© 2016 SomaLogic, Inc.

The SOMAscan® assay and SOMAmer® reagents:

Translatable tools from high-throughput biomarker discovery to targeted assays

Sheri K Wilcox, PhDAugust 18, 2016

1

Measuring Genes and Proteins

Genomics Proteomics

2

Measuring proteins provides the knowledge needed for real-time

health management

RISK STATUS

Historical Challenges Of Proteomics Technology

• To characterize biology, many proteins must be measured over a 1 billion-fold range of concentrations

• Existing technologies will measure:

• EITHER: a large number of proteins on a small number of samples

• Typically higher abundance

• OR: a small number of proteins on a large number of samples

• Typically lower abundance

• But NOT both

3

The Solution• SomaLogic has developed a protein measurement solution

that addresses both breadth and depth:

– SOMAmer® reagents, proprietary DNA-based molecules that bind to proteins with high affinity and specificity

– SOMAscan® assay, a highly sensitive platform that simultaneously measures >1000 analytes in biological samples

4

SOMAmer reagents: Superior protein-binding reagents

• Chemically synthesized modified DNA

• DNA modifications result in:– Increased chemical diversity– High affinity (sub-nM)– Unique binding profiles enabled

by hydrophobic interactions

• A variety of 5’ functional group additions possible (e.g., fluorophores, biotin, amine)

• Current public menu of over 1300 SOMAmer binding reagents to a wide variety of proteins

5

Modifications to nucleic acids

6

Bn-dU Nap-dU Trp-dU iBu-dU

Incorporation of amino acid-like side-chains, examples above, increases physico-chemical diversity

Modifications are compatible with standard methods, including PCR and hybridization

The use of modified nucleotides in the SELEX (Systematic Evolution of Ligands by EXponentialenrichment) process significantly increases the success rate of new SOMAmer discovery compared to unmodified aptamers

Ligands: Protein vs. Nucleic Acids

Favors proteinsFour bases vs. 20 amino acidsDiversity of amino acid side chains is greater

7

Favors nucleic acidsHigher conformational flexibility per monomer (7 vs. 2 rotatable bonds)Huge starting libraries

Modified nucleotides reduce the diversity gap

Enhanced Diversity Improves SELEX

8

• Focus on side chains that resemble amino acids

overrepresented in CDRs and privileged fragments of small

molecule drugs

• SOMAmer (Slow Off-rate Modified Aptamer) reagents

Unmodified DNA Library

(success rate ~30%)

One base uniformly modified

(success rate >80%; Kd<10 nM)

AA CC GG TT

Mod1

AA CC GG UU

Gold et al. (2010) PloS ONE 5, e15004

Modified Nucleotides Create Novel Intramolecular Motifs

9

Benzyl Zipper Motif (NGF SOMAmer)

Jarvis et al. (2015) Structure 23 ePub May 13

PDGF SOMAmer Reagent Mimics Receptor Binding

10

PDGF-BB:PDGFRβ

co-crystal structure

Kd = 200 pM

PDGF-BB:SOMAmer reagent

co-crystal structure

Kd = 20 pM

Davies et al. (2012) PNAS 109, 19971

IL-6 SOMAmer Reagent Occupies Binding Sites of Both Receptors: IL-6Rα and gp130

11

Gelinas et al. (2014) J. Biol. Chem. 289, 8720

THE SOMASCAN® ASSAY

12

The Highly Multiplexed SOMAscan Assay: Unbiased Interrogation of the Proteome

• Uses unique SOMAmers to simultaneously measure 1,310 proteins (current version)

• High dynamic range (>8 logs), sensitivity (40 fM median LLOD in buffer) and precision (~5% median CV)

• Low sample requirement (60 µL plasma or serum)

13

Agilent microarray

SOMAscan Multiplex Proteomic AssayConverting a protein measurement problem into a DNA measurement solution

14

Gold L. et al (2010) PLoS One, Kraemer S. et al (2011) PLoS One, Rohloff J. et al (2014) Molecular Therapy

SOMAscan Assay Experience To Date

• >65,000 samples run (since 2012)

• >700 studies (since 2012)

• 39 different matrices– 6 different species

– many tissue types • blood

• tissue homogenates

• cell lysates and media

• other bodily fluids

15

Metabolic Disease

Cardiology/Vascular Disease

Gastroenterology

Obstetrics/Gynecology

Musculoskeletal/ Rheumatology

Immunology/ Infectious Disease

NeurologyPharmacology/ Toxicology/

Nutrition

Oncology

Pulmonary/Respiratory

NUMBER OF SAMPLES PER THERAPEUTIC AREA

SNP Detection at the Protein Level

16

FCGR2A contains a frequent SNP that changes His at position 167 in the protein sequence to Arg.

The SOMAmer reagent was selected against H167R FCGR2A.

A bimodal distribution was observed in healthy individuals.

The SOMAmer reagent selected to bind FCGR2B, which is 93% identical to FCGR2A, including Arg

at the equivalent position, does not display a bimodal distribution of RFU values in the same

population.

Specificity Confirmed for Recombinant Targets

17

The affinity for the H167R mutant is two orders of magnitude higher than that for the wild-type

protein, illustrating a striking specificity among two proteins with a single amino acid substitution.

The reagent selected against FCGR2B is able to bind both proteins (93% identity), but the reagent

selected against FCGR2A only binds the FCGR2A with high affinity.

The H167 form of FCGR2A does not bind well to either SOMAmer reagent, suggesting that this

mutation results in a significant structural change in the protein.

Duchenne’s Muscular Dystrophy

• Cases were males with DMD and no other diagnosis• Ages ranged from 2 to 27 years

– 50% between 9 and 11

• Controls were siblings or healthy children from DMD community

18

126 Proteins are significantly increased

19

101 Proteins are significantly decreased

20

Protein Changes in Human Lung Tumor Tissue

21

Very little difference

between distant and

adjacent non-tumor

tissue

Tumor

Adjacent

Normal

Distant

Normal

Mehan et al. (2012) PLoS ONE 7, e35157

Consistent differences

between tumor normal

tissues

36 proteins with largest difference:• 20 higher in tumor• 16 lower in tumor• 13 previously

unreported in the literature

• 12 in common with serum biomarkers

Non-small Cell Lung Cancer Tissue

• Thrombospondin 2 levels (among other proteins) are elevated in tumor tissue compared to adjacent normal tissue

22

Discovery

(8 samples, Seattle)

Verification

(63 samples, Denver)

RF

U

log

10R

FU

Control Adeno Squamous

Tumor Profiling

23

Individual phenotypes stratify

populations for targeted therapy

Common phenotypes are observed across

standard sub-classifications

Meehan et al., 2012

PLOS ONE 7:4 (e35157)

Biomarkers lead to histochemical probes:

Stain with fluorophore-labeled thrombospondin-2 SOMAmer reagent

Metabolism “Enhancement”: A Project For The World Anti- Doping Agency

(WADA)

• 10 subjects were treated with human growth hormone (hGH) for 14 days and 10 subjects received placebo

• Many proteins responded to hGH, some persisted or even continued changing beyond the treatment period

24

Example protein from left to right placebo, day 0 treatment, day 7, day

14 and 7 days post-treatment (day 21)

Pharmacodynamic Patterns Of Human Growth Hormone

25

Proteins that peak early: n=18 proteins

Proteins that peakat end of hGH: n=12 proteins

Proteins that peak later: n=51 proteins

Growth hormone was injected in 10 human

volunteers daily from day 1 to day 14 (box);

SOMAscan assay was applied to serum

samples.

A collaboration with World Anti-Doping Association

SOMAmer Reagents as Enrichment Tools

• Enrichment using SOMAmer reagents upfront of MS has been demonstrated by several other groups – The Broad Institute, Novartis, Merck

• Novartis demonstrated that SOMAscan measurements across 8 cell lines correlate well with SOMAmer-enriched MRM results

• Correlations with RNAseq data were also observed, albeit not as strong as protein-protein correlations, as expected

• Translatability from discovery on the SOMAscan platform to targeted-MS with SOMAmer-enrichment is expected to be high

26

Figure adapted from Finkel, et al 2016 poster | US HUPO 2016Figure adapted from Finkel, et al 2016 poster | US HUPO 2016

SOMAmer Reagents as Enrichment Tools

• Merck recently published data demonstrating utility of the anti-PCSK9 SOMAmer reagent for enrichment

• Shown are chromatograms of eluent digestions of normal human plasma after mAb-enrichment (A) or SOMAmer-enrichment (B)

• The authors commented on the “unanticipated benefit” of significantly less background using the SOMAmer reagent

27

10.4155/bio-2016-0046

Gupta et al., 2016, Bioanalysis (Epub ahead of print)

10.4155/bio-2016-0046

Antibody-enrichment

SOMAmer-enrichment

SOMAmer Reagents as Enrichment Tools

• The Broad Institute recently published data demonstrating utility of the anti-ERBB1 SOMAmer reagent for enrichment in a paper accompanying SOMAscan data generated at MGH

• ERBB1 was detected with and without spiked protein in plasma, using 3 peptides

28

DOI: 10.1161/CIRCULATIONAHA.116.021803

Ngo et al., 2016, Circulation. 2016;134:270–285.

DOI: 10.1161/CIRCULATIONAHA.116.021803

No spike Spike

Discovery cell SELEX can generate reagents to novel targets

• Discovery cell SELEX experiment was performed on different cell lines with and without a drug treatment

• SOMAmer reagents were discovered and found to be specific to treated cells• SOMAmer-enrichment followed by mass spectrometry can be used to identify

the differential proteins

29

SELEX

Cell Only SOMAmers

Bind,

Amplify

Naive SOMAmer Library (1015)

Don’t bind

Amplify

NIBR | Whitehead Forum 2014

Slide Adapted from Vic Myer

NIBR | Whitehead Forum 2014

MF

I o

n t

rea

ted

ce

lls

MFI on cells with vehicle

These SOMAmer reagents are

binding to epitopes more dominant

after treatment

Ways to Access our Technology

• Profile samples at SomaLogic in our Assay Services department

• Become a SOMAscan Service Provider to run the assay for your own lab or as a service at your institution

• Obtain individual reagents for use in targeted enrichment assays and other applications (http://estore.somalogic.com/)

• Obtain novel reagents for targets of interest through our SOMAmer Discovery Service

• For more details, contact Andy Keys at akeys@somalogic.com

30

Agilent products described are For Research Use Only. Not for use in diagnostic

procedures.

31

© 2016 SomaLogic, Inc.

Thank you!

Questions?

32

© 2016 SomaLogic, Inc. 33