{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T13:15:41Z","timestamp":1785244541294,"version":"3.55.0"},"reference-count":55,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2024,6,27]],"date-time":"2024-06-27T00:00:00Z","timestamp":1719446400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FDA intramural research funds"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development\/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E\/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity. We confirm that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Furthermore, we delineated the cellular mechanisms underlying the restored ADCC activity following the recovery cultivation. We observed that CD69, an early marker of NK cell activation, was upregulated and a new subset CD56dim\/CD16dim population was dramatically increased in the recovered NK cells, which led to an increase in expression and secretion of perforin, granzyme B, and cytokine production. This study provides comprehensive technical insights into ADCC bioassay optimization to inform trastuzumab biosimilar development. The knowledge gained from this study can also be leveraged to guide bioassay development for therapeutic mAbs with ADCC as the primary mechanism of action.<\/jats:p>","DOI":"10.3390\/cancers16132367","type":"journal-article","created":{"date-parts":[[2024,6,27]],"date-time":"2024-06-27T13:26:43Z","timestamp":1719494803000},"page":"2367","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance"],"prefix":"10.3390","volume":"16","author":[{"given":"Hanjing","family":"Peng","sequence":"first","affiliation":[{"name":"Division of Pharmaceutical Quality Research III (OPQR III), Office of Pharmaceutical Quality Research (OPQR), Office of Pharmaceutical Quality (OPQ), Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukinori","family":"Endo","sequence":"additional","affiliation":[{"name":"Division of Pharmaceutical Quality Research III (OPQR III), Office of Pharmaceutical Quality Research (OPQR), Office of Pharmaceutical Quality (OPQ), Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5063-0957","authenticated-orcid":false,"given":"Wen Jin","family":"Wu","sequence":"additional","affiliation":[{"name":"Division of Pharmaceutical Quality Research III (OPQR III), Office of Pharmaceutical Quality Research (OPQR), Office of Pharmaceutical Quality (OPQ), Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1038\/s41523-021-00265-1","article-title":"HER2-positive breast cancer and tyrosine kinase inhibitors: The time is now","volume":"7","author":"Schlam","year":"2021","journal-title":"NPJ Breast Cancer"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1038\/s41571-019-0268-3","article-title":"HER2-targeted therapies\u2014A role beyond breast cancer","volume":"17","author":"Oh","year":"2020","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1056\/NEJM200103153441101","article-title":"Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2","volume":"344","author":"Slamon","year":"2001","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"610","DOI":"10.1056\/NEJMoa1914510","article-title":"Trastuzumab Deruxtecan in Previously Treated HER2-Positive Breast Cancer","volume":"382","author":"Modi","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1673","DOI":"10.1056\/NEJMoa052122","article-title":"Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer","volume":"353","author":"Romond","year":"2005","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"S32","DOI":"10.37765\/ajmc.2020.42900","article-title":"Integrating trastuzumab biosimilars and HER2-directed therapies into HER2-positive breast cancer management","volume":"26","author":"Cuellar","year":"2020","journal-title":"Am. J. Manag. Care"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1007\/s10549-021-06197-5","article-title":"Final overall survival analysis of the phase 3 HERITAGE study demonstrates equivalence of trastuzumab-dkst to trastuzumab in HER2-positive metastatic breast cancer","volume":"188","author":"Rugo","year":"2021","journal-title":"Breast Cancer Res. Treat."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1038\/s41416-019-0480-z","article-title":"The arrival of biosimilar monoclonal antibodies in oncology: Clinical studies for trastuzumab biosimilars","volume":"121","author":"Barbier","year":"2019","journal-title":"Br. J. Cancer"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4224","DOI":"10.3390\/curroncol29060337","article-title":"A Review of Trastuzumab Biosimilars in Early Breast Cancer and Real World Outcomes of Neoadjuvant MYL-1401O versus Reference Trastuzumab","volume":"29","author":"Yang","year":"2022","journal-title":"Curr. Oncol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1038\/s41416-018-0340-2","article-title":"PF-05280014 (a trastuzumab biosimilar) plus paclitaxel compared with reference trastuzumab plus paclitaxel for HER2-positive metastatic breast cancer: A randomised, double-blind study","volume":"120","author":"Pegram","year":"2019","journal-title":"Br. J. Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.ejca.2018.01.072","article-title":"A phase III study comparing SB3 (a proposed trastuzumab biosimilar) and trastuzumab reference product in HER2-positive early breast cancer treated with neoadjuvant-adjuvant treatment: Final safety, immunogenicity and survival results","volume":"93","author":"Pivot","year":"2018","journal-title":"Eur. J. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1016\/S1470-2045(17)30434-5","article-title":"CT-P6 compared with reference trastuzumab for HER2-positive breast cancer: A randomised, double-blind, active-controlled, phase 3 equivalence trial","volume":"18","author":"Stebbing","year":"2017","journal-title":"Lancet Oncol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1016\/S1470-2045(18)30241-9","article-title":"Efficacy and safety of ABP 980 compared with reference trastuzumab in women with HER2-positive early breast cancer (LILAC study): A randomised, double-blind, phase 3 trial","volume":"19","author":"Colleoni","year":"2018","journal-title":"Lancet Oncol."},{"key":"ref_14","unstructured":"(2024, April 16). Biosimilar Development, Review, and Approval, Available online: www.fda.gov\/drugs\/biosimilars\/biosimilar-development-review-and-approval#indication."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1056\/NEJMra043186","article-title":"Trastuzumab\u2014Mechanism of action and use in clinical practice","volume":"357","author":"Hudis","year":"2007","journal-title":"N. Engl. J. Med."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Maadi, H., Soheilifar, M.H., Choi, W.S., Moshtaghian, A., and Wang, Z. (2021). Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma. Cancers, 13.","DOI":"10.3390\/cancers13143540"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Li, F., and Liu, S. (2022). Focusing on NK cells and ADCC: A promising immunotherapy approach in targeted therapy for HER2-positive breast cancer. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.1083462"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"105","DOI":"10.21037\/atm.2019.01.42","article-title":"NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: Biological evidence and clinical perspectives","volume":"7","author":"Macagno","year":"2019","journal-title":"Ann. Transl. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.jim.2014.07.010","article-title":"Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies","volume":"414","author":"Cheng","year":"2014","journal-title":"J. Immunol. Methods"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.jim.2005.09.011","article-title":"Maximizing the retention of antigen specific lymphocyte function after cryopreservation","volume":"308","author":"Disis","year":"2006","journal-title":"J. Immunol. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5224","DOI":"10.1038\/s41467-020-19094-0","article-title":"Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells","volume":"11","author":"Mark","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"313","DOI":"10.3390\/cells1030313","article-title":"Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies","volume":"1","author":"Ramachandran","year":"2012","journal-title":"Cells"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1002\/jcla.1860050406","article-title":"Cryopreservation and long-term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis: Effects on cell subset proportions and fluorescence intensity","volume":"5","author":"Tollerud","year":"1991","journal-title":"J. Clin. Lab. Anal."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Lee, S., Joo, Y., Lee, E.J., Byeon, Y., Kim, J.H., Pyo, K.H., Kim, Y.S., Lim, S.M., Kilbride, P., and Iyer, R.K. (2024). Successful expansion and cryopreservation of human natural killer cell line NK-92 for clinical manufacturing. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0294857"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Oyer, J.L., Croom-Perez, T.J., Dieffenthaller, T.A., Robles-Carillo, L.D., Gitto, S.B., Altomare, D.A., and Copik, A.J. (2022). Cryopreserved PM21-Particle-Expanded Natural Killer Cells Maintain Cytotoxicity and Effector Functions. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.861681"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/0022-1759(93)90102-D","article-title":"Human natural killer cells: A convenient purification procedure and the influence of cryopreservation on cytotoxic activity","volume":"165","author":"Voshol","year":"1993","journal-title":"J. Immunol. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jim.2014.01.017","article-title":"Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in Cr-51-release and CD107a assays","volume":"406","author":"Mata","year":"2014","journal-title":"J. Immunol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0022-1759(84)90154-6","article-title":"The standardization of NK cell assays for use in studies of biological response modifiers","volume":"68","author":"Pross","year":"1984","journal-title":"J. Immunol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1007\/978-1-4939-8979-9_12","article-title":"Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay","volume":"1913","author":"Avila","year":"2019","journal-title":"Immune Checkp. Blockade: Methods Protoc."},{"key":"ref_30","unstructured":"Shimasaki, N. (2022). NK Cell Isolation and Cytotoxicity by Radioactive Chromium Release Assay and DELFIA-EuTDA Cytotoxicity Assay. Natural Killer (NK) Cells: Methods and, Protocols, Springer."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Wieckowski, S., Avenal, C., Orjalo, A.V., Gygax, D., and Cymer, F. (2020). Toward a Better Understanding of Bioassays for the Development of Biopharmaceuticals by Exploring the Structure-Antibody-Dependent Cellular Cytotoxicity Relationship in Human Primary Cells. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.552596"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1007\/978-1-62703-327-5_19","article-title":"Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement","volume":"988","author":"Broussas","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"102224","DOI":"10.1016\/j.xpro.2023.102224","article-title":"A co-culture model system to quantify antibody-dependent cellular cytotoxicity in human breast cancer cells using an engineered natural killer cell line","volume":"4","author":"Vincken","year":"2023","journal-title":"STAR Protoc."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1186\/1479-5876-11-307","article-title":"Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic HER2\/neu breast cancer patients","volume":"11","author":"Petricevic","year":"2013","journal-title":"J. Transl. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"19772","DOI":"10.1038\/srep19772","article-title":"A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells","volume":"6","author":"Yamashita","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e296","DOI":"10.1002\/cpz1.296","article-title":"Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays","volume":"1","author":"Garvin","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"810","DOI":"10.4161\/onci.20447","article-title":"Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells","volume":"1","author":"Kute","year":"2012","journal-title":"Oncoimmunology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2815","DOI":"10.4049\/jimmunol.179.5.2815","article-title":"Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen","volume":"179","author":"Tang","year":"2007","journal-title":"J. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.jcyt.2014.09.007","article-title":"Challenges of cancer therapy with natural killer cells","volume":"17","author":"Klingemann","year":"2015","journal-title":"Cytotherapy"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1189\/jlb.0611308","article-title":"Natural killer cell-produced IFN-\u03b3 and TNF-\u03b1 induce target cell cytolysis through up-regulation of ICAM-1","volume":"91","author":"Wang","year":"2012","journal-title":"J. Leukoc. Biol."},{"key":"ref_41","unstructured":"Ackerman, M.E., and Nimmerjahn, F. (2014). Antibody Fc: Linking Adaptive and Innate Immunity, Academic Press."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1046\/j.1365-2567.1999.00738.x","article-title":"CD69 is a stimulatory receptor for natural killer cell and its cytotoxic effect is blocked by CD94 inhibitory receptor","volume":"97","author":"Borrego","year":"1999","journal-title":"Immunology"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"331","DOI":"10.2147\/OTT.S357326","article-title":"An Innovative Site-Specific Anti-HER2 Antibody-Drug Conjugate with High Homogeneity and Improved Therapeutic Index","volume":"15","author":"Hui","year":"2022","journal-title":"Oncotargets Ther."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/S1074-7613(00)80441-0","article-title":"Signal transduction during natural killer cell activation: Inside the mind of a killer","volume":"6","author":"Leibson","year":"1997","journal-title":"Immunity"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"109","DOI":"10.5114\/ceji.2014.42135","article-title":"Perforin: An important player in immune response","volume":"39","author":"Osinska","year":"2014","journal-title":"Cent. Eur. J. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1128\/CDLI.9.5.1049-1056.2002","article-title":"Spontaneous cytokine production and its effect on induced production","volume":"9","author":"Walker","year":"2002","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4844","DOI":"10.4049\/jimmunol.150.11.4844","article-title":"Response of human NK cells to IL-6 alterations of the cell surface phenotype, adhesion to fibronectin and laminin, and tumor necrosis factor-alpha\/beta secretion","volume":"150","author":"Rabinowich","year":"1993","journal-title":"J. Immunol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1002\/eji.201646837","article-title":"CD69: From activation marker to metabolic gatekeeper","volume":"47","author":"Cibrian","year":"2017","journal-title":"Eur. J. Immunol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1084\/jem.178.2.537","article-title":"Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM\/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors","volume":"178","author":"Santis","year":"1993","journal-title":"J. Exp. Med."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1643","DOI":"10.1002\/eji.1830230737","article-title":"Molecular characterization of the early activation antigen CD69: A type II membrane glycoprotein related to a family of natural killer cell activation antigens","volume":"23","author":"Ziegler","year":"1993","journal-title":"Eur. J. Immunol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"928a","DOI":"10.1182\/blood.V106.11.3322.3322","article-title":"CD69 is required for activated NK cell-mediated killing of resistant targets","volume":"106","author":"Marden","year":"2005","journal-title":"Blood"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1016\/S1471-4906(01)02060-9","article-title":"The biology of human natural killer-cell subsets","volume":"22","author":"Cooper","year":"2001","journal-title":"Trends Immunol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3599","DOI":"10.1182\/blood-2012-04-425397","article-title":"NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17)","volume":"121","author":"Romee","year":"2013","journal-title":"Blood"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"3267","DOI":"10.1083\/jcb.201712085","article-title":"Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells","volume":"217","author":"Srpan","year":"2018","journal-title":"J. Cell Biol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Ziegler, S., Weiss, E., Schmitt, A.L., Schlegel, J., Burgert, A., Terpitz, U., Sauer, M., Moretta, L., Sivori, S., and Leonhardt, I. (2017). CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells. Sci. Rep. 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