Multiplex autoantibody profiling for autoimmune diseases and cancer

Profile antibodies to over 10,000 human proteins using Luminex xMAP™

technology

April, 2, 2014

Jim Lazar, Ph.D.VP, Assay Development

OriGene Introduction

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

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.

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.

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.

Biomarker Discovery• Proteomic /2D gel methods• Proteomic Mass Spec methods• High Density Protein Microarrays

Biomarker Screening & Quantitative Evaluation• Luminex Bead Arrays• Mid-Density Multiplexed methods

Clinical Assays

Identification of many targets from a small number of samples

• Luminex Bead Arrays• Low-Density Multiplexed

methods

Large sample #sRefine target listROC calculations

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

Available AntigensAny of OriGene’s >10,000 purified proteins can be custom-coupled to Luminex beads

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

Assay Principle

Protein-coupled Luminex Beads

Sample containing human IgG

Mix with Anti-human PE Conjugate

+

+

Read Signal in Luminex

Product Configuration

TruePlex™ Human Antibody Profiling KitAssay Reagents, Control Bead Mix, Positive Control

combine with

TruePlex™ Antibody Profiling Array

(pre-defined array)

Custom TruePlex™ Antibody Profiling

Arrayand/or

Multiple profiling arrays can be mixed together and analyzed as a larger multiplex

Assay ProtocolDilute sample

Mix with beads, incubate

Wash, add conj. & incubate

WashRead in Luminex

Analyze Data

Total time – about 2.5 hours for 96 samples

Up to 100 results per well

Quantitative results

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),

Available TruePlex™ Profiling ArraysPanel Symbol Name

Human Autoimmune I

SNRPA U1-snRNP A Protein [SNRPA 31 kD]SSB La/SS-B (Sjogren syndrome antigen B))

TROVE2 Ro/SS-A (TROVE domain family, member 2)

SNRNP70 U1-snRNP 68 Protein (68 kDa)SNRPC U1-snRNP C Protein [SNRPC 18 kd]

Human Autoimmune II

TRIM21 Ro/SS-A (52 kDa) (Tripartite motif-containing 21)PCNA Proliferating Cell Nuclear Antigen (PCNA)XRCC6 Ku (p70/p80)CENPA Centromere Protein A (CENP-A)AARS Alanyl-tRNA Synthetase (PL-12)

Human Autoimmune III

APOH Apolipoprotein HGIF Intrinsic Factor (Gastric)

PRTN3 Proteinase 3FTCD Formiminotransferase Cyclodeaminase (LC1)

CYP2D6 Cytochrome P450 2D6 (LKM 1)

Human Autoimmune IV

CENPB Centromere Protein B, 80 kD (CENP-B) MPO Myeloperoxidase

RPLP0 Ribosomal Phosphoprotein P0

IFIH1 Melanoma differentiation-associated protein 5 (MDA5)SNRPB U-snRNP B/B'

Autoimmune profiles in serum

TRIM

21SN

RPASS

BAPOH GIF

PCNAPRTN

3RPLP

OFT

CD

CYP2D6

TROVE2

SNRNP70

XRCC6CEN

PASN

RPCMPO

SNRPB

0

5000

10000

15000

20000

25000

30000

35000

Lupus (SLE)

Scleroderma

Sjorgren's Syndrome

Antigen bound to Luminex bead

Med

ian

Fluo

resc

ent I

nten

sity

Autoimmune sera – data from SSB and TRIM21

SLE1

SLE2

SLE3

SLE4

SLE5

SLE6

SLE7

SLE8

SLE9

SLE1

0

SCL-

1

SCL-

2

SCL-

3

SCL-

4

SCL-

5

SJO

-1

SJO

-2

SJO

-3

SJO

-4

SJO

-5

0

5000

10000

15000

20000

25000

30000

35000

SSB (La/SS-B)

TRIM21 (Ro/SS-A)

Med

ian

Fluo

resc

ent I

nten

sity

Normal (non-disease) Sera

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

50100150200250300350400450500 SSB

Sample Number

MFI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

50100150200250300350400450500 SNRPA

Sample Number

MFI

Dynamic Range

Control Results

anti-human Ig

G

Human IgG

Albumin

BSA-Tag

Donkey Ig

G

SNRP70

0

5000

10000

15000

20000

25000

30000

Positive Control

Negative Serum

Lupus (SLE) Serum

Antigen Coupled to Bead

Med

ian

Fluo

resc

ent I

nten

sity

Breast cancer samples tested with 20-plex tumor antigen panel

001_DBT

002_PDCIM

1

004_MYC

006_SF3

A1

009_PDCD6IP

010_PPIA

011_BCCIP

012_EIF4

A3

013_CSNKIE

014_NR2E3

015_ERBB2

017_PRDX2

018_BIRC5

021_IGF2

BP2

022_HSP

D1

024_MUC1

027_CDKN2A

028_HSPA14

029_CTAG1A

044_TP53

0

500

1000

1500

2000

2500

3000

3500

4000

4500

BC-4

BC-5

BC-7

BC-9

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

• Prostate Cancer– 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

Extensive Bibliography AvailableAutoantibodies to tailor-made panels of tumor-associated antigens in breast carcinoma.E Piura and B PiuraJ Oncol, Jan 2011; 2011: 982425.

Multiple serological biomarkers for colorectal cancer detection.CC Chan, et al. Int J Cancer, Apr 2010; 126(7): 1683-90.

A Distinct Repertoire of Autoantibodies in Hepatocellular Carcinoma Identified by Proteomic AnalysisFrançois Le Naour, et al.Mol. Cell. Proteomics, Mar 2002; 1: 197 - 203.

Seromic profiling of ovarian and pancreatic cancer. S. Gnjatic S, et al.PNAS U S A. Mar 2010 16;107(11):5088-93.

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 discoveriesImprove human health

Thank you for attending !

Questions ?

Contact us: jlazar@origene.com

assays@origene.com

240-620-0237 (direct)

www.origene.com/assays