Development of Novel Immunotherapeutics Against

Cancer

Abdelkader AshourDepartment 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

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

Project II: Development of DNA Vaccines against Mutant K-ras

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

B.

Flt3L and CCL21 as Immunotherapeutics against Breast Cancer

A.

Flt3L and CCL21 as Immunotherapeutics against Pancreatic Cancer

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

III A. Flt3L and CCL21 as Immunotherapeutics against Subcutaneous Pancreatic tumors

Flt3L treatment resulted in a trend toward longer survival compared to Flt3L/CCL21 treatment or CCL21 treatment

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

Orthotopic pancreatic tumors were established by injecting 104 Panc-02 cells into the pancreas

After 13 days (a period required to get 2.5-3.0 mm diameter tumors), PG-Flt3L (15 g/dose) or Pro-Gelz™ were given i.m. on Day 1 and 6

Flt3L in an Orthotopic Pancreatic Tumor Model

Intramuscular Administration of Flt3L Increases Survival of Mice With Orthotopic Panc02 Tumors

Flt3L in an orthotopic pancreatic tumor model

0 10 20 30 40 50 600

20

40

60

80

100ProGelz-PBSProGelz-Flt3L

Days Post Start of Treatment

Per

cen

tag

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urv

ival

P <0.0001

Efficacy of Ad-CCL21 Accompanied with Surgery (Ad-CCL21/Neoadjuvant) against Mammary Carcinoma

III B. Flt3L and CCL21 as Immunotherapeutics against Breast Cancer

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)

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.

Experimental Plan

Ad-CCL21/Surgical Resection of Mammary Tumors

Survival Data: Ad-CCL21/surgical tumor removal

0 25 50 75 1000

20

40

60

80

100Surgery onlyNo RxAd-z singleAd-z neoadjAd-z adjAd-CCL21 singleAd-CCL21 neoadjAd-CCL21 adj

Days Post Start of Treatment

Per

cen

tag

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Survival of Data: Ad-CCL21 + Surgical tumor removal

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100No RxAd-CCL21 neoadj

Day Post Start of Treatment

Per

cen

tag

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urv

ival

P = 0.0460

CCL21 given before or concurrently with surgical resection elicits an effective immune response against

residual disease and metastases

Survival Data: Ad-CCL21 vs control

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100Ad-z singleAd-CCL21 neoadj

Days Post Start of Treatment

Per

cent

age

Sur

viva

l

P = .0454

Survival Data: Surgery vs no treatment

0 10 20 30 40 50 600

20

40

60

80

100Surgery onlyNo Rx

Days Post Start of Treatment

Per

cen

tag

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urv

ival

P = 0.06

Survival Data: Ad-CCL21 neoadj. vs surgery only

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100Surgery onlyAd-CCL21 neoadj

Days Post Start of Treatment

Per

cen

tag

e S

urv

ival

P = 0.1717

Survival Data: Ad-CCL21 neoadj. vs control

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

100Ad-z neoadjAd-CCL21 neoadj

Days Post Start of Treatment

Per

cen

tag

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urv

ival

P = 0.3142

Results, contd.

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 Ad-CCL21 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

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

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

Experimental Plan

Survival Data: Ad-CCL21 + Surgical tumor removal

0 10 20 30 40 50 600

50

100No RxSurgery OnlyAd-control neoadj.Ad-CCL21 neoadj

Days Post Start of Treatment

Per

cen

tag

e S

urv

ival

Ad-CCL21????

Surgery Enhanced the survival of Mice with Mammary tumors

Results of the Study, contd.

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..

Multiple injections of Ad-CCL21 after a single dose of Ad-Flt3L are toxic (lethal) to the mice. However, the tumor growth in Ad-Flt3L/Ad-CCL21-treated mice was inhibited compared to without treatment.

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 g CCL21/mouse. A second group of 10 mice was left untreated.

Ad-Flt3L/Hy-CCL21 against Breast Cancer

0 10 20 300

20

40

60

80

100No RxAd-Flt3L/Hy-CCL21

Days Post Start of Treatment

Per

cen

tag

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P = 0.0025

I.v. Ad-Flt3L/i.t. Hy-CCL21 significantly enhanced survival of mice with breast cancer

Publications; Project III

• 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

• 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, 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.

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 adenovirus-Flt3 ligand injection. Submitted to Cancer Gene Therapy

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

Publications; Project III, contd.

In addition to oral and poster presentations at regional and international conferences

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

Ongoing Experiments

HY-CCL21 in neo-adjuvant setting

Nigella sativa and thymoquinone as promising immunotherapeutics

Kinetics of CCL21 activity in Panc-02 tumor-bearing C57Bl/6 mice

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 # of Mice Notes

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 g 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

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

List of Tables and List of Figures ---not yet

Most Abbreviations and part of the Acknowledgements

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

Dissertation, contd.

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

0 10 20 30 40 50 600

20

40

60

80

100No RxPG-PBSPG-Flt3L

Days Post Start of Treatment

Per

cen

tag

e S

urv

ival

P = 0.0025

0 10 20 300

20

40

60

80

100No RxAd-Flt3L/Hy-CCL21

Days Post Start of Treatment

Per

cen

tag

e S

urv

ival

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 g CCL21/mouse. A second group of 10 mice was left untreated.

Ad-Flt3L/Hy-CCL21 against Breast Cancer

P = 0.0025