1. SEREX Screening for Colorectal Cancer Antigens Jennifer Newton, MD April 5, 2011 Research Presentation

2. Colorectal Cancer (CRC) Jemal, A. et al. CA Cancer J Clin 2010;60:277-300.

3. Objectives for the Research Block Collaborate with TVG to create a Multi- Antigen Vaccine to prevent CRC –Identify potential immunogenic CRC antigens –Verify cancer-specificity –Purify and Isolate the antigens –Sequence the antigens –Literature review

4. 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

5. Rationale Methodology validated in breast cancer Disis ML, et al. J Clin Oncol. 2002;20:2624-32.

6. Disis ML, et al. J Clin Oncol. 2009;27:4685-92. XX

7. SEREX Serological Analysis of Recombinant cDNA Expression Libraries Testis/TumormRNAcDNAλ-phage cDNA library + XL Blue E.coli 30 min at 41 C + top agar NZY Agar 5 hours at 41 C + IPTG-soaked Nitrocellulose membrane

8. SEREX Expression of antigen protein and transfer to membrane overnight at 37 C Wash & Block Membrane Immunoscreening with cancer mice sera Incubation with secondary antibody Color Development + AP-Goat- Anti-Mouse IgG + NBT- BCIP Primary Screening

9. SEREX Immunoscreening with CANCER mice sera Immunoscreening with CONTROL mice sera Secondary Screening

10. 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 λ-phage cDNA libraries –Mouse-testis –MC38 tumor cell line –APC tumor –AOM tumor

11. 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

12. Results APC Sera & Mouse Testis cDNA

13. SEREX Data Secondary Screening –48 clones –15 positive results Single-clone Excision, PCR & Sequencing –No positive results

14. Results TF3-32 #2 1:100 #4 #3 #2 #1 Clones #1-4  SCE  PCR

15. 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

16. Acknowledgements Special Thanks –TVG –Elizabeth Broussard, MD –Mei Wu, PhD –Erik Espling

17. References 1. 1. Jemal A, et al. Cancer Statistics, 2010. CA Cancer J Clin. 2010 Sep- Oct;60(5):277-300. 2. 2. Galon J, et al. Type, density and location of immune cells within human colorectal tumors predict clinical outcome. Science 313, (2006);1960-64. 3. 3. Pages F, et al. Effector Memory T cells, Early Metastasis, and Survival in Colorectal Cancer. N Engl J Med 2005;353:2654-66. 4. 4. Pages F, et al. Immune infiltration in human tumors: a prognostic factor that should not be ignored. Oncogene 2010;29:1093-1102. 5. 5. 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. 6. 6. 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;107:477- 84. 7. 7. 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. 8. 8. Disis ML, et al. Concurrent trastuzumab and HER2/neu-specific vaccination in patients with metastatic breast cancer. J Clin Oncol. 2009;27:4685-92.

18. Modifications AOM: no positive results –Varying dilutions of sera –0, 1, and 2 –times Pseudoscreening Secondary Screening –1:100 dilutions Stopped Pseudoscreening Sera

19. Results AOM Sera & Mouse Testis Library –Few positive results on Primary Screen –No positive results on Secondary Screen

20. Results – PCR (Courtesy of Erik) 1.0 1.5 2.0 3.0 TF3-322 #1 TF3-322 #2TF3-322 #3TF3-322 #4TF3-323 #1TF3-324 #1TF3-321 #1