a surrogate for visceral disease burden inaccessible to PV-­‐10 injec$on. Both tumors were measured in two dimensions with calipers in intervals of 2-­‐5 days ajer IL-­‐treatment (tumors that were eschar or no longer palpable were recorded as “0”). Animals were euthanized if on a measurement day the total tumor burden exceeded 300 mm2, with last measurement captured. Sta$s$cally significant p-­‐values were only reported in tumor burden measurement comparisons between groups if neither groups’ survival medians had been reached. Low Dose 9H10 Experiment: 9 days ajer bilateral tumor induc$on (treatment day 0), a single IL injec$on of 50-­‐100 μL of PV-­‐10 (dose based on tumor size) or 100 μL IL saline control was administered into the larger of the two lesions. For 9H10 treatment groups, 100 μg of 9H10 was administered IP on day 0, and 50 μg each on days 3 and 6 [7]. Differences in tumor burden at day 0 (shown below) were not significant. Mid Dose 9H10 Experiment: Waitz et al. reported a murine model of prostate cancer where cryoabla$on of a single TRAMP C2 tumor (5-­‐8 mm in diameter) did not prevent growth of a secondary tumor given on the opposite flank 1 day post abla$on. The combina$on of cryoabla$on with CTLA-­‐4 blockade was successful at preven$ng growth of the delayed bystander tumor. The tumor was cryoablated on day 0, and 9H10 regimen administered was 200 μg on day 1 followed by 100 μg each on days 4, 7 and 10 [8]. Mid Dose 9H10 Experiment: Although this delayed tumor challenge model was not aggressive enough for evalua$ng the PV-­‐10 plus 9H10 combina$on, the 9H10 dosing regimen was adapted for use in a synchronous B16-­‐F10 bilateral tumor model. The mid dose experiment was iden$cal to the low dose experiment except that intralesional saline or PV-­‐10 was given 8 days ajer bilateral tumor induc$on (treatment day 0), and for 9H10 treatment groups 200 μg of 9H10 was administered IP on day 0, and 100 μg each on days 3, 7 and 10. The treatment day selec$on was necessary to confirm existence of a substan$al tumor on both flanks prior to treatment. Differences in tumor burden at day 0 (shown below) were not significant. High Dose 9H10 Experiment: Demaria et al. reported that local radia$on in a murine model of breast cancer was only effec$ve at delaying single flank tumor growth in established 4T1 tumors (mean diameter 5 mm) but treatment did not extend overall survival. A combina$on of local radia$on in established solitary tumors with CTLA-­‐4 blockade increased survival in this murine model. An aggressive 9H10 dosing regimen was given, consis$ng of 200 μg on days 1, 4 and 7 ajer local radia$on. [5] High Dose 9H10 Experiment: This dose regimen was slightly altered to give the same dose of 9H10, but at days 0, 3, 7 to coincide with IL PV-­‐10 treatment on day 0. Otherwise this experiment was iden$cal to both the low and mid dose experiments with the excep$on that the IL interven$ons were given 10 days ajer tumor cell implanta$on to ensure all animals had measurable bilateral tumors on treatment day 0. Differences in tumor burden at day 0 (shown below) were not significant. Treatment
Mean Total Tumor
Standard
2
Burden (mm )
Deviation
IL-PV-10
38.1
16.7
IL-PV-10 + 9H10
43.7
14.4
Lung Metastasis Results: These results corroborate previously reported data showing robust suppression of synchronous lung metastases upon PV-­‐10 abla$on of an established flank tumor [2-­‐3]. While the magnitude of the effect of PV-­‐10 alone makes it impossible to unequivocally conclude whether there is synergy in this model combina$on, absence of apparent increased toxicity coupled with suppression of lung metastases indicates minimal likelihood of interference between the component therapies. Comparison of Bilateral Flank Model Results: Despite delay in ini$a$on of therapy un$l onset of palpable tumors in a very aggressive melanoma model, PV-­‐10 and PV-­‐10 + 9H10 exhibited robust response in both injected and uninjected tumors. As expected, PV-­‐10 + 9H10 combina$on therapy exhibited maximum benefit. This was most evident in the low dose model, where advantages were pronounced in both tumor growth and survival data. Overlay of curves for the respec$ve combina$on groups shows that survival in the Survival
Analysis
low dose group (red) was significantly longer than that of the high dose group (blue, p = 0.03). Treatment
Mean Total Tumor
Burden (mm2)
Standard
Deviation
Treatment
Mean Total Tumor
Burden (mm2)
Standard
Deviation
IL-PV-10
33.3
14.8
IL-PV-10
48.7
12.5
IL-PV-10 + 9H10
30.7
12.8
IL-PV-10 + 9H10
53.7
14.0
IL-Saline
33.7
16.3
IL-Saline
53.1
26.8
IL-Saline
45.0
20.6
IL-Saline + 9H10
31.1
11.4
IL-Saline + 9H10
48.3
17.8
IL-Saline + 9H10
37.4
16.5
Bilateral Flank Model: Mid Dose 9H10 (B16-­‐F10) 2D Graph
IL 6 PV-­‐10 (x1) IL PV-10 vs IL PV-10 + IP 9H10
Total Tumor Burden
IL PV-10 vs IL PV-10 + IP 9H10
Total Tumor Burden
IL PV-10 vs IL PV-10 + IP 9H10
Total Tumor Burden
0.0
0
10
6
6
<8LesionsUntreated
6
4
TNC/StageIV
Tumor Size (mm2)
Tumor Size (mm2)
Tumor Size (mm2)
100
100
0
-10
-5
0
5
10
15
20
25
30
35
40
Days Post IL Treatment
4
1Ͳ2BystandersUntreated
Mean Survival
20
0
-5
0
5
Tumor Size (mm2)
ssection microscope. 2Paraffin-embedded lung sections were
chose to combine CTLA-4 blockade with GM-CSF–proned
with hematoxylin–eosin
using standard
procedures.
For
Lung Metastasis Results: which
Mice that was
received IL PV-­‐10 + IP 9H10 exhibited a drama$c reduc$on in the number of ducing
irradiated
whole
cell vaccine,
e. Paraffin-embedded
lung
sections
were
chose
to combine
CTLA-4
blockade
with
GM-CSF–promicroscope.
Paraffin-embedded
lung sections
wereB16-BL6
chose
to
combine
CTLA-4
blockade
with
GM-CSF–pro0a dissection
4
synchronous l
ung m
etastases (
p =
0
.004 )
. M
ice t
reated with IL PV-­‐10 + IP 9H10 had a mean of 3.2 lung �
10
B16-F10
cells
were
injected
intravival
experiments,
5
ssection
microscope.
Paraffin-embedded
lung
sections
were
chose
to
combine
CTLA-4
blockade
with
GM-CSF–prodescribed
by
others
as
the
most
effective
prophylactic
vaclin–eosin using standard
procedures.
For
stained
with
hematoxylin–eosin
using
standard
procedures.
For
ducing
irradiated
B16-BL6
whole
cell
vaccine,
which
was
ducing
irradiated
B16-BL6
whole
cell
vaccine,
which
CRusing
PRday. procedures.
SD4
NEV+PD
metastases (range 0 to 6). Control mice treated with IL Saline + IP was
9H10 had a mean of 84.0 lung metastases ned
with
hematoxylin–eosin
standard
For
ously
and
treatment
started
the next
4
ducing
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cine
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described
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described
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xtensive cine
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mpact w
ithin i
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oten$al f
or function
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from mice
B16-BL6
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vaccination
enhance might
their attract host APCs and enhance their
� 106 spleen
ic
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afterrejecting
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coculture.
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7.1 orsystemic a mixture
of B16-F0
and
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might
attract
host
APCs
and
enhancetreated
their
vitro
with
B16-BL6/B7.1
or
a
mixture
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B16-F0
and
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ffects, m
aking P
V-­‐10 a
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arac$ve c
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ombina$on w
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cells
subcutaneously
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with mixed
B16-BL6/B7.1
or a mixture
of B16-F0
and the den(16,000
rads) stimulator
cells,
sowere
with 10 irradiated
function
in
vivo.
C57BL/6
mice
were
challenged
with
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function
in
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C57BL/6
mice
were
challenged with 104
6
4
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� 10
spleen
after overnight coculture.
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�
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spleen
dritic
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after
overnight
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function
in
vivo.
C57BL/6
mice
were
challenged
with
10
6
starting on the and
samesubsequently
day
or 4–12 cells
d later.
A representative
� 10 spleen subcutaneously
icrecombinant
line DC2.4
after
overnight
coculture.
human
IL-2
was added
to a final5B16-BL6
concentration
treated
5 cell
B16-BL6
subcutaneously
and subsequently treated
5 irradiated cells
irradiated
(16,000
rads)
stimulator
cells,
0
(16,000
rads)
stimulator
cells,
cells
were
mixed
with
10
B16-BL6
cells
subcutaneously
and
subsequently
treated
5
irradiated
(16,000 rads)
stimulator
cells,
s0were
mixed
with
10cells
experiment
is
shown
in
Fig.
1.
Administration
of
anti–
IL
Saline
+
9H10
vs. IL PV-10 + IP 9H10
IU/ml.
After
7 d,
were collected
and purified
by
Hisstarting
on
the to
same
dayconcentration
or
4–12
d later.
Aorrepresentative
starting
on dthe
same
day
or 4–12IP
d later.
A representative
nrecombinant
IL-2 was added
to
a
final
concentration
and
recombinant
human
IL-2
was
added
a
final
starting
on
the
same
day
4–12
later.
A
representative
human gradient
IL-2 wascentrifugation.
added to a final
concentration
aque (Sigma-Aldrich)
Live
cells (2.5 �is shown
CTLA-4
antibody
9H10 or control
hamster IgG by themexperiment
in
Fig.
1.
Administration
of
anti–
cells
were
collected
and
purified
by
Hisexperiment
is
shown in Fig.
1. Administration
of anti–
Flank Tumor
of
30
IU/ml.
After
7
d,
cells
were
collected
and
purified
by
His4
experiment
iseffect
shown
in
Fig. of
1. B16-BL6
Administration
ofVaccianti–
0 IU/ml.
After stimulated
7 d, cells were
and
by well)
Hisper
well) were
with collected
target cells
(5 purified
� 10 per
selves
had
no
on
growth
tumors.
) gradient
centrifugation.
Live centrifugation.
cells (2.5 � Live
CTLA-4
antibody
9H10
control
by
themtopaque
gradient
centrifugation.
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cells
(2.5 �hamster
CTLA-4
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9H10
hamster IgG by them�
aque
(Sigma-Aldrich)
cells
(2.5
CTLA-4
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9H10
orIgG
control
hamster
IgGor
bycontrol
them6-well
round-bottomgradient
plates
for(Sigma-Aldrich)
24 h, after which
supernatant
300
nation
with
irradiated
GM-CSF–producing
B16-BL6
cells
4
5
4
ulated
with
target
cells10
(5 �
10well)
perwere
well)
per
with
target
cells
(5 �
10 no
perof
well)
selves
hadwell)
no effect
on
growth
B16-BL6
tumors.
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had
effect on
growth
of B16-BL6 tumors. Vacciper
well)
were
stimulated
with
target
cells
(5 �
per
selves
had
effect
on growth
of no
B16-BL6
tumors.
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by4 sandwich
collected
and tested
for the
presence
of stimulated
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with
control
antibody
delayed
growth
when
initiated
m
plates
for
24
h,
after
which
supernatant
A1
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round-bottom
plates
for
24 irradiated
h, after
which
supernatant
nation
with
GM-CSF–producing
B16-BL6
cells
nation
with irradiated
GM-CSF–producing
B16-BL6 cells
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round-bottom in
plates
for 24
h, after which
supernatant
SA
(PharMingen).
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with
irradiated
GM-CSF–producing
B16-BL6
cells
at
the
time
of
tumor
implantation
but
had
no
effect
when
� presence
by sandwich
d collected
for the presence
of for
IFNA2
� by sandwich
wasthe
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and
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of IFN�for
bythe
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and tested
of tested
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control
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with
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when
initiated
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no effect
when
at the
time of
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at the
time of tumor
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but
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200
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of
treatment
was
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of
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A5
duced rejection of all tumors injected the same day or 4 d
GM-CSF–producing
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duced rejection ofduced
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wasrejected
also delayed
by
the combination
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y derived
murine
including
CTLA-4
blockade.
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growth
of tumors
estab6-BL6
wasfrom
originally
derived
the
spontaneous
murine
including
CTLA-4
blockade.
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growth
of
tumors
estabB16-BL6
was
originally
derived
from
the spontaneous
murine
including
blockade.
The
growth of tumors estabeness
(21).
Boththe
thespontaneous
parentalfrom
line
and
its variant
express
treatment,
although
rejection
wasCTLA-4
not
obtained.
When
the
B5
B16-F0
by
inline
vivo
selection
invalished
12invad earlier
wasfrom
alsofor
by10
the
combination
lanoma
cell
inMHC
vivo
selection
forby
12
dadelayed
earlier
was
also
delayed
by
the
combination
melanoma
cell
line
B16-F0
in
vivo lished
selection
inva- of
lished
12 d earlier
was
also delayed
b B16-F0
and
Db, by
andfor
class
II
is undetectw
levels of
H-2K
data
series
experiments
were
combined,
an0 by the combination
B6
he
line and
its
variant
express
treatment,
although
wasrate
notof
obtained.
When
the
eness
(21).cytometry
Both
thein
parental
line
and
its
variant
express
although
rejection
was
not
obtained.
When
the
siveness
(21).
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the
parental
line
andtreatment,
itsrejection
variant
express
treatment,
although
rejection
was
not obtained. When the
e byparental
flow
vitro
and
ex
vivo
(data
not
shown).
overall
success
combination
treatment
of
80%
was
-10
-5
0
5
10
15
Group A Mean
b and Db, and MHC class
b, of
b and
bfrom
II
is
undetectw
levels
H-2K
data
from
a
series
of
10
experiments
were
combined,
an
and
D
and
MHC
class
II
is
undetectdata
a
series
of
10
experiments
were
combined,
an
D
,
and
MHC
class
II
is
undetectlow
levels
of
H-2K
data
from
a
series
of
10
experiments
were
combined,
an
ccination with irradiated B16-BL6 does not protect
achieved (68/85 mice cured) when treatment was begun at
Group B Mean
eyinst
by
flow
cytometry
in
vitro
and
ex
vivo
(data
not
shown).
overall
success
ratetreatment
of combination
of These
80%Days
was
in vitro
and
ex
vivo
(data
not
shown).
overall
success
rate
of
ofsuccess
80%treatment
was
able
by
flow
cytometry
in
vitro
and
ex
vivo
(data
overall
rate
of combination
treatment
of 80% was
IL Treatment
subsequent challenge with live B16-BL6 cells, nor
day
0combination
ornot
4 shown).
d after
tumor
implantation
(Table
I).
re-Post
ccination
with irradiated
B16-BL6
not Inaccessible protect
achieved
mice
cured)
when
treatment
was begun
at treatment was begun at
radiated
B16-BL6
(68/85
cured)
when
treatment
was
begun
atmice blockade
Vaccination
withdoes
irradiated
B16-BL6
does
not
protect
achieved
(68/85
cured)
when
o expression
Temporarily Adoes
rrest in
Tnot
umor Growth in Dachieved
isease o IL PV-­‐10 a(68/85
nd/or es
B7.1
result
anyprotect
significant
change
in
tu- tmice
sults
corroborate
the finding
that
CTLA-4
and
inst subsequent
challenge
with
live
B16-BL6
cells,
nor
day
0 orimplantation
4cells,
d after
tumor
implantation
(Table
I). implantation
These re- (Table I). These rehallenge
with
live
B16-BL6
cells,
nor
day
0
or
4
d
after
tumor
(Table
I).
These
reagainst
subsequent
challenge
with
live
B16-BL6
nor
day
0
or
4
d
after
tumor
o 
Amplify T
umor-­‐Specific I
mmune R
esponse u
pon P
V-­‐10 A
blaEon r growth in vivo (20, 22; our unpublished results). By
GM-CSF–producing vaccinesSynchronous act synergistically
cause
Flank Tumor Rto
esults: The IL saline + IP 9H10 group tumor size means were 13 mm2 at onset of esresult
B7.1
expression
result
in
any
significant
change
in
tusults
corroborate
the
finding
that
CTLA-4
blockade
and
in
any
significant
in tu- immunogenic
sults corroborate
the finding
that
CTLA-4
and
does
B7.1 poorly
expression
result
in any
significant
change
in tusults blockade
corroborate
finding
blockade
2 19 days that
se criteria,
B16-BL6
is achange
very
turejection
of poorly
immunogenic
tumors
(17).
treatment and 1the
94 mm
ajer cCTLA-4
ell implanta$on, individual and
animals are shown in thin lines above. The Prior t
o c
linical i
nves$ga$on o
f a
s
orafenib +
P
V-­‐10 c
ombina$on i
n h
epatocellular c
arcinoma (
HCC) u
nder r(20,
growth
in vivo
(20,
22;growth
our unpublished
results).
Byunpublished
GM-CSF–producing
vaccines
act
synergistically
to
cause
22;
our
unpublished
results).
By
GM-CSF–producing
vaccines
act
synergistically
to
cause
mor
in
vivo
(20,
22;
our
results).
By
GM-CSF–producing
vaccines
act
synergistically
to
cause
tumors in the PV-­‐10 combo gadminisroup were not measurable ajer abla$on. Consistent with previous studies, the r. Inprotocol previous
experiments,
had found
that CTLA-4
A single
dose
of GM-CSF–producing
vaccine
PB16-BL6
V-­‐10-­‐LC-­‐01, ais similar cwe
ombina$on w
as modeled (PV-­‐10 tu+ 5-­‐fluorouracil) in mice. poorly
This combina$on se
criteria,
a
very
poorly
immunogenic
rejection
of
immunogenic
tumors
(17).
L6
is a was
verynot
poorly
immunogenic
turejection
of poorly
poorlytered
immunogenic
tumors
(17).
these criteria,
B16-BL6
is aincluding very
immunogenic
turejection
of
poorly
tumors
(17).is sta$s$cally significant. reduc$on in flank immunogenic
tumor sufficient
burden in the PV-­‐10 group ckade
therapeutically
effective
poorly
on
the same
day as
tumor
challenge
was
to
showed a synergis$c effect in a we
HCC b
ilateral flank against
m
odel, CTLA-4
a longer $A
me single
to progression in r.
In
previous
experiments,
had
found
that
dose
of
GM-CSF–producing
vaccine
adminiseriments,
wetumors had found
that
CTLA-4
Afound
single
dose
of
GM-CSF–producing
vaccine
adminis mice
mor.
In
previous
experiments,
we
had
found
that
CTLA-4
single
dose
of GM-CSF–producing
vaccine adminis In this munogenic
such
as
B16-BL6.
also
that
eradicate
in all
when
combined with
untreated tumors
[3].
w
ork systemic We
immune s$mula$on was modeled using the tumors
murine analog of ofAthe
ckade
was
not
therapeutically
effective
against
poorly
tered
on
the
same
day
as
tumor
challenge
was
sufficient
to
erapeutically
effective
against
poorly
tered
on thean$-­‐CTLA-­‐4 sameCTLA-4
day
as tumor
wasSimilarly,
sufficient
was
not
effective
against
poorly
on
the same
day dose
as tumor
challenge was sufficient to
cination
withtherapy, irradiated
cells
inV-­‐10. combination
blockade
(Fig.tered
2).
ato
single
of anti–
an$-­‐CTLA-­‐4 9blockade
H10, iB16-BL6
n combina$on with therapeutically
P
Successful combina$ons in challenge
murine munogenic
tumors rsuch
asin B16-BL6.
We
also
found
that
eradicate
tumors
in
all eradicate
ofcombined
the mice
when
with when combined with
as B16-BL6.
We
also
eradicate
tumors
in
allalso
of
the
with
immunogenic
tumors
such
as B16-BL6.
We
that
tumors
in combined
all of the mice
models have been eported pfound
rostate (data
(that
9H10 + cryoabla$on) [4] and breast cancer cell found
lafter
ines mice
(9H10 +when
vaccinations
hsuch
anti–CTLA-4
was
ineffective
not
shown).
We
CTLA-4
three
with
GM-CSF–produccination
with
cells
combination
CTLA-4
blockade
2). Similarly,
a single
dose
of anti– a single dose of anti–
diated
B16-BL6
in B16-BL6
combination
CTLA-4
blockade
(Fig.
2).
Similarly,
a single
dose blockade
ofB16-BL6
anti–
radia$on) [5], irradiated
athis
nd cells
B16-­‐OVA mbe
elanoma (to
9H10 + rin
adiofrequency abla$on) cells
[6]. None of these msufficient
odels l(Fig.
ej vaccination
irradiated
B16-BL6
in
combination
CTLA-4
(Fig.
2). Similarly,
pothesized
that
might
duewith
insufficient
presening
cells
was
to induce
rejection
(not
h anti–CTLA-4
waswith
ineffective
(data
not
shown).
We
CTLA-4
after
three
vaccinations
with
GM-CSF–producuntreated fl
ank t
umor b
urden t
o m
odel v
isceral d
isease, r
ather i
nduced l
ung m
etastases w
ere c
ounted o
r was
ineffective
(data
not
shown).
We
CTLA-4
after
three
vaccinations
with
GM-CSF–producineffective
not shown).
We production
CTLA-4
three vaccinations
with GM-CSF–producon of tumor antigens byanti–CTLA-4
host APCs. was
Therefore,
we (data
shown).
GM-CSF
byafter
the vaccine
was found to
pothesized
that
this
might
be
due
to
insufficient
presening
cells
was
sufficient
to
induce
B16-BL6
rejection
(not
mice w
ere r
e-­‐challenged w
ith t
umor. E
lsas a
nd c
oworkers r
eported r
educ$on i
n s
ingle fl
ank t
umor b
urden s might be due to insufficient
presencellsbewas
to induce
B16-BL6
(not
hypothesized
that this ing
might
duesufficient
to insufficient
presening rejection
cells
sufficient
to oinduce
B16-BL6
rejection
(not
Lung was
Metastases: Number f lung metastases on the anterior and posterior sides of each lung were enumerated on
of
tumor
antigens
by
host
APCs.
Therefore,
we
shown).
GM-CSF
production
by
the
vaccine
was
found
to
compared t
o c
ontrols f
or a
B
16-­‐BL6 v
accine w
ith 9
H10 u
pon i
ni$a$on o
f t
reatment e
ither 8
o
r 1
2 d
ays a
jer 2 2 of tumor $ssue counted as 1 lung from standardized photographs uby
sing the
a 1 m
m grid, wwas
ith each 1 mmto
igens by host APCs.
Therefore,
we antigens
shown).
production
by we
the vaccine
was
found
tation
of tumor
by GM-CSF
host APCs.
Therefore,
shown).
GM-CSF
production
vaccine
found
Figure
1.toSuccessful
treatment
of
Poten$al for Complementary Therapy 50
60
Low
Dose
Mid
Dose
High
Dose
PV-10
16.4
15.3
19.2
PV-10 + 9H10
21.8
17.8
Saline
15.3
Saline + 9H10
20.8
Median Survival
Low
Dose
Mid
Dose
High
Dose
PV-10
17.0
15.0
18.0
14.6
PV-10 + 9H10
21.5
20.0
15.0
9.6
11.4
Saline
15.5
9.0
12.0
13.1
13.4
Saline + 9H10
22.0
13.5
12.0
15
20
25
30
35
40
-10
-5
0
5
Total Tumor Burden: A representa$on of PV-­‐10 alone (green) and the combina$on of PV-­‐10 + 9H10 (red) total tumor burden is shown above. The blue triangles represent 9H10 doses (PV-­‐10 or saline injected IL only on day 0). Once again both these groups are significantly smaller than total tumor burden in the non-­‐ablated saline and 9H10 controls. The PV-­‐10 + 9H10 and PV-­‐10 alone groups aren’t sta$s$cally different from one another in total tumor burden. 10
15
20
25
30
35
40
Days Post IL Treatment
Total Tumor Burden: A representa$on of PV-­‐10 alone (green) and the combina$on of PV-­‐10 + 9H10 (red) total tumor burden is shown above. The blue triangles represent 9H10 doses (PV-­‐10 or saline injected IL only on day 0). Both groups are once again smaller than total tumor burden in the non-­‐ablated saline and 9H10 controls. The PV-­‐10 + 9H10 and PV-­‐10 alone groups aren’t sta$s$cally different from one another in total tumor burden. P-­‐
values for the difference between these groups and the 9H10 alone group (given in both groups at the high dose) are < 0.001 for days 3 ,6, 8 and 12. Variability in response across dose levels may be due to toxicity of 9H10 at elevated doses, with addi$onal differences arising from the effect of tumor burden. In these experiments, uninjected tumor burden and dose of 9H10 varied between studies, and the impact of this is illustrated below (similar results were noted when data were ploaed against total tumor burden). Saline alone (black) and PV-­‐10 alone (green) exhibited a linear rela$onship between tumor burden and survival whereas 9H10 (blue) had a highly nonlinear response that featured a marked reduc$on in survival at the high dose. Combina$on of PV-­‐10 with 9H10 (red) drama$cally improved survival at the mid dose despite higher tumor burden. This was repeated at the high dose, although apparent toxicity of 9H10 in these groups negated much of the advantage of the combina$on. 25
Mean Uninjected Tumor Burden
Mean Uninjected Tumor Burden
Mean Uninjected Tumor Burden
400
400
400
IL Saline
300
IL Saline + IP 9H10
IL PV-10
IL PV-10 + IP 9H10
200
300
100
-5
IL PV-10
200
100
300
0
5
10
15
20
25
30
35
40
-10
-5
IL PV-10
IL PV-10 + IP 9H10
200
100
0
0
5
10
15
20
25
30
35
40
-10
-5
0
Days Post IL Treatment
Modeled Visceral Disease (Uninjected Flank Tumors): A representa$on of all groups in the experiment, including 9H10 (blue) and saline (black) controls, is shown above (truncated at survival medians). The combina$on of PV-­‐10 + 9H10 (red) beaer controlled un-­‐injected tumor burden than PV-­‐10 alone (green), aaaining sta$s$cal significance at day 12, 14, 17. (p = 0.008, p = 0.001, p < 0.0001). IL-­‐Saline + 9H10 un-­‐injected tumors were also significantly smaller than PV-­‐10 alone at days 12, 14 and 17. Column 1
Column 4
Column 7
Column 10
120512.01A
Overall Survival
15
20
25
30
35
40
Column 7
Column 10
012913.01A
Column 1
Column 4
Column 7
Column 10
Survival Analysis
Overall Survival
IL PV-10 + IP 9H10
20
IL PV-10
15
IL Saline + IP 9H10
Mid Dose
10
IL Saline
High Dose
12.0
14.0
16.0
18.0
20.0
22.0
Uninjected Tumor Burden (mm2)
Modeled Visceral Disease (Uninjected Flank Tumors): A representa$on of all groups in the experiment, including 9H10 (blue) and saline (black) controls, is shown above. The combina$on of PV-­‐10 + 9H10 (red) was no different in un-­‐injected tumor burden than PV-­‐10 alone (green). Perhaps due to the aggressive 9H10 regimen, this experiment showed no group clear difference or trend in untreated tumor burden. Overall Survival
1.0
1.0
Conclusions Examples of successful treatment of established B16 tumors in murine models are rare. As a single agent, a single dose of IL PV-­‐10 can be highly effec$ve in reducing overall tumor burden in both murine models and clinical trial pa$ents. However, when substan$al inaccessible tumor burden is present, single injec$on may be insufficient to rapidly control uninjectable disease. It is in these cases that systemic therapy in combina$on with PV-­‐10 may be indicated. Overall, these results demonstrate: •  PV-­‐10 is highly effec$ve when all exis$ng tumor is accessible for injec$on, providing rapid reduc$on in tumor burden (both in animal models and clinically in cancer pa$ents) •  Tumor abla$on with PV-­‐10 induces tumor-­‐specific immunity 0.8
0.8
0.8
0.6
0.6
0.6
0.4
10
Days Post IL Treatment
Modeled Visceral Disease (Uninjected Flank Tumors): A representa$on of all groups in the experiment, including 9H10 (blue) and saline (black) controls, is shown above. The combina$on of PV-­‐10 + 9H10 (red) was no different in un-­‐injected tumor burden than PV-­‐10 alone (green). Perhaps due to the large size of ini$al tumors rela$ve to Column
1
the other experiments, this experiment showed no group sta$s$cal difference or trend in untreated tumor Column 4
burden. 1.0
5
Low Dose
5
10.0
0
Days Post IL Treatment
Survival
IL Saline + IP 9H10
IL PV-10 + IP 9H10
0
-10
IL Saline
IL Saline + IP 9H10
IL Saline
Sta$s$cal Methods tumor implanta$on [7]. At earlier $me points (not shown), the mice never manifested palpable flank metastasis. The MB16-BL6
ann-­‐Whitney rank sum test was used to analyze for differences in group medians. preestablished
using
anti–
tumors (only 4 of the 5 mice treated with the combina$on at day 8 had a measureable tumor). 1. Successful
treatment of
Figure
CTLA-4
Figure 1. Successful
treatment and
of GM-CSF–producing
1. Successful treatment of
preestablished
B16-BL6
usingFigure
anti–
BL6
vaccine.
C57BL/6
female
mice
Tumor A
rea: T
reated, u
ntreated a
nd t
otal tumor burden (length x width) were analyzed using an Analysis of preestablished B16-BL6CTLA-4
using anti–
preestablished
B16-BL6
using anti–
and
GM-CSF–producing
(five
per
group)
were
injected
with
Covariance (ANCOVA) unequal slopes or parallel lines model, with pre-­‐treatment Study Day 0 values used as the CTLA-4 and GM-CSF–producing
and GM-CSF–producing
4 vaccine.
BL6
C57BL/6
femaleCTLA-4
mice
B16-BL6
cells
subcutaneously
10
covariate, tmice
o adjust for baseline dBL6
ifferences. Normality was assessed by various plots of residuals and by the BL6 vaccine. C57BL/6
female
vaccine.
C57BL/6
female
mice
(five
perback,
group)
were
injected
with
on
the
on
the
same
day
(A)
or
test, cells
while subcutaneously
homogenous roup variance was assessed using Levene’s test at Day 0 (assessments at (five per group) wereShapiro-­‐Wilk injected
with
(five gper
group)
were
injected
with
4 B16-BL6
10
4,
8,
or
12
d
(B–D)
before
treatment
4
4
subcutaneously
10 B16-BL6 cells the 1% level of son
ignificance aday
nd v(A)
isual inspec$on ocells
f residual plots). Consistent viola$ons of either assump$on B16-BL6
subcutaneously
10 or
on
the
back,
the sameconsisted
was
started.
Treatment
of
on the back, on the same
day
(A)
or
on thein back,
on the same
daytransforma$on (A) or
across $orme oints would have resulted an appropriate data (e.g. log, rank, etc.) to correct the 4,
8,
12pd
(B–D)
before
treatment
three
consecutive
injections
(in
a
4, 8, or 12 d (B–D) before
treatment
4,
8,
or
12
d
(B–D)
before
treatment
problem. was
started.
Treatment
consisted
of
6-d
time
frame
as indicated
in Matewas started. Treatment
consisted
of
was
Treatment consisted of
consecutive
injections
(in started.
a
three
rials and
Methods)
of
anti–CTLA-4
three consecutive injections
(in
a
three
consecutive
injections
(in alog-­‐rank test to compute nonparametric 6-d
time
frame
as
indicated
inuMateSurvival: Survival was aintraperitoneally
nalyzed sing the Kaplan-­‐Meier product-­‐limit antibody
9H10
6-d time frame as indicated
in
Mate6-d time frame as indicated in Materials
Methods)
of IgG
anti–CTLA-4
survivor func$on es$mates. (�), and
control
hamster
(100, 50,
rials and Methods) of anti–CTLA-4
rials and Methods) of anti–CTLA-4
antibody
intraperitoneally
BL6/g
50 �g; �),9H10
or 106 irradiated
antibody 9H10 intraperitoneally
antibody
9H10 intraperitoneally
(�),
control hamster in
IgG
(100,
50,
cells C
subcutaneously,
combination
Group omparisons: P
airwise g
roup d
ifferences for all sIgG
ta$s$cal were assessed using the Tukey-­‐Kramer 6
(�), control hamster IgG
(100,
50,or 10 irradiated BL6/g
(�),
control hamster
(100,analyses 50,
50
�g;
�),
with
9H10
(�)
or
hamster
IgG
(�).
mul$ple comparisons a2djustment, hich �),
is based on ukey’s Studen$zed 6 T
BL6/g
50 �g; �), or 106 irradiated
irradiated
BL6/g Range Test. All sta$s$cal tests of 50w�g;
or 10
cells
subcutaneously,
in
combination
)
was
scored
by
Tumor
growth
(mm
pairwise treatments ifferences wcells
ere conducted two-­‐sided, at the 5% level of significance. cells subcutaneously, in
combination
with
9H10
(�)
ordhamster
IgG
(�).
subcutaneously,
in combination
measuring
perpendicular
diameters
2
with 9H10 (�) or hamster
IgG averaged
(�). (mm
was
scored
Tumor
withby9H10 (�) or hamster IgG (�).
and wasgrowth
for )all
mice
within
2
was
by
Tumor growth (mm )Group measuring
perpendicular
was dscored
by for evalua$ons of the PV-­‐10 + 9H10 Tumor growth
(mmg2)roup Comparisons Experiments: Pairwise ifferences eachscored
group.
In between some diameters
treatment
and
was only
averaged
for
mice
within
measuring perpendicular
diameters
measuring
perpendicular
diameters
combina$ons across the all
three dose levels were validated using the Kruskal-­‐Wallis one way Analysis of Variance groups,
a fraction
of the
mice
group.
In
some
treatment
and was averaged for (ANOVA) alleach
mice
within
and
was
averaged
for
all
mice
within
on Rbetween
anks with brackets)
pairwise develmul$ple comparison (Tukey Test) on baseline tumor burden (treated, untreated (indicated
groups,
only
a
fraction
of
the
each group. In some
treatment
each
group.two-­‐sided, In some
and total tumor burden). All were mice
conducted at ttreatment
he 5% level of significance. oped
a tumor.
(indicated
between
brackets)
develgroups, only a fraction of the mice
Adapted from Elsas et al. groups, only a fraction of the mice
oped a develtumor.
(indicated between brackets)
(indicated between brackets) develal.a single PV-­‐10 injec$on into a flank tumor 357
Researchers at Moffit Cancer Center hvan
ave sElsas
hown et
that reduce opedcan a tumor.
oped a tumor.
lung metastases in B16 melanoma, b
ut n
ot i
n m
odeled m
etastases i
n s
ynchronous u
ntreated B
16 fl
ank van Elsas et al.
357
tumors (the less aggressive MT-­‐901 breast cancer model did show a significant reduc$on in untreated van Elsas et al.
357
van Elsas et al.
357
bystander tumors) [2]. We sought to combine the CTLA-­‐4 blockade with PV-­‐10 in the most aggressive models of exis$ng tumor burden to ensure that any systemic treatment was poten$ally safe in combina$on with PV-­‐10 and efficacy signals from the combina$on could be discriminated from those of PV-­‐10 alone. 10
Days Post IL Treatment
Total Tumor Burden: A representa$on of PV-­‐10 alone (green) and the combina$on of PV-­‐10 + 9H10 (red) total tumor burden is shown above. The blue triangles represent 9H10 doses (PV-­‐10 or saline injected IL only on day 0). Both treatment groups are significantly smaller than total tumor burden in the non-­‐ablated saline and 9H10 controls. While the PV-­‐10 + 9H10 group appears favorable to PV-­‐10 alone, the groups aren’t sta$s$cally different from one another in total tumor burden. 40
0
0
-10
30
In general, the combina$on of PV-­‐10 + 9H10 was favored, while PV-­‐10 yielded increased survival over saline in each case. At the high dose level of 9H10, PV-­‐10 proved to be more effec$ve than the combina$on. 200
Survival
AllLesionsTreated
300
Tumor Size (mm2)
IL PV-10 + IP 9H10
200
Survival
3
40
+
12
100
300
Tumor Size (mm2)
0
60
200
20
Days Post IL Treatment
300
Tumor Size (mm2)
0
14
(N=80)
0.6
0.4
400
9H
10
*death prior to day 19
80
+
>100
Phase2MelanomaStudyPVͲ10ͲMMͲ02
ITTSubjects
>100
Sa
lin
e
IL Saline + IP 9H10
100
-1
0
>100
120
PV
85
140
9H
10
37
Number of Lung Metastases (N=6)
Number of Lung
Metastases
UntreatedTumorBurdenvs.ObjectiveResponse
8
0.8
400
Clinical Background 10
IL PV-10 + IP 9H10 (Low Dose)
IL PV-10 + IP 9H10 (Mid Dose)
IL PV-10 + IP 9H10 (High Dose)
0.2
400
Rose bengal disodium (PV-­‐10) is a small molecule immuno-­‐chemoabla$ve agent directly injected into tumors that is rapidly cleared from systemic circula$on. Intermiaent intralesional exposure to PV-­‐10 in Phase 2 tes$ng (limited to 4 doses over 16 weeks) resulted in locoregional disease control (CR+PR+SD) in 69% of pa$ents. Complete response was observed in 54% of pa$ents who had all of their tumor burden treated whereas those pa$ents who entered the study with stage IV disease exhibited a substan$ally less robust response, typically due to progression prior to repeat dosing with PV-­‐10. 1.0
Median Survival (Days Post IL Treatment)
Rose bengal disodium (PV-­‐10) is an inves$ga$onal small molecule abla$ve agent currently entering pivotal phase 3 clinical tes$ng as a monotherapy for locoregional control of cutaneous metasta$c melanoma. Upon intralesional (IL) administra$on, PV-­‐10 localizes to the injected tumor $ssues while clearing rapidly from healthy $ssue. Tumor infiltra$on with PV-­‐10 leads to rapid necrosis of the injected lesion, with complete resolu$on common within 2-­‐8 weeks. In phase 2 tes$ng in 80 pa$ents with Stage IIIB-­‐IV (M1c) melanoma, IL PV-­‐10 elicited an objec$ve response in injected tumors in 51% of pa$ents (CR:26%, PR:25%) ajer 1-­‐4 treatment cycles. In addi$on to this direct abla$ve effect on injected tumors, some pa$ents achieved an objec$ve response in their monitored untreated tumors (CR:26%, PR:7% in 42 subjects with monitored untreated lesions) in an apparent immune-­‐mediated bystander response that highly correlated with successful abla$on of their injected tumors. Treatment was generally well tolerated, with adverse events confined mainly to the injec$on site and no grade 4 or 5 adverse events associated with use of PV-­‐10. Recent nonclinical tes$ng in the B16-­‐F10 murine melanoma tumor line has confirmed that PV-­‐10 abla$on induces tumor-­‐specific immunity, resul$ng in marked suppression of synchronous lung metastases upon abla$on of a flank tumor and tumor-­‐specific IFN-­‐γ produc$on. In this study we assess poten$al benefit of combina$on of PV-­‐10 immuno-­‐chemoabla$on with the hamster an$-­‐murine CTLA-­‐4 an$body 9H10 in bilateral flank and lung metastasis models (B16-­‐F10 melanoma in C57BL/6 mice). Results from these models are reported and support clinical development of combina$on therapy in advanced melanoma pa$ents, such as stage IV pa$ents with substan$al tumor burden in loca$ons inaccessible to PV-­‐10 injec$on. The rapid reduc$on in tumor burden and tumor specific immunologic s$mula$on provided by PV-­‐10 appears to complement the immune s$mula$on of an$-­‐CTLA-­‐4 an$bodies such as ipilimumab without increased toxicity. Bilateral Flank Model: Low Dose 9H10 (B16-­‐F10) Survival
Abstract 10 min
For addi$onal informa$on: Eric Wachter [email protected] 0.4
•  Rapid, permanent local abla$on in combina$on with induc$on of tumor-­‐specific immunity afforded by PV-­‐10 comprises a uniquely powerful approach that may be complementary to many systemic therapies, while its dis$nc$ve adverse effect profile and pharmacology minimizes poten$al for drug interac$on •  For visceral or other inaccessible disease, the combina$on of PV-­‐10 with CTLA-­‐4 blockade has important poten$al for synergy •  Apparent toxicity of high dose 9H10 markedly reduced the effect of both 9H10 alone and the combina$on of PV-­‐10 + 9H10, although the combina$on remained more effec$ve than 9H10 alone at the high dose 0.4
•  Further studies are underway to confirm the apparent synergy of PV-­‐10 with CTLA-­‐4 blockade 0.2
0.2
0.0
0.0
0
10
20
30
40
Days
Overall Survival: The survival of all groups in the low dose experiment is represented above. Events included animals euthanized for excessive tumor burden. A single animal in the PV-­‐10 + 9H10 arm was censored (sacrificed with no evidence of disease on day 56). The PV-­‐10 + 9H10 combina$on (red) showed sta$s$cally significant survival over PV-­‐10 alone (green, p = 0.052) and saline alone (black, p = 0.01), but not against 9H10 alone (blue, p = 0.13). 0.2
0.0
0
10
20
30
40
Days
Overall Survival: The survival of all groups in the mid dose experiment is represented above. Events included animals euthanized for excessive tumor burden. No animals were censored in this experiment. The PV-­‐10 alone group (green) and the PV-­‐10 + 9H10 combina$on (red) showed sta$s$cally significant survival over saline alone (black, p = 0.02 and 0.0002 respec$vely) but the PV-­‐10 + 9H10 combina$on again did not reach significance against 9H10 alone (blue, p = 0.17). 0
10
20
30
40
Days
Overall Survival: The survival of all groups in the high dose experiment is represented above. Events included animals euthanized for excessive tumor burden. A single animal in the PV-­‐10 alone arm was censored on day 37 (with a slowly regressing tumor on the untreated flank). The PV-­‐10 alone group (green) showed sta$s$cally significant survival over saline (black, p = 0.02), but not against 9H10 alone (blue, p = 0.24). •  A phase 1/2 an$-­‐CTLA-­‐4 dose escala$on trial with PV-­‐10 is warranted References 1. Wachter et al. Func$onal imaging of photosensi$zers using mul$photon microscopy. SPIE 2002; 4630:143-­‐7. 2. Pilon-­‐Thomas et al. Intralesional injec$on with PV-­‐10 induces a systemic an$-­‐tumor immune response in murine models of breast cancer and melanoma. 2013 AACR Poster 1248. 3. Dees et al. Genera$on of an an$tumor response and immunity using a small molecule drug (PV-­‐10). SITC Mee$ng 26-­‐28 October 2012, Poster 1452582. 4. Waitz et al. CTLA-­‐4 blockade synergizes with cryoabla$on to mediate tumor rejec$on. Oncoimmunology 2012; 1: 544-­‐546. 5. Demaria et al. Immune-­‐mediated inhibi$on of metastases ajer treatment with local radia$on and CTLA-­‐4 blockade in mouse model of breast cancer. Clin Cancer Res 2005; 11: 728-­‐734. 6. den Brok et al. Efficient loading of dendri$c cells following cryo and radiofrequency abla$on in combina$on with immune modula$on induces an$-­‐tumor immunity. Br. J. of Cancer 2006; 95: 896-­‐905. 7. van Elsas et al. Combina$on immuno-­‐therapy of B16 melanoma using an$-­‐cytotoxic T lymphocyte-­‐ associated an$gen 4 (CTLA-­‐4) and granulocyte/macrophage colony-­‐s$mula$ng factor (GM-­‐CSF)-­‐producing vaccines induces rejec$on of subcutaneous and metasta$c tumors accompanied by autoimmune depigmenta$on. J Exp Med 1999; 190: 355–366. 8. Waitz et al. Potent induc$on of tumor immunity by combining tumor cryoabla$on with an$-­‐CTLA-­‐4 therapy. Cancer Research 2012; 72: 430-­‐439.