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Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid ChromatographyMass Spectrometry: Method Development and Qualification Eugene Ciccimaro, Yongxin Zhu, Dmitry Ostanin, Suzanne Suchard, Jamus MacGuire, Qing Xiao, Ashok R. Dongre, Anjaneya Chimalakonda, Timothy V. Olah, and Petia Shipkova Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00688 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 10, 2017
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Analytical Chemistry
Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography-Mass Spectrometry: Method Development and Qualification Eugene Ciccimaro, Yongxin Zhu, Dmitry Ostanin, Suzanne Suchard, Jamus MacGuire, Qing Xiao, Ashok Dongre, Anjaneya Chimalakonda, Timothy Olah, Petia Shipkova Bristol-Myers Squibb, Princeton, New Jersey 08543, United States ABSTRACT: We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.
Introduction Treating the right patient, with the right drug, at the right dose is a goal of modern medicine that requires a deep understanding of disease biology, patient population, and drug pharmacology. Acknowledging this, the pharmaceutical industry has focused its research efforts to ensure a detailed understanding of drug effect on the target of interest by conducting preclinical translational studies and earlier incorporation of exploratory clinical testing1. These focused activities are designed to expedite and hopefully increase the success rate of drug discovery and development2. Currently, the highest rate of failure for drug progression from discovery to patient is during proof of concept (Phase II) clinical trials where the industry median success rate is only 29%, and the majority of failures are due to lack of efficacy3. An in-depth evaluation of this barrier revealed that the likelihood of success was highest among projects that supplied a fundamental understanding of the nominated molecule’s pharmacology, including drug exposure at the target site of action, drug binding to the target, and functional modulation of the target4. Techniques capable of supplying these measures in a timely manner during early clinical testing are essential and help guide critical clinical decisions, such as dose selection5. Therefore there is a clear need to develop sensitive, selective, and clinically applicable methods to calculate tissue target engagement for new and emerging biologics therapies. Here we present an analytical technique that provides a comprehensive and direct confirmation of drug reaching the site of action at therapeutically relevant concentrations, and of target engagement (TE) by the dosed material at the observed exposure. In addition to the %TE, the absolute measurement of total target and total drug allows a determination of the molar relationship of exposure with target and discern any modulation of target resulting from dosing. These data are critical to understanding PK/PD, and may potentially guide human dose projection and patient stratification6.
There have been significant advances in technologies allowing the determination of target engagement (TE) of small molecule drugs5. Techniques based on competition with radio labeled tracers are widely utilized to assess TE for small molecule drugs 7 . Most recently, liquid chromatography–mass spectrometry (LC-MS) was successfully employed to measure TE of small molecules without radioligands using chemical probes in vitro8 and by a thermal shift assay in tissues9. Furthermore, Receptor Occupancy (RO) for small molecules and biologics is often measured in blood by flow cytometry. However, application of this method to tissues is technically challenging to implement in the clinic10. One major limitation is the requirement for viable cells, thus requiring special instrumentation and trained personnel to process fresh tissues at collection sites, which severely limits its clinical applicability. Soluble protein biomarkers can successfully be measured by ligand binding assays (LBA)11. The “Hybrid” LBA-LC-MS assay is emerging as a powerful technology platform capable of measuring dosed biologics and endogenous proteins with unsurpassed selectivity12. Although the technique relies on an affinity extraction (AE) step to improve sensitivity, the selectivity of the measurement is less dependent on the specificity of the affinity reagent, but rather on the highly specific and selective detection by LC-MS. Antibodies specific for target proteins1315 , selected peptides16,17, or with broader specificity for protein classes18, post-translational modifications19, and peptide motifs20 have all been utilized as capture reagents for LC-MS quantitation. Additionally non-antibody based reagents such as endogenous ligands, chemical probes21, small molecule drugs22, and immobilized metal23,24 can serve as the “affinity reagents” for LC-MS analysis. TE determination by AE-LC-MS hybrid assays have been demonstrated in cell based systems by measurement of downstream phosphorylation events following kinase inhibition25,26. Additionally, recent AE-LC-MS studies allowed the simultaneous in vivo quantification of a dosed antibody drug and soluble
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target, indicating the potential for multiplexed pharmacokinetic/pharmacodynamic (PK/PD) bioanalysis27. In this report, we aim to extend the application of AE-LC-MS to provide a novel and very important link between PK/PD measurements by providing a direct antibody drug-target engagement measurement in tissue. Absolute quantification of antibody drugs in tissue is challenging due to low levels of exposure relative to circulation, extraction inefficiency, and matrix complexity. However, advances in affinity based sample preparation steps and mass spectrometric detection have improved these measurements18,28. Alternatively, radio-labeled antibody can be used to evaluate tissue distribution in pre-clinical species29.
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baseline and desired time points, mimicking the clinical tissue collection protocol during the longitudinal analysis. All tissue samples were immediately frozen in liquid nitrogen (for LC-MS) or placed in PBS and kept on ice for flow cytometry (Fluorescence-activated cell sorting, FACS). Blood from the same animals was also collected for RO measurements and for PK evaluation. Figure 1 highlights the overall scheme for sample collection and distribution for analysis. Furthermore, Figure 1 indicates the methodology used (LBA, FACS or AE-LC-MS) together with the intended use for calculation of PK and PD.
We demonstrate feasibility of measuring total antibody drug and target levels as well as measuring the amount of drug-target complex at the site of therapeutic action (colon) using a technology (AE-LC-MS) that is applicable to pre-clinical and clinical sample analysis. The assay discussed here was developed to measure an anti-CD40 antibody and the membrane bound receptor (CD40, Tumor necrosis factor receptor superfamily member 5) in colon biopsies of cynomolgus monkeys. CD40 is a member of the tumor necrosis factor receptor superfamily and a type 1 transmembrane protein30 critical to immune regulation31 and expressed on multiple cell types32. The colon biopsy collection and storage were done following a standard clinical protocol, since the ultimate goal is to apply this approach to clinical studies where flow cytometry assessment is not practical due to limitations of sample collection and subsequent freezing prior to shipping. In this proof-of-concept study, the biopsy AE-LC-MS measurements were compared to traditional blood and tissue PK/PD assessments of drug exposure and receptor occupancy in order to generate a comprehensive understanding of assay performance and drug pharmacology. Importantly, this evaluation allowed comparison of the AE-LC-MS approach for target engagement to an orthogonal flow cytometry assessment of biopsies, currently the gold standard in TE estimation10. We highlight steps taken to address the challenges of accurately quantifying drug bound-target in tissue33 and address the early stages of fit-for-purpose assay qualification for an exploratory biomarker of target engagement34. Consistent with biomarker assay development, full validation is not applicable for exploratory studies, however a number of important checks were performed as part of fit-for-purpose qualification to ensure the quality of the measurements were suitable and reliable for these proof-of-concept experiments35.
Experimental
Tissue collection. In addition to normal non-disease cynomolgus (cyno) monkeys (non-colitic), animals with a history of chronic, intermittent, non-pathogenic diarrhea, unresponsive to standard treatment regimens which include fiber diet supplementation, antacid/antidiarrheal therapy, and antibiotic treatment (colitic cynos) were also selected as their colons are likely similar to those of colitic human patients. All animals were injected (i.v.) with a single dose of 3 mg/kg (for longitudinal study) or a 5 mg/kg anti-CD40 antibody (for method development samples). The 5 mg/kg dose was selected based on previous flow cytometry data (not shown) indicating a close to 100% target engagement (TE) even in a single dose regiment. Tissues (pinch) biopsies were collected with endoscopic forceps from anesthetized animals at
Figure 1: Overview of PK/PD Strategy in Proof-of-Concept Experiment using Multiple Technology Platforms. Comprehensive PK/PD experimental design allowing for a comparison of drug exposure and receptor occupancy in the periphery with measurements made in tissue. Biopsy tissue homogenization for AE-LC-MS analysis. Biopsies were homogenized using the Precellys Evolution chilled bead mill processor (Bertin Technologies). Homogenization and extraction was done in 0.5 mL tissue lysis tubes containing 1.4 mm ceramic (zirconium oxide) beads (Bertin Technologies) and 200 µL tissue extraction buffer (T-PER, pH = 7.6) containing HALT protease inhibitor (Thermofisher Scientific). Samples were shaken on the bead mill at 5,500 RPM for 30 seconds, twice, maintaining 4 ⁰C. Following centrifugation (14,000 g, 4 ⁰C) a 75 µL portion of the supernatant was removed for both total capture and bound capture as described in the Results and Discussion section. The remainder of tissue homogenate was used for total protein measurement. AE-LC-MS sample preparation and total protein. The AELC-MS strategy is summarized in Figure 2. Reagents used in the analysis were characterized and screened for assay performance. Based on in vitro testing, a binding epitope for the capture reagent used for CD40 was distinguished from the binding epitope of the anti-CD40 drug on CD40 (data not shown). Additionally, the CD40 capture reagent was selected for optimal
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recovery of spiked CD40 into serum and found non-competitive to spiked drug (data not shown). The CD40 capture antibody was conjugated to magnetic beads. The anti-CD40 was captured with Protein G magnetic beads. Total CD40 and Total anti-CD40 Ab drug capture was accomplished by incubating with a “mixed bead” approach referred hereon as “total capture” using anti-CD40 conjugated magnetic beads and protein G magnetic beads. In addition to this mixed bead total capture, an equal portion of tissue homogenate from the same sample was affinity extracted with protein G magnetic beads alone. Incubations were performed in 96-well plates. Samples were washed and captured protein was then eluted from beads using acid and heat. The final eluent was transferred to a new deepwell 96-well plate and dried. Dried samples were resuspended, reduced and alkylated, and digested with the addition of trypsin. After digestion, isotopically labeled synthetic reference peptides (peptide IS) were added to samples at 50 nM concentration. Finally, samples were transferred to a 96-well AS injection plate. A detailed description of bead capture processing is supplied in the supplemental experimental procedure. The total protein measurements were done using a micro BCA protein assay kit (ThermoFisher Scientific) following manufacturer’s instruction. Calibration and QC standards preparation. To generate calibration standard samples, cyno CD40 reference material (NP_001252791.1, extracellular domain Met1-Arg193 expressed with a C-terminal polyhistidine-tag in human cells, Sino Biological) and the antibody drug (anti-CD40 antibody, Ab) were spiked into rat colon homogenate to generate a 7 point calibration curve. A sequence alignment showing human (accession P25942), rhesus macaque (accession F6SNI, sequence
is identical to cynomolgus monkey), and rat (accession Q4QQW2) is provided (Figure S-1), indicating tryptic peptides used for analysis. QC samples were treated similar to study samples, with the exception that tissue homogenate from separate cyno (non-dosed and dosed) were mixed prior to capture. A detailed description of the calibration standard sample preparation and description of tissue processing for method development and QC purposes is provided in the supplemental experimental procedure. LC-MS acquisition and analysis. Data acquisition was conducted by injecting 30 µL sample onto an LC-MS system consisting of an Acquity I-Class UPLC (Waters) interfaced to a 6500 QQQ (AB Sciex). Quantitative analysis was done using Analyst 1.6 software (AB Sciex). For tissue measurements, concentrations were expressed in total molar amounts normalized to total protein in homogenate (fmol/mg). CD40 quantified in the total capture (considered total CD40) was compared to CD40 measured in the bound capture (considered bound CD40), allowing calculation of percent target engagement. Figure 2 displays the overall measurement strategy and calculation of %TE. A detailed description of LC-MS acquisition and analysis parameters are provided in the supplemental experimental procedure and all MRM parameters are shown in Table S-1. Error for measurements are shown as standard deviation (S.D.) throughout results. Blood and Tissue Flow Cytometry RO assay. Preparation of blood and intestinal lamina propia (LP) cells for RO were isolated according to standard protocols. A detailed description of the assay is provided in the supplemental experimental procedure.
Figure 2: AE-LC-MS TE Experimental Overview. A) Total Capture (Free CD40, Free Drug and CD40-Drug complex) and B) Bound Capture (Free Drug and CD40-drug complex). Total target and drug are measured in capture A), while bound target, recovered bound to drug, is measured in capture B). The ratio of bound- to total-target is used to calculate the percent target engagement.
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During the method development of a quantitative target engagement protein assay in tissue there are a number of important evaluations that are essential for generating meaningful measurements. These include 1) choosing a surrogate matrix that mimics the sample matrix; 2) designing QCs that properly reflect the measurement of the endogenous protein(s) of interest; 3) evaluating choice of buffers, dilutions and homogenization protocols to assure minimum (or no) disruption of the drug-target complex during sample preparation; and, 4) ensuring sample integrity is maintained within the limits necessary for the assay during tissue collection and storage. A full evaluation of the stability of the target-drug complex under the assay conditions were considered and results are discussed. Detection, LLOQ and general considerations in choosing quantitation strategies. Calibration curves were created in rat colon homogenate (as a surrogate matrix) with both recombinant CD40 reference material and drug added exogenously prior to the AE step. Surrogate tissue homogenate was chosen over buffer to normalize the affinity extraction and provide similar background interference between standards and samples. Internal standard peptides where spiked to control for auto-sampler variability, LC-MS performance, and matrix effect differences across samples and do not control for any of the early sample preparation steps. The response from the internal standard peptides were consistent throughout the analysis (CV ~ 10%) and showed similar response between bound and total capture samples, as well as between calibrants and biopsies (Figure S-2). Total and bound CD40 was measured from the total capture approach (Figure 2a) and the achieved LLOQ was 0.78 ng/mL (26 pM). Total drug quantification was conducted in both the total and bound captures (Figure 2a and b) with similar performance. Importantly, drug could be readily measured in all early time point biopsy samples with the achieved LLOQ of 7.8 ng/mL (100.3 pM). Typical standard calibration performance for the total target and drug are shown (Table S-2). Results indicate error across standards at the LLOQ were below 16%. Results for calibration curve performance are consistent to unpublished protein biomarker and published dosed Ab assays conducted in our laboratory using similar technique18. Example chromatograms for total CD40 and total anti-CD40 Ab drug at LLOQ and in biopsy samples are shown in Figure 3 (chromatograms showing IS response and qualifying peptide response are provided in Figure S-3). Of note, during method development, the strategy of measuring bound CD40 was found analytically superior to measuring bound drug (data not shown). Additionally, the direct measurement of the bound target was considered a primary measure of engagement for CD40, while free target would only serve as a surrogate end point. Due to these considerations, the measurement of bound CD40 was used for calculating % TE. Method development utilizing colon tissue from animals dosed with anti-CD40 Ab. The method development discussed here were based on analyses of colon tissue obtained from 2 cynomolgus monkeys 48 hrs following an i.v. dose of 5 mg/kg drug, as well as colon tissue from non-dosed cyno. The resected colon tissue from dosed animals served as a source of material for method development, and also provided multiple measurements from a stored tissue in a form of incurred (patient) sample reanalysis (ISR). Within individual dosed animals, CD40 measured 294.5 (± 13.5) and 155.5 (± 40.5) fmol/ mg total protein, and drug measured 865 (± 55) and 618 (± 37.7)
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fmol/ mg total protein respectively in animals. Measurements of total CD40 and total drug as well as bound CD40 were relatively consistent from these samples. Finally, levels of total and bound target were not statistically different between regions of the colon (data not shown), indicating clinical sampling of the distal colon would reflect %TE throughout the large intestine. The cyno anti-CD40 Ab described in this study was selected to have high binding affinity (Kd = 30%) a sample would be flagged. For standards, values were typically well within 30% deviation between the two capture approaches (~20%). In the longitudinal data set, of the 84 biopsies measured, 4 samples failed this QC criteria (Table S-3) and were excluded from further data analysis. In samples used for analysis in the longitudinal study, excluding the 4 flagged samples, the average discrepancy in drug measurement between the capture approaches was 12 ±8.5%. Longitudinal biopsy analysis. Four cynomolgus monkeys (non-colitic cyno 1,2 and colitic cyno 1,2) where studied in a longitudinal study quantifying drug engagement in the colon during a 37 day time course. Pinch mucosal biopsies were collected from live animals using endoscopy forceps. Six biopsies were collected from each animal beginning a day prior to dosing (day 0, baseline control) and thereafter at days 2, 9, 16, 23, 30, and 37. Three biopsies were used for the AE-LC-MS analysis, while 3 were used to measure RO using a flow cytometry
Total CD40, total drug, and bound CD40 where measured in all samples using the described AE-LC-MS methodology. Example chromatograms for representative analysis of total and bound CD40 in biopsy homogenate are shown in Figure 6. The mean values for all measurements within each animal from multiple biopsies collected per time point are shown in Figure 7 (individual biopsy values are provided in Table S-3). More detailed chromatograms for these biopsy samples showing all transitions monitored for CD40 and anti-CD40 Ab including IS peptides are provided in Figure S-6 for total capture and Figure S-7 for bound capture. Additionally, chromatograms illustrating the response for the CD40 qualifying peptide are provided in Figure S-8. Peptide IS for CD40 and anti-CD40 Ab were consistent throughout analysis and between total and bound samples (Figure S-2). At day 2, total CD40 was observed in individual animals ranging from ~100 fmol/mg to ~300 fmol/mg. These values were in agreement with those observed in the method development tissues collected from animals dosed 5 mg/kg. Bound CD40 was less than 100% of total CD40 across all animals and averaged 70.3 (±6.1)% on day 2. Total drug measured in both capture techniques from individual samples were compared and samples failing to pass threshold criteria were excluded as discussed above (Table S-3).
Figure 6: Example Chromatograms of Total and Bound CD40 in Biopsy Homogenate. Total and Bound CD40 measured in biopsies collected prior to dosing and at days 2, 16, and 23 following dosing. Data from Non-colitic Cyno 1. Drug exposure was reproducible across three of the four animals dosed (non-colitic cynos 1 & 2 and colitic cyno 2). Percent engagement profiles within individual animals showed agreement to total drug observed in these animals and time points. For example, total drug was observed in colitic cyno 1 to fall below quantitative limits at day 9, while the target engagement profile for this animal was the shortest lived, showing no engagement by day 16. Conversely, non colitic cyno 2 and colitic cyno 2 had the highest sustained drug exposure (in that order) and, similarly, the longest sustained target engagement (Figure 7). Profiles for the FACS based tissue %RO and AE-LC-MS %TE where in agreement, with %RO in colitic cyno 1 falling below 50% by day 9 and colitic cyno 2 having the highest sustained %RO (Figure 7). Interestingly, in both blood and biopsy
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RO measurement, colitic cyno 1 showed a comparatively accelerated drug elimination (Figure 7). Blood PK values for drug exposure (data not shown) were in agreement to tissue target engagement profiles and drug exposure measured in the colon by AE-LC-MS, showing colitic cyno 1 to present a precipitous drop in exposure between days 2 and 9, and colitic cyno 2 with the highest sustained exposure (Figure 7). Total CD40 measured prior to dosing was similar to measurements made during
the time course of the PK analysis, with no clear trends observed, and no difference between colitic and normal animals was noted (Figure 7). RO and %TE by AE-LC-MS were in good agreement, for example, maximum target engagement was observed in day 2-9 samples as reflected in both blood RO, tissue RO, and tissue TE measurements (Figure 7). However, the absolute %RO was observed at greater values on days 2 and 9, when compared to AE-LC-MS %TE and will require more experimentation to determine the cause.
Figure 7: Target and Drug Measurements across Technology Platforms in Circulation and Tissue. Biopsies (n=3) and blood were collected from cyno (n=4) prior to dosing and at days 2, 9, 16, 23, 30, and 37. Biopsy PK shows the average Total-Drug measured in the biopsy using AE-LC-MS and represented as fmol/mg (drug/total protein). Biopsy total CD40 shows the average Total-Target measured in the biopsy using AE-LC-MS and represented as fmol/mg (target/total protein). Blood RO shows the relative %RO determined in blood using FACS. RO in biopsy determined using FACS (-----) is overlayed with the TE measurement made using AE-LC-MS ( ).
Conclusion The success of this analysis relied on the availability and understanding of the drug-target interaction. Importantly, the target (CD40) is a single-pass transmembrane protein with relatively high expression in colon tissue, being expressed on multiple cell types32 and having relatively low shed soluble amounts in circulation38. In this study, CD40 was measured within colon mucosal pinch biopsies. The colon lamina propria is a cell rich mucosal layer of loose connective tissue that may be sampled through a colonoscopy pinch biopsy. The selective harvesting of this outer layer will result in a tissue sample enriched in epithelial, lymphocytes, plasma, and mast cells, as supported by histological and proteomic studies39-41. Blood contamination is a concern when measuring drug in tissue due to the high concentration in circulation relative to tissue levels, however, with the consideration of CD40 expression within the tissue and the selective sampling technique employed, it is likely that measurement of drug-target complex as described would reflect target engagement within the lamina propria.
Two important caveats of the analysis make target engagement by AE-LC-MS a distinct measurement from receptor occupancy determined by flow cytometry. First, the %TE calculation by AE-LC-MS is determined by comparing bound target associated with captured drug to total target as detected by the presence of tryptic peptides in the processed sample. This is in contrast to a secondary detection of drug on isolated cells by flow cytometry. Second, the AE-LC-MS measurement can potentially measure all target in a sample. In the tissue homogenate, all CD40 regardless of cell type or surface expression was potentially recovered and measured as part of the total target pool. In comparison, classic receptor occupancy determined by flow cytometry considers only surface expressed target on selected cell types. In this way, if total CD40 is greater than surface expressed CD40 and includes a portion not accessible to drug, than %TE will measure below %RO. Although the %RO and target engagement profiles for the longitudinal study presented are in good agreement, absolute values disagree 20-30% at days 2 and 9. Further experimentation is needed to confirm
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the cause of this discrepancy and ultimately determine how values correlate with clinical efficacy. Supplemental Information Supplemental experimental procedures. Figure S-1: sequence alignment showing Human, Monkey (Rhesus/Cyno), and Rat CD40. Table S-1: MRM experimental conditions. Figure S-2: LCMS area response from CD40 and anti-CD40 Ab internal standards (IS). Table S-2: Example accuracy and precision of calibration standards. Figure S-3: Representative detailed chromatograms for CD40 and Anti-CD40 Ab. Figure S-4: effect of detergent in homogenization buffer on total and bound measurements. Table S-3: AE-LC-MS/MS quantitation for individual biopsies. Figure S-5: total protein recovered from biopsies in longitudinal study. Figures S-6, 7, and 8: example detailed chromatograms of total and bound CD40 and anti-CD40 Ab in biopsy homogenate.
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Analytical Chemistry
Author Contributions
ACKNOWLEDGMENT
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Authors would like to thank Bogdan Sleczka, Asoka Ranasinghe, John Mehl, Alexander Kozhich, and Robert Neely for technical discussions. For support with reagents, the authors thank Aaron Yamniuk. Serum PK analysis was performed by Uma Kavita.
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