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Abstracts of Oak Ridge Posters |
Department of Clinical Immunology, Amgen, Inc., Thousand Oaks, CA;
aaddress correspondence to this author at: Department of Clinical Immunology, Amgen, Inc., Thousand Oaks, CA 91320-1799; fax 805-480-1306, e-mail mmoxness{at}amgen.com)
Monitoring the immune response against human therapeutic monoclonal antibodies is an important component of preclinical and clinical trials to assess drug exposure, efficacy, and safety (1)(2). Detection of antibodies (analyte) directed against therapeutic antibodies (drug) is difficult because of the similarity of structure between drug and analyte and the high concentrations of drug present in serum, particularly during preclinical toxicology studies (3)(4). The purpose of this study was to develop a robust assay format applicable across species and drug compounds for rapid method development and validation.
We developed 5 assays to detect antibodies against 5 distinct monoclonal antibody therapeutic drug products. Detection was dependent on bivalent antibodies binding both biotin- and ruthenium-conjugated drug compounds (for a schematic of the assay, see Fig. 1 of the Data Supplement that accompanies the online version of this abstract at http://www.clinchem.org/content/vol51/issue10/). This "bridging" assay format eliminated the need for species–specific secondary antibodies and provided assay consistency in different serum matrices as a drug product moves from preclinical to clinical development. In addition, these assays theoretically detected all classes of immunoglobulins as long as they were functionally multivalent. We used 4 different serum matrices (mouse, monkey, rat, and human). Each drug was conjugated with a ruthenium complex (Meso Scale Discovery) that produces light [electrochemiluminescence (ECL)] on application of an electric potential. A separate sample of the drug was also conjugated with biotin (Pierce). A rabbit polyclonal affinity-purified antibody specific for each drug was generated and used as a surrogate analyte. Labeling ratios were optimized to retain immunochemical reactivity, which was determined by surface plasmon resonance measurements (Biacore 3000; Biacore) of the binding between conjugated drugs and the rabbit anti-drug antibody. Relative binding was required to fall within 80%–120% of binding of the native drug compound. Typically, label-to-protein molar ratios were found to be optimal in the range of 2:1 to 5:1. Sera were diluted to 10%, and 25 µL was incubated overnight with 50 µL of biotin- and ruthenium-conjugated drug, each at a concentration of 500 µg/L. The mixture was then added to plates equipped with electrodes and coated with streptavidin (Meso Scale Discovery) to capture complexes formed between biotin-labeled drug, analyte, and ruthenium-labeled drug. After a 2-h incubation, the plates were washed, and the ECL signals were measured on an MSD Sector PR analyzer (Meso Scale Discovery). Optimization included maximizing conjugated drug concentrations and incubation times. Samples were pretreated with acid in some assays to disrupt immune complexes before analysis. Acid pretreatment enhanced the detection of analyte in the presence of high drug concentrations (5). Assay precision, limits of detection (LOD), and tolerance to excess soluble drug were determined for each assay (Table 1
). Analyte stability was also determined for each assay. As expected, antibodies withstood up to 10 freeze–thaw cycles and up to 24 h at room temperature without substantial loss of activity (data not shown).
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The wide dynamic range of the ECL technology allowed for a linear signal response between analyte concentrations of 10 and 10 000 µg/L. The upper limit of a 95% one-sided reference interval (threshold value) was estimated for each assay by measuring at least 60 individual serum samples from the designated species (Bioreclamation). All samples were from drug-naive individuals or animals. Each response was normalized against a pooled species-specific serum (Bioreclamation) run in every assay. The frequency distribution was analyzed for gaussian shape, and the data were transformed by log or square-root function if necessary. The LOD was determined by adding a known amount of analyte (a unique rabbit anti-drug–specific antibody generated for each assay) to at least 30 donor samples (6). The normalized frequency distribution was examined, and the expected minimal response (mean – 2 SD) at the LOD was required to fall above the threshold value. The LOD varied between 5 and 63 µg/L; however, this variability probably depended primarily on the use of different drug-specific antibodies with differing affinities.
Tolerance to the presence of excess drug was determined by mixing known amounts of drug and analyte in pooled species-specific serum and incubating for at least 1 h. Samples were analyzed and determined to be positive if the response was above the threshold value (Fig. 1
).
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In summary, ECL bridging assays for the detection of antibodies directed against 5 different human therapeutic antibodies were developed and validated in sera of multiple species. The high sensitivity and reproducibility of this technique allowed for low LOD (5–63 µg/L), as well as for good precision (CV <20%), dynamic range (10–10 000 µg/L), and tolerance to excess drug present in serum (drug concentrations up to 100 000 µg/L tolerated at an analyte concentration of 500 µg/L). These assays can be used to detect antibodies against therapeutic antibody drugs from preclinical development through clinical trials and for postmarketing immunogenicity surveillance.
Acknowledgments
Amgen, Inc. supported all work described in this report. All authors are also employed by Amgen, Inc.
References
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), 500 (
), 1000 (
), and 5000 (
) µg/L analyte, respectively.