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Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples Jia Duo, Camelia Chiriac, Richard Y-C Huang, John Timothy Mehl, Guodong Chen, Adrienne A Tymiak, Peter Sabbatini, Renuka Pillutla, and Yan J. Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05277 • Publication Date (Web): 31 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Analytical Chemistry

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Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples

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Jia Duo1,*, Camelia Chiriac1, Richard Y.-C. Huang2, John Mehl2, Guodong Chen2, Adrienne Tymiak2, Peter Sabbatini1, Renuka Pillutla1, and Yan Zhang1

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*Corresponding author:

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Phone: +1 609 252 6999

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Fax: +1 609 252 7768

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Translational Medicine, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA

Email: [email protected]

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ACKNOWLEDGEMENTS

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We sincerely thank Carol Gleason for providing statistical guidance to this work and Uma Kavita for the scientific review and discussion. We also thank Sheri Wilcox and Michelle Guyer from SomaLogic for supplying the glypican-3 SOMAmer reagent, and Rangan Vangipuram, Chin Pan, and Ayesha Nazeer from Biologics Discovery California, Bristol-Myers Squibb for supplying the anti-GPC3 antibodies.

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ABSTRACT:

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Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging due to the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (Slow Off-Rate Modified Aptamer) is a novel reagent which binds, with high affinity, to a far distinct epitope of glypican-3 when compared to all available antibody reagents generated in house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development especially when traditional antibody reagents cannot meet assay requirements.

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Glypican-3 (GPC3), a member of the glypican family of glycosylphosphatidylinositol (GPI)-anchored cellsurface heparan sulfate proteoglycans, has become a key biomarker in hepatocellular carcinoma (HCC) diagnosis in recent years.1,2 GPC3 is expressed on the cell surface as a ~70 kDa protein which can be cleaved by furin to generate a 40 kDa N-terminal protein and a 30 kDa C-terminal cell membrane bound protein. In HCC patients, GPC3 is known to be overexpressed on cell surfaces, and elevated soluble GPC3 (sGPC3) has also been reported in the serum of ~50% of HCC patients compared with healthy individuals.3,4 Moreover, when sGPC3 levels are assessed in conjunction with levels of alpha-fetoprotein (AFP), the sensitivity for HCC diagnosis is increased relative to either biomarker alone.5 Although the use of sGPC3 as an HCC diagnostic tumor marker is growing, it is still not quite clear that, once shed from the cell surface, the sGPC3 is present as a full-length 70 kDa protein, its N-terminal fragment, its C-terminal fragment, or all. While studies have shown that furin cleavage is involved in GPC3 processing and functioning in vivo,6 the efficiency of furin cleavage is not yet fully evaluated in tumors.7 The 14 conserved cysteine residues within GPC3 are considered responsible for intramolecular disulfide bond formation which may hold the N- and C-terminal subunits together even after the potential furin cleavage. Therefore, characterizing the biochemical structure of serum GPC3 is critical for validating sGPC3 as a serological biomarker of HCC and other GPC3-expressing tumors.

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The reported methods used for sGPC3 measurement in clinical applications rely on immunoassays which are based on antibodies that can specifically bind GPC3.3,4,5,8,9,10,11 Anti-GPC3 monoclonal and polyclonal antibodies are both commercially available for immunochemistry applications. However, it is quite interesting that a vast majority of these commercial antibodies are Cterminus specific based on the epitope mapping results provided by the suppliers if available. The most popular GPC3 monoclonal antibody, hybridoma clone 1G12, that has been used in different immunochemistry applications and been reported in many publications is also a C-terminus specific antibody. At Bristol-Myers Squibb, we have also generated numerous GPC3 monoclonal antibodies 2 ACS Paragon Plus Environment

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using recombinant GPC3 as an immunogen. However, all these antibodies are extensively specific to the regions within the C-terminus of the GPC3 protein. A possible explanation to this is that the intramolecular disulfide bonds derived from the 14 cysteine residues within GPC3 result in a rigid and complex protein structure that prevents the N-terminus from being exposed and serving as an epitope for antibody generation. However, a firm answer cannot be drawn until the three-dimensional structure of GPC3 is resolved. Although some laboratories have reported use of in-house generated N-terminus specific antibodies for sGPC3 immunoassay development,4,9,11 limited access to these antibodies prohibits analytical scientists from fully understanding the forms of sGPC3 that are present in circulation and utilizing sGPC3 as a validated HCC biomarker.

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Nucleic acid aptamers are alternative protein recognition reagents that have gained increasing interest in a wide range of applications including biomarker discovery12,13,14,15, biomarker validation16, and bioanalysis.17,18,19,20 Different from antibody reagents which are proteins, aptamers are structured nucleic acid polymers synthesized and selected through an in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX). Due to the different nature and production method, aptamers, when used as the novel reagents for immunoassay development, provide many advantages over antibodies including higher chemical and thermal stability,21,22 better batch-to-batch reproducibility,23 easier tagging process,24,25 and faster production turnaround.26 A successful SELEX process is highly dependent on the quality of the initial synthetic DNA/RNA library (normally ~1015 unique sequences) from which the aptamers are selected and enriched. In the past decade of aptamer research, efforts have been devoted to the modification of libraries by introducing artificial riboses and nucleobases to generate aptamers with enhanced nuclease resistance and binding affinity.12,27 Using their library with functionalized nucleotides, combined with a strategy of slow off-rate selection, SomaLogic has successfully developed the slow off-rate modified aptamers (SOMAmers) against thousands of protein targets with high specificity and binding affinity.28,29,30 Additionally, since SOMAmers are smaller molecules with different physico-chemical properties from antibodies, they may exhibit distinct binding characteristics in terms of affinity, kinetics, and specificity. Investigations have been implemented and reported in the literature in order to understand the structural rules that govern the specific and high-affinity binding characteristics of aptamer–protein interactions.31,32,33

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In view of the lack of GPC3 N-terminus specific antibodies, we evaluated the utility of a SOMAmer reagent that binds to an N-terminal domain of GPC3 with high affinity and specificity. A hydrogen/deuterium exchange mass spectrometry (HDX-MS) method34,35,36 was developed to establish the unique N-terminus binding epitope of the SOMAmer reagent. This is the first aptamer ligand developed for GPC3 protein that shows a distinct binding epitope on GPC3 from our in-house anti-GPC3 antibodies. To evaluate the feasibility of using the SOMAmer as an alternative immunoassay reagent, a SOMAmer-based electrochemiluminescence (ECL) assay was developed for sGPC3 measurement in HCC patient samples. The SOMAmer-based assay utilized a similar strategy as published before where an aptamer and a paired antibody served as the capture and detection reagents, respectively.37,38 Systematic assay optimization and qualification demonstrated good assay sensitivity and performance. Owing to the unique binding epitope of the SOMAmer reagent, the SOMAmer-based assay, when combined with our previously developed antibody-based immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, helped confirm the clinically relevant sGPC3 forms in vivo. Implementing this integrated analytical strategy of reagent characterization and orthogonal methods for complementarity, this work demonstrates that an aptamer, owing to its distinct 3 ACS Paragon Plus Environment


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biochemical properties, can be used as an alternative reagent for immunoassay development to answer questions that cannot be answered by the traditional antibody reagents alone.

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EXPERIMENTAL SECTION

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Materials and Equipment. The recombinant GPC3 (amino acid 25-559 with six C-terminal histidine residues and a spacer) was in-house generated and used as the reference standard in the three assays developed in this work. The anti-GPC3 monoclonal and polyclonal antibodies (mAbs and pAbs) used in this work were either Bristol-Myers Squibb Company proprietary or commercially available (MAB2119, R&D Systems, Inc., Minneapolis, MN; sc-65443/mAb 1G12, Santa Cruz Biotechnology, Inc., Dallas, TX) materials. The GPC3 SOMAmer was produced and supplied by SomaLogic (Boulder, CO) with a single biotin molecule incorporated into the 5’-end during production. Binding affinity (KD) of the SOMAmer to GPC3 was determined to be 0.1 nM using a Zorbax affinity method by SomaLogic. Biotinylation of the anti-GPC3 antibodies was performed using the EZ-Link Sulfo-NHS-LC-Biotin and Zeba™ Spin Desalting Columns (molecular weight cutoff of 7 kDa) from Thermo Scientific (Rockford, IL) according to the manufacturer’s instructions. Ruthenium-labeled anti-GPC3 antibodies was prepared using the Sulfo-TAG NHS-Ester labeling kit from Meso Scale Discovery (MSD, Rockville, MD) according to the manufacturer’s instructions. MULTI-ARRAY 96-well streptavidin-coated MSD standard plates and the MSD read buffer T were also purchased from MSD (Rockville, MD). The wash buffer (PBST) used for plate washes was PBS (10 mM phosphate buffer containing 2.7 mM KCl, 137 mM NaCl) with 0.05% (v/v) Tween 20, pH 7.4 at 22°C. The in-house prepared assay buffer (PTB) contained the same composition as the wash buffer with 1% (w/v) bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) added. The other assay buffers evaluated in this work included MSD Diluent 3 purchased from MSD (Rockville, MD), SuperBlock™, StartingBlock™, and Blocker™ Casein purchased from Thermo Scientific (Rockford, IL). All plate washing steps were performed on the BioTek EL × 405 microplate washer (BioTek Instruments, Inc., Winooski, VT).

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Screening of Antibodies on Octet and MSD for SOMAmer Pairing. Fifteen anti-GPC3 monoclonal and polyclonal antibodies were screened on the Octet RED384 label-free biosensor (Pall ForteBio LLC, Fremont, CA) for their pairing with the GPC3 SOMAmer. First, 5 µg/mL of the biotinylated SOMAmer was captured onto the streptavidin biosensor and used to bind 1 µg/mL of the recombinant GPC3. Next, the bound GPC3 was allowed to interact with 5 µg/mL of each antibody for 5 min followed by another 5 min with PTB for dissociation. The pairing criteria is the observation of significant increase of instrumental response (≥ 0.1 nm) for antibody association and no complete response decrease during dissociation.

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Antibodies that met the criteria were selected as potential pairing antibodies, and their pairing with the SOMAmer was further confirmed on the final assay platform, MSD. Fifty microliter of the biotinylated SOMAmer (1 µg/mL) was first added onto the streptavidin MSD plate and incubated at 22 °C for 1 h. After washing the plate four times, 50 µL of the recombinant GPC3 (1000, 100, 10, 1, 0.1, and 0 ng/mL in PTB) was added and incubated at 22 °C for 1 h. After washing the plate four times, 50 µL of the selected antibodies (ruthenium labeled, 1 µg/mL) was added and incubated at 22 °C for 1 h. After the last four washes, 150 µL of the 1x MSD read buffer T was added and the plate was read on an MSD SECTOR Imager 6000. The best pairing antibody was selected based on the lowest assay background and the highest signal-to-background (S/B) ratio at the lowest GPC3 concentration tested.


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HDX-MS. Prior to epitope mapping experiments, non-deuterated experiments were carried out to generate a list of common peptides for recombinant GPC3 and protein complexes of GPC3 with SOMAmer or mAbs (1:1 molar ratio). In the HDX-MS experiment, 5 µL of each sample (GPC3 alone, GPC3 with SOMAmer, or GPC3 with either mAb) was diluted into 55 µL of D2O buffer (10 mM phosphate buffer, D2O, pD 7.0) to start the labeling reactions. The reactions were carried out for different periods of time: 1 min, 10 min and 240 min. By the end of each labeling reaction period, the reaction was quenched by adding quenching buffer (100 mM phosphate buffer with 4 M guanidine hydrocloride and 0.4 M tris(2-carboxyethyl)phosphine, pH 2.5, 1:1, v/v), and 50 µL of the quenched sample was injected into Waters HDX-MS system for analysis as described previously.39

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Mass spectra were obtained with a Waters Synapt G2si Q-TOF equipped with standard ESI source (Waters Corp., Milford, MA). The instrument configuration was the following: capillary was 3.5 kV, sampling cone at 35 V, source temperature of 80 °C and desolvation temperature of 175 °C. Mass spectra were acquired over an m/z range of 260 to 2000.

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Peptic peptides identification was accomplished through a combination of exact mass analysis and MSE using ProteinLynx Global Server 3.0.2 (Waters Corp., Milford, MA). The deuterium uptake levels of common peptic peptides were monitored in the absence/presence of SOMAmer or each mAb using Waters DynamX 3.0TM software. All assignments, deuterated spectra, and data processing were manually checked and verified. All experiments were performed in triplicate.

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ECL Immunoassay. SOMAmer-Based. The MSD streptavidin plate was blocked with 200 µL/well of SuperBlock™ and incubated at 22 °C with 400 rpm shaking for 1 h. After washing four times with 300 µL/well of PBST, the plate was coated with 50 µL/well of biotinylated GPC3 SOMAmer diluted in SuperBlock™ and incubated at 22 °C with 400 rpm shaking for 1 h followed by another washing step as above. Reference standards (with a technical range of 0.1 to 100 ng/mL) and quality controls (QCs) were prepared in the standard matrix, aliquoted, and frozen at -70 °C before use. During the day of analysis, the frozen standards, QCs, and samples were thawed at room temperature, diluted two-fold in SuperBlock™ supplemented with 1 µM of the SomaLogic proprietary polyanionic competitor, and added to the plate at 50 µL/well in duplicate. The plate was incubated at 22 °C with 400 rpm shaking for 1 h followed by another washing step. The ruthenium-labeled mAb, GPC3.4, was then added at 50 µL/well, and the plate was incubated at 22 °C with 400 rpm shaking for 1 h. After the washing step, 150 µL/well of the 1x MSD read buffer T was added, and the plate was read on an MSD SECTOR® Imager 6000. The assay standard curve was generated based on the ECL values using a 4-parameter regression model with 1/y2 weighting on the Softmax Pro 5.4.1 Software (Molecular Devices, Sunnyvale, CA).

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Antibody-Based. The antibody-based assay had the same assay procedure as the SOMAmerbased assay except for the following differences. MSD Diluent 3 was used as the buffer for both MSD streptavidin plate blocking and sample/reagent dilution. Biotinylated mAb GPC3.4 (5 µg/mL) and ruthenium-labeled mAb 1C2 (0.5 µg/mL) were used as the capture and detection antibodies, respectively, in this assay.

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Accuracy and precision of both assays were determined by running both spiked QC samples at five concentration levels (upper limit of quantification, ULOQ; high QC; medium QC; low QC; lower limit of quantification, LLOQ) and endogenous QC samples (three levels) on different days. The assay LLOQ was determined as the lowest concentration at which sGPC-3 could be reliably detected with acceptable accuracy and precision, and was defined as the assay sensitivity. 5 ACS Paragon Plus Environment


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Immunocapture Liquid Chromatography tandem Mass Spectrometry (IC-LC-MS/MS) Assay. GPC3 levels were also measured using an IC-LC-MS/MS method that is similar to a previously described method.40 Briefly, M280 magnetic streptavidin beads (Thermo Fisher Scientific, Waltham, MA) coated with biotinylated mAb GPC3.4 were used for immunocapture. The capture beads were prepared by loading 100 µg of biotinylated mAb GPC3.4 for every 1 mL of streptavidin beads (10 mg/mL of bead concentration). To measure sGPC3 in patient samples, 20 µL of beads were combined with 50 µL of serum sample in the presence of 75 µL of PBS containing 0.05% (w/v) of 3-12 Zwittergent and 0.1% (w/v) of BSA in a 96-deepwell plate. After incubation at room temperature for 1.5 h, the beads were washed three times with 200 µL of ammonium bicarbonate, pH 8.2 containing 0.05% (w/v) of 3-12 Zwittergent. Following the final wash, the beads were transferred to a clean 96-deepwell plate and resuspended in 20 µL of acetonitrile to denature the captured GPC3 protein. Then, 80 µL of 50 mM ammonium bicarbonate, pH 8.2 containing 1 µg of trypsin was added to each sample for overnight digestion. The LC-MS/MS system was comprised of an ACQUITY UPLC (Waters Corp, Milford, MA) coupled to a SCIEX 6500 triple quadrupole (SCIEX, Framingham, MA). The column used was an ACQUITY 1.0 x 50 mm HSS T3, 1.8 µm (Waters Corp, Milford, MA), at 60 °C using a flow rate of 250 µL/min. Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in 95% acetonitrile. Gradient elution was used, starting from 0% to 40 %B over 3.6 min and from 40% to 95%B over 1.5 min. Twenty-five microliter of sample was injected for LC-MS/MS detection of GPC3 selective peptides, 34LQPGLK39 (328.1 – 414.5 m/z, CE 16) and 196VFGNFPK202 (404.7-565.3 m/z, CE 17). Data analysis was performed using the Analyst software, version 1.6.2 (SCIEX, Framingham, MA). Quadratic curve fitting with weighting (1/x) was applied for quantification.

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HCC Patient Sample Analysis. To evaluate the sGPC3 forms in vivo, serum samples collected from 106 HCC patients were analyzed using both the SOMAmer-based and the antibody-based assays. Correlation analyses between sGPC3 concentrations measured on both assays were conducted by a Deming regression and a Bland-Altman plot.41,42,43 Data analysis and result download was done through a published interactive website for analytical method comparison and bias estimation.44 Where possible, JMP software (JMP Version 10, SAS Institute, Inc., Cary, NC) was used to verify some of the results obtained from the website. Additionally, a concordance correlation coefficient was independently computed in SAS (SAS 9.2 Base and SAS/STAT Version 9.2, SAS Institute, Inc., Cary, NC) according to published methods.45

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RESULTS AND DISCUSSION

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SOMAmer Antibody Pair Screening. A sandwich-based immunoassay requires two reagents that can specifically bind the analyte, one as the capture reagent and one as the detection reagent. A typical immunoassay uses two antibodies that can bind two different epitopes on the analyte to form the immune complex for analyte detection. In this work, we used the SOMAmer to replace one of the two antibodies. To build the sandwich for GPC3 detection, another antibody that can pair with the SOMAmer was needed. Initial antibody screening for SOMAmer pairing was done on the Octet RED384 biosensor which allows for label-free and real-time monitoring the binding interactions between the SOMAmer, GPC3, and the antibodies. The screening format on the biosensor is identical to the format that will be used on the final assay platform, MSD, where the SOMAmer capture reagent is immobilized onto a solid surface via the biotin-streptavidin interaction and the GPC3 antibody is used as the detection reagent. Of the fifteen in-house and commercial anti-GPC3 antibodies, eleven showed significant increase of instrumental responses on Octet RED384 during incubation with GPC3 captured 6 ACS Paragon Plus Environment

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on the SOMAmer immobilized biosensor (data not shown). This result indicates these antibodies recognize a different epitope on GPC3 and can potentially form a pair with the SOMAmer in a sandwichbased immunoassay format. To confirm this, the eleven antibodies were labeled with MSD Sulfo-TAG and used as detection reagents for GPC3 measurement on the MSD platform. Biotinylated SOMAmer was immobilized onto the MSD plate through the biotin-streptavidin interaction and used to capture GPC3 from the samples. All 11 antibodies can form a pair with the SOMAmer on the MSD platform and detect GPC3 across the concentration range tested (0.1-1000 ng/mL), however, with different sensitivities. Figure S1, presented in Supporting Information, shows the background ECL response and the S/B ratio at 0.1 ng/mL of GPC3 when each SOMAmer/antibody pair was used to set up the assay. A high S/B ratio at the low GPC3 concentration together with a low assay background tends to indicate a good assay sensitivity that can be achieved. The top three performers were identified to be mAb 5D2, mAb GPC3.2, and mAb GPC3.4 based on the highest S/B ratios at 0.1 ng/mL of GPC3 they provided with relatively low assay background. We finally chose mAb GPC3.4 as the detection reagent for the following work due to the wide availability of this antibody we have in stock.

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GPC3 Binding Epitope Characterization by HDX-MS. HDX-MS has been widely utilized in the biopharmaceutical industry for higher order structure characterization of therapeutic proteins in discovery and development.34,35 A major application of this technique is for the epitope mapping of therapeutics.46,47 HDX-MS provides information on the protein conformational dynamics in solution when exchange kinetics is monitored.48 The extent of exchange reflects the degree of solvent accessibilities and hydrogen bonds formation, which are subject to change upon protein-protein interaction. In this study, HDX-MS was applied for the epitope mapping of mAb GPC3.4, mAb 1C2, and the SOMAmer. mAb GPC3.4 and mAb 1C2 were Bristol-Myers Squibb generated and used as the capture and the detection antibody, respectively, in our previously developed antibody-based sGPC3 immunoassay as described in the experimental section. mAb GPC3.4 was also used as the detection antibody in the SOMAmer-based assay. HDX-MS coupled with proteolytic digestion provided ~87% sequence coverage of GPC3. Pairwise comparison of the HDX profiles of the peptic peptides revealed only four peptide regions in GPC3 were affected upon SOMAmer/mAbs binding (Figure 1). Compared to mAbs, the SOMAmer binds distinctively to the N-terminal domain of GPC3 in which at least three amide hydrogens in region 42FQRLQPGL49 were occluded from the solvent upon SOMAmer binding at the 20 sec exchange time period. Both regions 130FKNNYPSLTPQAFE143 and 548EISTFHNLGNVHTGTET564 showed significant HDX reduction upon mAb GPC3.4 binding in which at least one hydrogen in both regions was occluded from HDX at the 20 sec exchange time period and the level of reduction slightly increased for longer exchange period. The binding of mAb GPC3.4 to the full-length GPC3 (the recombinant GPC3 reference standard used in this work) and a recombinant GPC3 C-terminal fragment (amino acid 360559 with six C-terminal histidine residues and a spacer) was further evaluated by the Octet RED384 biosensor (Figure S2). The Octet results revealed similar binding of mAb GPC3.4 to both full-length and C-terminal fragment of GPC3, indicating the epitope of mAb GPC3.4 is mainly in the C-terminal domain of GPC3. The HDX reduction observed in 130FKNNYPSLTPQAFE143 upon mAb GPC3.4 binding could be an outcome of the protein conformational change that was induced by mAb GPC3.4 binding. mAb 1C2 exhibited overlapping epitope region with mAb GPC3.4 in 548EISTFHNLGNVHTGTET564 and minor solvent protection in 476MSMPKGRVLDKNL488. The observation of HDX reduction in multiple regions in GPC3 upon mAb 1C2 binding is likely the outcome of its conformational epitope, whereas SOMAmer exhibited a linear epitope that is distinctively in the very N-terminal region, indicating the potential application of SOMAmer as a unique immunoassay reagent. 7 ACS Paragon Plus Environment


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Immunoassay Development for sGPC3 Measurement. Our method development for the SOMAmer-based assay started with detection reagent screening as described above and standard matrix selection, followed by the optimization of several parameters for the best assay sensitivity, including the capture and detection reagent concentrations, the assay buffer, and the polyanionic competitor concentration. S/B ratio at the lowest GPC3 concentration tested (i.e., 0.14 ng/mL) was used as the criterion for assay sensitivity assessment. For conditions with similar S/B ratios, the one providing lower assay background is always preferred. The optimal assay minimal required dilution (MRD) was then determined during the assay qualification. Experimental details and figures supporting the assay optimization results are reported in Supporting Information, Supplemental experiments and Figure S3 AE.

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First, the matrix used to prepare the assay standard curve was studied. The selected standard matrix should best represent the sample matrix for accurate quantification. Therefore, the best standard matrix for clinical sGPC3 serum sample analysis is the human serum preferably with no detectable sGPC3. Recombinant GPC3 was prepared in three commercial normal human serum pools as well as three general immunoassay buffers including PTB, SuperBlock™, and SeraSub™. As shown in Figure S3 A, the recombinant GPC3 performs identically in the three serum pools, and the response curves generated in the serum pools are in parallel with those generated in the three immunoassay buffers. The ECL responses in the serum pools are consistently lower than those in the buffers across the entire assay range (0.1-100 ng/mL) with no sign of detectable endogenous sGPC3 in either serum pool. The ideal performance of the serum pools allowed for the selection of one pool (BioreclamationIVT Lot BRH1197315) as the standard matrix for more accurate sGPC3 measurement than choosing the artificial buffers.

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Second, the concentrations of the capture and detection reagents were optimized in order to achieve the best assay sensitivity. As shown in Figure S3 B, a combination of 0.5 µg/mL of SOMAmer capture and 0.12 µg/mL of mAb GPC3.4 detection provides the highest S/B ratio (S/B = 1.5 ± 0.2, n =3) at the lowest GPC3 concentration (0.14 ng/mL) studied. The typical capture antibody concentration on the streptavidin MSD plate is in the range of 1.0-5.0 µg/mL. Considering the smaller size of the SOMAmer (a molecular weight of 18 kDa) as compared to antibodies, the molar amount of SOMAmer used for capture is in the same range as antibodies.

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In order to further improve the assay sensitivity, one in-house prepared and four commercial buffers were studied to see if the S/B ratio could be increased when they are used as assay buffers for sample dilution. The analysis was done using the above optimized capture and detection concentrations. As shown in Figure S3 C, the SuperBlock™ buffer provides the highest S/B ratio (S/B = 2.1 ± 0.2, n = 3) among the five buffers tested in this study. The S/B ratio improvement was attributed to the signal boost at the lowest GPC3 concentration (0.14 ng/mL) tested since similar assay background signals were provided by the SuperBlock™ buffer and the PTB buffer used in the above capture/detection concentration optimization (background ECL response of 97 and 92, respectively).

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Including the polyanionic competitor into the assay buffer for sample dilution was found to be effective in improving the S/B ratio at low GPC3 concentrations and thus the assay sensitivity. Figure S3 D shows the S/B ratio obtained when increasing concentration of the polyanionic competitor is included in the optimized assay buffer, SuperBlock™. Comparing with no polyanionic competitor, the assay S/B ratio was significantly increased when the polyanionic competitor was used, and the highest S/B ratio 8 ACS Paragon Plus Environment

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(S/B = 2.4 ± 0.2, n = 3) was achieved when 1 µM of the polyanionic competitor was included. Further increasing the polyanionic competitor concentration didn’t affect the S/B ratio significantly. The polyanionic competitor serves as a charge blocker to prevent any positively charged components within serum from non-specifically binding to the negatively charged SOMAmer oligonucleotide under the physiological conditions. The polyanionic competitor used in this work was a proprietary product from SomaLogic. Dextran, which is also recommended by SomaLogic, of 15 and 40 KDa molecular weights at concentrations above 1 µM also worked though in a less effective way than the SomaLogic polyanionic competitor (data not shown).

322 323 324 325 326 327 328 329 330 331

The assay MRD was determined by assessing the dilution linearity of sera (no detectable endogenous sGPC3) spiked with 30 ng/mL of GPC3 and sera containing endogenous sGPC3. Figure S3 E shows the back-calculated GPC3 concentrations against the dilution factors in each serum. For the spiked sera, except for one serum (Lot BRH1095929), the back-calculated concentrations of samples diluted from 2- to 16-fold are within 75-125% of the nominal concentration, suggesting no significant matrix effect in this dilution range. The sera containing endogenous sGPC3 also dilute linearly from 2- to 16-fold. Therefore, a two-fold MRD was selected for the SOMAmer-based assay to maintain the same MRD as required for the antibody-based assay. Dilutional linearity of the sera containing endogenous sGPC3 also demonstrates good assay parallelism and the capability of accurately measuring endogenous sGPC3 using the recombinant GPC3 as a reference standard.

332 333 334 335 336 337 338

In summary, the final SOMAmer working concentration used for GPC3 capture was 0.5 µg/mL, and the mAb GPC3.4 working concentration for detection was 0.12 µg/mL. Samples need to be diluted at least two-fold in the SuperBlock™ buffer supplemented with 1 µM of the polyanionic competitor. The SuperBlock™ buffer was also used to block the MSD plate and dilute the capture and detection reagents to their working concentrations for the assay. The assay calibrators were prepared in a normal human serum pool with no detectable sGPC3 levels. The optimized assay conditions for the antibody-based assay are described in the experimental section.

339 340 341 342 343 344 345 346 347

With these optimized conditions, both the SOMAmer-based and the antibody-based assays can detect 0.1 to 100 ng/mL of sGPC3 in human serum. Figure 2 shows the representative standard curves of both assays with defined assay ranges. The technical range of a standard curve is the lowest and highest points with acceptable accuracy and precision (total % CV ≤ 15% and % Dev ≤ 15%). The assay accuracy and precision results are summarized in Table 1 using five spiked and three endogenous QC samples spanning the assay range. For the SOMAmer-based assay, the % Dev was within 10.8% and the between-run, within-run precision and total variation (% CV), were below 11.8%, 10.8%, and 13.9%, respectively. For the antibody-based assay, the % Dev was within 7.8% and the between-run, within-run and total % CV, were below 12.1%, 11.2%, and 14.0%, respectively. .

348 349 350 351 352 353 354 355

sGPC3 Measurement in HCC Patient Samples. Knowing the distinct GPC3 binding epitopes of the SOMAmer and other GPC3 antibodies inspired us to use the SOMAmer-based immunoassay for sGPC3 measurement in clinical serum samples. Two advantages are proposed owing to the distinct SOMAmer binding epitope that has never been reached by our available GPC3 antibodies. First, recognizing amino acid sequences on two ends of a protein with 70 kDa MW raises the possibility of having binding epitopes spatially far from each other. This was proved by the large number of antibodies (11 out of 15) that could pair with the SOMAmer during pair screening. Second, using the SOMAmer as a capture reagent allows us to develop an assay that measures a different context of sGPC3 species than the 9 ACS Paragon Plus Environment


356 357 358 359 360

antibody-based assay. As illustrated in Figure 3 (A and B), since the antibody binding epitopes are both located close to the C-terminus of GPC3, the antibody-based assay is able to measure both full-length GPC3 and the C-terminal fragment if any in the serum. By contrast, the SOMAmer-based assay only measures the full-length GPC3 in serum because the capture and detection reagents bind to the two ends of the protein.

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387

To investigate the possible sGPC3 forms in vivo, both the SOMAmer-based and our previously developed antibody-based assays were used to analyze the same set of HCC patient samples. Out of the 106 samples, sGPC3 was detectable in 41 samples (38.7% of total) by either assay and in 31 samples (29.2% of total) by both assays. Figure 4A shows the Deming regression of the sGPC3 concentrations in the 31 samples measured by both assays. The 95% confidence interval (CI) is calculated with the Jackknife method.42 The Deming regression, which fits the parameters a (intercept) and b (slope) of the linear equation y = a + bx, is a statistical model generally used in clinical chemistry for method comparison with “y” responses from one method and “x” responses from the other. For the two methods to be considered as comparable, the CI for the intercept should contain the value “0” and the CI for the slope should contain the value “1”. This corresponds to the concept of the identity line, which has a slope of “1” and an intercept of ‘”0”. If the “x” and “y” results for the methods are the same, the plot falls on this line. The concordance correlation coefficient is a measurement of how well the two sets of ”x” and “y” results compare as relative to the identity line.45 Rejection of either null hypothesis indicates a possible systematic difference or a possible proportional difference, respectively, between the two methods. The inclusion of both value “0” and value “1” in the 95% CIs of the intercept and the slope suggests our two assays are comparable for sGPC3 measurement. Considering the different context of sGPC3 species these two assays may measure, this means there is a low chance of different sGPC3 species present in these samples. In order to further evaluate the comparability of the two assays, differences between assay results as a function of the mean of the results were displayed as a Bland-Altman plot for the samples with measured sGPC3 concentrations below 1.0 ng/mL (n = 20). As shown in Figure 4B, differences of the measured sGPC3 concentrations by both assays randomly scatter around “0” over the mean concentration range on the Bland-Altman plot showing no significant bias toward either assay, which indicates the absence of different sGPC3 forms in these samples. It should be also noted that, although the measurable samples showed good agreement between the two immunoassays, a large portion of the samples were still below the assay quantification limit. Improving assay sensitivity and making more samples measurable especially at the low concentration range should provide us more insights into the sGPC3 forms in vivo.19

388 389 390 391 392 393 394 395 396 397 398

IC-LC-MS/MS Assay for sGPC3 Measurement. The high specificity and multiplex capability of LC-MS/MS was also used to measure sGPC-3 in HCC patient serum. The GPC3.4 mAb was used for immunoaffinity enrichment of sGPC3 from serum before trypsin digestion to improve assay sensitivity. By monitoring two tryptic peptides originating from the N-terminus of GPC-3 (34LQPGLK39 and 196VFGNFPK202), the assay was able to confirm the presence of the full-length sGPC3 in serum (Figure 3C). The assay sensitivity was determined to be 1.0 ng/mL for both peptides, 34LQPGLK39 and 196VFGNFPK202. As shown in Table 2, good assay accuracy and precision was demonstrated using five spiked QC samples spanning the assay range (1.0 - 32 ng/mL). In order to confirm the in vivo sGPC3 forms, the same set of HCC patient samples (48 samples) was analyzed by both the IC-LC-MS/MS assay and the antibody-based immunoassay. Although sGPC3 was only measurable in 11 samples by the IC-LC-MS/MS assay due to limited assay sensitivity, the comparable results between these two assays as reported in Figure 5 10 ACS Paragon Plus Environment

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399 400 401

indicate that the full-length protein should be the dominant sGPC3 form present in circulation. Using the IC-LC-MS/MS assay as an orthogonal method, we were able to confirm our observation above when the two immunoassays were used for the same set of sample analysis.

402

CONCLUSION

403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

Here, we report an integrated analytical approach to immunoassay reagent characterization and qualification including 1) epitope mapping to aid the rational selection of key reagents for assay development, 2) SOMAmer-based immunoassay development for sGPC3 quantification in HCC patient samples, and 3) orthogonal immunoassay and IC-LC-MS/MS assays for complementarity. Using the SOMAmer as a capture reagent paired with an antibody detection reagent, the immunoassay demonstrated good sensitivity (0.1 ng/mL limit of quantification) and performance (% Dev within 10.8% and total variation within 13.9%) for sGPC3 measurement. The major advantage of the SOMAmer-based assay is that it could measure a different context of sGPC3 species in vivo than the antibody-based assay owing to the distinct N-terminus binding capability of the SOMAmer reagent. Integrated analytical approach including HDX-MS epitope mapping revealed a unique SOMAmer binding domain located on the N-terminus of GPC3 which is different from antibody binding domains. By comparing sGPC3 concentrations measured by the SOMAmer-based immunoassay, the antibody-based immunoassay, and the IC-LC-MS/MS assay, we were able to confirm that the full-length protein should be the dominant sGPC3 form present in vivo. The work presented here indicates that, SOMAmer, when used as a novel immunoassay reagent, may allow the research community to answer questions and move forward in areas where we may not have had adequate tools in the past. Aptamers should be considered as an alternative affinity platform of immunoassay reagents especially when traditional antibody reagents cannot meet assay requirements.

421



422

FIGURES

423 424 425 426

Figure 1. HDX kinetics of four peptic peptide regions upon SOMAmer/mAbs binding. Results indicate that SOMAmer binds distinctively to the N-terminal region of GPC3, whereas mAb GPC3.4 and mAb 1C2 share overlapping epitope in the C-terminal domain.

427

428 429 430

Figure 2. Representative standard curves of the SOMAmer-based and the antibody-based immunoassays with defined assay ranges.

431


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432 433 434

Figure 3. Schematic representation of different possible sGPC3 species that can be measured by (A) the antibody-based immunoassay, (B) the SOMAmer-based immunoassay, and (C) the IC-LC-MS/MS assay.

435

436 13 ACS Paragon Plus Environment


437 438 439 440 441 442 443 444 445

Figure 4. sGPC3 measurement comparison between the antibody-based and the SOMAmer-based assays. (A) Correlation of sGPC3 concentrations in 31 HCC patient samples determined by Deming regression. The dotted line indicates the identity line. The solid line indicates the Deming regression line. The shaded area represents the 95% CI calculated by the Jackknife method. (B) Bland-Altman plot of the mean sGPC3 concentration of the two assays (x axis) and the difference in sGPC3 concentrations between the two assays (y axis) on the samples with measured sGPC3 concentrations below 1.0 ng/mL by both assays (n = 20). The dotted lines represent the 95% CI estimated by the mean difference (MEAN) and the standard deviation of the differences (SD).

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90.0

Antibody-based immunoassay IC-LC-MS/MS assay "LQPGLK" IC-LC-MS/MS assay "VFGNFPK"

80.0

sGPC3 concentration (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 1

2

3

4

5

6

7

8

9

10

11

HCC patient sample 447 448 449 450 451

Figure 5. sGPC3 measurement comparison between the antibody-based immunoassay and the IC-LCMS/MS assay. The IC-LC-MS/MS assay sensitivity was determined to be 1.0 ng/mL by the two peptides, 34 LQPGLK39 and 196VFGNFPK202. Sample 11 was below the detection limit of the IC-LC-MS/MS assay with the signature peptide, 196VFGNFPK202.

452


Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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453

TABLES

454 455

Table 1. Accuracy and Precision Results of (A) the SOMAmer-Based Immunoassay and (B) the Antibody-Based Immunoassay for sGPC3 Measurement in Human Serum

nominal concentration (ng/mL)

ULOQ (100)

high QC (75)

Endogenous QC 1

middle (7.5)

QC Endogenous QC 2

low QC (0.75) Endogenous QC 3

LLOQ (0.1)

4.77

0.751

0.181

0.0892

11.8

-10.8 10.0

(A) mean observed concentration (ng/mL) % Dev between-run precision (% CV) within-run precision (% CV) total variation (% CV) n number of runs

103

76.9

50.4

7.37

2.5 7.1

2.5 6.8

7.5

-1.7 5.0

3.9

0.1 5.6

1.7

2.6

3.5

2.4

4.9

3.3

3.1

10.8

6.4

6.4

7.5

4.9

6.0

5.8

11.0

13.9

13 4

13 4

6 3

13 4

6 3

13 4

6 3

13 4

4.57

0.808

0.177

0.105

12.1

7.8 6.0

3.7

4.8 7.1

(B) mean 105 observed concentration (ng/mL) % Dev 4.8 between-run 5.6 precision (% CV)

77.4

3.1 5.1

52.1

7.72

5.7

2.9 5.0


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within-run precision (% CV) total variation (% CV) n number of runs

3.4

3.5

2.7

3.4

9.0

2.8

3.0

11.2

5.9

5.6

5.8

5.6

14.0

5.9

4.5

12.8

13 4

13 4

6 3

13 4

6 3

13 4

6 3

13 4

456 457

Table 2. Accuracy and Precision Results of the IC-LC-MS/MS assay for sGPC3 Measurement in Human Serum

nominal concentration (ng/mL)

ULOQ (32)

high QC (12.8)

middle QC (6.4)

LLOQ (1.0)

2.08

1.14

4.2 10.4

14.4 3.0

3

3

1.86

1.03

34

mean observed concentration (ng/mL) % Dev within-run precision (% CV) n

30.5

12.33

-4.6 2.7

-3.6 7.2

3

3

LQPGLK39 6.95

low QC (2.0)

8.7 6.4 3 VFGNFPK202 6.03

196

mean observed concentration (ng/mL) % Dev within-run precision (% CV) n

33.0

12.56

3.2 4.4

-1.9 6.8

-5.9 7.4

-6.9 7.3

3.2 10.5

3

3

3

3

3

458 459



460

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