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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 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. 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 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. Ubiquitin-K ras TAT-PTD-K ras mTAT-PTD-K ras Leader-K ras Leader Sequence: Ubiquitin: Modified HIV TAT-PTD (mTAT-PTD): HIV TAT protein transduction domain (TAT-PTD): An endoplasmic reticulum insertion/signal Targets cellular toamino the proteasome for TAT-PTD with a proteins modified acid sequence mediates the delivery of peptides and sequence derived from the adenovirus E3/19K to enhance degradation. proteins intopeptide targetedtransduction. cells (Ho et al., 2001). glycoprotein. 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 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 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. Vaccination with K ras-D DNA Test K ras-D peptide against K ras-V peptide Proliferation to Ras D 16000 cpm 12000 8000 4000 0 pcDNA Ras D Ras DUbiquitin Ras D-Tat Ras D-PTD Ras D-Leader Vaccine Ras D (ug/ml) 1 Ras D (ug/ml) 0.1 Ras D (ug/ml) 0.01 Proliferation to Ras V 16000 cpm 12000 8000 4000 0 pcDNA Ras D Ras D-Ubiquitin Ras D-Tat Ras D-PTD Ras D-Leader Vaccine Ras V 1 Ras V 0.1 Ras V 0.01 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 Vaccination with K ras-V DNA Test K ras-V peptide against K ras-D peptide Proliferation to Ras V 6000 cpm 4000 2000 0 pcDNA Ras V Ras V-Ubiquitin Ras V-Tat Ras V-PTD Ras V-Leader Vaccine Ras V 1 Ras V 0.1 Ras V 0.01 Proliferation to Ras D 6000 cpm 4000 2000 0 pcDNA Ras V Ras VUbiquitin Ras V-Tat Ras V-PTD Ras V-Leader Vaccine Ras D (ug/ml) 1 Ras D (ug/ml) 0.1 Ras D (ug/ml) 0.01 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. Acknowledgments Solheim Lab Members Dr. Adrian Reber Dr. Jason Petersen Dr. Chantey Morris Committee Members Heth Turnquist Dr. Surinder Batra Rachael Turnquist Dr. Stanley Cox Kris Seipel Dr. Tony Hollingsworth Mary McIlhaney Dr. Myron Toews Advisor Dr. Joyce Solheim