Transcript MSBRF Omaha 2003 - Home - KSU Faculty Member websites

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