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Development of a Cell-based Neutralizing Antibody Assay for Zinpentraxin Alfa: Challenges and Mitigation Strategies

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Abstract

Therapeutic protein drugs can potentially induce immune responses in patients and result in the production of anti-drug antibodies (ADAs), including a subset of ADAs called neutralizing antibodies (NAbs) that might cause loss of efficacy by inhibiting clinical activities of the drug. Herein, we describe the unique challenges encountered during the development of a fit-for-purpose cell-based NAb assay for a new protein modality, zinpentraxin alfa, including our strategies for assay design to overcome various matrix interferences and improve assay drug tolerance. We demonstrated that a typical biotin-drug extraction with acid dissociation (BEAD) approach alone was not sufficient to eliminate matrix interferences in this assay. Instead, the combination of the BEAD and ZebaTM spin size exclusion plate (SEP) was required to achieve the desirable assay performance. We also demonstrated that appropriate acidic buffers were critical in sample pretreatment to improve assay drug tolerance, which not only dissociated the drug/NAb immune complex but also effectively and irreversibly denatured the free drug. The final assay performed well with confirmed assay robustness and suitability for the clinical applications.

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References

  1. Boehncke WH, Brembilla NC. Immunogenicity of biologic therapies: causes and consequences. Expert Rev Clin Immunol. 2018;14(6):513–23. https://doi.org/10.1080/1744666X.2018.1468753.

    Article  CAS  PubMed  Google Scholar 

  2. Koren E, Zuckerman LA, Mire-Sluis AR. Immune responses to therapeutic proteins in humans--clinical significance, assessment and prediction. Curr Pharm Biotechnol. 2002;3(4):349–60. https://doi.org/10.2174/1389201023378175.

    Article  CAS  PubMed  Google Scholar 

  3. Shankar G, Pendley C, Stein KE. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol. 2007;25(5):555–61. https://doi.org/10.1038/nbt1303.

    Article  CAS  PubMed  Google Scholar 

  4. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et al. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal. 2008;48(5):1267–81. https://doi.org/10.1016/j.jpba.2008.09.020.

    Article  CAS  PubMed  Google Scholar 

  5. Gunn GR 3rd, Sealey DC, Jamali F, Meibohm B, Ghosh S, Shankar G. From the bench to clinical practice: understanding the challenges and uncertainties in immunogenicity testing for biopharmaceuticals. Clin Exp Immunol. 2016;184(2):137–46. https://doi.org/10.1111/cei.12742.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Gupta S, Devanarayan V, Finco D, Gunn GR 3rd, Kirshner S, Richards S, et al. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. J Pharm Biomed Anal. 2011;55(5):878–88. https://doi.org/10.1016/j.jpba.2011.03.038.

    Article  CAS  PubMed  Google Scholar 

  7. Nahtman T, Jernberg A, Mahdavifar S, Zerweck J, Schutkowski M, Maeurer M, et al. Validation of peptide epitope microarray experiments and extraction of quality data. J Immunol Methods. 2007;328(1-2):1–13. https://doi.org/10.1016/j.jim.2007.07.015.

    Article  CAS  PubMed  Google Scholar 

  8. Wu B, Chung S, Jiang XR, McNally J, Pedras-Vasconcelos J, Pillutla R, et al. Strategies to determine assay format for the assessment of neutralizing antibody responses to biotherapeutics. AAPS J. 2016;18(6):1335–50. https://doi.org/10.1208/s12248-016-9954-6.

    Article  CAS  PubMed  Google Scholar 

  9. FDA Guidance Document. Immunogenicity testing of therapeutic protein products - developing and validating assays for anti-drug antibody detection. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-and-validating-assays-anti-drug. Accessed Feb 2019.

  10. Zhong ZD, Clements-Egan A, Gorovits B, Maia M, Sumner G, Theobald V, et al. Drug target interference in immunogenicity assays: recommendations and mitigation strategies. AAPS J. 2017;19(6):1564–75. https://doi.org/10.1208/s12248-017-0148-7.

    Article  CAS  PubMed  Google Scholar 

  11. Zoghbi J, Xu Y, Grabert R, Theobald V, Richards S. A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays. J Immunol Methods. 2015;426:62–9. https://doi.org/10.1016/j.jim.2015.08.002.

    Article  CAS  PubMed  Google Scholar 

  12. Bourdage JS, Cook CA, Farrington DL, Chain JS, Konrad RJ. 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(1-2):10–7. https://doi.org/10.1016/j.jim.2007.07.004.

    Article  CAS  PubMed  Google Scholar 

  13. Smith HW, Butterfield A, 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. Regul Toxicol Pharmacol. 2007;49(3):230–7. https://doi.org/10.1016/j.yrtph.2007.07.005.

    Article  CAS  PubMed  Google Scholar 

  14. Xu W, Sank M, Cummings J, Carl S, Juhel M, Gleason C, et al. Bead-extraction and heat-dissociation (BEHD): a novel way to overcome drug and matrix interference in immunogenicity testing. J Immunol Methods. 2018;462:34–41. https://doi.org/10.1016/j.jim.2018.08.003.

    Article  CAS  PubMed  Google Scholar 

  15. Lofgren JA, Wala I, Koren E, Swanson SJ, Jing S. Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein. J Immunol Methods. 2006;308(1-2):101–8. https://doi.org/10.1016/j.jim.2005.10.007.

    Article  CAS  PubMed  Google Scholar 

  16. Xu W, Cummings J, Sank M, Juhel M, Li X, Gleason C, et al. Development and validation of a functional cell-based neutralizing antibody assay for ipilimumab. Bioanalysis. 2018;10(16):1273–87. https://doi.org/10.4155/bio-2018-0109.

    Article  CAS  PubMed  Google Scholar 

  17. Wu B, Schnarr M, Devlin JL, Brown S, Yang TY. Approaches to improve drug tolerance and target tolerance in the assessment of neutralizing anti-drug antibodies. Bioanalysis. 2019;11(22):2061–74. https://doi.org/10.4155/bio-2019-0184.

    Article  CAS  PubMed  Google Scholar 

  18. Luong M, Wang Y, Berasi SP, Buhlmann JE, Yang H, Gorovits B. Development of a cell-based assay for the detection of neutralizing antibodies to PF-06730512 using homogenous time-resolved fluorescence. AAPS J. 2020;22(2):56. https://doi.org/10.1208/s12248-020-0431-x.

    Article  CAS  PubMed  Google Scholar 

  19. Xu W, Jiang H, Titsch C, Haulenbeek JR, Pillutla RC, Aubry AF, et al. Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays. J Immunol Methods. 2015;416:94–104. https://doi.org/10.1016/j.jim.2014.11.005.

    Article  CAS  PubMed  Google Scholar 

  20. Jiang Z, Kamerud J, Zhang M, Ruiz CC, Guadiz C, Fichtner A, et al. Strategies to develop highly drug-tolerant cell-based neutralizing antibody assay: neutralizing antidrug antibodies extraction and drug depletion. Bioanalysis. 2020;12(18):1279–93. https://doi.org/10.4155/bio-2020-0091.

    Article  CAS  PubMed  Google Scholar 

  21. Wickramarachchi D, Wagner J, Woo T, Ferrari F, Steinmetz T, Helmy R, et al. A novel neutralization antibody assay method to overcome drug interference with better compatibility with acid-sensitive neutralizing antibodies. AAPS J. 2023;25(1):18. https://doi.org/10.1208/s12248-023-00783-9.

    Article  CAS  PubMed  Google Scholar 

  22. Raghu G, Hamblin MJ, Brown AW, Golden JA, Ho LA, Wijsenbeek MS, et al. Long-term evaluation of the safety and efficacy of recombinant human pentraxin-2 (rhPTX-2) in patients with idiopathic pulmonary fibrosis (IPF): an open-label extension study. Respir Res. 2022;23(1):129. https://doi.org/10.1186/s12931-022-02047-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Raghu G, van den Blink B, Hamblin MJ, Brown AW, Golden JA, Ho LA, et al. Long-term treatment with recombinant human pentraxin 2 protein in patients with idiopathic pulmonary fibrosis: an open-label extension study. Lancet Respir Med. 2019;7(8):657–64. https://doi.org/10.1016/S2213-2600(19)30172-9.

    Article  CAS  PubMed  Google Scholar 

  24. Dillingh MR, van den Blink B, Moerland M, van Dongen MG, Levi M, Kleinjan A, et al. Recombinant human serum amyloid P in healthy volunteers and patients with pulmonary fibrosis. Pulm Pharmacol Ther. 2013;26(6):672–6. https://doi.org/10.1016/j.pupt.2013.01.008.

    Article  CAS  PubMed  Google Scholar 

  25. Sen JW, Recke C, Rahbek L, Skogstrand K, Heegaard NH. Structural, quantitative and functional comparison of amyloid P component in sera from patients with systemic lupus erythematosus and healthy donors. Scand J Immunol. 2002;56(6):645–51. https://doi.org/10.1046/j.1365-3083.2002.01178.x.

    Article  CAS  PubMed  Google Scholar 

  26. Duffield JS, Lupher ML Jr. PRM-151 (recombinant human serum amyloid P/pentraxin 2) for the treatment of fibrosis. Drug News Perspect. 2010;23(5):305–15. https://doi.org/10.1358/dnp.2010.23.5.1444206.

    Article  CAS  PubMed  Google Scholar 

  27. Verstovsek S, Mesa RA, Foltz LM, Gupta V, Mascarenhas JO, Ritchie EK, et al. Phase 2 trial of PRM-151, an anti-fibrotic agent, in patients with myelofibrosis: stage 1 results. blood. 2014;124(21):713.

    Article  Google Scholar 

  28. Verstovsek S, Hasserjian RP, Pozdnyakova O, Veletic I, Mesa RA, Foltz LM, et al. PRM-151 in myelofibrosis: efficacy and safety in an open label extension study. Blood. 2018;132(Supplement 1):686.

    Article  Google Scholar 

  29. Raghu G, van den Blink B, Hamblin MJ, Brown AW, Golden JA, Ho LA, et al. Effect of recombinant human pentraxin 2 vs placebo on change in forced vital capacity in patients with idiopathic pulmonary fibrosis: a randomized clinical trial. JAMA. 2018;319(22):2299–307. https://doi.org/10.1001/jama.2018.6129.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lu J, Marnell LL, Marjon KD, Mold C, Du Clos TW, Sun PD. Structural recognition and functional activation of FcgammaR by innate pentraxins. Nature. 2008;456(7224):989–92. https://doi.org/10.1038/nature07468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Castano AP, Lin SL, Surowy T, Nowlin BT, Turlapati SA, Patel T, et al. Serum amyloid P inhibits fibrosis through Fc gamma R-dependent monocyte-macrophage regulation in vivo. Sci Transl Med. 2009;1(5):5ra13. https://doi.org/10.1126/scitranslmed.3000111.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lu J, Mold C, Du Clos TW, Sun PD. Pentraxins and Fc receptor-mediated immune responses. Front Immunol. 2018;9:2607. https://doi.org/10.3389/fimmu.2018.02607.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nakagawa N, Barron L, Gomez IG, Johnson BG, Roach AM, Kameoka S, et al. Pentraxin-2 suppresses c-Jun/AP-1 signaling to inhibit progressive fibrotic disease. JCI Insight. 2016;1(20):e87446. https://doi.org/10.1172/jci.insight.87446.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Bottazzi B, Garlanda C, Teixeira MM. Editorial: The role of pentraxins: from inflammation, tissue repair and immunity to biomarkers. Front Immunol. 2019;10:2817. https://doi.org/10.3389/fimmu.2019.02817.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Du Clos TW. Pentraxins: structure, function, and role in inflammation. ISRN Inflamm. 2013;2013:379040. https://doi.org/10.1155/2013/379040.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Moreira AP, Cavassani KA, Hullinger R, Rosada RS, Fong DJ, Murray L, et al. Serum amyloid P attenuates M2 macrophage activation and protects against fungal spore-induced allergic airway disease. J Allergy Clin Immunol. 2010;126(4):712–21 e7. https://doi.org/10.1016/j.jaci.2010.06.010.

    Article  CAS  PubMed  Google Scholar 

  37. Evans TC Jr, Nelsestuen GL. Dissociation of serum amyloid P from C4b-binding protein and other sites by lactic acid: potential role of lactic acid in the regulation of pentraxin function. Biochemistry. 1995;34(33):10440–7. https://doi.org/10.1021/bi00033a016.

    Article  CAS  PubMed  Google Scholar 

  38. Breathnach SM, Kofler H, Sepp N, Ashworth J, Woodrow D, Pepys MB, et al. Serum amyloid P component binds to cell nuclei in vitro and to in vivo deposits of extracellular chromatin in systemic lupus erythematosus. J Exp Med. 1989;170(4):1433–8. https://doi.org/10.1084/jem.170.4.1433.

    Article  CAS  PubMed  Google Scholar 

  39. Hamazaki H. Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate. J Biol Chem. 1987;262(4):1456–60.

    Article  CAS  PubMed  Google Scholar 

  40. Li XA, Yutani C, Shimokado K. Serum amyloid P component associates with high density lipoprotein as well as very low density lipoprotein but not with low density lipoprotein. Biochem Biophys Res Commun. 1998;244(1):249–52. https://doi.org/10.1006/bbrc.1998.8248.

    Article  CAS  PubMed  Google Scholar 

  41. Omtvedt LA, Wien TN, Myran T, Sletten K, Husby G. Serum amyloid P component in mink, a non-glycosylated protein with affinity for phosphorylethanolamine and phosphorylcholine. Amyloid. 2004;11(2):101–8. https://doi.org/10.1080/13506120410001728063.

    Article  CAS  PubMed  Google Scholar 

  42. Ying SC, Gewurz AT, Jiang H, Gewurz H. Human serum amyloid P component oligomers bind and activate the classical complement pathway via residues 14-26 and 76-92 of the A chain collagen-like region of C1q. J Immunol. 1993;150(1):169–76.

    Article  CAS  PubMed  Google Scholar 

  43. Schwalbe RA, Dahlback B, Nelsestuen GL. Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes. J Biol Chem. 1990;265(35):21749–57.

    Article  CAS  PubMed  Google Scholar 

  44. Zahedi K. Characterization of the binding of serum amyloid P to type IV collagen. J Biol Chem. 1996;271(25):14897–902. https://doi.org/10.1074/jbc.271.25.14897.

    Article  CAS  PubMed  Google Scholar 

  45. de Beer FC, Baltz ML, Holford S, Feinstein A, Pepys MB. Fibronectin and C4-binding protein are selectively bound by aggregated amyloid P component. J Exp Med. 1981;154(4):1134–9. https://doi.org/10.1084/jem.154.4.1134.

    Article  PubMed  Google Scholar 

  46. Bharadwaj D, Mold C, Markham E, Du Clos TW. Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis. J Immunol. 2001;166(11):6735–41. https://doi.org/10.4049/jimmunol.166.11.6735.

    Article  CAS  PubMed  Google Scholar 

  47. Mold C, Baca R, Du Clos TW. Serum amyloid P component and C-reactive protein opsonize apoptotic cells for phagocytosis through Fcgamma receptors. J Autoimmun. 2002;19(3):147–54. https://doi.org/10.1006/jaut.2002.0615.

    Article  PubMed  Google Scholar 

  48. Park EK, Jung HS, Yang HI, Yoo MC, Kim C, Kim KS. Optimized THP-1 differentiation is required for the detection of responses to weak stimuli. Inflamm Res. 2007;56(1):45–50. https://doi.org/10.1007/s00011-007-6115-5.

    Article  CAS  PubMed  Google Scholar 

  49. Lund ME, To J, O'Brien BA, Donnelly S. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus. J Immunol Methods. 2016;430:64–70. https://doi.org/10.1016/j.jim.2016.01.012.

    Article  CAS  PubMed  Google Scholar 

  50. Teillaud C, Galon J, Zilber MT, Mazieres N, Spagnoli R, Kurrle R, et al. Soluble CD16 binds peripheral blood mononuclear cells and inhibits pokeweed-mitogen-induced responses. Blood. 1993;82(10):3081–90.

    Article  CAS  PubMed  Google Scholar 

  51. Jiang H, Xu W, Titsch CA, Furlong MT, Dodge R, Voronin K, et al. Innovative use of LC-MS/MS for simultaneous quantitation of neutralizing antibody, residual drug, and human immunoglobulin G in immunogenicity assay development. Anal Chem. 2014;86(5):2673–80. https://doi.org/10.1021/ac5001465.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We want to thank Dr. Audrey Arjomandi and Dr. Tao Sun for providing support on critical reagents generation, Dr. Florian Cymer and Dr. Sebastien Wieckowski for helpful discussions.

Funding

All work was funded by Genentech Inc., a member of the Roche group.

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Authors and Affiliations

  1. BioAnalytical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA

    Zhaojun Yin, Joyce Guerrero, Rachel Melendez, Ben Andrews & Kun Peng

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  1. Zhaojun Yin
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  2. Joyce Guerrero
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All authors contributed to the experimental design and data analysis. ZY, JG, BA, and RM did the experimental work. ZY and KP drafted the manuscript.

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Correspondence to Zhaojun Yin.

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Yin, Z., Guerrero, J., Melendez, R. et al. Development of a Cell-based Neutralizing Antibody Assay for Zinpentraxin Alfa: Challenges and Mitigation Strategies. AAPS J 25, 75 (2023). https://doi.org/10.1208/s12248-023-00841-2

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