title: False-positive immunogenicity responses are caused by CD20+ B cell membrane fragments in an anti-ofatumumab antibody bridging assay.
abstract: An electrochemiluminescent (ECL) bridging assay to detect anti-ofatumumab antibodies (ADA) in human serum samples was developed and validated. Using this assay format, clinical samples were first screened to identify potential ADA positive samples, which were then further tested by adding excess drug, confirming the positive signals as drug specific. However, when the method was implemented into clinical studies for ADA testing, a high positive rate was observed in the pre-dose samples collected from patients with chronic lymphocytic leukemia (CLL). Since the positive signals were not associated with ofatumumab (Ofa) treatment, and diminished after treatment, it was suspected that matrix interference might be responsible, resulting in false-positive responses. We performed a series of experimental investigations to identify, characterize, minimize or eliminate the possible false-positive responses. One possible source was identified to be CD20 (the target of Ofa) present on cell membrane fragments (CMFs). The false-positive responses caused by CD20(+) CMFs could be reduced by solid-phase immunodepletion, ultracentrifugation, or inhibited by adding another anti-CD20 antibody (rituximab). As a consequence, the ADA method was modified to minimize the matrix interference caused by CD20(+) CMFs and, then, validated for sample testing.
authors: Clinical Immunology, Biopharm R&D, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19406, USA. Keguan.x.chen@gsk.com
authors: Keguan
authors: K
authors: Chen
authors: Jerry G
authors: JG
authors: Page
authors: Ann M
authors: AM
authors: Schwartz
authors: Thomas N
authors: TN
authors: Lee
authors: Stephen L
authors: SL
authors: DeWall
authors: Daniel J
authors: DJ
authors: Sikkema
authors: Catherine
authors: C
authors: Wang
mesh_terms: D000906:Antibodies / Q000032:analysis*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized; D018951:Antigens, CD20 / Q000032:analysis*; D001402:B-Lymphocytes / Q000276:immunology*; D005189:False Positive Reactions; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology*; D018613:Microscopy, Confocal
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018951:Antigens, CD20; D008565:Membrane Proteins; C527517:ofatumumab
keywords: ADA; CD20; CLL; CMFs; Cell membrane fragments; Drug target interference; ECL; Immunogenicity; MSD; MVs; Meso-Scale Discovery; NC; Ofa; Ofatumumab; PC; Pre-dose positives; RTX; anti-drug antibody; cell membrane fragments; chronic lymphocytic leukemia; electrochemiluminescent; microvesicles; negative control; positive control; rituximab