Multiplex autoantibody profiling for autoimmune diseases and cancer

1. Multiplex autoantibody profiling for autoimmune diseases and cancer April, 2, 2014 Profile antibodies to over 10,000 human proteins using Luminex xMAP™ technology Jim Lazar, Ph.D. VP, Assay Development

2. OriGene Introduction • OTI Locations • Rockville (HQTs/R&D) • Seattle (Blue Heron) • Delaware (SDIX) • Maine (SDIX) • Foster City (BioCheck) • Beijing • Wuxi

4. Antibodies as Biomarkers • The immune system functions as a an early warning system. • Malignant transformation, infections, and many diseases can trigger an immune response • Antibodies are abundant, easily detectable and stable in serum.

5. Autoantibodies to Tumor Antigens • Tumors typically over-express multiple normal and mutant antigens • Autoantibodies to tumor antigens can be detected much earlier than other cancer biomarkers • Frequency of autoantibody responses to an individual tumor antigen is typically 15-20%. • Responses to a panel of antigens can be higher than 90% • Multiplexed autoantibody detection to a panel of antigens is necessary. What is EarlyCDT–Lung? EarlyCDT-Lung is a simple, physician-ordered, diagnostic blood test that measures the presence of autoantibodies to a panel of seven lung cancer-associated antigens.

6. Autoimmune Diseases • Autoimmune diseases affect an estimated 3% of the world population. • Over 150 recognized autoimmune diseases. • Each disease is characterized by autoantibodies to multiple antigens. • Profiles may help classify disease variants and indicate organ-specific involvement. • Autoantibody profiles may help guide the development of antigen-specific tolerizing therapy.

7. Biomarker Discovery • Proteomic /2D gel methods • Proteomic Mass Spec methods • High Density Protein Microarrays Identification of many targets from a small number of samples Biomarker Screening & Quantitative Evaluation Large sample #s Refine target list ROC calculations • Luminex Bead Arrays • Mid-Density Multiplexed methods Clinical Assays • Luminex Bead Arrays • Low-Density Multiplexed methods

8. 10,000+ Full Length Human Proteins • Produced from TrueORF Gold™ cDNA clones • Fully sequenced • Expression validated • Expressed in HEK293 cells • Human-expressed • Affinity purified • Native presentation • Optimal preservation of protein structure, • post-translational modifications

9. Available Antigens Any of OriGene’s >10,000 purified proteins can be custom-coupled to Luminex beads

10. Luminex xMAP™ technology • Multiplex Bead Array • Efficient high-throughput microplate format • Up to 100 targets per well • OriGene is a Luminex partner and Certified Assay Developer

11. Assay Principle + Protein-coupled Luminex Beads Sample containing human IgG + Mix with Anti-human PE Conjugate Read Signal in Luminex

12. Product Configuration • TruePlex™ Human Antibody Profiling Kit • Assay Reagents, Control Bead Mix, Positive Control combine with • Custom TruePlex™ Antibody Profiling Array • TruePlex™ Antibody Profiling Array • (pre-defined array) and/or Multiple profiling arrays can be mixed together and analyzed as a larger multiplex

13. Assay Protocol Dilute sample Mix with beads, incubate Total time – about 2.5 hours for 96 samples Up to 100 results per well Quantitative results Wash, add conj. & incubate Wash Read in Luminex Analyze Data

14. Stringency Options • Sample Diluent can be prepared at different stringencies • High, medium or low • High-stringency Sample Diluent will minimize non-specific signals and is recommended for initial sample testing. • The medium- or low-stringency Sample Diluent may give better results for samples • with low autoantibody levels, • in which the antibody affinity may be low • for higher sample dilutions (1:500 or greater),

15. Available TruePlex™ Profiling Arrays

16. Autoimmune profiles in serum

17. Autoimmune sera – data from SSB and TRIM21

18. Normal (non-disease) Sera

19. Dynamic Range

20. Control Results

21. Breast cancer samples tested with 20-plex tumor antigen panel

22. Available cancer tumor antigens known to generate autoantibodies • Breast Cancer • ASB-9, BAT4, BDNF, Survivin, Livin, BMX, BRCA2, P16, CSNK1E, NY-ESO-1/LAGE1, CTBP1, DBT, EIF3E, HER2, Fibulin, FKBP4, GIPC-1, HSP-90, HSPA4, HSP-27, HSP-60, IMP1 , P62, IMP2, MUC1, C-Myb, c-myc, PDCD6IP, PPIA, PRDX2, RAB5A, RAC3, Lipophilin B, SERPINH1, SF3A1, SOX2, p53, TRIM32, UBAP1 • ProstateCancer • Caldesmon 1, Clusterin, c-myc, HER2/neu, HSP70, HSP71, IMP1, MCP1, NY-ESO-1/LAGE1, p53, p62, p90, PARK7, PSA, RACE, SSX2, SSX4, Survivin, TARDBP, TTLL12 • Pancreatic Cancer • CTDSP1, DNAJB1,EIF4A3 (DDX48), ELAC1,GAS2, HCFC1R1,HERPUD1, MAPK9, NR2E3, NR2E3, PDLIM1 (CLP36), PGK1,PPARG, PTPRA, RNF213 (KIAA1618), ROR2, SHOC2, SMOX, TMOD1, TMSB10, ULK4 • Antigens for other cancers and diseases available including • Ovarian cancer, melanoma, hepatocellular carcinoma, lung cancer, diabetes, COPD, multiple sclerosis http://www.origene.com/Luminex/Proteinarray

23. Extensive Bibliography Available Autoantibodies to tailor-made panels of tumor-associated antigens in breast carcinoma.E Piura and B PiuraJ Oncol, Jan 2011; 2011: 982425. Seromic profiling of ovarian and pancreatic cancer. S. Gnjatic S, et al. PNAS U S A. Mar 2010 16;107(11):5088-93. A Distinct Repertoire of Autoantibodies in Hepatocellular Carcinoma Identified by Proteomic Analysis François Le Naour, et al. Mol. Cell. Proteomics, Mar 2002; 1: 197 - 203. Multiple serological biomarkers for colorectal cancer detection. CC Chan, et al. Int J Cancer, Apr 2010; 126(7): 1683-90.

24. Summary • Autoantibodies are useful biomarkers for cancer and autoimmune diseases. • OriGene’s TruePlex profiling kits are optimally suited for biomarker screening and validation. • Choose from over 10,000 proteins expressed in human cells. • The assay procedure is simple and fast. • Make great discoveries • Improve human health

25. Thank you for attending ! Questions ? Contact us: jlazar@origene.com assays@origene.com 240-620-0237 (direct) www.origene.com/assays