Clinical Therapeu tics /Volume 32, Number 9, 2010 Subset Analysis of Patients Experiencing Clinical Events of a Potentially Immunogenic Nature in the Pivotal Clinical Trials of Tocilizumab for Rheumatoid Arthritis: Evaluation of an Antidrug Antibody ELISA Using Clinical Adverse Event- Driven Immunogenicity Testing Kay Stubenrauch, PhD'; Uwe Wessels, DP; Herbert Birnboeck, PhD2; Francisco Ramirez, PhD3; AngelikaJahreis, MD, PhD4; and Julia Schleypen, PhD' 1 Department of Bioanalytics, Pharma Research Penzberg, Roche Diagnostics GmbH, Pemberg, Germany; 2Non-Clinical Safety, F Hoffmann-La Roche AG, Basel, Switzerland; 3Department of Biostatistics, Pharma Development, Roche Products Ltd. , Welwyn, United Kingdom; and "Product Development Inflammation, Genentech Inc. , South San Francisco, California ------ - _. ABSTRACT Background: Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody directed against the interleukin-6 receptor. In Europe, TCZ is approved for use in combination with methotrexate in the treatment of adult patients with moderate to severe rheumatoid arthritis (RA) who have failed to respond to or were unable to tolerate previous therapy with one or more disease-modifying antirheumatic drugs or tumor necrosis factor (TNF) antagonists; in the United States, it is ap- proved for the treatment of adult patients with moderate to severe active RA who have failed to respond to one or more TNF antagonists. As part of the Phase III clinical development program, the immunogenicity ofTCZ was evaluated using a bridging ELISA; however, this assay is considered limited in detect ing low-affinity or immuno- globulin G4 subisotype antidrug antibodies (ADAs). Objective: This study assessed the validity of the ELISA for detecting anti-TCZ ADAs by using comple- mentary bioanalytic assays to test samples from a sub- group of patients with clinical adverse events (AEs) of a potentially immunogenic nature, who were consid- ered highly likely to have ADAs.The goal was to deter- mine whether use of these additional assays led to detection of ADAs not found on the ELISA, thu s min i- mizing the risk of false-negat ive results. Methods: The Phase III program for TCZ consisted of 5 core studies in which adult patients with RA received either TCZ 4 or 8 mg/kg IV or control every 4 weeks, with or without concomitant antirheumatic therapy. Blood samples obtained at baseline and at regular in- August 2010 tervals thereafter were tested using the ELISAfor ADA screening and confirmation. Regardless of the results on ADAscreening, samples from patients who developed clinical AEs of a potentially immunogenic nature (ie, falling within predefined system organ classes, occurring during or within 24 hours ofTCZ infusion, considered related to TCZ therapy, or leading to study withdrawal) were subjected to add itional testing with a surface plasmon resonance (SPR) assay for isoryping and epitope localization and a standard ImmunoCAP immunoglobu- lin E (lgE) assay made specific for TCZ. Results: The 5 core studies and their open-label, long- term extension studies enrolled a total of 4199 adult patients with RA (82.1% female; 74.0% white; mean age, 52 .0 years [range, 18-89 years]; mean weight, 73.4 kg [range, 35- 150 kg]); 292 8 patients received TCZ and 1271 received control. Of the 2816 samples from TCZ- treated patients tested, 64 (2.3%) had samples that tested positive at least once on the ELISAscreening and confirmation assay, 48 (75.0% ) of them at baseline. A clinical AE of a potentially immunogenic nature occurred during TCZ treatment in 21 patients, 8 of whom had an anaphylactic reacti on. Eleven of the samples from Th is work was presented in part as a post er at th e Annua l Scientific Meeting of the American College of Rhe um atol ogy, O ctobe r 24-29, 2008 , in Sa n Fran cisco , Ca lifornia . Acc epted fo r publicationjune 24, 20 10. Express Track online publication July 29, 2010. doi:l 0.1016/j.c1inthera.201 0.07.021 0149-2918/$ - see front matt er © 2010 Excerpta Medica Inc. All rights reserved. 1597