- Email: [email protected]
A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay for Ixekizumab using Affinity Capture Elution
Accepted Manuscript A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay for Ixekizumab using Affinity Capture Elution Talia M. Muram, John H. Sloan, Jana S. Chain, Wendy J. Komocsar, Bruce I. Meiklejohn, Andrew Blauvelt, Kim Papp, Michael P. Heffernan, Yue-Wei Qian, Robert J. Konrad PII:
S0022-202X(16)31014-4
DOI:
10.1016/j.jid.2016.01.040
Reference:
JID 275
To appear in:
The Journal of Investigative Dermatology
Received Date: 18 August 2015 Revised Date:
4 January 2016
Accepted Date: 18 January 2016
Please cite this article as: Muram TM, Sloan JH, Chain JS, Komocsar WJ, Meiklejohn BI, Blauvelt A, Papp K, Heffernan MP, Qian Y-W, Konrad RJ, A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay for Ixekizumab using Affinity Capture Elution, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.01.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay
RI PT
for Ixekizumab using Affinity Capture Elution
Talia M. Muram1*, John H. Sloan1, Jana S. Chain1, Wendy J. Komocsar1, Bruce I. Meiklejohn1, Andrew Blauvelt2, Kim Papp3, Michael P. Heffernan1, Yue-Wei Qian1, and Robert J. Konrad1
SC
From 1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, 2Oregon
M AN U
Medical Research Center, Portland, OR, 3Probity Medical Research, Waterloo, Ontario, Canada *To whom correspondence should be addressed at:
Eli Lilly and Company, Indianapolis, IN 46285, USA Phone: (317) 433-6247, Fax: (317) 276-5281
TE D
E-mail: [email protected]
Key words: ixekizumab, IL-17A, drug tolerance, immunogenicity, affinity capture elution (ACE), mesoscale discovery (MSD), anti-drug antibodies
ACE:
affinity
AC C
Abbreviations:
EP
Running Title: Improved drug-tolerant immunogenicity assay for ixekizumab capture
elution,
ADA:
anti-drug
antibody,
ECLU:
electrochemiluminescent units, ELISA: enzyme linked immunosorbent assay, IL-17A: interleukin-17A, MSD: MesoScale Discovery, OD: optical density, SEM: standard error of the mean
Word Count: 985 words
ACCEPTED MANUSCRIPT
To The Editor Ixekizumab is a humanized monoclonal antibody directed against interleukin-17A (IL-
RI PT
17A), and is currently under investigation in phase 3 trials for the treatment of psoriasis and psoriatic arthritis. As with any therapeutic antibody, ixekizumab can be immunogenic (MireSluis et al, 2004, Sauerborn et al, 2010) and induce anti-drug antibodies (ADA). Individual
SC
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
M AN U
ineffective (Mire-Sluis et al, 2004, Sauerborn et al, 2010, Büttel et al, 2011, Casadevall et al, 2002). To identify whether antibodies to a therapeutic protein have been generated, screening immunogenicity assays are developed to detect the presence of ADA in patient serum samples. However, not all assay formats are equivalent (Wadhwa et al, 2003, Butterfield et al, 2010, Moxness et al, 2009, Bourdage et al 2005, Büttel et al, 2011, Liang et al, 2011, Sickert et al
TE D
2008, Smith et al, 2007, Patton et al, 2005, Lofgren et al, 2007). Two immunogenicity assays were developed for ixekizumab, an affinity capture elution
EP
(ACE) immunogenicity assay with some modifications (Bourdage et al 2005, Butterfield et al, 2010) and a Mesoscale Discovery (MSD) bridging assay based on a previously described format
AC C
(Moxness et al, 2009), including an upfront acid dissociation. Assays were designed to produce approximately 5% putative positive results in non-exposed subjects, consistent with regulatory expectations (FDA guidance, 2009). 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-
ACCEPTED MANUSCRIPT
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,
RI PT
human anti-ixekizumab affinity-purified polyclonal antibody was similarly prepared from pooled serum obtained from patients found to be ADA positive during phase 2 studies. All procedures in this protocol had institutional approval and were in compliance with the U.S. Department of
M AN U
of Health, Office of Laboratory Animal Welfare.
SC
Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3) and the National Institutes
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 affinitypurified anti-ixekizumab antibody spiked into normal human serum. This level of affinity-
TE D
purified antibody was chosen based on the minimal level of acceptable sensitivity for clinical immunogenicity screening assays (FDA guidance, 2009). MSD software and SigmaPlot version 8.0 were used for fitting MSD bridging assay and ACE assay ELISA calibration curves,
EP
respectively. Raw data were plotted using Microsoft Excel (2010).
AC C
The ACE assay and MSD bridging assay formats were similar in terms of sensitivity. Both formats demonstrated sensitivity better than 10 ng/mL (Figure 1), far exceeding the recommended sensitivity level of 250-500 ng/mL (FDA guidance, 2009). 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 to monkey antibody.
ACCEPTED MANUSCRIPT
With monkey antibody, the MSD bridging assay demonstrated 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 demonstrated a drug tolerance of better
RI PT
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
M AN U
antibody obtained from hyperimmune serum.
SC
polyclonal human antibody having lower affinity for ixekizumab than the monkey polyclonal
In summary, two different immunogenicity screening assays for the detection of antiixekizumab antibodies, an ACE assay and a MSD bridging assay, were developed and directly compared to one another. While many different characteristics of immunogenicity assays should be evaluated, sensitivity and drug tolerance are two of the most critical features and were the
TE D
focus of this study. For ixekizumab, this is particularly important, as its high-affinity binding to its target may affect both the sensitivity and the drug tolerance of an assay designed to measure
EP
ADA directed against it.
Sensitivity results obtained for the ixekizumab ACE assay were similar to those of the
AC C
ixekizumab MSD bridging assay, with both assays demonstrating 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.
ACCEPTED MANUSCRIPT
To maximize 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
RI PT
ensure adequate drug tolerance could lead to false negative results (Wang et al, 2012). 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
SC
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
M AN U
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, 2012).
In light of the the excellent sensitivity and superior drug tolerance of the ACE format,
TE D
this method was selected to support ongoing phase 3 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
EP
immunogenicity that may be associated with ixekizumab.
AC C
Conflicts of Interest: This research was funded by Eli Lilly and Co. Talia M. Muram, John H. Sloan, Jana S. Chain, Wendy J. Komocsar, Bruce I. Meiklejohn, Michael Heffernan, Yue-Wei Qian, and Robert J. Konrad are all employees and stockholders of Eli Lilly and Co.. Andrew Blauvelt is a consultant and investigator for Abbvie, Amgen, Boehringer Ingelheim, Celgene, Janssen, Eli Lilly and Co., Merck, Novartis, Pfizer and Sandoz. Kim Papp is a consultant, investigator and speaker for Eli Lilly and Co.
ACCEPTED MANUSCRIPT
References Bourdage JS, Cook CA, Farrington DL, Chain JS, Konrad RJ. An affinity capture elution
RI PT
(ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug. J Immunol Methods 2007;327:10-17.
Büttel IC, Chamberlain P, Chowers Y, Ehmann F, Greinacher A, Jefferis R, et al. Taking
SC
immunogenicity assessment of therapeutic proteins to the next level. Biologicals 2011;39:100-109.
M AN U
Butterfield AM, Chain JS, Ackermann BL, Konrad RJ. Comparison of assay formats for drug-tolerant immunogenicity testing. Bioanalysis 2010;2:1961-69. Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, et al. Pure red-cell apalasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin.
TE D
N Engl J Med 2002;146:469-75.
FDA Draft Guidance for Industry: Assay Development for Immunogenicity Testing of
EP
Therapeutic Proteins. http://www.fda.gov/downloads/Drugs/.../Guidances/UCM192750.pdf (accessed 23 March 2016).
AC C
Liang M, Klakamp SL, Funelas C, Lu H, Lam B, Herl C, et al. Detection of high-and lowaffinity antibodies against a human monoclonal antibody using various technology platforms. Assay Drug Dev Tech 2007;5:655-62. Lofgren, JA, Dhandapani S, Pennucci JJ, Abbott CM, Mytych DT, Kaliyaperumal A, et al. Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of Panitumumab. J of Immunol 2007;178:7467-72.
ACCEPTED MANUSCRIPT
Mire-Sluis AR, Barrett YC, 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: 1-16.
RI PT
Moxness M, Tatrewicz S, Weerartne D, Murakami N, Wullner D, Mytych D, et al. Immunogenicity testing by electrochemiluminescent detection for antibodies directed against
SC
therapeutic human monoclonal antibodies. Clin. Chem 2005;51:1983-85.
Patton A, Mullenix MC, Swanson SJ, Koren E. An acid dissociation bridging ELISA for
Immunol Methods 2005;304:189-95.
M AN U
detection of antibodies directed against therapeutic proteins in the presence of antigen. J of
Sauerborn M, Brinks V. Jiskoot W, Schellekens H. Immunological mechanism underlying the immune response to recombinant human protein therapeutics. Trends Pharmacol Sci
TE D
2010;31:53-9.
Sickert D. Kroeger K, Zickler C, Chokote E, Winkler B, Grenet JM, et al. Improvement of drug tolerance in immunogenicity testing by acid treatment on Biacore. J of Immunol
EP
Methods. 2008;334:29-36.
AC C
Smith HW, Butterfield A, and Sun D. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA. Regulatory Tox and Pharmacol 2007;49:230-37.
Wadhwa M, Bird C, Dilger P, Gaines-Das R, Thorpe R. Strategies for detection, measurement, and characterization of unwanted antibodies induced by therapeutic biologicals. J Immunol Methods 2013;278:1–17.
ACCEPTED MANUSCRIPT
Wang, YC, Fang L, Zhou L, Wang J, Ahn HY. A Survey of Applications of Biological
AC C
EP
TE D
M AN U
SC
RI PT
Products for Drug Interference of Immunogenicity Assays. Pharm Res 2012;29:3384-92.
ACCEPTED MANUSCRIPT
Figure Legends
RI PT
Figure 1: Sensitivity of ACE assay compared to MSD bridging assay – Affinity-purified antiixekizumab antibody from monkeys or humans was spiked into normal human serum at concentrations ranging from 2-2000 ng/mL. Samples were analyzed on the ACE and MSD
SC
bridging assays, with results shown as the mean ± SEM (n = 2). The MSD bridging assay reports
M AN U
in ECL units (ECLU), while the ACE assay generates an OD450 readout.
Figure 2: Drug tolerance of ACE assay compared to MSD bridging assay – Affinity-purified anti-ixekizumab antibody from monkeys or humans was spiked into normal human serum at a concentration of 500 ng/mL. Samples were also spiked with concentrations of ixekizumab
TE D
ranging from 0.1-500 µg/mL and analyzed on the ACE and MSD bridging assays. Results are shown as the mean ± SEM (n = 2). The MSD bridging assay reports in ECL units (ECLU), while
AC C
EP
the ACE assay generates an OD450 readout.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-202X(16)31014-4
DOI:
10.1016/j.jid.2016.01.040
Reference:
JID 275
To appear in:
The Journal of Investigative Dermatology
Received Date: 18 August 2015 Revised Date:
4 January 2016
Accepted Date: 18 January 2016
Please cite this article as: Muram TM, Sloan JH, Chain JS, Komocsar WJ, Meiklejohn BI, Blauvelt A, Papp K, Heffernan MP, Qian Y-W, Konrad RJ, A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay for Ixekizumab using Affinity Capture Elution, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.01.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
A Highly Sensitive and Drug-Tolerant Anti-Drug Antibody Screening Assay
RI PT
for Ixekizumab using Affinity Capture Elution
Talia M. Muram1*, John H. Sloan1, Jana S. Chain1, Wendy J. Komocsar1, Bruce I. Meiklejohn1, Andrew Blauvelt2, Kim Papp3, Michael P. Heffernan1, Yue-Wei Qian1, and Robert J. Konrad1
SC
From 1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, 2Oregon
M AN U
Medical Research Center, Portland, OR, 3Probity Medical Research, Waterloo, Ontario, Canada *To whom correspondence should be addressed at:
Eli Lilly and Company, Indianapolis, IN 46285, USA Phone: (317) 433-6247, Fax: (317) 276-5281
TE D
E-mail: [email protected]
Key words: ixekizumab, IL-17A, drug tolerance, immunogenicity, affinity capture elution (ACE), mesoscale discovery (MSD), anti-drug antibodies
ACE:
affinity
AC C
Abbreviations:
EP
Running Title: Improved drug-tolerant immunogenicity assay for ixekizumab capture
elution,
ADA:
anti-drug
antibody,
ECLU:
electrochemiluminescent units, ELISA: enzyme linked immunosorbent assay, IL-17A: interleukin-17A, MSD: MesoScale Discovery, OD: optical density, SEM: standard error of the mean
Word Count: 985 words
ACCEPTED MANUSCRIPT
To The Editor Ixekizumab is a humanized monoclonal antibody directed against interleukin-17A (IL-
RI PT
17A), and is currently under investigation in phase 3 trials for the treatment of psoriasis and psoriatic arthritis. As with any therapeutic antibody, ixekizumab can be immunogenic (MireSluis et al, 2004, Sauerborn et al, 2010) and induce anti-drug antibodies (ADA). Individual
SC
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
M AN U
ineffective (Mire-Sluis et al, 2004, Sauerborn et al, 2010, Büttel et al, 2011, Casadevall et al, 2002). To identify whether antibodies to a therapeutic protein have been generated, screening immunogenicity assays are developed to detect the presence of ADA in patient serum samples. However, not all assay formats are equivalent (Wadhwa et al, 2003, Butterfield et al, 2010, Moxness et al, 2009, Bourdage et al 2005, Büttel et al, 2011, Liang et al, 2011, Sickert et al
TE D
2008, Smith et al, 2007, Patton et al, 2005, Lofgren et al, 2007). Two immunogenicity assays were developed for ixekizumab, an affinity capture elution
EP
(ACE) immunogenicity assay with some modifications (Bourdage et al 2005, Butterfield et al, 2010) and a Mesoscale Discovery (MSD) bridging assay based on a previously described format
AC C
(Moxness et al, 2009), including an upfront acid dissociation. Assays were designed to produce approximately 5% putative positive results in non-exposed subjects, consistent with regulatory expectations (FDA guidance, 2009). 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-
ACCEPTED MANUSCRIPT
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,
RI PT
human anti-ixekizumab affinity-purified polyclonal antibody was similarly prepared from pooled serum obtained from patients found to be ADA positive during phase 2 studies. All procedures in this protocol had institutional approval and were in compliance with the U.S. Department of
M AN U
of Health, Office of Laboratory Animal Welfare.
SC
Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3) and the National Institutes
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 affinitypurified anti-ixekizumab antibody spiked into normal human serum. This level of affinity-
TE D
purified antibody was chosen based on the minimal level of acceptable sensitivity for clinical immunogenicity screening assays (FDA guidance, 2009). MSD software and SigmaPlot version 8.0 were used for fitting MSD bridging assay and ACE assay ELISA calibration curves,
EP
respectively. Raw data were plotted using Microsoft Excel (2010).
AC C
The ACE assay and MSD bridging assay formats were similar in terms of sensitivity. Both formats demonstrated sensitivity better than 10 ng/mL (Figure 1), far exceeding the recommended sensitivity level of 250-500 ng/mL (FDA guidance, 2009). 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 to monkey antibody.
ACCEPTED MANUSCRIPT
With monkey antibody, the MSD bridging assay demonstrated 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 demonstrated a drug tolerance of better
RI PT
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
M AN U
antibody obtained from hyperimmune serum.
SC
polyclonal human antibody having lower affinity for ixekizumab than the monkey polyclonal
In summary, two different immunogenicity screening assays for the detection of antiixekizumab antibodies, an ACE assay and a MSD bridging assay, were developed and directly compared to one another. While many different characteristics of immunogenicity assays should be evaluated, sensitivity and drug tolerance are two of the most critical features and were the
TE D
focus of this study. For ixekizumab, this is particularly important, as its high-affinity binding to its target may affect both the sensitivity and the drug tolerance of an assay designed to measure
EP
ADA directed against it.
Sensitivity results obtained for the ixekizumab ACE assay were similar to those of the
AC C
ixekizumab MSD bridging assay, with both assays demonstrating 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.
ACCEPTED MANUSCRIPT
To maximize 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
RI PT
ensure adequate drug tolerance could lead to false negative results (Wang et al, 2012). 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
SC
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
M AN U
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, 2012).
In light of the the excellent sensitivity and superior drug tolerance of the ACE format,
TE D
this method was selected to support ongoing phase 3 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
EP
immunogenicity that may be associated with ixekizumab.
AC C
Conflicts of Interest: This research was funded by Eli Lilly and Co. Talia M. Muram, John H. Sloan, Jana S. Chain, Wendy J. Komocsar, Bruce I. Meiklejohn, Michael Heffernan, Yue-Wei Qian, and Robert J. Konrad are all employees and stockholders of Eli Lilly and Co.. Andrew Blauvelt is a consultant and investigator for Abbvie, Amgen, Boehringer Ingelheim, Celgene, Janssen, Eli Lilly and Co., Merck, Novartis, Pfizer and Sandoz. Kim Papp is a consultant, investigator and speaker for Eli Lilly and Co.
ACCEPTED MANUSCRIPT
References Bourdage JS, Cook CA, Farrington DL, Chain JS, Konrad RJ. An affinity capture elution
RI PT
(ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug. J Immunol Methods 2007;327:10-17.
Büttel IC, Chamberlain P, Chowers Y, Ehmann F, Greinacher A, Jefferis R, et al. Taking
SC
immunogenicity assessment of therapeutic proteins to the next level. Biologicals 2011;39:100-109.
M AN U
Butterfield AM, Chain JS, Ackermann BL, Konrad RJ. Comparison of assay formats for drug-tolerant immunogenicity testing. Bioanalysis 2010;2:1961-69. Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, et al. Pure red-cell apalasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin.
TE D
N Engl J Med 2002;146:469-75.
FDA Draft Guidance for Industry: Assay Development for Immunogenicity Testing of
EP
Therapeutic Proteins. http://www.fda.gov/downloads/Drugs/.../Guidances/UCM192750.pdf (accessed 23 March 2016).
AC C
Liang M, Klakamp SL, Funelas C, Lu H, Lam B, Herl C, et al. Detection of high-and lowaffinity antibodies against a human monoclonal antibody using various technology platforms. Assay Drug Dev Tech 2007;5:655-62. Lofgren, JA, Dhandapani S, Pennucci JJ, Abbott CM, Mytych DT, Kaliyaperumal A, et al. Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of Panitumumab. J of Immunol 2007;178:7467-72.
ACCEPTED MANUSCRIPT
Mire-Sluis AR, Barrett YC, 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: 1-16.
RI PT
Moxness M, Tatrewicz S, Weerartne D, Murakami N, Wullner D, Mytych D, et al. Immunogenicity testing by electrochemiluminescent detection for antibodies directed against
SC
therapeutic human monoclonal antibodies. Clin. Chem 2005;51:1983-85.
Patton A, Mullenix MC, Swanson SJ, Koren E. An acid dissociation bridging ELISA for
Immunol Methods 2005;304:189-95.
M AN U
detection of antibodies directed against therapeutic proteins in the presence of antigen. J of
Sauerborn M, Brinks V. Jiskoot W, Schellekens H. Immunological mechanism underlying the immune response to recombinant human protein therapeutics. Trends Pharmacol Sci
TE D
2010;31:53-9.
Sickert D. Kroeger K, Zickler C, Chokote E, Winkler B, Grenet JM, et al. Improvement of drug tolerance in immunogenicity testing by acid treatment on Biacore. J of Immunol
EP
Methods. 2008;334:29-36.
AC C
Smith HW, Butterfield A, and Sun D. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA. Regulatory Tox and Pharmacol 2007;49:230-37.
Wadhwa M, Bird C, Dilger P, Gaines-Das R, Thorpe R. Strategies for detection, measurement, and characterization of unwanted antibodies induced by therapeutic biologicals. J Immunol Methods 2013;278:1–17.
ACCEPTED MANUSCRIPT
Wang, YC, Fang L, Zhou L, Wang J, Ahn HY. A Survey of Applications of Biological
AC C
EP
TE D
M AN U
SC
RI PT
Products for Drug Interference of Immunogenicity Assays. Pharm Res 2012;29:3384-92.
ACCEPTED MANUSCRIPT
Figure Legends
RI PT
Figure 1: Sensitivity of ACE assay compared to MSD bridging assay – Affinity-purified antiixekizumab antibody from monkeys or humans was spiked into normal human serum at concentrations ranging from 2-2000 ng/mL. Samples were analyzed on the ACE and MSD
SC
bridging assays, with results shown as the mean ± SEM (n = 2). The MSD bridging assay reports
M AN U
in ECL units (ECLU), while the ACE assay generates an OD450 readout.
Figure 2: Drug tolerance of ACE assay compared to MSD bridging assay – Affinity-purified anti-ixekizumab antibody from monkeys or humans was spiked into normal human serum at a concentration of 500 ng/mL. Samples were also spiked with concentrations of ixekizumab
TE D
ranging from 0.1-500 µg/mL and analyzed on the ACE and MSD bridging assays. Results are shown as the mean ± SEM (n = 2). The MSD bridging assay reports in ECL units (ECLU), while
AC C
EP
the ACE assay generates an OD450 readout.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
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EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT