1. IMMUNIZATION WITH DNA ENCODING A MUTANT K-RAS PEPTIDE PLUS A LEADER SEQUENCE EVOKES A SUPERIOR T CELL RESPONSE Abdel-kader Ashour, Adrian J. Reber, Jason L. Petersen, Mary M. McIlhaney, and Joyce C. Solheim; University of Nebraska Medical Center, Omaha, NE

2. Introduction
The ras p21 (K-ras) proto-oncogenes code for proteins that are involved in cellular proliferation and differentiation.
Point mutations that occur at codons 12, 13, or 61 cause the ras proteins to become oncogenic.
These mutated proteins are targets for immunotherapy.

3. Introduction, cont.d
About 90% of pancreatic adenocarcinomas have a K-ras gene mutated at codon 12 (usually Gly12Asp or Val).
Peptides derived from mutated K-ras can be presented at the cell surface by major histocompatibility complex class I (MHC-I) for recognition by cytotoxic T lymphocytes

4. Objective
Improve the delivery/immune stimulation of K-ras vaccines containing either the Asp or Val point mutations.
Hypothesis
Fusion of the K-ras mutant peptides to leader sequence, transduction domain sequence, or ubiquitin would facilitate the generation of a T cell response against the mutant peptides.

5. Targets cellular proteins to the proteasome for degradation.
Ubiquitin-K ras
TAT-PTD-K ras
mTAT-PTD-K ras
Leader-K ras
Leader Sequence:
An endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein.
Ubiquitin:
Targets cellular proteins to the proteasome for degradation.
Modified HIV TAT-PTD (mTAT-PTD):
TAT-PTD with a modified amino acid sequence to enhance peptide transduction.
HIV TAT protein transduction domain (TAT-PTD):
TAT-PTD mediates the delivery of peptides and proteins into targeted cells (Ho et al., 2001).

6. Vaccination Protocol
BALB/c mice (5/group) were vaccinated i.m. with 100 mg of plasmid DNA encoding:
- pcDNA (control)
- K-ras alone
- ubiquitin + K-ras
- TAT-PTD + K-ras
- mTAT-PTD + K-ras
- Leader sequence + K-ras
Mice were boosted 3X, 2 weeks apart

7. Measuring Vaccine Efficacy
Mice were sacrificed and spleen cells were harvested 2 weeks after last boost
Proliferation of spleen cells in response to K-ras mutant peptides (Ras D and Ras V) was tested in vitro by the lymphocyte proliferation assay

8. Lymphocyte Proliferation Assay
Proliferation assays were performed by incubating harvested spleen cells with purified mutant K-ras peptides.
Lymphocytes recognizing the mutant K-ras peptides become activated and proliferate.
Proliferation is assessed by monitoring the uptake of 3H-thymidine into cellular DNA.
The general ability of the T lymphocytes to proliferate was confirmed by the use of the mitogen Concanavalin A.

9. Vaccination with K ras-D DNA K ras-D peptide
K ras-V peptide
Test
against

12. Conclusion
Modification of K-ras D by the leader sequence, TAT-PTD, or mTAT-PTD increased the proliferative response of mouse lymphocytes to K-ras D peptide.
Leader sequence conjugated to K-ras D produced the highest proliferation, followed by mTAT-PTD, then TAT-PTD .
This response was cross reactive with K-ras V

13. Vaccination with K ras-V DNA K ras-V peptide
K ras-D peptide
Test
against

16. Conclusion
Modification of K-ras V by the ubiquitin, TAT-PTD, mTAT-PTD, or leader sequence increased the proliferative response of mouse lymphocytes to K-ras V peptide.
Ubiquitin and TAT-PTD conjugated to K-ras V produced the highest proliferation, followed by leader sequence and TAT-PTD .
This response was cross reactive with K-ras D.

17. Acknowledgments Advisor Dr. Joyce Solheim Committee Members
Dr. Surinder Batra
Dr. Stanley Cox
Dr. Tony Hollingsworth Dr. Myron Toews
Solheim Lab Members
Dr. Adrian Reber
Dr. Jason Petersen
Dr. Chantey Morris
Heth Turnquist
Rachael Turnquist
Kris Seipel
Mary McIlhaney