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CCL21 AND FLT3 LIGAND AS IMMUNOTHERAPEUTICS FOR PANCREATIC CANCER
Abdelkader Ashour1,2, H. R. Turnquist1,3, A. J. Reber1, J. Kampf1, S. Kurz3, D. LaFace4, M. A. Hollingsworth1,3, R. K. Singh1,3, J. E. Talmadge1,3, and J. C. Solheim1,2,3.
Eppley Institute1, Department of Biochemistry and Molecular Biology2 and Department of Pathology and Microbiology3 University of Nebraska Medical Center, Omaha, NE; Canji Inc.4
Abstract
100
Tumor Growth of panc02 in C57BL/6 mice
Tumor Growth of panc02 in C57BL/6 RAG2-/- /Pfp-/- mice
100
100
PBS (n=6)
PBS
(n=6) (n=6)
90SLC (n=7)
PBS
60
50
5
10
15
20
Figure 3. Untreated tumors are inhibited in growth following intratumoral
treatment of separate tumors with CCL21. Panc02 cells were inoculated into
both flanks of C57BL/6 female mice, and upon development of palpable right
and left flank tumors, mice were administered intratumoral CCL21 (n = 12) or
PBS alone (n = 11) on days 1, 2, 3, 8, 9, and 10 in the right flank tumor, and
the growth of both right and left flank tumors was monitored. (A) Change in
CCL21-or PBS-treated right flank tumor volumes over time. (B) Change over
time in untreated left flank tumor volumes in CCL21- or PBS-treated mice.
The asterisks indicate statistical significance (p≤0.05).
35
0
0
5
10
15
20
25
30
0
5
10
20
10
15
20
25
30
35
40
45
Figure 5. Intramuscular administration of Flt3L slows
tumor growth in mice bearing Panc02.Neo tumors.
Differences in time until death from sacrifice upon
reaching a 1.2 cm tumor size were assessed with logrank statistical analysis (Graph Pad Prism) and are
displayed on a Kaplan-Meier survival curve.
35
15
5
Days Post Startof Therapy
Days
0 Post Start of T herapy
25
30
35
900
Pro-Gelz(i.m.)/Pro-Gelz
(i.m.)/Pro-Gelz(i.t.)
(i.t.)
Pro-Gelz
800
pFlt3L (i.m.)/pCCL21
pFlt3-L
(i.m)/pSLC (i.t.)(i.t.)
700
pSLC
(i.t.)(i.t.)
pCCL21
600
pFlt3L (i.m.)
pFlt3-L
(i.m)
Survival and tumor growth were examined in C57BL/6 mice challenged s.c. with 1 X 106 Panc02 cells
Mice with tumors 1.5 - 3.5 mm were treated in groups:
 Flt3L (15 mg/dose-Day 1 and 6) in Pro-Gelz™ i.m. + CCL21 in Pro-Gelz™ (3 mg/dose- Day 1 and 8) i.t.
 CCL21 (3 mg/dose-Day 1 and 8) in Pro-Gelz™ i.t.
 Flt-3L (15 mg/dose-Day 1 and 6) in Pro-Gelz™ i.m.
 Pro-Gelz™ i.m. (Day 1 and 6) + Pro-Gelz™ (Day 1 and 8) i.t.
Day 6
pFlt-3L i.m.
0
4
7
11
16
19
Day 1
Adv-Flt-3L IV
1.5 to 3.5 mm Tumors
pFlt3L
pFlt-3(n=8)
(n=8)
Adv-CCL21 i.t.
Day 6
p
i.m.(n=10)
(n=10)
p i.t./p
it/p im
10
20
30
1.
2.
3.
Figure 8. Percentage CD11c+ CD11b- LDC
LN -- rCCL21
SLC
PBS
Percentage Total Cells
rSLC
rCCL21
6
Figure 13.
5.
Groups of C57BL/6 mice were given 1X s.c. injections in the right flank with 50 ml of:
 PBS,
 PBS containing 10 mg recombinant CCL21 protein (rCCL21),
 1X1011 adenoviral-CCL21 (Adv-CCL21),
 or 1X1011 adenoviral control (Ad-control)
 Lymph nodes (LNs) and spleens were harvested at different time points and examined by flow cytometry for DCs
 Injection sites were histologically examined
8
7
4.
Experimental Plan
5
4
6.
7.
3
Adv-CCL21 – Day 21 10X
40X
2
1
Figure 2. A typical CCL21-treated Panc02 tumor has necrotic areas infiltrated with many
lymphocytes and some neutrophils, as shown by hematoxylin and eosin staining of a paraffinembedded tumor section.
0
Day 4
Day 8
8.
Day Post Rx
5
4
3
2
Percentage of Control
rCCL21
rSLC
6
PBS
4
Percentage Total Cells
rCCL21
rSLC
3.5
3
2.5
2
1.5
1
0.5
1
0
0
Day 4
Day 4
Day 8
700%
600%
500%
400%
300%
200%
4
200%
12.
Day 21
Day 4
Days post-injection of Adv-Flt3L
12
Cells/ml (X1E4)
10
cells (X1E6)
Figure 15.
0
20
15
10
5
280
240
200
160
120
80
40
0
2
4
6
8
10
Days post-injection of Adv-Flt3L
12
25
20
15
10
5
0
4
6
Days post-injection
8
10
12
15.
Figure 17.
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expression on murine T cells in lymphoid tissues. Immunol 2003; 110: 170-179.
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Acknowledgments
0
2
14.
Peripheral Blood DC1
(CD11c+ CD11b- CD8+)
Figure 16.
0
Day 21
13.
0
0
Day 8
Day
Peripheral Blood DC2 (CD11c+ CD11b+)
25
1
8
300%
Day
Spleen DC2 (CD11c+ CD11b+)
2
6
400%
0%
3
4
11.
500%
0%
BALB/c mice (4/group) were given 1X i.v. injection with 1x1011 Adv-Flt3L (shown) or with PBS (not shown)
 Mice were sacrificed, and spleen and peripheral blood were harvested at day 0, 2, 4, 6, 8, 10, or 12.
 DC populations (DC1 and DC2) were examined by flow cytometry.
Figure 14.
2
AD-SLC (CD11c+
(DC CD1
1c+ LN
CD1
Adv-CCL21
CD11b+
) 1b+ L N)
600%
100%
Day 8
rSL C (CD11c+
(DC CD11
c+ LN
C11b+
L N)
rCCL21
CD11b+
)
700%
Experimental Plan
5
0
800%
100%
Day 4
Day 8
10.
AD-SLC (CD11c+
(DC CD1
1c+ Spleen
CD1 1b+
Sple en)
Adv-CCL21
CD11b+
)
Kinetics of Flt3L Activity
Spleen DC1
(CD11c+ CD11b- CD8+)
cells (X1E6)
800%
Day Post Rx
Day Post Rx
strong
immune
response
900%
900%
Cells/ml (X1E4)
Percentage of Total Cells
4.5
PBS
9.
rSL C (CD11c+
(DC CD11
c+ Spleen
CD11)b+ Splee n)
rCCL21
CD11b+
1000%
rCCL21
(CD11c+
CD8+CD8+
Spleen
)
rSLC (Lymph
CD11c+
Spleen)
Adv-CCL21
(CD11c+
CD8+
Spleen
)
AD-SLC (Lymph
CD11c+
CD8+
Spleen)
rCCL21
(CD11c+
CD8+CD8+
LN ) LN)
rSLC (Lymph
CD11c+
Adv-CCL21
(CD11c+
CD8+
LN ) LN)
AD-SLC (Lymph
CD11c+
CD8+
1000%
8
7
, DC2DC2DC2
Kinetics
of S.C.
SC
SLC
Figure
12. 12.
Kinetics
of S.C.
rCCL21,
Figure
Kinetics
of
rCCL21,
Kinetics
of SC
SLCrCCL21, DC1
Figure 11.
Kinetics
of S.C.
Percentage CD11c+ CD11b+ mDC
LN --rCCL21
SLC
Percentage of Control
Figure 10.
Figure 9. Percentage CD11c+ CD11b- CD8+
LN -- rCCL21
SLC
Day 20
References
40
Day Post Start of Therapy
Day 8
Kinetics of CCL21 Activity
Day 13
 Examine the possible synergistic effect of agents that can stimulate
maturation of DCs, such as CD40 ligand and CpG DNA on CCL21/Flt3L
immunotherapy
23
Days Post Start of Therapy
Day 1
pSLC (n=10)
pCCL21
(n=10)
0
pCCL21 i.t.
0
The disease of pancreatic adenocarcinoma is a highly progressive and
lethal cancer, with only 4% of patients surviving five years after diagnosis. It
is the fifth leading cause of cancer-related death in the U.S., and during the
last decade its incidence has increased worldwide (1). Treatments
lengthening the lives of those developing this disease are drastically
needed. Given the resistance of pancreatic cancer to both chemo- and
radiotherapy, we are focusing on the development of immunotherapy for this
disease. The ability of the immune system to respond to malignancies relies
on the presentation to T lymphocytes of peptides derived from new or over
expressed protein sequences in tumor cells. DCs present peptide antigens
derived from tumor cells on MHC class I molecules to naïve CD8+ T cells to
activate them against the tumor (2). Therefore, to initiate a robust immune
response, DCs need to gain access to the tumor, where they can obtain
antigen from apoptotic cells (3). The DCs then traffic to the regional lymph
nodes where they present the tumor antigens to T cells. The chemokine
CCL21 (SLC) acts as a strong chemoattractant for both DCs and Th1 cells
in vitro and in vivo (4, 5). In a murine model of lung cancer, intratumoral
injection of SLC enhanced the antitumor effect of DCs (6). Similarly, in
murine models of melanoma and mammary cancer, injection of recombinant
SLC inhibited tumor growth (7). In the melanoma model, the inhibition was
accompanied by infiltrating DCs, CD4+ T cells, and CD8+ T cells and was
dependent on CD8+ T cells but not CD4+ T cells (7). Flt3L is a
hematopoietic growth factor that can expand DCs in the blood and in
lymphoid and parenchymal tissues (8-10). In ability to expand DCs, Flt3L is
superior to granulocyte macrophage-colony stimulating factor (11).
Expansion of DCs by Flt3L, alone or combined with attraction of DCs by
intratumoral CCL21 administration, should be a viable approach to
increasing the immune response against pancreatic tumors.
 Inject Adv-CCL21 intratumorally 6-8 days after intravenous Adv-Flt3L.
pCCL21/pFlt3L
pSLC/pFlt-3L(n=9)
(n=9)
100
90
80
70
60
50
40
30
20
10
0
Treatments
1.5 to 3.5 mm
Tumors
 Administration of Adv-Flt3L significantly expands both the DC1 and
DC2 populations within the spleen
 The expansion of DCs by Adv-Flt3L occurs by as much as 10 fold over
the basal level with the peak of the expansion at 6-8 days postinjection.
Figure 7. Survival following pCCL21 (SLC)/pFlt3L
Experimental Plan
Day 1
 Intratumoral administration of CCL21 in a pancreatic tumor model
causes significant immune cell infiltration of the tumor mass and
delays growth of both directly treated and untreated tumors
Future Directions
Combination CCL21/Flt3L Immunotherapy
100
Figure 1.
30
Figure 4. Intratumoral administration of CCL21 slows tumor
growth in mice that were injected with Panc02.Neo tumors.
Differences in time until death from sacrifice upon reaching a 1.2
cm tumor size were assessed with log-rank statistical analysis
(Graph Pad Prism) and are displayed on a Kaplan-Meier survival
curve.
0
500
Introduction
Figure 1. CCL21 (secondary lymphoid tissue
ckemokine, SLC) acts as a strong chemoattractant
for both DCs and Th1 cells. Colocalization is
possible due to expression of the shared G-protein
coupled receptor, CCR7, on both cell types (12,13).
This allows both cells to migrate on chemical
gradients formed on the extracellular matrix (ECM)
of endothelial cells of the high endothelial venules
which constitutively secrete CCL21. Also, CCL21
binding to CCR7 results in intracellular signaling
which initiates integrin-mediated firm adhesion that
allows cellular migration and extravasation into the T
cell zones of the lymphoid tissue (14). In addition to
aiding cellular homing, CCL21 has been found to
produce rapid endocytosis in mature DC (15).
25
Days Post Start of T herapy
1000
 Increasing DCs through systemic Flt-3L treatment significantly
increased survival
 Recombinant CCL21 delivered intratumorally produces immunemediated tumor growth inhibition
10
Days Post Start of T herapy
Figure 6. Tumor growth following pCCL21/pFlt3L
 Treatment with intratumoral CCl21 (SLC) in Pro-Gelz™ is therapeutic
for pancreatic cancer
20
0
0
Conclusions
 Two effective immunotherapy modalities for pancreatic cancer
30
0
Figure 2. Tumor growth is inhibited by intratumoral CCL21 treatment of Panc02 tumors
in wild type mice, but not in mice lacking fully functional T, B, and NK lymphocytes.
Tumor cells (1x106 Panc02 cells) were injected subcutaneously above the scapulae in
female wild type C57BL/6 mice (A) or C57BL/6 RAG2-/-Pfp-/- mice (B). Following
development of palpable tumors, mice were treated on days 1, 2, 3, 8, 9, and 10 with
intratumoral injections of 1 µg recombinant murine CCL21 in PBS+0.05% NMS (n=8)
or PBS+0.05% NMS alone (n=8). The graphs display the changes in tumor volumes
over time, with the first day of treatment as day 1.
Conclusions and Future Directions
40
50
50
p=0.0124
50
p=0.0124
200
DC
p it/p im (n=10)
pFlt-3 (n=8)
p=0.0223
80
70
p=0.0124
300
CCL21
attracts DCs
& T cells
ProGelzTM (n=10)
 Flt3L in ProGelzTM (n=8)
SLC (n=7)
400
Flt3-L
increases
DC numbers
PBS (n=6)
SLC (n=7)
100
CCL21 (n=7)
mm3
Pancreatic adenocarcinoma is a devastating disease, characterized by
resistance to current therapies and extremely poor prognosis. New, effective
treatments are urgently needed. Cellular immune responses can be initiated
via specialized antigen presenting cells, such as dendritic cells (DCs) that
stimulate naive T cells in regional draining lymph nodes. DC and T cell
trafficking is regulated by chemokines, such as CCL21. Our data indicate
that intratumoral administration of CCL21, a chemoattractant for DCs, as
well as for natural killer (NK) cells and T cells, slows the growth of
pancreatic tumors. Furthermore, we have data that suggests intratumoral
CCL21 injection can delay the growth of distant tumors. The number of DCs
present in the blood and lymphoid organs is controlled by hematopoietic
growth factors, for example, fms-like tyrosine kinase-3 ligand (Flt3L). In this
study, we tested the hypothesis that sustained DC expansion by Flt3L and
DC attraction by CCL21 will result in increased therapeutic activity against
pancreatic cancer. We tested this hypothesis by examining the anti-tumor
effect of simultaneous administration of Flt3L and CCL21, as well as both
individually,
against
pancreatic
adenocarcinomas
established
subcutaneously in C57BL/6 mice. Increasing DC numbers by intramuscular
Flt3L treatment significantly delayed tumor growth. Similarly, treatment with
intratumoral CCL21 reduced the rate of tumor growth. Surprisingly, the
combination of both systemic Flt3L and intratumoral CCL21 was not more
effective than either cytokine alone. To begin to determine whether
manipulation of the timing of Flt3L and CCL21 treatments, relative to each
other, would improve therapeutic efficacy, we analyzed the kinetics of Flt3L
and CCL21 activity. In these experiments, adenoviral delivery of Flt3L and
CCL21 was used, because it permits high transduction efficiency and
transgene expression in vivo. We observed that subcutaneous injection of
Adv-CCL21 was superior to recombinant CCL21 (rCCL21) not only in
increasing DC, NK cell, and T cell populations in the local lymph node, but
also in prolonging the duration of that increase (21 days in Adv-CCL21
injected mice versus 8 days in rCCL21 injected ones). Furthermore, AdvFlt3L significantly expanded DC populations in the spleen with the peak of
the expansion at 6-8 days post-injection. Overall, these observations
suggest that 1) both CCL21 and Flt3L may be therapeutic for pancreatic
cancer, and 2) peak activities for Adv-Flt3L and Adv-CCL21 are observed at
6-8 days and 7-9 days, respectively. Our subsequent study will be to inject
Adv-CCL21 intratumorally 6-8 days after intravenous Adv-Flt3L.
CCL21 and Flt3L Can Individually Delay Pancreatic Tumor Growth
2
4
6
8
Days post-injection
10
12
This work was supported by an LB595/Cattlemen’s Ball Grant (to J.C.S.),
by the Nebraska Research Initiative Program in Molecular Therapeutics
(to J.E.T., J.C.S., R.K.S. and M.A.H.), and the NIH Specialized Program
in Research Excellence Grant P50 CA72712 (to M.A.H. and J.C.S.). A.A.
received support from a UNMC Graduate Fellowship. H.R.T. received
support from the NIH Training Grant T32 CA09476, UNMC Graduate
Fellowships, and a UNMC Presidential Fellowship.