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An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody

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Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743 ng/mL and can detect 1500 ng/mL of a surrogate positive control antibody in the presence of 1500 ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

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All supporting data for this study is securely stored at Genentech and will be immediately available upon request.


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Authors would like to thank Patty Siguenza for her support and guidance, Justin Lowe for providing the goat anti-ocrelizumab polyclonal antibody, Genentech Antibody Engineering for providing anti-ocrelizumab monoclonal antibodies, and Genentech Biological Technology for providing the NK cell line. We would also like to thank Anshin BioSolutions for assistance with the preparation of the manuscript.


These studies were sponsored by Genentech, Inc. and F. Hoffmann-La Roche Ltd.

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Authors and Affiliations



VN and SC conceptualized and implemented the assay strategy. VN and AC participated in the assay validation. VN, RH, and SC undertook data analysis and interpretation. VN, RH, KP, and SC wrote and reviewed the manuscript. All authors critically reviewed and edited draft versions of the paper and approved the final version.

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Correspondence to Van Nguyen.

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All authors are employees, past employees, and stockholders of Genentech, Inc. and F. Hoffmann-La Roche Ltd.

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Nguyen, V., Cheung, A., Hendricks, R. et al. An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody. AAPS J 25, 97 (2023).

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