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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 1. 2. 3. 4. 5. 6. 7. 8. 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) 3.0 2.0 1.5 1.0