Assessment of human antihuman antibodies to eculizumab after long‐term treatment in patients with paroxysmal nocturnal hemoglobinuria
Authors: Peter Hillmen, Petra Muus, Jeffrey Szer, Anita Hill, Britta Höchsmann, Austin Kulasekararaj, Antonio M. Risitano, Eric Van Den Neste, Maria Liljeholm, Kurt S. Ebrahim, Camille L. Bedrosian, Xiang Gao, Donna Ames, Gérard Socié
Journal: American Journal of Hematology (2015)

To the Editor: Eculizumab (Soliris®; Alexion Pharmaceuticals, Cheshire, CT, USA)―a humanized monoclonal antibody (mAb) that displays high-affinity binding to complement protein C5―has been approved by US, European, and other regulatory agencies for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). The administration of any large-molecule therapeutic, such as a mAb, potentially induces an unwanted immune response through the development of antidrug antibodies (ADAs) 1-3. In clinical studies of eculizumab in patients with PNH, the development of HAHAs was transient and very infrequent with minimal impact on clinical response 4-6. This current laboratory study, M07-003 (registered at http://www.clinical trials.gov as NCT01412047), addresses a postapproval commitment to the FDA and the European Medicines Agency (EMA) to examine immunogenicity of eculizumab after long-term treatment. The development of neutralizing and non-neutralizing HAHAs associated with eculizumab use was assessed in patients with PNH who participated in the open-label, long-term study E05-001 7. Briefly, the study included 195 patients with PNH who participated in three prospective trials: a phase two pilot study 5; and two phase three studies 4, 6. The E05-001 study comprised a 104-week treatment period and a 16-week post-treatment follow-up period for patients who terminated treatment early 7. The M07-003 study included patients from E05-001 who had eculizumab-naïve blood samples for comparison. The primary study endpoint was the proportion of patients who developed neutralizing HAHAs during treatment with commercialized eculizumab or who had discontinued treatment. Secondary endpoints were the proportion of patients who developed non-neutralizing HAHAs and the proportion that developed neutralizing HAHAs with evidence of increased hemolysis. Study procedure-related adverse events (AEs), defined as any AE not present prior to protocol-required blood draw and occurring as a result of the blood draw, were monitored and recorded from informed consent until site notification of final HAHA results. AEs were coded using current Medical Dictionary for Regulatory Activities (MedDRA) terminology. The full analysis set included patients with informed consent who had a blood sample collected; the safety analysis included patients with informed consent. Analyses were based on the full analysis set using descriptive statistics for patient disposition and demographics. A validated electrochemiluminescence (ECL) ADA detection method tested for evidence of anti-eculizumab antibody formation―the BTM-0048, analysis of human antibodies against eculizumab in human serum using the Meso Scale Discovery® SECTOR Imager 2400 Validation (Meso Scale Discovery, Rockville, MD).assay. A total of 75 patients were enrolled in the current study; the mean age (standard deviation [SD]) was 48.4 (13.14) years, and most were female (57.3%). At the initial visit, 97.3% (73/75) of patients were receiving eculizumab, most (89.3%; 67/75) at the maintenance dosage of 900 mg infused biweekly; six patients were receiving eculizumab 1200 mg biweekly. The median (range) duration of treatment from the initial dose in study E05-001 to the last treatment before Visit 1 of the current study was 90.2 (77.3–129.0) months, or 7.5 (6.5–10.8) years, for the 72 patients who had treatment data available. Most patients (88%) on treatment received their previous infusion 13–15 days before Visit 1. Antieculizumab HAHA assay findings showed no positive test results for HAHAs. Thus, no assay for confirmation of antibody formation was performed, and no follow-up visits or samples were needed for assessment of hemolysis or pharmacodynamics. Serum samples from 72 patients currently receiving eculizumab and from two patients no longer receiving eculizumab were assayed for serum eculizumab levels (Table 1). Blood samples from two patients on treatment were not collected at trough per protocol and were considered out-of-window. Exclusion of patients with out-of-window measurements had no meaningful impact on results. The median trough level for the on-treatment group, excluding patients with out-of-window measurements, was 113.3 µg/ml (range 31.1–427.4). 75 patients were included in the safety population. A total of three patients experienced eight AEs. One patient experienced sepsis, a serious AE, with no cultured organism. The second patient experienced four AEs: an arterial hemorrhage, a serious AE and mild epistaxis, moderate wound infection, and a skin lesion. A third patient experienced three mild AEs: fatigue, arthralgia, and headache. None of the eight AEs was considered by the investigators to be related to study procedure. The current study demonstrated that long-term eculizumab treatment (>6.0 years) at 900 or 1200 mg biweekly resulted in no development of HAHAs. This analysis provides sufficient evidence to support long-term lack of immunogenicity of commercial eculizumab in the treatment of PNH 8. Moreover, these findings are consistent with shorter-term studies in which HAHAs were infrequent, occurring in 2% (3/196) of patients 9. No sequelae, including rebound hemolysis, were linked to the detection of ADAs in these studies or HAHAs in the present study. Additionally, in the extension study, E05-001, 5 of 161 patients (3.1%) experienced transient positive results on HAHA assays after a follow-up of >66 months, without a discernible impact on clinical response or pharmacodynamic effects of eculizumab, which continued to block complement activity 7, 9. Of these five patients, three have participated in the present HAHA study; testing for antibody formation was negative at the time of enrollment. The frequencies of humanized antitherapeutic antibodies observed in other FDA-approved biologics range from <1 to 9% 10. Enzyme-linked immunosorbent assay (ELISA) was used to assess HAHAs in the three pivotal studies, and an ECL bridging assay was used in the extension and present study 4-6. ELISA has important limitations when assessing HAHA reactivity, as it lacks an acceptable detection reagent for humanized mAbs and is considered complex and time consuming 3. The current study utilized a validated and quantitative ECL bridging assay that was specifically designed to allow detection of an antibody response to any region of the eculizumab molecule, buttressing confidence in the study findings. No study procedure related–AEs occurred. Our study was not designed to assess non–study procedure-related AEs. However, investigators related three mild AEs (fatigue, arthralgia, and headache) in one patient as probably related to eculizumab; these AEs were not unexpected since they have been reported in previous clinical studies 4-7. In summary, this analysis demonstrates that long-term treatment with commercial eculizumab in patients with PNH is associated with minimal risk for the development of HAHAs. Writing assistance in the development of this manuscript was provided by Jim Kesslick and Nicholas C. Stilwell of Connexion Healthcare (Newtown, PA); editorial support provided by Tmirah Haselkorn, PhD of EpiMetrix (Los Altos, CA). Alexion Pharmaceuticals provided funding for these services. The authors meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE) and were fully responsible for all content and editorial decisions, and were involved at all stages of correspondence development. Peter Hillmen1* Petra Muus2 Jeffrey Szer Anita Hill4 Britta Höchsmann5 Austin Kulasekararaj6 Antonio M. Risitano7 Eric Van Den Neste8 Maria Liljeholm9 Kurt S. Ebrahim10 Camille L. Bedrosian11 Xiang Gao11 Donna Ames11 Gérard Socié12 1 Section of Experimental Haematology, St James's University Hospital, Leeds, United Kingdom; 2Department of Hematology, Radboud University Medical Centre, Nijmegen, The Netherlands; 3Department of Clinical Haematology and BMT Service, The Royal Melbourne Hospital, Melbourne, Australia; 4Department of Haematology, St James' Institute of Oncology, Leeds Teaching Hospitals, Leeds, United Kingdom; 5Institute of Transfusion Medicine, University of Ulm, Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen, and University Hospital Ulm, Ulm, Germany; 6King's College Hospital, Hill, Denmark, London, United Kingdom; 7Dipartimento Di Medicina Clinica E Chirurgia Dell' Università Degli Studi Di Napoli Federico II, Napoli, Italy; 8Hematology Department, Cliniques Universitaires UCL Saint-Luc, Brussels, Belgium; 9Hematology Department, University Hospital of Umea, Umea, Sweden; 10New England Cancer Specialists, Scarborough, Maine; 11Alexion Pharmaceuticals, Inc, Cheshire, Connecticut; 12AP-HP Hematology Hospital Saint Louis, Paris, France