From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR).This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD.This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA.This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing

Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.

<p>Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.</p

EGFR SOMamer assay detects cetuximab and panitumumab-unbound fraction of EGFR.

<p>Normal serum samples were incubated with varying amounts of panitumumab [PANEL A] or cetuximab [PANEL B] for 30 min at room temperature. These samples were then diluted 30 fold, followed by EGFR SOMAmer capture and quantitative PCR (qPCR) (gray bars). EGFR ELISA was also performed on drug-treated samples on the same day (white bars). Triplicate samples were tested at each condition. The vertical lines at each bar represent standard deviations among replicates.</p

Spiked-in purified EGFR to serum is accurately detected by the EGFR SOMAmer assay.

<p>Thirty-fold diluted serum samples were appropriately spiked with three different amounts of purified EGFR ECD (final concentration of 30, 150, and 300 ng/mL plus the endogenous serum EGFR ECD). We then performed the EGFR SOMAmer assay followed by the qPCR. The percent recovery is calculated by dividing SOMAmer-measured EGFR by the expected EGFR level (spiked plus endogenous serum EGFR level), multiplied by one hundred. The codes on the X-axis represent sample number and concentration of spiked EGFR. For example, S_1_300 is serum sample #1 with 300 ng/mL of spiked purified EGFR protein. Each sample was measured three times. The error bars represent standard deviations.</p

EGFR SOMAmer specificity test.

<p>EGFR SOMAmer reagent was incubated with one of the ErbB proteins followed by labeling of bound protein with Alexa fluor 647. After extensive washing, photocleavage dissociates SOMAmer:protein complex from the bound beads, and the complex is separated by polyacrylamide gel electrophoresis under denaturing conditions. Lane 1, pulled down EGFR; Lane 2, EGFR standard; lane 3, pulled down ErbB2; lane 4, ErbB2 standard; lane 5, pulled down ErbB3; lane 6, ErbB3 standard; lane 7, pulled down ErbB4; lane 8, ErbB4 standard, and lane 9, MW size standards The EGFR SOMAmer showed limited cross-reactivity with ErbB family proteins. Relative to EGFR, the SOMAmer “pulled down” limited amounts of ErbB2 (0.4%), ErbB3 (6.9%), and ErbB4 (1.3%).</p

Comparison of ELISA and SOMAmer EGFR levels.

<p>Serum EGFR ECD levels were measured using EGFR SOMAmer assay (y-axis) and ELISA (x-axis). Black diamond data points showed correlation between the two methods and were used for R² calculation. Grey diamond data points showed much reduced EGFR SOMAmer levels and were not used for R² calculation.</p

Intra and inter assay variability of EGFR ECD SOMAmer assay.

<p>Inter-assay variability was determined from 20 runs each of control samples with low (∼27 ng/mL), middle (∼53 ng/mL), or high (∼310 ng/mL) concentrations of EGFR. Intra-assay variability was determined by testing each control sample at least 20 times in a single run.</p