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Development of CCL21 as a
Promising Immunotherapeutic for
Breast Cancer
Abdelkader Ashour, Ph.D.
Postdoctoral Research Associate
Eppley Institute for Research in Cancer and Allied Diseases
University of Nebraska Medical Center
Overview
A. Introduction
B. Kinetics of Immune Cell Infiltration
Induced by CCL21
C. CCL21 as an Immunotherapeutic Against
Breast Cancer
Breast Cancer
 Breast cancer strikes one in eight women in America. Over 200,000
new cases arise each year
 The majority of the 40,000 annual breast cancer deaths result from
complications arising from metastasic disease rather than a
consequence of the primary breast tumor
 the spread of cancerous cells to the bone marrow and lymphatics occurs early in
the development of the disease
 The development of these micrometastases into metastatic breast cancer
establishes a disease state currently considered incurable, as the 5-year survival
rate falls below 25%
 The recurrence of disease following surgical resection or the
development of metastases greatly increases mortality
 A major reason for this lethality is the limited effectiveness of current therapies
Breast Cancer, contd.
 Current chemotherapeutic treatments target rapidly proliferating cells in
the primary tumor and fail to remove disseminated cells, as a majority of
these rest in the non-proliferative phase of the cell cycle (G0)
 An ideal therapy would be one directed at the easily identifiable primary tumor,
but also able to remove micrometastases and any metastatic disease
 Immunotherapies, or treatments stimulating the body’s own immune
system to attack cancerous cells, may be viable options to target both
the primary tumor and any disseminated disease, typically with minimal
side effects
Immune Responses to Cancer
 Cytotoxic T cell (CTL) CD8+

Primary effector cells in tumor rejection

Perforin, granzyme, FasL, TNFa
 T helper cell (TH) CD4+


TH1 - IFN-g - Cellular responses (Type 1)
TH2 - IL-4, -5 - Humoral responses (Type 2)
 B cells (plasma cells)

Antibodies against tumor antigens
 Natural killer (NK) cells

Innate anti-tumor immunity

Surveillance and control of hematogenous spread of tumor cells
Initiators and Regulators of Tumor Immunity:
Dendritic Cells (DCs)
What is a DC:
 White blood cells that capture and process
antigen for presentation to T cells, resulting
in primary immune response
 Trigger an innate immune response to
tumors by activating macrophages, natural
killer (NK) cells
 DCs link the innate immune system with the
adaptive immune system
Most potent antigen presenting cell:
 Small numbers of DCs with low levels of antigen
can stimulate a significant T cell response
 Naïve and memory T cells
 Express co-stimulatory and adhesion molecules
 Processes and presents exogenous antigen on
high levels of MHC class I/II
 Highly effective at cross-priming
Requirements for T cell activation
B7
CD28
CD4/8
APC
Antigenic
Peptide
MHC
I/II
Naïve
T Cell
TCR
Requirements for T cell activation
Naive
T Cell
APC
Requirements for T cell activation
Activated
T Cell
APC
Requirements for T cell activation
APC
T Cell
Proliferation and
Differentiation
DC Functions are Linked to Maturation
 Immature DC scan peripheral tissue and acquire antigen
 Maturation signals
 Cytokines
 Necrotic materials
 T helper cell signaling
 DC maturation
 Migrate to secondary
lymphoid organs and
display antigen
 Cytokine secretion
Intrinsic Differences in Murine DC Subtypes in Their
Ability to Influence Immune Responses
 Lymphoid DC (DC1)
– CD11c+ CD11b- CD8+
– Direct TH1 -- IFN-g and IL-12 secretion
 Myeloid DC (DC2)
– CD11c+ CD11b+ CD8– Direct TH2 – IL-10 secretion
 Plasmacytoid DC (pDC)
– CD11c+ CD11b- B220+ Gr-1+
– TGF-b and IFN-a production
CCL21 as a Potential Immunotherapeutic
 Expressed in T-cell
zones of spleen and
lymph nodes
 Multiple functions to
facilitate T cell
responses
 Strong chemoattractant
for DCs, NK cells, and T
cells
 Induces anti-apoptotic
signaling in DCs
 Stimulates DC
endocytosis
CCL21 gradient
Overview
A. Introduction
B. Kinetics of Immune Cell Infiltration
Induced by CCL21
C. CCL21 as an Immunotherapeutic Against
Breast Cancer
Kinetics of Immune Cell Infiltration Induced by
CCL21
Experimental Plan

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 control virus particles (Adv-control)
1X1011 adenoviral-CCL21 virus particles (Adv-CCL21)
Injection sites were histologically examined
Lymph nodes and spleens were harvested at various time points
and examined by flow cytometry for DCs and CTLs
Injection of CCL21 Resulted in Local Foci of Mononuclear
Cells that Predominantly Had Lymphocytic Morphology
Adv-CCL21 Day 21 400x
Ashour &
Turnquist
CCL21 Significantly Increased the Frequency of DCs
and Effector CTLs in Draining Lymph Nodes
Ashour & Turnquist
Adv-CCL21 Not Only Increases DC Numbers But
Also the Duration of the Increase
of
SLC
Kinetics
ofKinetics
S.C.
CCL21,
DC2
Kinetics
of SC
SC
SLC
Kinetics
of S.C.
CCL21,
DC1
rSL C (CD11c+
(DC CD11
c+ Spleen
CD11)b+ Splee n)
rCCL21
CD11b+
rCCL21
(CD11c+
CD8+CD8+
Spleen
)
rSLC (Lymph
CD11c+
Spleen)
Adv-CCL21
(CD11c+
CD8+
Spleen
)en)
AD-SLC (CD11c+
(DC
CD1
1c+
CD1
1b+
Sple
AD-SLC
(Lymph
CD11c+
Spleen)
Adv-CCL21
CD11b+
Spleen
) CD8+
rCCL21
(CD11c+
CD8+CD8+
LN )
(Lymph
CD11c+
rSL C (CD11c+
(DCrSLC
CD11
c+ LN
C11b+
L N) LN)
rCCL21
CD11b+
)
Adv-CCL21
(CD11c+
CD8+
LN ) LN)
AD-SLC (Lymph
CD11c+
CD8+
AD-SLC (CD11c+
(DC CD1
1c+ LN
CD1
Adv-CCL21
CD11b+
) 1b+ L N)
1000%
Percentage
PercentageofofControl
Control
1000%
900%
900%
800%
800%
700%
700%
600%
600%
500%
500%
400%
400%
Figure 8.
Figure 13.
300%
300%
200%
200%
100%
100%
Figure 10.
Figure 9.
Figure 11.
, DC1
0%0%
Day
Day
4 4
Day
Day
88
Day21
21
Day
Day
Day
Ashour & Turnquist
Conclusions, I
 S.c. injection of CCL21 resulted in local foci of mononuclear cells that
predominantly had lymphocytic morphology
 CCL21 significantly increased the frequency of DCs and effector
CTLs in draining lymph nodes
 Adv-CCL21 not only increases DC numbers but also the duration of
the increase
Overview
A. Introduction
B. Kinetics of Immune Cell Infiltration
Induced by CCL21
C. CCL21 as an Immunotherapeutic Against
Breast Cancer
CCL21 Effect on Orthotopic Mammary Tumor Growth
 Cl-66: aggressive mammary adenocarcinoma cell line of BALB/c
origin
– derived from a spontaneously arising mammary tumor
– consistently produces metastases to the bone marrow and other organs
 BALB/c mice were injected in the fourth inguinal mammary fat pad
with 1X105 Cl-66 cells.
 Once tumors were palpable, the mice received intratumoral
implantation of 6 μg CCL21 in HydronTM, or PBS-HydronTM
 Tumor growth and survival were monitored
 HydronTM is a commercially available hydrogel polymer
 sustained release drug delivery system
 utilized in several ongoing FDA-approved clinical trials, but has not been
examined as a means to deliver CCL21 intratumorally
Hy-CCL21 Effectively Inhibited Tumor Growth
2800
2600
2400
Tumor Size (mm3)
2200
No Treatment
2000
PBS-Hydron
1800
CCL21-Hydron
1600
1400
1200
1000
800
600
400
200
0
Day 0
Day 4
Day 7
Day 10
Day 15
Day 19
Day 22
Day 25
Days Post Start of Therapy
Group
Tumor Vol. (mm3) day 25
Group
Tumor Vol. (mm3) day 25
PBS-Hydron
2709.3
No Treatment
2595
CCL21-Hydron
1635.4
CCL21-Hydron
1635.4
P value
<0.0001
P value
<0.0001
Hy-CCL21 Significantly Enhanced Survival of
Mammary Tumor-Bearing Mice
100
Percentage Surviving Mice
Percentage Surviving Mice
100
90
80
70
60
50
PBS-Hydron
40
CCL21-Hydron
30
20
P<0.0001
10
0
90
80
70
60
50
No Treatment
40
30
CCL21-Hydron
20
P<0.0001
10
0
0
5
10
15
20
25
30
35
40
45
50
55
Days Post Start of Therapy
0
5
10
15
20
25
30
35
40
45
50
55
Days Post Start of Therapy
Group
Median Survival
Group
Median Survival
PBS-Hydron
35.5
No Treatment
34
CCL21-Hydron
46
CCL21-Hydron
46
CCL21 and Surgery Against Mammary Carcinoma
surgical resection
immunosuppression created by the tumor
protective immunity
necrosis and inflammation at the surgery site
+
attraction of immune cells into the region of the primary tumor by
CCL21
intensified local immune response that removes residual and
metastatic disease
Testing Surgery Followed by Hy-CCL21 (Hy-CCL21
Adjuvant) Against Mammary Carcinoma
Experimental Plan
 Orthotopic mammary tumors were established by the injection of 1 X
105 cl-66 cells into the fourth inguinal mammary fatpad of 20 female
BALB/c mice
 Once tumors reached 60 mm3 (Day 0), they were resected
 Hy-PBS or Hy-CCL21 (6 mg) was implanted in the surgical site
immediately following resection of the primary tumor
 Survival was monitored
Percentage Surviving Mice
Efficacy of Hy-CCL21 (Adjuvant) Against Mammary
Carcinoma
100
90
80
70
60
50
40
30
20
10
0
PBS-Hydron (Adjuvant)
CCL21-Hydron (Adjuvant)
P=0.8623
p=0.8623
0 10 20 30 40 50 60 70 80 90 100 110
Days Post Start of Therapy
Testing Hy-CCL21 Accompanied by Surgery (HyCCL21 Neoadjuvant) Against Mammary Carcinoma
Experimental Plan
 Orthotopic mammary tumors were established by the injection of 1 X
105 cl-66 cells into the fourth inguinal mammary fatpad of 180 female
BALB/c mice
 Once tumors reached 60 mm3 (Day 0), Hy-PBS or Hy-CCL21 (6 mg of
CCL21/mouse) was implanted i.t.
 Four days following initial treatment, surgical resection of the tumors
was carried out. Survival was monitored
 On Day 100, surviving mice, as well as 20 naïve BALB/c mice, were
re-challenged with 1 X 105 cl-66 cells injected into the fourth inguinal
mammary fatpad on the opposite side
Efficacy of Hy-CCL21 (Neoadjuvant) Against
Mammary Carcinoma
Percentage Surviving Mice
100
90
80
70
60
No Treatment
50
Surgery Only
40
PBS-Hydron (No Surgery)
30
CCL21-Hydron (No Surgery)
20
PBS-Hydron (Neoadj uv ant)
10
CCL21-Hydron (Neoadj uv ant)
0
0
10
20
30
40
50
60
70
80
Days Post Start of Therapy
90 100 110
100
100100
100
90
9090
Percentage Surviving Mice
Percentage
Surviving
Mice
PercentageSurviving
Surviving
Mice
Percentage
Percentage
Surviving
Mice
Mice
Efficacy of Hy-CCL21 (Neoadjuvant) Against
Mammary Carcinoma
90
80
8080
8070
70
70
70
60
6060
60
6050
No
Treatm ent
CCL21-Hydron
(No Surgery)
50
50
50
5040
CCL21-Hydron
(Neoadjuvant)
40
40
CCL21-Hydron
(Neoadjuvant)
40
PBS-Hydron
(No
Surgery)
4030
30
30
PBS-Hydron
p=0.0464
Surgery Only(Neoadjuvant)
30
3020
CCL21-Hydron (Neoadj uv ant)
20
p=0.0193
20
CCL21-Hydron (Neoadjuvant) p<0.0001
20
2010
p<0.0001
p<0.0001
10
10
10 0
10
00 0 10 20 30 40 50 60 70 80 90 100 110
0
00 10
10 20
20 30
30 40
40 50
50 60
60 70
70 80
80 90
90 100
100 110
110
0 10
20
30
40
50
60
80
90
100
110
Days
Post
Start
of 70
Therapy
Days
DaysPost
PostStart
Startof
ofTherapy
Therapy
Days
Post
Start
of
Therapy
Efficacy of Hy-CCL21 (Neoadjuvant) Against
Mammary Carcinoma
Metastasis
 In the Surgery Only group, out of 13 dead mice, externally visible
tumors in areas other than the primary site were found in 4 mice
 In the Hy-PBS Neoadjuvant group, out of 13 dead mice, externally
visible tumors in areas other than the primary site were found in 4
mice. One of these mice had also a tumor at the primary site
 In the Hy-CCL21 Neoadjuvant group, no tumors were externally
visible at either the primary site or remote sites
Metastasis (Control Mice)
Metastasis, contd.
Lung of a
control
mouse,
completely
infiltrated with
metastases
Liver of a control mouse,
black, shrinking and
infiltrated with a huge tumor
Lung of HyCCL21
Neoadjuvant
mouse
Liver of Hy-CCL21
Neoadjuvant mouse
Hy-CCL21 Neoadjuvant-Treated Mice Had Significantly
Inhibited Tumor Growth after Tumor Re-challenge
130
120
110
Tumor Size (mm3)
100
90
80
Naiv e
70
Surge ry Only
60
PBS-Hydron (Ne oadjuv ant)
50
CCL21-Hydron (Ne oadjuv ant)
40
30
20
10
0
Day 0
Day 9
Day 13
Day 16
Days Post Re-challenge with Cl-66
Group
PBS-Hy (Neo)
Tumor Vol. (mm3)
day 16
100.5338
Group
Tumor Vol. (mm3)
day 16
Group
Tumor Vol. (mm3)
day 16
Surgery Only
88.453
Naive
123.704
CCL21-Hy (Neo)
29.698
CCL21-Hy (Neo)
29.698
CCL21-Hy (Neo)
29.698
P value
<0.0001
P value
0.0013
P value
<0.0001
Conclusions, II
 Sustained CCL21 delivery resulted in primary mammary tumor
growth inhibition, and enhanced the survival of treated mice
 Sustained CCL21 did not increase the length of survival when
administered as an adjuvant immediately following tumor resection
 Sustained CCL21 delivery used as a neoadjuvant prior to tumor
resection significantly increased the duration of survival and appears
to inhibit metastasis
 Re-challenged mice that had previously received CCL21 neoadjuvant
had delayed tumor onset and inhibited tumor growth, compared to
control mice.
 mice developed an immunologic memory against cl-66 tumor
Future Directions
 Test CCL21 as a potential breast cancer vaccine adjuvant
 Examine the possible synergistic effect of agents that can stimulate
maturation/activation of DCs, such as GM-CSF, CD40 ligand and
CpG DNA on CCL21 immunotherapy
 Test the effect of Flt3L and CCL21 treatments, alone and in
combination, in a neadjuvant setting
 Thus our model may approximate the human clinical setting, in which the
primary tumor is surgically removed
Acknowledgments
 Dr. Joyce Solheim
Collaborators
Committee members
Dr. M. A. Hollingsworth
Surinder K. Batra, PhD
G. Stanley Cox, PhD
Michael A. Hollingsworth, PhD
Myron L. Toews, PhD
Current lab members
Dr. Xiaojian Wang
Dr. Xuede Lin
Nicole Burns
Amit Tuli
-Chunhui Yi
-Judy Anderson
Dr. James Talmadge
Dr. Rakesh Singh
-Anguraj Sadanandam
-Michelle Varney
Former lab members
Dr. Heth Turnquist
Dr. Adrian Reber
Dr. Jason Petersen
Dr. Chantey Morris
Kris Siepel
Jack Kampf
Carrie Mislivec
Mary McIlhaney
 Department of Biochemistry and
Molecular Biology
Acknowledgments
UNMC-Eppley Facilities
Supported by:
Cell Analysis Facility
To A.E.A
UNMC Research Assistantship
UN Presidential Fellowship
Dr. Charles Kuszynski
Linda Wilkie
Victoria Smith
Animal Facility
Connie Arnold
Douglas Eicher
Histology Department
Karen Dulany
Maureen Harmonthe
Molecular Biology Core
DOD Breast Cancer Research
Program Predoctoral Traineeship
Norman and Bernice Harris
To J.C.S
NRI Molecular Therapeutics Program
NIH Grant GM57428
LB595/Cattlemen’s Ball Grant
NIH SPORE P50 CA72712
Developmental Grant
Questions?
•
%
Targets
with
Granzy
me B
100
80
60
40
20
0
50/1
17/1
Effector/Target Ratio
5.7/1
1.9/1