ccl21 and flt3l for pancreatic cancer treatment figure 9. figure 11., dc1figure 10. abdelkader...
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CCL21 AND FLT3L FOR PANCREATIC CANCER
TREATMENT
Figure 9. Figure 11. , DC1Figure 10.
Abdelkader AshourAbdelkader AshourEppley Institute for Research in Cancer and Allied DiseasesEppley Institute for Research in Cancer and Allied Diseases
Department of Biochemistry and Molecular BiologyDepartment of Biochemistry and Molecular BiologyUniversity of Nebraska Medical Center, Omaha, NE, USAUniversity of Nebraska Medical Center, Omaha, NE, USA
4th leading cause of cancer-related death in the U.S.A.
Median survival of ≤ 6 months
5-year survival rate of 3%
Asymptomatic development and limited diagnostic methods
No current effective treatment
Immunotherapies may be a possible method to lengthen life
and increase survival
Pancreatic CancerPancreatic Cancer
T cell and dendritic cell (DC) infiltration of T cell and dendritic cell (DC) infiltration of tumor correlates with increased survivaltumor correlates with increased survival
Dallal Dallal et alet al. 2002. . 2002. SurgerySurgery 131: 135 131: 135
Fukunago Fukunago et alet al. 2004. . 2004. PancreasPancreas 28(1): 26 28(1): 26
– Pancreatic adenocarcinoma resection samplesPancreatic adenocarcinoma resection samples
– Patients whose samples were CD8Patients whose samples were CD8++/CD4/CD4++ had had increased survival post surgical resection increased survival post surgical resection (p=0.0098) (p=0.0098)
– CD4CD4++/CD8/CD8++ tumor samples displayed increased tumor samples displayed increased DC infiltrateDC infiltrate
Tumor Infiltrate (n=80) 5-year Sur.
CD4-/CD8+ (n=3) 0%
CD4+/CD8- (n=34) 10.1%
CD4-/CD8- (n=30) 4.6%
CD4+/CD8+ (n=13) 48.4%
CCL21????
NaïveT Cell
APC
MHC
Antigenic Peptide
Requirements for T cell activationRequirements for T cell activation
CD4/8
TCR
Proliferation and Differentiation
Initiators and regulators of tumor immunity: Initiators and regulators of tumor immunity: dendritic cells (DCs)dendritic cells (DCs)
Most potent antigen presenting cell Most potent antigen presenting cell • Small numbers of DCs with low levels of antigen can Small numbers of DCs with low levels of antigen can
stimulate a significant T cell responsestimulate a significant T cell response– Naïve and memory T cellsNaïve and memory T cells
• Antigen capture and migration to 2Antigen capture and migration to 2o o lymph node and lymph node and optimizes T cell exposure to antigen optimizes T cell exposure to antigen
• Process and present exogenous antigen on high Process and present exogenous antigen on high levels of MHC Class I/IIlevels of MHC Class I/II
• Co-stimulatory and adhesion molecules Co-stimulatory and adhesion molecules
Dendritic Cell MaturationDendritic Cell Maturation
PPSC
Bone Marrow
Immature or Indeterminate
Cell
GM-CSF & IL4 or Flt3L CD40L
TNF LPS CPG
Antigen processing Antigen
presentation
CD86, CD80, CD11c
Class I/II Cytokines
APC and T cellsAPC and T cells
IFN-, IL-2,
TNF-, IL-18, IL-24, GM-CSF
Cell Mediated
IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-22, IL-26 Ab mediated
Th0
Th1
Th2
CD154LFA-1
CD28CD152
CD40ICAM
CD80CD86
IL-10
IL-22
IL-12
IL-18
DC2
DC1
Pre DC
IFN- and no co-stim.
Treg
CD4+CD25+
TGF-2
Intrinsic differences in murine DC subtypes in Intrinsic differences in murine DC subtypes in their ability to influence immune responsestheir ability to influence immune responses
Lymphoid DC– CD11c+ CD11b- CD8+
– Direct Th1 – IFN- and IL-12 secretion
Myeloid DC– CD11c+ CD11b+ CD8-
– Direct Th2 - Lack of IFN- and IL-12
Plasmacytoid (pDC)– CD11c+ CD11b- B220+ Gr-1+
– IFN-production
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
CCL21 as a potential immunotherapeuticCCL21 as a potential immunotherapeutic
• Secondary lymphoid tissue chemokine (SLC)
• Expressed in T-cell zones of spleen and lymph nodes
• Multiple functions to facilitate T cell responses
• Angiostatic properties in murine tumor models (via CXCR3)
• Flt3L is a potent hematopoietic growth factor capable of expanding and mobilizing many types of hematopoietic progenitor and stem cells
• Cells that express message for Flt3 receptor (Flt3R) include B cells, T cells, NK cells, peritoneal macrophages, and monocytes (primarily the immature stages of these cells)
• These diverse effects, especially its effects on the hematopoietic and dendritic cell populations, have prompted the use of Flt3L as a potential vaccine adjuvant and therapeutic agent.
Flt3L as a potential immunotherapeuticFlt3L as a potential immunotherapeutic
Pro-Gelz™Pro-Gelz™
• Matrix of 22% Poloxamer-407/5% hydroxypropylmethyl cellulose
• Practical means for clinical delivery
• Sustained release drug delivery system
• Reversible thermal gelation
Experimental designExperimental design
• Panc02: weakly immunogenic C57BL/6 pancreatic carcinoma cell line Panc02: weakly immunogenic C57BL/6 pancreatic carcinoma cell line • Growth properties and immune response at subcutaneous and orthotopic sites are Growth properties and immune response at subcutaneous and orthotopic sites are
well characterizedwell characterized
• C57BL/6 C57BL/6 (n=8) or(n=8) or C57BL/6 RAG2C57BL/6 RAG2-/--/- PFP PFP-/--/- (n=8) mice were challenged s.c. with 1 X (n=8) mice were challenged s.c. with 1 X 101066 Panc02 cells Panc02 cells
• Intratumoral injection of 1Intratumoral injection of 1g of CCL21 or PBS g of CCL21 or PBS
• Tumor growth rate to 1100mmTumor growth rate to 1100mm33 was examined was examined
Average tumor volume; diameter range at 1st treatmentAverage tumor volume; diameter range at 1st treatmentPBS = 10.1PBS = 10.1++3.9 mm3.9 mm33; 1.5 to 4 mm; 1.5 to 4 mmCCL21 = 11.4CCL21 = 11.4++2.6 mm2.6 mm33; 1.5 to 3.5 mm; p=0.78; 1.5 to 3.5 mm; p=0.78PBS = 14.2PBS = 14.2++3.2 mm3.2 mm33; 2 to 4 mm; 2 to 4 mmCCL21 = 12.5CCL21 = 12.5++ 2.5 mm 2.5 mm33; 2 to 4 mm; p=0.73; 2 to 4 mm; p=0.73
Day 80
CCL21 or PBS i.t.CCL21 or PBS i.t.
Day 1-2-3Day 1-2-3 8-9-108-9-10
Palpable Tumors
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Tumor Growth of panc02 in C57BL/6 mice Tumor Growth of panc02 in C57BL/6 RAG2-/- /Pfp-/- mice
Tumor growth is inhibited by intratumoral CCL21 treatment of Tumor growth is inhibited by intratumoral CCL21 treatment of Panc02 tumors in wild type mice, but not in mice lacking fully Panc02 tumors in wild type mice, but not in mice lacking fully
functional T, B, and NK lymphocytes.functional T, B, and NK lymphocytes.
Recombinant CCL21 delivered intratumorally produces immune-mediated tumor growth inhibition
Experimental designExperimental design
• C57BL/6 mice (n=8 to 10) were challenged s.c. with 2 X 10C57BL/6 mice (n=8 to 10) were challenged s.c. with 2 X 1066 Panc02 cells Panc02 cells in right flank and 1 X 10in right flank and 1 X 1066 Panc02 in the left flank Panc02 in the left flank
• Intratumoral injections of 1mg of CCL21 or PBS into the right tumor (Days Intratumoral injections of 1mg of CCL21 or PBS into the right tumor (Days 1, 2, 3 and 8, 9, 10)1, 2, 3 and 8, 9, 10)
• Left flank remained untreatedLeft flank remained untreated
• Tumor growth rateTumor growth ratewas examinedwas examined
Average tumor volume at 1st treatmentAverage tumor volume at 1st treatment
PBS (n=12); RT 19.6 PBS (n=12); RT 19.6 ++3.3 mm3.3 mm33
LF 7.7LF 7.7++2.6 mm2.6 mm33
CCL21 (n=11); RT 17.8CCL21 (n=11); RT 17.8++3.7 mm3.7 mm33
LF 4.2LF 4.2++ 1.5 mm 1.5 mm33
1 X 101 X 1066 Panc02 Panc02
No TreatmentNo Treatment
22 X 10X 1066 Panc02 Panc02
CCL21 or PBSCCL21 or PBS
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Untreated tumors are inhibited in growth following Untreated tumors are inhibited in growth following intratumoral treatment of separate tumors with CCL21intratumoral treatment of separate tumors with CCL21
Intratumoral CCL21 injection can delay the growth of distant tumors
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Intratumoral administration of CCL21 increases survival Intratumoral administration of CCL21 increases survival of mice that were injected with Panc02 tumorsof mice that were injected with Panc02 tumors
Treatment with intratumoral CCl21 (SLC) in Pro-Gelz™ is therapeutic for pancreatic cancer
0 5 10 15 20 25 30 350
50
100 PBS (n=6)SLC (n=7)
p=0.0124
Days Post Start of Therapy
0 5 10 15 20 25 30 350
50
100 PBS (n=6)SLC (n=7)
p=0.0124
Days Post Start of Therapy
CCL21 (n=7)
PBS (n=6)
Flt3L ImmunotherapyFlt3L Immunotherapy
Experimental PlanExperimental PlanSurvival 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 g/dose-Day 1 and 6) in Pro-Gelz™ i.m.
Pro-Gelz™ i.m. (Day 1 and 6)
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Intramuscular administration of Flt3L increases survival Intramuscular administration of Flt3L increases survival of mice that were injected with Panc02 tumorsof mice that were injected with Panc02 tumors
Treatment with intratumoral Flt3L in Pro-Gelz™ is therapeutic for pancreatic cancer
0 5 10 15 20 25 30 35 40 450
10
20
30
40
50
60
70
80
90
100
p it/p im (n=10)
pFlt-3 (n=8)
Days Post Startof Therapy
ProGelzTM (n=10) Flt3L in ProGelzTM (n=8)
p=0.0223
0 5 10 15 20 25 30 350
50
100 PBS (n=6)SLC (n=7)
p=0.0124
Days Post Start of Therapy
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Hypothesis
Sustained DC expansion by Flt3L and DC attraction by CCL21 will have therapeutic activity against
pancreatic cancer
CCL21 attracts DCs
& T cells
Flt3-L increases
DC numbers
strong immune
response DC
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Combination CCL21/Flt3L ImmunotherapyCombination CCL21/Flt3L Immunotherapy
Experimental PlanExperimental PlanSurvival 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 g/dose-Day 1 and 6) in Pro-Gelz™ i.m. + CCL21 in Pro-Gelz™ (3 g/dose- Day 1 and 8) i.t. CCL21 (3 g/dose-Day 1 and 8) in Pro-Gelz™ i.t. Flt-3L (15 g/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.
Treatments
Day 1
pCCL21 i.t.
Day 1
Day 6
pFlt3L i.m.1.5 to 3.5 mm
Tumors
Day 8
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
The combination of both systemic Flt3L and intratumoral The combination of both systemic Flt3L and intratumoral CCL21 is not more effective than either cytokine alone!CCL21 is not more effective than either cytokine alone!
0
100
200
300
400
500
600
700
800
900
1000
0 4 7 11 16 19 23
Days Post Start of Therapy
mm
3
Pro-Gelz (i.m.)/Pro-Gelz (i.t.)
pFlt3-L (i.m)/pSLC (i.t.)
pSLC (i.t.)
pFlt3-L (i.m)
pCCL21 (i.t.)
pFlt3L (i.m.)/pCCL21 (i.t.)
Pro-Gelz (i.m.)/Pro-Gelz (i.t.)
pFlt3L (i.m.)
0 10 20 30 400
10
20
30
40
50
60
70
80
90
100 pSLC/pFlt-3L (n=9)
p it/p im (n=10)
pSLC (n=10)pFlt-3 (n=8)
Day Post Start of Therapy
pCCL21 (n=10)
pCCL21/pFlt3L (n=9)
pFlt3L (n=8)
p i.t./p i.m. (n=10)
whether manipulation of the timing of Flt3L and CCL21
treatments, relative to each other, would improve therapeutic
efficacy?
QuestionQuestion
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Kinetics of CCL21 ActivityKinetics of CCL21 Activity
Experimental PlanExperimental PlanGroups of C57BL/6 mice were given 1X s.c. injections in the right flank with 50 l of: PBS, PBS containing 10 g 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
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
CCL21 increases numbers of dendritic cells by day 4CCL21 increases numbers of dendritic cells by day 4
The duration of activity??
Percentage CD11c+ CD11b+ (mDC) LN -- rCCL21
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Day 4 Day 8
Day Post Rx
Perc
enta
ge T
ota
l C
ells
PBS
rSLCrCCL21
Percentage CD11c+ CD11b- CD8+ (lDC) LN -- rCCL21
0
1
2
3
4
5
6
7
8
Day 4 Day 8
Day Post Rx
Perc
enta
ge o
f T
ota
l Cells
PBS
rSLCrCCL21
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Adv-CCL21 not only increases DC numbers but also the Adv-CCL21 not only increases DC numbers but also the duration of the increaseduration of the increase
Kinetics of SC SLC
0%
100%
200%
300%
400%
500%
600%
700%
800%
900%
1000%
Day 4 Day 8 Day 21
Day
Per
cent
age
of C
ontr
ol
rSLC (Lymph CD11c+ CD8+ Spleen)AD-SLC (Lymph CD11c+ CD8+ Spleen)rSLC (Lymph CD11c+ CD8+ LN)AD-SLC (Lymph CD11c+ CD8+ LN)
rCCL21 (CD11c+ CD8+ Spleen )
Kinetics of S.C. rCCL21, DC1
Adv-CCL21 (CD11c+ CD8+ Spleen )rCCL21 (CD11c+ CD8+ LN )Adv-CCL21 (CD11c+ CD8+ LN )
Kinetics of SC SLC
0%
100%
200%
300%
400%
500%
600%
700%
800%
900%
1000%
Day 4 Day 8 Day 21
Day
Perc
enta
ge o
f C
ontr
ol
rSLC (DC CD11c+ CD11b+ Spleen)
AD-SLC (DC CD11c+ CD11b+ Spleen)
rSLC (DC CD11c+ C11b+ LN)
AD-SLC (DC CD11c+ CD11b+ LN)
rCCL21 (CD11c+ CD11b+ Spleen )
rCCL21 (CD11c+ CD11b+ LN )
Adv-CCL21 (CD11c+ CD11b+ Spleen )
Adv-CCL21 (CD11c+ CD11b+ LN )
Kinetics of S.C. rCCL21, DC2
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Kinetics of Flt3L ActivityKinetics of Flt3L ActivityExperimental PlanExperimental PlanBALB/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.
Spleen DC1 (CD11c+ CD11b- CD8+)
0
1
2
3
4
5
0 2 4 6 8 10 12Days post-injection of Adv-Flt3L
cells
(X
1E
6)
Spleen DC2 (CD11c+ CD11b+)
0
5
10
15
20
25
0 2 4 6 8 10 12Days post-injection of Adv-Flt3L
cells
(X1E
6)
Peripheral Blood DC1 (CD11c+ CD11b- CD8+)
0
5
10
15
20
25
0 2 4 6 8 10 12Days post-injection
Cel
ls/m
l (X
1E4)
Peripheral Blood DC2 (CD11c+ CD11b+)
04080
120160200240280
0 2 4 6 8 10 12
Days post-injection
Cel
ls/m
l (X
1E4)
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
ConclusionsConclusions
Treatment with intratumoral CCl21 (SLC) in Pro-Gelz™ is therapeutic for pancreatic cancer
Increasing DCs through systemic Flt3L treatment significantly increased survival
Two effective immunotherapy modalities for pancreatic cancer
Recombinant CCL21 delivered intratumorally produces immune-mediated tumor growth inhibition
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Conclusions, contd.Conclusions, contd.
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 post-injection
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Future DirectionsFuture Directions
Inject Adv-CCL21 intratumorally 6-8 days after intravenous Adv-Flt3L
Treatments
Day 15
Adv- CCL21 i.t.
Day 1
Adv-Flt-3L I.V.1.5 to 3.5 mm
Tumors
Day 8
Day 23
Figure 8.
Figure 9. Figure 11.
Figure 13.
, DC1Figure 10.
Future Directions, contd.Future Directions, contd.
CCL21 treatment of orthotopic pancreatic cancer
Examine the possible synergistic effect of agents that can stimulate maturation of DCs, such as CD40 ligand and CpG DNA on CCL21/Flt3L immunotherapy
AcknowledgmentsAcknowledgments
Dr. Joyce SolheimCurrent lab membersDr. Chantey MorrisDr. Xiaojian WangHethTurnquistMary McIlhaneyCarrie Mislivec
Former lab membersDr. Adrian ReberDr. Jason PetersenKris SiepelJack Kampf
CollaboratorsDr. M. A. HollingsworthDr. James Talmadge - Scott KurzDr. Rakesh SinghDrake LaFace (Canji inc.)
AllahMy parents
Thank you all
