Transcript SEREX screening for colorectal cancer antigens

SEREX Screening for
Colorectal Cancer Antigens
Jennifer Newton, MD
April 5, 2011
Research Presentation
Colorectal Cancer (CRC)
Jemal, A. et al. CA Cancer J Clin 2010;60:277-300.
Objectives for the Research Block
Collaborate with TVG to create a MultiAntigen Vaccine to prevent CRC
– Identify potential immunogenic CRC antigens
– Verify cancer-specificity
– Purify and Isolate the antigens
– Sequence the antigens
– Literature review
Rationale
CRC is immunogenic
Th1-type immune response is associated
with improved prognosis
Suggests CRC-specific Th1 immunity may
prevent disease or disease recurrence
HYPOTHESIS
Certain CRC antigens may trigger a Th1
immune response that could be used to develop
a multi-antigen vaccine
Rationale
Methodology validated in breast cancer
Disis ML, et al. J Clin Oncol. 2002;20:2624-32.
X
X
Disis ML, et al. J Clin Oncol. 2009;27:4685-92.
SEREX
Serological Analysis of Recombinant
cDNA Expression Libraries
Testis/Tumor
+
XL Blue
E.coli
mRNA
30 min
at 41 C
cDNA
5 hours
at 41 C
+ top agar
NZY Agar
λ-phage cDNA library
+ IPTG-soaked
Nitrocellulose
membrane
SEREX
overnight
at 37 C
Wash & Block
Membrane
Immunoscreening with
cancer mice sera
Expression of antigen protein
and transfer to membrane
+ AP-GoatAnti-Mouse
IgG
+ NBTBCIP
Incubation with
secondary antibody
Color Development
Primary
Screening
SEREX
Immunoscreening with
CANCER mice sera
Secondary
Screening
Immunoscreening with
CONTROL mice sera
Mouse Models
Serum
– AOM-induced tumor in FVB mice
– APC/Min in Black6 mice
– Rejection Sera: MC38 injected in FVB mice
λ-phage cDNA libraries
– Mouse-testis
– MC38 tumor cell line
– APC tumor
– AOM tumor
SEREX Data…aka “what did I do
for 8 weeks???”
Primary Screening: 215 membranes and
955,000 plaques
– cDNA libraries
Mouse Testis: 515,000 plaques
MC38: 300,000 plaques
APC-F12B: 140,000 plaques
– Sera
AOM: 470,000 plaques
APC: 295,000 plaques
FVB-MC38 Rejection: 190,000 plaques
Results
APC Sera & Mouse Testis cDNA
SEREX Data
Secondary Screening
– 48 clones
– 15 positive results
Single-clone Excision, PCR & Sequencing
– No positive results
Results
Clones #1-4
#1
#3
#2
#4
TF3-32 #2 1:100
 SCE
 PCR
Future Projects
Screen AOM sera against AOM tumor cDNA
library
Screen additional APC/APC & MC38/Rejection
Trial of varying dilutions for APC & Rejection
Sera
Using anti-IgA secondary Antibody
Create mouse-vaccine model using peptides
from literature review
Human CRC SEREX
Acknowledgements
Special Thanks
– TVG
– Elizabeth Broussard, MD
– Mei Wu, PhD
– Erik Espling
References
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Jemal A, et al. Cancer Statistics, 2010. CA Cancer J Clin. 2010 SepOct;60(5):277-300.
Galon J, et al. Type, density and location of immune cells within human colorectal
tumors predict clinical outcome. Science 313, (2006);1960-64.
Pages F, et al. Effector Memory T cells, Early Metastasis, and Survival in
Colorectal Cancer. N Engl J Med 2005;353:2654-66.
Pages F, et al. Immune infiltration in human tumors: a prognostic factor that
should not be ignored. Oncogene 2010;29:1093-1102.
Disis ML, et al. Generation of T-cell immunity to the HER-2/neu protein after active
immunization with HER-2/neu peptide-based vaccines. J Clin Oncol.
2002;20:2624-32.
Knutson KL, et al. Immunization with a HER-2/neu peptide vaccine generates
HER-2/neu CD8 T-cell immunity in cancer patients. J Clin Invest. 2001;107:47784.
Goodell V, et al. Level of HER-2/neu protein expression in breast cancer may
affect the development of endogenous HER-2/neu-specific immunity. Mol Cancer
Ther. 2008;7:449-54.
Disis ML, et al. Concurrent trastuzumab and HER2/neu-specific vaccination in
patients with metastatic breast cancer. J Clin Oncol. 2009;27:4685-92.
Modifications
AOM: no positive results
– Varying dilutions of sera
– 0, 1, and 2 –times Pseudoscreening
Secondary Screening
– 1:100 dilutions
Stopped Pseudoscreening Sera
Results
AOM Sera & Mouse Testis Library
– Few positive results on Primary Screen
– No positive results on Secondary Screen
Results – PCR
(Courtesy of Erik)
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2.0
1.5
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