the somascan assay and somamer reagents - agilent · gold l. et al (2010) plos one, kraemer s. et...
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© 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
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Measuring Genes and Proteins
Genomics Proteomics
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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
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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
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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
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Modifications to nucleic acids
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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
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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
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• 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
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Benzyl Zipper Motif (NGF SOMAmer)
Jarvis et al. (2015) Structure 23 ePub May 13
PDGF SOMAmer Reagent Mimics Receptor Binding
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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
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Gelinas et al. (2014) J. Biol. Chem. 289, 8720
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)
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Agilent microarray
SOMAscan Multiplex Proteomic AssayConverting a protein measurement problem into a DNA measurement solution
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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
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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
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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
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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
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Protein Changes in Human Lung Tumor Tissue
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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
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Discovery
(8 samples, Seattle)
Verification
(63 samples, Denver)
RF
U
log
10R
FU
Control Adeno Squamous
Tumor Profiling
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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
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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
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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
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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
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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
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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
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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 [email protected]
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