Results of Long-Term Therapy with a Biosimilar of Eculizumab in Patients with Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal hematopoietic stem cell disease that presents with hemolytic anemia, thrombosis, and smooth muscle dystonias, and in some cases is associated with bone marrow failure [1]. The disease is associated with the expansion of hematopoietic stem cell clones with a somatic mutation of the PIGA gene. This impairs the synthesis of the carbohydrate moiety of the glycosylphosphatidylinositol (GPI) anchor of GPI-anchored proteins, including membrane complement inhibitors CD55 and CD59 [2, 3]. The absence of CD59 on PNH red blood cells leads to uncontrolled formation of membrane attack complexes (MACs) [4, 5], which results in red blood cell hemolysis, release of hemoglobin into the plasma, nitric oxide (NO) consumption, and platelet activation [6, 7].

The inhibition of MAC formation using eculizumab, a humanized anticomplement component C5 antibody, remains the mainstay of treatment for PNH [7, 8]. Several studies have shown successful control of intravascular hemolysis and prevention of life-threatening complications both in clinical trials and in real-world clinical practice [9‒11].

Elizaria^® developed as a biosimilar has been used in the Russian Federation since 2019 for the treatment of PNH and atypical hemolytic uremic syndrome [12]. The biosimilar product was developed in accordance with the regulatory requirements of the Eurasian Economic Union (EAEU) and the European Medicines Agency (EMA) [12‒15]. In the early stages of clinical development of biosimilar, phase I and Ib clinical trials were conducted to confirm sufficient safety and tolerability of the product, as well as to study the pharmacokinetic (PK) and pharmacodynamic (PD) properties of investigated product following its single-dose administration in healthy volunteers and as induction therapy in treatment-naive PNH patients [16]. A phase III clinical trial (NCT04463056 on clinicaltrial.gov) has demonstrated non-inferiority of the investigational biosimilar product to the originator product Soliris [17]. This clinical trial ECU-PNH-III-X (NCT04679103, clicaltrials.gov) was carried out to assess the long-term safety, efficacy, and immunogenicity of eculizumab biosimilar in patients with PNH.

An open-label, prospective, multicenter study of the long-term safety, immunogenicity, PKs, PDs, and efficacy of eculizumab biosimilar in patients who had previously completed the ECU-PNH-III and ECU-PNH-Ib clinical trials was conducted at 8 study sites (https://clinicaltrials.gov/study/NCT04671810). After signing the informed consent form, 30 patients with PNH aged 23–75 years were enrolled in the clinical trial. Prior to this study, 25 patients received Elizaria^® (Group A), and 5 patients received Soliris^® (Group B). All patients were vaccinated against meningococcal infection with a tetravalent vaccine (Neisseria meningitidis serotypes A, C, Y, and W135), and also agreed to use reliable methods of contraception throughout the study period and for 10 weeks after the last dose of the investigational product.

Patients with hypersensitivity to the investigational product and its components, as well as active ingredients and components of the meningococcal vaccine, patients with a history of infectious diseases caused by N. meningitidis, and patients with a concomitant disease or condition that the investigator believed could endanger the patient’s safety during the study, were excluded.

A patient could be excluded during the study for the following reasons: ≥2 events of grade 3 toxicity, or ≥1 event of grade 4 toxicity according to the CTCAE 4.03, which the investigator considered as associated with the investigational product; diagnosis of cancer; meningococcal infection; treatment with immunosuppressants, except for eculizumab, glucocorticosteroids, androgens, erythropoietin products, or adjusted-dose warfarin; refusal to participate or noncompliance with study procedures; significant deviations from the protocol (ver. 4.0 dated 13.07.2018). Any outcome data were collected for participants who could discontinue or deviate from intervention protocols.

The open-label, prospective, multicenter study included the following periods: the end of study visit of previous trials (Switching Visit), which was the first visit of this study; the treatment period (up to 104 weeks); and the follow-up period (2 weeks). If the product became commercially available to an individual patient, the patient would withdraw from the study.

The patients received the biosimilar at a maintenance dose of 900 mg every 2 weeks. The maximum duration of treatment was 104 weeks. During the study, patients received from 18 to 44 injections of the biosimilar, the average number of injections was 32.33 ± 6.74). In previous clinical trials, all patients were immunized with a meningococcal vaccine at least 14 days before the first eculizumab infusion or received preventive antibacterial therapy for at least 2 weeks after vaccination. Re-immunization during the clinical study was possible, if necessary.

The study procedures included physical examination, anthropometry, assessment of vital signs, electrocardiography, and Doppler echocardiography with an assessment of the mean pulmonary artery pressure and the degree of pulmonary hypertension. Blood samples were taken for red blood cell/granulocyte PNH clone tests (according to the high-sensitivity flow cytometry protocol [18]), complete blood count and blood chemistry with glomerular filtration rate (GFR) calculation, coagulation profile, and D-dimer test. Urine samples were taken for routine urinalysis.

The PK analysis (C_min, which is the minimum product concentration at the end of the dosing interval) included the data of 5 patients from group B. Blood samples for the PK/PD studies were taken at visit 1 and after 2, 4, and 6 weeks of treatment, 5 min pre-dose. The analysis was performed using Octet^® QKe System interferometer (Pall ForteBio). All calculations were performed automatically using Octet^® Software, v.10.0 (Pall ForteBio). The analytical range for quantitative determination of eculizumab in serum samples was 6.25–1,200 μg/mL. The PD analysis included the measurement of membrane attack complex serum concentration (MAC, C5b-9) by enzyme-linked immunosorbent assay (MicroVue Complement SC5b-9 Plus EIA [Quidel, San Diego, CA, USA]) at the same time periods as for the PK analysis.

The therapy safety was assessed by the incidence and severity of adverse reactions (ARs) according to symptoms, physical examination, assessment of vital signs, electrocardiography, laboratory and instrumental findings, and patient diaries. All adverse events (AEs) reported in the study were analyzed by their association with the investigational products (definite, probable, or possible), system organ classes, preferred terms, and treatment groups. The AEs and ARs were coded using the Medical Dictionary for Regulatory Activities (MedDRA), version 21.0, and presented with the aggregation of preferred terms by System Organ Classes.

Immunogenicity was determined by the level of antidrug antibodies (ADAs), including the neutralizing activity of antibodies to eculizumab, by bridging enzyme-linked immunosorbent assay. The assay was carried out at Weeks 12, 24, 36, 48, 60, 72, 84, and 102 during the treatment period. The immunogenicity analysis was performed considering the total duration of therapy, including the results of previous studies. The analysis results before the first dose of the product in previous clinical studies (ECU-PNH-Ib and ECU-PNH-III) were considered as baseline.

Efficacy variables included changes in the levels of lactate dehydrogenase (LDH), hemoglobin, reticulocytes, and red blood cell/granulocyte PNH clone. In addition, the number of patients requiring blood transfusions was evaluated.

R 4.0.3 [19] was used for statistical analysis, which evaluated 2 patient populations. All patients enrolled in the study comprised the full analysis set (FAS). This population was used to assess the efficacy, safety, and immunogenicity, as well as to describe baseline characteristics, including patient demographics, anthropometric parameters, and underlying and concomitant diseases. The population for PK and PD analysis (PK/PD population) included all patients with data available to assess at least one of the prespecified PK/PD parameters. The analysis was performed in two groups of patients depending on the previous therapy: group A included patients who continued receiving the biosimilar, and group B included patients who switched from the originator product to the biosimilar product.

Qualitative attributes were described using the absolute number of observations, percentages, and two-sided 95% CIs for percentages. Nonparametric Mann-Whitney and Friedman tests were used to compare quantitative parameters between groups and changes in these parameters between visits in each group, respectively, since all study populations and groups were represented by small subsets.

The study enrolled 30 patients, of which 25 (83%) and 5 (17%) previously received biosimilar (group A) and originator (group B), respectively. All patients completed the study according to the protocol when the biosimilar became commercially available, each at their own visit. As a result, the data at the end of study visit were actually collected at several different time points. The PK/PD population accounted for 16.67% of the FAS population (5 patients) and included only patients from group B.

The median age of patients was 37 years (39 years in group A and 33 years in group B). The study included 12 male and 18 female patients. Patients in groups A and B were comparable in terms of all baseline characteristics (shown in Table 1).

The most common concomitant diseases in the study subjects were vascular diseases (50% of patients) and cardiac disorders (36.7% of patients). There were no significant differences in the manifestations of PNH and incidence of various comorbidities between the groups (p > 0.05).

In group B, where patients switched from the originator to the biosimilar prior to enrollment in this study, the mean serum concentration of eculizumab was 127.77 ± 63.33 μg/mL at visit 1, 128.62 ± 57.96 μg/mL at visit 2, 112.54 ± 48.84 μg/mL at visit 3, and 126.54 ± 62.73 μg/mL at visit 4. High variability in the product concentration was observed at all time points; the coefficient of variation was close to 50% but did not exceed this value (shown in Fig. 1).

At the end of each dosing interval, the mean eculizumab concentration 5 min pre-dose at all visits exceeded 35 μg/mL, which is the minimum concentration sufficient for complete inhibition of intravascular hemolysis in patients with PNH [20, 21]. The PD analysis showed stable MAC levels in the blood at all visits after switching from the originator product to the biosimilar product. The concentration of MAC was 70.25 ± 34.86 ng/mL at visit 1, 57.03 ± 32.87 ng/mL at visit 2, 59.73 ± 29.08 ng/mL at visit 3, and 63.24 ± 31.44 at Visit 4 (p = 0.66) (shown in Fig. 1).

The median LDH activity ranged from 226 (197.8; 300) U/L to 320 (235.8; 377.5) U/L, and median changes in LDH activity from baseline (visit 1) ranged from −3.5 (−84.3; 130.8) U/L to −98.5 (−128.3; −68.8) U/L. The analysis of changes in LDH activity showed that the median LDH activity in the FAS population was stable throughout the study (p = 0.21). There was an insignificant increase in the LDH activity at week 64 due to acute respiratory viral infection in 1 patient, and at week 68 due to an exacerbation of chronic hepatitis in another patient. The intergroup comparison demonstrated no significant differences in LDH activity at each visit, and there were no significant changes in LDH activity from baseline (visit 1) (p > 0.05) (shown in Fig. 2).

Both groups demonstrated comparable positive mean changes in the hemoglobin level at the end of the study, which were 3.7 ± 13.6 g/L in group A and 1 ± 2.5 g/L in group B (p = 0.36). There were no significant changes in the hemoglobin level at each visit compared to baseline (visit 1) both in the general population (p > 0.05) and in groups A (p = 0.090) and B (p = 0.54) (shown in Table 2).

The reticulocyte count in the general population was stable throughout the study; there were no significant changes compared to visit 1 (p = 0.06). The intergroup comparison showed no significant differences in the reticulocyte count at each visit, as well as compared to baseline (visit 1) (p > 0.05). At the end of the study, both groups showed insignificant changes in the reticulocyte count: −1.48 ± 7.1% in group A and 0.45 ± 5.56% in group B (p = 1.00).

With regard to changes in PNH type II + III red blood cells and granulocytes compared to baseline values at screening, similar changes in mean values were observed in both groups. At the end of the study, groups A and B demonstrated an insignificant decrease in type II + III red blood cells, which was −3.2 ± 18.42% and −1.08 ± 16.73%, respectively (p = 0.79), as well as a decrease in the percentage of PNH granulocytes, which was −1.81 ± 27.69% in group A and −0.79 ± 4.36% in group B (p = 0.65).

Breakthrough hemolysis was recorded in 4 patients in group A (shown in Table 2). In 2 patients, it occurred after a respiratory infection. In another 2 patients, only darkening of urine was observed, which did not require treatment adjustment.

Eight (27%) patients received red blood cell transfusions from the start of therapy to the end of the study, with no significant differences between groups A (28%) and B (20%). No new thrombotic events were reported in the study.

During the study period, none of the patients developed a meningococcal infection or other infections potentially associated with long-term terminal complement inhibition. None of the evaluated patients had SAEs associated with hemolysis.

Among the reported 362 AEs, only two episodes in two patients had at least a possible association with the investigational product: alopecia in group A (4%) and elevated aspartate aminotransferase in Group B (20%) (shown in online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000542294). These ARs were mild (elevated aspartate aminotransferase) and moderate (alopecia) according to the NCI CTCAE 4.03 and did not require drug treatment.

Throughout the study, binding ADAs were detected in 7/30 (23.3%) patients: 6/25 (24%) in group A and 1/5 (20%) in group B. According to the immunogenicity analysis, there were no significant differences between the groups with regard to the frequency of ADA detection at any study visit (p > 0.05). The ADAs detected in patients of group A did not show neutralizing activity or affect the efficacy and safety parameters in these patients. Neutralizing antibodies were detected in 1 patient who received the originator product (group B) prior to enrollment in this study. However, the formation of neutralizing antibodies in this patient did not affect the efficacy and safety parameters.

Currently, data on the efficacy and safety of new biosimilars of eculizumab in PNH are being actively published [12, 22‒25]. However, the data on long-term therapy with biosimilars of eculizumab are limited by this study.

The stable LDH activity and hemoglobin levels observed in this study indicate long-term treatment efficacy due to a decrease in hemolytic activity. The persistent need for RBC transfusions is consistent with previously published data on the long-term use of the originator product Soliris (18–29%), including the population of Russian patients [10, 11].

According to the PK/PD analysis, mean eculizumab concentration 5 min pre-dose at all visits exceeded 35 μg/mL, which is the minimum concentration sufficient for complete inhibition of intravascular hemolysis in patients with PNH. At the same time, a persistent decrease in the mean serum MAC concentration confirms the efficacy of terminal complement activation control [21, 26]. The mean eculizumab and serum MAC concentrations were stable and showed no significant differences between visits after switching from the originator to the biosimilar in group B.

The overall safety profile of the investigational product was consistent with the findings of previous studies of originator [9, 10]. The percentage of patients with reported ARs and changes in laboratory and investigational findings during long-term therapy with biosimilar was comparable regardless of previous therapy with the biosimilar or originator.

The main limitation of this clinical study was the low number of PNH patients, as well as the orphan status of eculizumab, which made it difficult to include more patients in the study, and especially in group B, given the decreased use of the originator. In addition, the study did not implement the recently proposed criteria for response to complement inhibitor therapy [27]. The study did not evaluate the prevalence of extravascular C3-mediated hemolysis, which significantly affects the quality of the erythroid response during therapy with eculizumab in PNH patients [7, 27, 28].

The anti-C5 monoclonal antibody eculizumab has been used in the treatment of PNH for 20 years. Eculizumab has revolutionized treatment, controlling intravascular hemolysis and thrombosis occurrence, with improved long-term survival [7, 29]. The development of biosimilars of eculizumab for the treatment of PNH is increasing interest and becoming more significant [12].

Therefore, this clinical study demonstrates that long-term use of biosimilar of eculizumab in the treatment of PNH patients ensures successful control of hemolytic activity of the complement system, which is the main pathogenetic mechanism of the disease. The study also confirms the safety of biosimilar in long-term treatment of PNH patients, with a low percentage of ARs and cases of ADA formation. The findings of the ECU-PNH-III-X clinical study conducted as part of the clinical development of the biosimilar confirm its efficacy and safety for long-term use in patients with PNH.

This clinical study was approved by the Ministry of Health of Russia, Approval No. 205 dated 08.05.2018. The study protocol was reviewed and approved by Ethics Council at the Ministry of Health of the Russia; meeting number 165 dated 13.03.2018. This study protocol was reviewed and approved by Ethics Committees at each of the participating sites. This full list of participating site and Ethics Committees can be found at https://clinicaltrials.gov/search?term=NCT04679103.

The study was conducted in accordance with the ethical principles of the World Medical Association (WMA) Declaration of Helsinki (last revised in 2013) and the standards of the Good Clinical Practice (GCP) Guidelines of the International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH), designed to monitor compliance with the interests and safety of the patients. Personal information about potential and enrolled participants was collected and kept in strict confidence before, during and after the study. Written informed consent to participate in the study was obtained by the investigators from all participants before their participation in the study.

Alexander D. Kulagin: Alexion Pharmaceuticals, Inc: consultancy, research funding; JSC GENERIUM: consultancy, honoraria, research funding. Vadim V. Ptushkin: Alexion Pharmaceuticals, Inc: consultancy, research funding; JSC GENERIUM: research funding; Janssen: consultancy; AbbVie: consultancy; Roche: consultancy. Elena A. Lukina: JSC GENERIUM: research funding; Sanofi Genzyme: honoraria, membership on an entity’s Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Other: travel reimbursement, research funding. Oksana А. Markova: JSC GENERIUM: employment. Eugene V. Zuev: JSC GENERIUM: employment.

The study was organized and conducted with the support of GENERIUM JSC.

Contribution: AD.K., V.V.P., and E.A.L. recruited patients, collected data, analyzed and interpreted the data, contributed to the manuscript, and approved the final version; I.L.D., A.V.K., T.S.K., E.Yu.K., N.V.M., T.A.M., O.U.К., E.G.A., and V.D.L. recruited patients, collected data, contributed to the manuscript, and approved the final version; O.A.M. and E.V.Z. developed the protocol, analyzed and interpreted the data, contributed to the manuscript, and approved the final version.

The data that support the findings of this study are not publicly available due to they containing information that could compromise the privacy of research participants but are available from the corresponding author Alexander Kulagin.