Transcript Effects of adenovirus delivered Flt

Development of Novel
Immunotherapeutics Against
Cancer
Abdelkader Ashour
Department of Biochemistry and Molecular Biology
Eppley Institute for Research in Cancer and Allied Diseases
University of Nebraska Medical Center, Omaha, NE, USA
Project I: Development of Idiotype DNA Vaccines
against Lymphoma
 We tested murine B cell 38C13 lymphoma idiotype DNA vaccines, to
which HIV Tat-derived transduction sequences were added, for ability
to prolong survival after tumor challenge
 Although the rate of tumor onset was similar for all the mouse
treatment groups, the survival was increased in the mice that received
idiotype+Tat transduction sequence vaccinations, in comparison with
those that received idiotype-alone vaccinations
 Thus, addition of transduction domains to an idiotype vaccine
improved the efficacy of the vaccine in this preclinical murine
lymphoma model
Publication
 Ashour, A. E., J. L. Petersen, M. M. McIlhaney, J. M. Vose, and J. C.
Solheim. Transduction domain sequence linked to a lymphoma
idiotype DNA vaccine prolongs survival after vaccination and tumor
challenge. Accepted for publication, contingent on revision, in
Leukemia and Lymphoma
 To be submitted by April 15, 2006 to Hybridoma
Project II: Development of DNA Vaccines against
Mutant K-ras
 K-ras genes are mutated in several human cancers, and are found in mutated
form in up to 90% of pancreatic adnocarcinomas
 To increase the magnitude of immune responses against mutant K-ras, we
tested modifications of K-ras mutant peptides in the form of DNA vaccines
(fusion of the K-ras mutant DNA sequences to a leader sequence, a
transduction domain sequence, or ubiquitin )
 For the DNA vaccine expressing the RasD epitope, the leader sequence was
most effective in elevating the T cell response against RasD and RasV, with
an altered, -helical Tat transduction sequence second
 linkage of the RasV 13-mer epitope to the ubiquitin and Tat sequences were
most effective in elevating the T cell response against the Ras 13-mers
 Overall, these observations suggest that:
1) the T cell response to mutant K-ras sequences can be increased by
modifications; this may be a promising immunotherapy approach for cancer
2) The type of modification that is optimal may differ depending on the epitope
sequence
Presentations at Conferences
 Ashour, A., A. J. Reber, J. L. Petersen, M. M. McIlhaney, and J. C.
Solheim. Immunization with DNA encoding a mutant K-ras peptide
plus a leader sequence evokes a superior T cell response. Oral
presentation at the Midwest Student Biomedical Research Forum,
February 2003
 Ashour, A. E., A. J. Reber, J. L. Petersen, M. M. McIlhaney, and J. C.
Solheim. Immunization with DNA encoding mutant K-ras peptides
modified with a leader, transduction domain, or ubiquitin sequence
evokes a superior T cell response. Poster presentation at the Keystone
Symposium: Rational Design of Vaccines and Immunotherapeutics,
January 2004.
 Keystone Symposia Scholarship
 Publication
Project III: Flt3L and CCL21 as Immunotherapeutics
against Cancer
A.
Flt3L and CCL21 as Immunotherapeutics against
Pancreatic Cancer
B.
Flt3L and CCL21 as Immunotherapeutics against
Breast Cancer
III A. Flt3L and CCL21 as Immunotherapeutics
against Subcutaneous Pancreatic tumors
 Treatment with intratumoral CCL21 (SLC) in Pro-Gelz™ is
therapeutic for pancreatic cancer
 Systemic Flt3L treatment significantly increased survival
 We examined the anti-tumor effect of simultaneous administration of
Flt3L and CCL21, and of each alone, against pancreatic
adenocarcinomas established subcutaneously in C57BL/6 mice
 Surprisingly, the combination of both systemic Flt3L and intratumoral
CCL21 was not significantly more effective than either cytokine alone
 Flt3L treatment resulted in a trend toward longer survival compared to
Flt3L/CCL21 treatment or CCL21 treatment
Flt3L in an Orthotopic Pancreatic Tumor Model
 Orthotopic pancreatic tumors were established by injecting 104 Panc02 cells into the pancreas
 After 13 days (a period required to get 2.5-3.0 mm diameter tumors),
PG-Flt3L (15 mg/dose) or Pro-Gelz™ were given i.m. on Day 1 and 6
 Survival was monitored for a period of 80 days post therapy initiation
(day 1). The survival endpoint was defined as death or appearance of
moribund behavior
Intramuscular Administration of Flt3L Increases
Survival of Mice With Orthotopic Panc02 Tumors
Flt3L in an orthotopic pancreatic tumor model
Percentage Survival
100
ProGelz-PBS
ProGelz-Flt3L
80
60
P <0.0001
40
20
0
0
10
20
30
40
50
Days Post Start of Treatment
60
III B. Flt3L and CCL21 as Immunotherapeutics
against Breast Cancer
Efficacy of Ad-CCL21 Accompanied with Surgery (AdCCL21/Neoadjuvant) against Mammary Carcinoma
Experimental Plan
 Orthotopic breast tumors were established by the injection of 1X105 Cl-66
cells into the fourth inguinal mammary fatpad of female BALB/c mice.
 Once tumors reached 60 mm3 (Day 0), the described treatments (see table
below) were initiated.
1. No Rx (tumor w/out treatment)
2. Surgery Only (surgery @ day 6)
3. Ad-Control (1x1011 Ad-zz vp, Single Agent)
4. Ad-Control (1x1011 Ad-zz vp, Neoadjuvant., surgery @ day 6)
5. Ad-Control (surgery @ day 6, 1x1011 Ad-zz vp, Adjuvant)
6. Ad-CCL21 (1x1011 Ad-CCL21 vp, Single Agent)
7. Ad-CCL21 (1x1011 Ad-CCL21 vp, Neoadjuvant., surgery @ day 6)
8. Ad-CCL21 (surgery @ day 6, 1x1011 Ad-CCL21 vp, Adjuvant)
Ad-CCL21/Surgical Resection of Mammary Tumors
Survival Data: Ad-CCL21/surgical tumor removal
Percentage Survival
100
Surgery only
No Rx
Ad-z single
Ad-z neoadj
Ad-z adj
Ad-CCL21 single
Ad-CCL21 neoadj
Ad-CCL21 adj
80
60
40
20
0
0
25
50
75
Days Post Start of Treatment
100
CCL21 given before or concurrently with surgical
resection elicits an effective immune response against
residual disease and metastases
Survival of Data: Ad-CCL21 + Surgical tumor removal
Survival Data: Ad-CCL21 vs control
Percentage Survival
Ad-z single
Ad-CCL21 neoadj
80
60
P = .0454
40
20
Percentage Survival
100
100
No Rx
Ad-CCL21 neoadj
80
60
P = 0.0460
40
20
0
0
0 10 20 30 40 50 60 70 80 90 100
Days Post Start of Treatment
0
10 20 30 40 50 60 70 80 90 100
Day Post Start of Treatment
Results, contd.
Survival Data: Ad-CCL21 neoadj. vs surgery only
100
80
Percentage Survival
Surgery only
Ad-CCL21 neoadj
60
P = 0.1717
40
20
0
Ad-z neoadj
Ad-CCL21 neoadj
80
60
P = 0.3142
40
20
0
0 10 20 30 40 50 60 70 80 90 100
0 10 20 30 40 50 60 70 80 90 100
Days Post Start of Treatment
Days Post Start of Treatment
Survival Data: Surgery vs no treatment
100
Percentage Survival
Percentage Survival
100
Survival Data: Ad-CCL21 neoadj. vs control
Surgery only
No Rx
80
60
P = 0.06
40
20
0
0
10
20
30
40
50
Days Post Start of Treatment
60
Conclusions
 Surgical removal of breast tumors resulted in a trend toward a better
survival, relative to no treatment (p = 0.06)
 Ad-CCL21 given before surgical resection enhanced survival, relative
to no treatment (p = 0.046) and adenoviral control (p = 0.045)
 Ad-CCL21 given before surgical resection resulted in a trend toward
a better survival, relative to adenoviral control + surgery (p = 0.31)
 After re-challenge of the mice with cl66, Ad-CCL21/neoadj prevented
development of breast tumor, while Ad-zz/ neoadj did not
 Therapeutic efficacy of Ad-CCL21/neoadj > Ad-CCL21/Adj and AdCCL21 single agent
Conclusions, contd
 Bigger number of mice--- Power Analysis—at least 7 more
mice/gp for better statistics
 Direct comparison of the efficacy of Ad-CCL21 vs Hy-CCL21
 Surgery and tumor implantation!
Adv-Flt3L/Adv-CCL21 as Immunotherapeutics for
Breast Cancer
This is a study to :
1. Examine the efficacy of Ad-CCL21/neoadjuvant against breast
cancer with the inclusion of larger cohorts
2. Determine the toxicity of multiple injections of Ad-CCL21 after a
single dose of Ad-Flt3L
Experimental Plan
 Orthotopic tumors were established by the injection of 1X105 Cl-66 cells
into the fourth inguinal mammary fatpad of female BALB/c mice
 Once tumors reach 60 mm3 (5 mm in diameter), the described treatments
(see table below) were initiated
Treatment
# of mice
1. No Rx (tumor w/out treatment)
10
2. Surgery Only
10
3. Ad-Control (Neoadjuvant, 1x1011 vp)
10
4. Ad-SLC (Neoadjuvant, 1x1011 vp)
10
5. Ad-Flt3L (2x1010 vp) i.t. + Ad-CCL21 i.t. (2x1010 vp) (1X)
5
6. Ad-Flt3L (2x1010 vp) i.t. + Ad-CCL21 i.t. (3.4x1010 vp) (3X)
10
8. Ad-Flt3L (1x1011 vp) i.v. + Ad-CCL21 i.t. (2x1010 vp) (1X)
5
9. Ad-Flt3L (1x1011 vp) i.v. + Ad-CCL21 i.t. (3.4x1010 vp) (3X)
10
Surgery Enhanced the survival of Mice with
Mammary tumors
Survival Data: Ad-CCL21 + Surgical tumor removal
Percentage Survival
100
No Rx
Surgery Only
Ad-control neoadj.
Ad-CCL21 neoadj
50
0
0
10
20
30
40
50
Days Post Start of Treatment
Ad-CCL21????
60
Results of the Study, contd.
 Multiple injections of Ad-CCL21 after a single dose of Ad-Flt3L are
toxic (lethal) to the mice. However, the tumor growth in AdFlt3L/Ad-CCL21-treated mice was inhibited compared to without
treatment.
Follow up
 Multiple adenoviral injections!!! ----- either cytokine in Adeno and the other in
another formulation that may provide long duration of activity
 Hydron delivery of CCL21..
Ad-Flt3L/Hy-CCL21 against Breast Cancer
 Orthotopic tumors were established by the injection of 1X105 Cl-66 cells into the fourth
inguinal mammary fatpad of female BALB/c mice.
 Once tumors reached 60 mm3 (5 mm in diameter), a group of 10 mice received 1x1011
Ad-Flt3L vp intravenously/mouse. Five days after, mice received Hy-CCL21 implants
containing 6 mg CCL21/mouse. A second group of 10 mice was left untreated.
Percentage Survival
100
No Rx
Ad-Flt3L/Hy-CCL21
80
60
P = 0.0025
40
20
0
0
10
20
30
Days Post Start of Treatment
I.v. Ad-Flt3L/i.t. Hy-CCL21 significantly enhanced survival of mice
with breast cancer
Publications; Project III
• Reber, A. J., A. E. Ashour, S. N. Robinson, J. E. Talmadge, and J. C.
Solheim. Flt3 ligand bioactivity and pharmacology in neoplasia.
Current Drug Target-Immune, Endocrine and Metabolic Disorders.
2004 Jun; 4(2):149-56.
• A. E. Ashour, Turnquist, H. R., M. A. Hollingsworth, R. K. Singh, J.
E. Talmadge, and J. C. Solheim. CCL21 induces specific cellular
immunity against a pancreatic tumor. Clin. Immunol. Immunother,
accepted contingent on revision
• A. E. Ashour, H. R. Turnquist, A. J. Reber, N. Burns, X. Wang, X.
Lin, A. Tuli, J. Kampf, S. Kurz, D. LaFace, M. A. Hollingsworth, R.
K. Singh, J. E. Talmadge, and J. C. Solheim. CCL21 and FLT3 ligand
as promising immunotherapeutics for pancreatic adenocarcinoma.
Manuscript in preparation to be submitted to Cancer Research.
Publications; Project III, contd.
 Solheim, J. C., A. J. Reber, A. E. Ashour, S. Robinson, M. Futakuchi, S.
G. Kurz, K. Hood, R. R. Fields, L. R. Shafer, D. Cornell, S. Sutjipto, S.
Zurawski, D. M. LaFace, R. K. Singh, and J. E. Talmadge. Spleen, but
not tumor, infiltration by dendritic and T cells is increased by adenovirusFlt3 ligand injection. Submitted to Cancer Gene Therapy
 Turnquist, H. R., A. E. Ashour, X. Lin, M. A. Hollingsworth, R. K. Singh, J.
E. Talmadge, and J. C. Solheim. Intratumoral CCL21 administration:
similarities and disparities in effects on mammary and pancreatic tumors.
Manuscript in preparation
 Ashour, A. E., H. R. Turnquist, X. Lin, D. LaFace, R. K. Singh, J. E.
Talmadge, and J. C. Solheim. Kinetics of immune cell infiltration induced
by CCL21. Manuscript in preparation
 In addition to oral and poster presentations at regional and international
conferences
Ongoing Experiments
 HY-CCL21 in neo-adjuvant setting
 Kinetics of CCL21 activity in Panc-02 tumor-bearing C57Bl/6 mice
 Nigella sativa and thymoquinone as promising immunotherapeutics
Efficacy of Hy-CCL21 Accompanied with Surgery
(Neoadjuvant) against Mammary Carcinoma III
 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 reach 60 mm3 (Day 0), Hy-PBS or Hy-CCL21 (6ug of
CCL21/mouse) was implanted i.t. Four days following initial
treatment, surgical resection of the tumors was carried out. Tumor
growth, as well as survival, was monitored
 Tumor regressors (if any), plus same number of naïve Balb/c mice,
will be challenged again by the injection of 1 X 105 Cl-66 cells in
order to examine whether these mice developed long lasting immune
response against the tumor. Time to tumor development and tumor
growth rate will be recorded
Efficacy of Hy-CCL21 Accompanied with Surgery
(Neoadjuvant) against Mammary Carcinoma, contd.
Treatment
Notes
# of Mice
1. No Rx (tumor w/out treatment)
20
2. Surgery Only
25
Surgical tumor removal 4 days after initiation of therapy in gp 4& 6
3. Hy-Control ( Single Agent), when tumor is 5 mm
20
No surgical tumor removal
4. Hy-Control (Neoadjuvant), when tumor is 5 mm
25
Surgical tumor removal 4 days after initiation of therapy in gp 4& 6
5. Hy-CCL21 ( Single Agent), when tumor is 5 mm
20
No surgical tumor removal
6. Hy-CCL21 (Neoadjuvant), when tumor is 5 mm
25
Surgical tumor removal 4 days after initiation of therapy in gp 4& 6
Total Number of Mice
135
Kinetics of CCL21 activity in Panc-02-bearing
C57Bl/6 Mice
 80 C57BL/6 mice were first given s.c. injections of 1x106 Panc02
cells dorsally between the scapulae.
 Once tumors become palpable (2-3 mm diameter), the mice were
randomized into treatment cohorts.
 Treatments of either 1 mg CCL21 or PBS were delivered i.t. on days 1,
2, and 3, followed by 4 days without treatment, and again for 3
consecutive days (on days 8, 9, and 10)
 On day 11, 13, 15, 17 and 19, the tumors, spleen and regional LNs
(from subsets of 5 mice/treatment) will be resected and minced
 Following treatment with collagenase and deoxyribonuclease I, the
mononuclear cells will be isolated with Lympholyte-M, and stained
for flow cytometric analysis
 The following antibodies will be utilized in the flow cytometric
analysis: CD3 (for T cells), CD11c (for DCs), NK1.1 (for NK cells)
Dissertation
 Face Page and the Abstract, and part of the Acknowledgements
 List of Tables and List of Figures ---not yet
 Most Abbreviations and part of the Acknowledgements
 Less than half of the Introduction (Chapter 1)
 Half of the Materials and Methods (Chapter 2)
 Development of Idiotype DNA Vaccines against Lymphoma (Chapter
3)
 Transduction Domain Sequence Linked to a Lymphoma Idiotype DNA Vaccine
Prolongs Survival After Vaccination and Tumor Challenge
 Summary and Future Directions--- done
Dissertation, contd.
 Development of DNA vaccines against mutant K-ras (Chapter 4)
 Immunization with DNA Encoding Mutant K-ras Peptides Modified with a
Leader Sequence, Transduction Domain Sequence or Ubiqitin Evokes a
Superior T Cell Response
 Summary and Future Directions--- done
 In vivo manipulation of immune cells to increase immune reponses
against pancreatic cancer cells (Chapter 5)
 Flt3L and CCL21 as Promising Immunotherapeutics against Pancreatic
Adenocarcinoma --- is being done
 Summary and Future Directions--- are being done
 In vivo manipulation of immune cells to increase immune reponses
against breast cancer cells (Chapter 6)
 CCL21 accompanied with Surgery as Promising Treatment for Breast Cancer --is being done
 Summary and Future Directions--- are being done
Dissertation, contd.
 Kinetics of immune cell infiltration induced by CCL21 (Chapter 7)--not yet
 Summary and Future Directions--- not yet
 Kinetics of Flt3L activity (Chapter 8)--- not yet
 Summary and Future Directions--- not yet
 References ----Cell
 Duesbery, N.S., Webb, C.P., Leppla, S.H., Gordon, V.M., Klimpel, K.R.,
Copeland, T.D., Ahn, N.G., Oskarsson, M.K., Fukasawa, K., Paull, K.D. et al.
(1998). Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.
Science 280, 734-737.
Awards Since September 2005
 Certificate of Achievement for outstanding performance (in both the
written and oral components of the Comprehensive Examination)
from the Dept. of Biochemistry and Mol. Biology at UNMC
 Harris Award for excellence in cancer research
Survival Data: Flt3L in an orthotopic pancreatic model
Percentage Survival
100
No Rx
PG-PBS
PG-Flt3L
80
60
40
20
0
0
10
20
30
40
50
Days Post Start of Treatment
60
Ad-Flt3L/Hy-CCL21 against Breast Cancer
 Orthotopic tumors were established by the injection of 1X105 Cl-66 cells into the fourth
inguinal mammary fatpad of female BALB/c mice.
 Once tumors reach 60 mm3 (5 mm in diameter), a group of 10 mice received 1x1011 Ad-Flt3L
vp intravenously/mouse. Five days after, mice received Hy-CCL21 implants containing 6 mg
CCL21/mouse. A second group of 10 mice was left untreated.
Percentage Survival
100
No Rx
Ad-Flt3L/Hy-CCL21
80
P = 0.0025
60
P = 0.0025
40
20
0
0
10
20
Days Post Start of Treatment
30