A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay for Ixekizumab using Affinity Capture Elution

Ixekizumab is a humanized monoclonal antibody directed against IL-17A, and it is currently under investigation in phase III trials for the treatment of psoriasis and psoriatic arthritis. As with any therapeutic antibody, ixekizumab can be immunogenic (Mire-Sluis et al., 2004Mire-Sluis A.R. Barrett Y.C. Devanarayan V. Koren E. Liu H. Maia M. et al.Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products.J Immuol Methods. 2004; 289: 1-16Crossref PubMed Scopus (539) Google Scholar, Sauerborn et al., 2010Sauerborn M. Brinks V. Jiskoot W. Schellekens H. Immunological mechanism underlying the immune response to recombinant human protein therapeutics.Trends Pharmacol Sci. 2010; 31: 53-59Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and induce anti-drug antibodies (ADAs). Individual immunogenic responses to therapeutic proteins vary greatly, ranging from having no effect on clinical efficacy or drug levels to neutralizing the therapeutic protein so as to render it ineffective (Büttel et al., 2011Büttel I.C. Chamberlain P. Chowers Y. Ehmann F. Greinacher A. Jefferis R. et al.Taking immunogenicity assessment of therapeutic proteins to the next level.Biologicals. 2011; 39: 100-109Crossref PubMed Scopus (120) Google Scholar, Casadevall et al., 2002Casadevall N. Nataf J. Viron B. Kolta A. Kiladjian J.J. Martin-Dupont P. et al.Pure red-cell apalasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin.N Engl J Med. 2002; 146: 469-475Crossref Scopus (1113) Google Scholar, Mire-Sluis et al., 2004Mire-Sluis A.R. Barrett Y.C. Devanarayan V. Koren E. Liu H. 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Strategies for detection, measurement, and characterization of unwanted antibodies induced by therapeutic biologicals.J Immunol Methods. 2003; 278: 1-17Crossref PubMed Scopus (104) Google Scholar). Two immunogenicity assays were developed for ixekizumab, an affinity capture elution (ACE) immunogenicity assay with some modifications (Bourdage et al., 2007Bourdage J.S. Cook C.A. Farrington D.L. Chain J.S. Konrad R.J. An affinity capture elution (ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug.J Immunol Methods. 2007; 327: 10-17Crossref PubMed Scopus (111) Google Scholar; Butterfield et al., 2010Butterfield A.M. Chain J.S. Ackermann B.L. Konrad R.J. Comparison of assay formats for drug-tolerant immunogenicity testing.Bioanalysis. 2010; 2: 1961-1969Crossref PubMed Scopus (26) Google Scholar) and a Meso Scale Discovery (MSD) (Meso Scale Diagnostics LLC, Rockville, MD) bridging assay based on a previously described format (Moxness et al., 2005Moxness M. Tatrewicz S. Weerartne D. Murakami N. Wullner D. Mytych D. et al.Immunogenicity testing by electrochemiluminescent detection for antibodies directed against therapeutic human monoclonal antibodies.Clin Chem. 2005; 51: 1983-1985Crossref PubMed Scopus (72) Google Scholar), including an upfront acid dissociation. Assays were designed to produce approximately 5% putative positive results in nonexposed subjects, consistent with regulatory expectations (United States Food and Drug AdministrationUnited States Food and Drug Administration. FDA draft guidance for industry: assay development for immunogenicity testing of therapeutic proteins, http://www.fda.gov/downloads/Drugs/…/Guidances/UCM192750.pdf (accessed 23 March 2016).Google Scholar). Positive control material was obtained by hyperimmunizing monkeys with ixekizumab. Anti-ixekizumab polyclonal antibody was affinity-purified by passing the hyperimmune serum over a column to which ixekizumab had been covalently coupled. After washing, bound anti-ixekizumab antibodies were eluted with acid, placed into neutral buffer, and quantitated via protein assay. Based on results from competition binding experiments with IL-17A, the monkey polyclonal antibody was observed to bind mainly the variable regions of ixekizumab. In addition, human anti-ixekizumab affinity-purified polyclonal antibody was similarly prepared from pooled serum obtained from patients found to be ADA positive during phase II studies. All procedures in this protocol had institutional approval and were in compliance with the U.S. Department of Agriculture’s Animal Welfare Act (9 code of federal regulations Parts 1, 2, and 3) and the National Institutes of Health, Office of Laboratory Animal Welfare. Sensitivity was tested by analyzing 2–2,000 ng/ml of affinity-purified anti-ixekizumab antibody spiked into normal human serum. Drug tolerance was assessed by varying the concentration of ixekizumab from 0.1 to 500 μg/ml in the presence of 500 ng/ml of affinity-purified anti-ixekizumab antibody spiked into normal human serum. This level of affinity-purified antibody was chosen based on the minimal level of acceptable sensitivity for clinical immunogenicity screening assays (United States Food and Drug AdministrationUnited States Food and Drug Administration. FDA draft guidance for industry: assay development for immunogenicity testing of therapeutic proteins, http://www.fda.gov/downloads/Drugs/…/Guidances/UCM192750.pdf (accessed 23 March 2016).Google Scholar). MSD software and SigmaPlot, version 8.0 (Systat Software, Inc. San Jose, CA), were used for fitting MSD bridging assay and ACE assay ELISA calibration curves, respectively. Raw data were plotted using Microsoft Excel 2010 (Microsoft, Redmond, WA). The ACE assay and MSD bridging assay formats were similar in terms of sensitivity. Both formats showed sensitivity better than 10 ng/ml (Figure 1), far exceeding the recommended sensitivity level of 250–500 ng/ml (United States Food and Drug AdministrationUnited States Food and Drug Administration. FDA draft guidance for industry: assay development for immunogenicity testing of therapeutic proteins, http://www.fda.gov/downloads/Drugs/…/Guidances/UCM192750.pdf (accessed 23 March 2016).Google Scholar). For the ACE assay, sensitivity was almost identical whether human or monkey affinity-purified anti-ixekizumab antibody was evaluated, whereas the MSD bridging assay showed slightly less sensitivity with human antibody compared with monkey antibody. With monkey antibody, the MSD bridging assay showed a modest drug tolerance of approximately 10 μg/ml, which decreased somewhat when human antibody was tested (Figure 2). In contrast, with monkey antibody, the ACE assay showed a drug tolerance of better than 200 μg/ml (far above any expected trough exposure levels), thus achieving roughly 20-fold greater drug tolerance than the MSD bridging assay. For both assays, the drug tolerance was somewhat lower when human antibody was evaluated (Figure 2). This may be due to the polyclonal human antibody having lower affinity for ixekizumab than the monkey polyclonal antibody obtained from hyperimmune serum. In summary, two different immunogenicity screening assays for the detection of anti-ixekizumab antibodies, an ACE assay and a MSD bridging assay, were developed and directly compared with one another. Although many different characteristics of immunogenicity assays should be evaluated, sensitivity and drug tolerance are two of the most critical features and were the focus of this study. For ixekizumab, this is particularly important, because its high-affinity binding to its target may affect both the sensitivity and the drug tolerance of an assay designed to measure ADA directed against it. Sensitivity results obtained for the ixekizumab ACE assay were similar to those of the ixekizumab MSD bridging assay, with both assays showing a sensitivity of better than 10 ng/ml. The drug tolerances of these two formats, however, were markedly different. Specifically, the drug tolerance of the ACE assay was roughly 20-fold greater than the drug tolerance of the MSD bridging format when evaluations were performed with monkey antibody and approximately 10-fold greater when human antibody was tested. To maximize the detection of clinically relevant levels of ADA during dosing, the immunogenicity assay for ixekizumab must be able to detect ADA in the presence of expected trough therapeutic drug levels of ixekizumab, which can be up to or exceed 10 μg/ml. Failing to ensure adequate drug tolerance could lead to false-negative results (Wang et al., 2012Wang Y.C. Fang L. Zhou L. Wang J. Ahn H.Y. A survey of applications of biological products for drug interference of immunogenicity assays.Pharm Res. 2012; 29: 3384-3392Crossref PubMed Scopus (33) Google Scholar). The MSD bridging assay for ixekizumab was no longer able to detect 500 ng/ml of ADA in the presence of greater than 10 μg/ml of ixekizumab. In contrast, the drug tolerance of the ACE format was sufficient to detect anti-ixekizumab antibodies at or even far above the anticipated circulating concentrations of ixekizumab. The importance of drug tolerance for clinical immunogenicity assays was recently illustrated by the Center for Drug Evaluation Research at the Food and Drug Administration, which highlighted the lack of drug tolerance of clinical immunogenicity assays supporting large-molecule registrations (Wang et al., 2012Wang Y.C. Fang L. Zhou L. Wang J. Ahn H.Y. A survey of applications of biological products for drug interference of immunogenicity assays.Pharm Res. 2012; 29: 3384-3392Crossref PubMed Scopus (33) Google Scholar). In light of the excellent sensitivity and superior drug tolerance of the ACE format, this method was selected to support ongoing phase III ixekizumab clinical trials. Importantly, by being able to detect clinically meaningful levels of ADA in the presence of expected concentrations of drug, this assay will improve our ability to characterize any clinical immunogenicity that may be associated with ixekizumab. This research was funded by Eli Lilly and Co. TMM, JHS, JSC, WJK, BIM, MPH, Y-WQ, and RJK are all employees and stockholders of Eli Lilly and Co. AB is a consultant and investigator for Abbvie, Amgen, Boehringer Ingelheim, Celgene, Janssen, Eli Lilly and Co., Merck, Novartis, Pfizer, and Sandoz. KP is a consultant, investigator, and speaker for Eli Lilly and Co.

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