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Expert Opinion on Investigational Drugs

ISSN: 1354-3784 (Print) 1744-7658 (Online) Journal homepage: http://www.tandfonline.com/loi/ieid20

Bioequivalence, safety and immunogenicity ofBI 695501, an adalimumab biosimilar candidate,compared with the reference biologic in arandomized, double-blind, active comparatorphase I clinical study (VOLTAIRE®-PK) in healthysubjects

Christopher Wynne, Mario Altendorfer, Ivo Sonderegger, Lien Gheyle, RodEllis-Pegler, Susanne Buschke, Benjamin Lang, Deepak Assudani, SandeepAthalye & Niklas Czeloth

To cite this article: Christopher Wynne, Mario Altendorfer, Ivo Sonderegger, Lien Gheyle, RodEllis-Pegler, Susanne Buschke, Benjamin Lang, Deepak Assudani, Sandeep Athalye & NiklasCzeloth (2016) Bioequivalence, safety and immunogenicity of BI 695501, an adalimumabbiosimilar candidate, compared with the reference biologic in a randomized, double-blind,active comparator phase I clinical study (VOLTAIRE®-PK) in healthy subjects, Expert Opinion onInvestigational Drugs, 25:12, 1361-1370, DOI: 10.1080/13543784.2016.1255724

To link to this article: http://dx.doi.org/10.1080/13543784.2016.1255724

Accepted author version posted online: 04Nov 2016.Published online: 18 Nov 2016.

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ORIGINAL RESEARCH

Bioequivalence, safety and immunogenicity of BI 695501, an adalimumab biosimilarcandidate, compared with the reference biologic in a randomized, double-blind, activecomparator phase I clinical study (VOLTAIRE®-PK) in healthy subjectsChristopher Wynnea, Mario Altendorferb, Ivo Sondereggerb, Lien Gheylec, Rod Ellis-Peglerd, Susanne Buschkeb,Benjamin Langb, Deepak Assudanib, Sandeep Athalyeb and Niklas Czelothb

aChristchurch Clinical Studies Trust, Christchurch, New Zealand; bBoehringer Ingelheim, Ingelheim, Germany; cSGS, Clinical Pharmacology Unit,Antwerp, Belgium; dAuckland Clinical Studies Limited, Auckland, New Zealand

ABSTRACTBackground: This Phase I study (VOLTAIRE®-PK) aimed to evaluate three-way pharmacokinetic similar-ity (bioequivalence), safety, and immunogenicity of BI 695501 (a Humira® [adalimumab] biosimilarcandidate) compared with US- and EU-approved Humira in healthy male subjects.Methods: Subjects (N = 327) were randomized 1:1:1 to receive one 40-mg subcutaneous dose ofBI 695501, US- or EU-approved Humira; safety was assessed for 70 days. Bioequivalence was evaluatedusing the average bioequivalence method to test if the 90% confidence intervals (CIs) of the geometricmeans (BI 695501 vs US- and EU-approved Humira) for the primary end points were within prespecifiedacceptance ranges (80–125%). Immunogenicity was assessed using a sensitive bridging method.Results: Bioequivalence between BI 695501 and US- and EU-approved Humira was demonstratedwith the 90% CIs of the ratios of all primary end points: Cmax, AUC0–inf, pred and AUC0–tz being withinthe prespecified acceptance ranges of 80–125%. Concentration vs time profiles were similar as werethe time course and frequency of immunogenic responses. All study drugs showed similar safety andtolerability results.Conclusions: Three-way bioequivalence of BI 695501 to US- and EU-approved Humira was demon-strated; safety and immunogenicity results of the three study drugs were also similar.

Clinical trial registration: 2013-003722-84 (EudraCT) and NCT02045979.

ARTICLE HISTORYReceived 31 August 2016Accepted 28 October 2016

KEYWORDSAdalimumab; BI 695501;bioequivalence; biosimilar;Humira; pharmacokinetics

1. Introduction

Patients with immune-mediated diseases, such as rheumatoidarthritis (RA) and psoriasis, often have significant comorbid-ities that impact their quality of life [1,2] and increase their riskof death [3,4]. The direct and indirect costs of treating andmanaging these patients can place a large financial burden onhealth-care systems, patients, and society; thus, financial fac-tors impact the availability of treatments [5].

Biologics are a treatment option available to patients withimmune-mediated diseases; one such example is Humira®(AbbVie Ltd; adalimumab), which is approved in the UnitedStates and Europe for the treatment of diseases such as RA,psoriasis, and Crohn’s disease [6,7]. Adalimumab is a tumornecrosis factor (TNF) antagonist that modulates biologicalresponses induced or regulated by TNF, including a reductionin inflammation [7].

A biosimilar is a biological product that is highly similar to,and has no clinically meaningful differences from, theapproved reference product [8]. Given that the cost of mana-ging immune-mediated diseases with biologics is rising, biosi-milars have the potential to offer significant cost savings toboth health-care systems and patients [9].

Complex antibody biologics, including biosimilars, are largeglycoproteins that undergo an intricate manufacturing processusing living cells [10]. The focus of biosimilar developmentand the regulatory approval process is the extensive physico-chemical and functional characterization to ensure biosimilar-ity in terms of structure, mechanism of action, andpharmacology to the reference product [8–10]. Additionally,demonstration of pharmacokinetic (PK) bioequivalence is keyto establishing biosimilarity [8–10].

Biologics can induce immunogenicity, an antibody-mediated immune response, and can instigate a hypersen-sitivity reaction. Immunogenicity can additionally impactPK, clinical efficacy and alter the safety profile of theagent. Evaluation of immunogenicity is therefore an impor-tant consideration in biosimilar development [11].Importantly, immunogenicity results reported from differentstudies can vary because of the differences in the perfor-mance/sensitivity of the assays used [12]. As such, a com-parison of immunogenicity to historical data would bemisleading; immunogenicity data should be directly com-pared to the reference data in the same study, using thesame method.

CONTACT Deepak Assudani deepak.assudani@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH & Co. KG, 55216 Ingelheim am Rhein,Germany

EXPERT OPINION ON INVESTIGATIONAL DRUGS, 2016VOL. 25, NO. 12, 1361–1370http://dx.doi.org/10.1080/13543784.2016.1255724

© 2016 Informa UK Limited, trading as Taylor & Francis Group

In the United States and Europe, the regulatory approvalprocess for biosimilars requires that a thorough physicochem-ical and functional characterization of the proposed biosimilarbe performed, and that PK and clinical efficacy and safety bedetermined in clinical studies. Typically, a Phase I PK study isperformed to compare the single-dose PK profiles in healthysubjects to ensure that the drug disposition of the proposedbiosimilar is equivalent to the reference product [8–10]. ThisPhase I study also offers the opportunity to evaluate theimmunogenicity profile of the proposed biosimilar in a sensi-tive population, without the effect of other confounding fac-tors (such as prior exposure to biologics, concomitantmedications, and immune status of patients).

BI 695501 is an Humira biosimilar candidate being devel-oped by Boehringer Ingelheim and is under investigation foruse in immune-mediated diseases, including RA. The Phase Istudy reported here (VOLTAIRE®-PK) aimed to evaluate three-way PK bioequivalence, safety, and immunogenicity of a singledose of BI 695501 compared with US- and EU-approvedHumira.

2. Subjects and methods

2.1. Study design

This Phase I clinical study was a randomized, double-blind,single-dose, parallel arm, active comparator study and wasconducted to assess three-way PK bioequivalence, safety,and immunogenicity of BI 695501 compared with US- and

EU-approved Humira in healthy subjects. Subjects wereenrolled at three sites in New Zealand and Belgium, stratifiedby site and randomized 1:1:1 to one of three treatment groups(Figure 1; EudraCT: 2013-003722-84; NCT02045979). Subjectsreceived one subcutaneous dose (40 mg/0.8 mL) of BI 695501,US- or EU-approved Humira on Day 1 of the study.

A single administration of the approved 40-mg dose wasdeemed to have an acceptable risk/benefit ratio when givento healthy subjects. The trial objective was to establish a three-way clinical PK equivalence bridge so that a single globalPhase III study could be conducted using only one of thereference products, whilst satisfying approval requirementsfor BI 695501 across countries globally, including the US andEU regions. Both US- and EU-approved Humira were used inthis Phase I clinical study to comply with US Food and DrugAdministration (FDA) and European Medicines Agency (EMA)guidelines stating that comparisons must be made to thelocally approved reference product [8,9].

Prior to the start of the study, the Independent EthicsCommittees (IECs; located in Belgium and New Zealand)reviewed and approved the study. The IECs met the require-ments of the International Conference on Harmonization (ICH)Harmonized Tripartite Guideline for Good Clinical Practice(GCP), the definition in 21 Code of Federal Regulations 312.3and of the participating countries. The study was performed incompliance with the protocol, the principles of the Declarationof Helsinki, ICH GCP, and the applicable regulatory require-ments. The standard operating procedures from the threeclinical research organizations involved in the study were

EnrolledN = 327

Subjects completedstudy (BI 695501)

n = 106

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Humira®)n = 107

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Humira®)n = 107

Excluded from PKpopulation

n = 1Reason: withdrawal

from study

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Reason:withdrew consent

Randomization

US-approvedHumira®

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EU-approvedHumira®

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n = 108(safety population)a

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n = 108(safety population)a

Non-completionn = 1

Reason:immigration issues

Randomized butnot treated

n = 1

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n = 0

Excluded from PKpopulation

n = 1Reason: irregularpre-dose results

Randomized butnot treated

n = 1

Randomized butnot treated

n = 1

Non-completionn = 1

Reason:withdrew consent

PK populationb

(US-approvedHumira®)n = 108

PK populationb

(EU-approvedHumira®)n = 107

Figure 1. Patient flow.PK: pharmacokinetic. aThe safety population consisted of subjects who received a single dose of study drug. bThe pharmacokinetic population consisted of allsubjects who received a single dose of study drug, had at least one evaluable PK end point and were without protocol deviations or violations likely to impact thePK of the study drugs.

1362 C. WYNNE ET AL.

followed and were consistent with Boehringer Ingelheim stan-dards, GCP, and local legislation requirements.

2.2. Study population

Healthy adult males (≥18–≤55 years) who passed a completemedical assessment, had a body mass index (BMI) of≥18.5–≤29.9 kg/m [2], and provided informed consent wereeligible to enroll in this study. Key exclusion criteria includedprevious exposure to a biologic, exposure to restricted drugswithin a defined time frame, specific lifestyles (including smok-ing and alcohol abuse), and an inability/unwillingness to com-ply with protocol requirements, including extensive blooddraws for PK.

2.3. Study objectives and end points

The primary objective of this study was to evaluate three-waybioequivalence between BI 695501, US- and EU-approvedHumira. The secondary objectives were to evaluate safetyand tolerability and other PK parameters of BI 695501.Immunogenicity was also assessed. The primary end pointswere area under the concentration–time curve (AUC) fromtime zero to predicted infinity (AUC0–inf, pred), AUC from timezero to the last measurable concentration (AUC0–tz), and themaximum observed drug concentration in plasma (Cmax).

2.4. PK methodology

All PK analyses were conducted on the PK population set,those subjects who received a single dose of study medica-tion, had at least one evaluable primary PK end point, andwere without protocol deviations thought to significantlyaffect PK assessments.

2.4.1. Blood sampling and bioanalytical assaysPK sampling was performed on Days 1–9, 14, 21, 28, 35, 44,56, and 71 (end of study). Plasma concentration was mea-sured using a validated, indirect enzyme-linked immunosor-bent assay (ELISA) method; a 96-well microtiter plate(Greiner, catalogue no. 655061) was coated with drug-bind-ing ligand, TNFα (R&D Systems, catalogue no. 210-TA-10),and the free drug was detected with a horseradish perox-idase-conjugated mouse–anti-human immunoglobulin GFcγ-specific antibody (Jackson Immunoresearch, catalogueno. 209-035-098). Tetramethylbenzidine was added to thewells to generate a chromophore and the development ofcolor was stopped by the addition of a stopping solution.The absorbance at 450 nm was measured using a 190microplate reader (Molecular Devices, Sunnyvale, California,USA) and BI 695501, US- and EU-approved Humira concen-trations were calculated using a four-parameter standardcalibration curve. The lower limit of quantification of theELISA was 25 ng/mL; values lower than this were notreported. The upper limit of quantification of the assaywas greater than that of the highest Cmax data pointrecorded. All samples were tested using the same techni-que, to ensure consistency in terms of methods used.

2.4.2. PK evaluationsPrimary end points were AUC0–inf, pred, AUC0–tz, and Cmax.Secondary end points were truncated AUCs (time zero to168, 312, 480, 648, and 1032 h postdose) and AUC from timezero to observed infinity (AUC0–inf, obs) based on the observedconcentration of the analyte in plasma at the time of the lastconcentration measurement. Other PK parameters assessedwere terminal half-life of the analyte in plasma (T1/2), timefrom administration to maximum observed concentration ofthe analyte in plasma (Tmax), terminal rate constant in plasma(λz), apparent volume of distribution during the terminal phasefollowing an extravascular dose (Vz/F), and apparent clearanceof the analyte in the plasma after extravascular administration(CL/F). Drug concentration measurements of BI 695501 werecompared with both US- and EU-approved Humira. PK para-meters were derived using non-compartmental methods withPhoenix WinNonlin Version 6.3 (Certara, St Louis, Missouri,USA). Graphics were prepared using SAS Version 9.2 (SAS,Cary, North Carolina, USA). Formal bioequivalence testingwas performed, and the primary analysis used a statisticalmodel to assess bioequivalence. In addition, all PK parameterswere summarized using descriptive statistics.

2.5. Immunogenicity assays

Immunogenicity evaluations were conducted on the safetypopulation. The proportion of antidrug antibody (ADA) posi-tivity, titer, and neutralizing capacity of confirmed ADA-positive samples was reported separately for all three treat-ment groups. Blood samples for ADA and neutralizing anti-body (nAb) analysis were taken prior to study start(predose) and during the study up to Day 71. The positivityof ADA status and titers was determined using a singlebridging electrochemiluminescence assay on the MSD plat-form (Meso Scale Diagnostics LLC, Rockville, Maryland, USA),applying BI 695501 labeled with sulfo-tag and biotin asdetection reagents. To improve drug tolerance in theassay, an acid dissociation step was introduced to detachexisting conjugates of ADAs with circulating drug. ADAs(against BI 695501, US- and EU- approved Humira) weredetected using a three-tiered approach, applying a screen-ing, a confirmation, and a titer assay. A statistical cut pointwas set to deliver a 5% false-positive rate for the screeningassay and a 0.1% false-positive rate for the confirmatoryassay. The confirmatory assay comprised the addition offree drug (BI 695501, US- and EU-approved Humira) toreduce the assay signal by drug interference, and sampleswith more than 34.9% reduction of the signal were consid-ered confirmed positive (confirmatory cut point).

Assay sensitivity was 50 ng/mL, and the drug tolerance wasat least 30 µg/mL (free drug) at the low positive control level.During the whole study, free drug levels did not exceed thisconcentration and, as such, the assay was deemed able todetect ADA responses at any time point during the study.

The existence of nAb against BI 695501, US- and EU-approved Humira was determined using a cell-based, anti-body-dependent cell-mediated cytotoxicity (ADCC) method,with a sensitivity of 1.5 µg/mL. The assay comprised

EXPERT OPINION ON INVESTIGATIONAL DRUGS 1363

membrane TNFα-expressing target cells and human peripheralblood mononuclear cells as effector cells. Cell killing wasinduced by the addition of 0.047 µg/mL BI 695501; the pre-sence of nAb was confirmed by a drop in ADCC activity. A cutpoint where a signal reduction was deemed to be a positivenAb result was statistically set to result in a 5% false-positiverate. Assay methodologies were fully validated in accordancewith ICH guidelines.

2.6. Safety evaluations

Safety evaluations were conducted on the safety population(subjects who received a single dose of study medication), andassessments were conducted continuously throughout thestudy. Adverse events (AEs) were classified by System OrganClass and Preferred Term using the Medical Dictionary forRegulatory Activities (MedDRA) Version 17.0. AEs were assessedfor severity and relationship to the study drug. AEs of specialinterest (AESIs) were hepatic injuries, anaphylactic reactions,serious infections, and hypersensitivity reactions. All incidencesof AEs during the study were collected, documented, andreported as per the protocol instructions. Any AEs occurringwithin 70 days of study drug administration were considered tobe on-treatment; AEs after that period were not recorded.

2.7. Statistical analysis

The analysis strategy used in this study is in line with thatrecommended by the FDA, EMA, and the ICH [13–15].

The sample size was calculated so that the power to concludethree-way PK bioequivalence (with an acceptance range of 80–125%, one-sided significance level 5%, and taking into account allthree comparisons) was approximately 90% assuming no treat-ment differences, and approximately 80% for a 5% treatmentdifference (ratio scale). The assumed variability was based on in-house as well as literature data on the approved versions ofHumira.

The ratios of the geometric means (BI 695501 vs. US- andEU-approved Humira) and their two-sided 90% confidenceintervals (CIs) were provided for all primary and secondaryPK end points; the analysis was conducted using an analysisof covariance (ANCOVA) model on the logarithmic scale.Dependent variables were the logarithm of AUC0–inf, AUC0–tz,and Cmax; independent variables were treatment, study site,age at baseline, and body weight at baseline. Treatment andstudy site were categorical variables; age and body weight atbaseline were continuous variables.

Bioequivalence was evaluated using the average bioequiva-lence method (using a statistical model) to test if the ratios ofthe geometric means (BI 695501 vs. US- and EU-approved Humira)for the primary end points were within the prespecified accep-tance range of 80–125%. All three comparisons (i.e. BI 695501 vs.EU-approved Humira, BI 695501 vs. US-approved Humira, and US-vs. EU-approved Humira) needed to show statistical significancefor bioequivalence to be declared; therefore, no adjustment of thetype 1 error rate was required. All PK parameters were analyzeddescriptively, with geometric coefficients of variation given tomeasure dispersion. Graphics and statistical analyses were gener-ated using SAS Version 9.2 (SAS, Cary, North Carolina, USA).

3. Results

3.1. Subject demographics and baseline characteristics

Overall 460 subjects were screened, and 327 were randomized tothe three treatment groups (n = 109 per treatment group,Figure 1). Subject demographics and baseline characteristicswere comparable between the three groups; the mean age was31 years (standard deviation [SD] 11), and the mean BMI was24.4 kg/m2 (SD 2.6 kg/m2).

3.2. PK

The median time at which the maximum plasma concentra-tions (Tmax) were detected was 132, 109, and 120 h (medianvalues) for BI 695501, US- and EU-approved Humira, respec-tively. Unadjusted geometric mean Cmax levels were 3.9, 3.9,and 4.1 µg/mL for BI 695501, US- and EU-approved Humira,respectively. Unadjusted geometric mean AUC0–inf, pred levelswere 2630, 2470, and 2650 µg*h/mL for BI 695501, US- andEU-approved Humira, respectively. The 90% CIs for theadjusted geometric mean ratios of BI 695501 to referenceproduct analyses, and between EU- and US-approvedHumira, for the primary PK end points were all within theprespecified acceptance range of 80–125%. The mean plasmaconcentration–time profiles for BI 695501, US- and EU-approved Humira were comparable over the entire profilingperiod (Figure 2(a)). Results of the statistical comparison ofprimary PK end points corroborated the similarity of the meanplasma concentration–time curves (Figure 2(b)). An overviewof the geometric mean and mean PK parameters obtained forthe individual treatment groups is provided in Table 1.

Point estimates of the adjusted geometric mean ratio and the90% CIs for the ratios of the primary PK end points for the compar-ison of BI 695501 and US-approved Humira were 108.6% (98.5–119.8%) for AUC0–inf, pred, 107.3% (98.5–117.0%) for AUC0–tz, and100.9% (95.2–106.9%) forCmax. The point estimates of the adjustedgeometric mean ratio and the 90% CIs for the ratios of the primaryPK end points for the comparison of BI 695501 and EU-approvedHumira were 101.3% (92.5–111.0%) for AUC0–inf, pred, 99.9% (92.2–108.4%) for AUC0–tz, and 96.4% (91.1–102.0%) for Cmax. The pointestimates of the adjusted geometric mean ratio and the 90% CIsfor the ratios of the primary PK end points for the comparison ofUS- and EU-approved Humira were 94.0% (86.0–102.8%) for AUC0–inf, pred, 93.7% (86.8–101.1%) for AUC0–tz, and 95.9% (90.8–101.3%)for Cmax (Figure 2(b)).

Three-way bioequivalence of BI 695501 to US- and EU-approved Humira was declared for all comparisons. The statis-tical comparison of secondary and further PK parametersshowed that the 90% CIs of the adjusted geometric meanratios were all within 80–125% limits and as such supportedsimilarity between BI 695501, US- and EU-approved Humira.

3.3. Immunogenicity

Overall, 3.4% of subjects (n = 11) had ADAs at baseline. Thefrequency of predose-positive samples was equally distributedacross the treatment groups with 3.7% (n = 4), 2.8% (n = 3),and 3.7% (n = 4) for BI 695501, US- and EU-approved Humira,

1364 C. WYNNE ET AL.

respectively. Median (range) titer values for the predose sub-jects were: 5 (1–512), 2 (1–128), and 5 (1–32) for the BI 695501,US- and EU-approved Humira groups, respectively. A rapiddevelopment of ADAs was observed with all three drugs,and 4 weeks after dosing, 46.7% (n = 50), 56.1% (n = 60),and 37.4% (n = 40) of subjects were confirmed as ADA-positivein the BI 695501, US- and EU-approved Humira groups,

respectively. The frequency of confirmed ADA-positive sub-jects further increased until the end of the study (Day 71)with 92.5% (n = 99), 88.0% (n = 95), and 84.3% (n = 91) ofthe subjects in the BI 695501, US- and EU-approved Humiragroups, respectively (Figure 3(a)).

Despite the rapid development of ADA response, mediantiter values remained low until the end of the study. At Day 28,

1 7 14 21 28 35 44 56 71

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Figure 2. Arithmetic mean plasma concentration–time profiles in healthy subjects after a single dose of study drug for (a) BI 695501, US- and EU-approved Humira,(b) Forest plot presenting point estimate and 90% confidence intervals for primary PK parameters for BI 695, 501, US- and EU-approved Humira (bioequivalence wasdeclared if the 90% confidence intervals were within prespecified acceptance ranges of 80–125%).AUC0–inf, pred: AUC of the analyte in plasma over the time interval from zero extrapolated to infinity; AUC0–tz: AUC of the analyte in plasma over the time intervalfrom time zero to the last measurable concentration; Cmax: maximum observed drug concentration in plasma; PK: pharmacokinetic.

EXPERT OPINION ON INVESTIGATIONAL DRUGS 1365

median (range) titer values were 2 (1–512), 4 (1–512), and 2(1–256) for the BI 695501 group, US- and EU-approved Humiragroups, respectively. At the end of the study (Day 71), mediantiter values remained at a relatively low level (16) for all groupswith ranges of 1–2048 (BI 695501), 1–1024 (US-approvedHumira), and 1–1024 (EU-approved Humira; Figure 3(b,c)).

Confirmed ADA-positive samples were further character-ized for their neutralizing capacity using an ADCC assay. Atpredose, 2.8% (n = 3), 0.9% (n = 1), and 1.9% (n = 2) ofsubjects in the BI 695501, US- and EU-approved Humiragroups, respectively, were nAb-positive. After 4 weeks,

11.2% (n = 12), 17.8% (n = 19), and 8.4% (n = 9) of thesubjects were nAb-positive in the BI 695501, US- and EU-approved Humira groups, respectively. By the end of thestudy, the percentage of nAb-positive subjects in the treat-ment groups had risen to 59.8% (n = 64), 63.9% (n = 69), and58.3% (n = 63) for BI 695501, US- and EU-approved Humira,respectively (Figure 4).

The development of nAb positivity was comparable acrossall three treatment groups. Taken together, these data suggestsimilar immunogenicity results between BI 695501, US- andEU-approved Humira.

Table 1. Summary of PK parameters in healthy subjects after a single dose of BI 695501, US- or EU-approved adalimumab.

BI 695501a US-approved adalimumabb EU-approved adalimumabc

Geometric mean(gCV%) Mean ± SD

Geometric mean(gCV%) Mean ± SD

Geometric mean(gCV%) Mean ± SD

AUC0–inf, pred(µg*h/mL)

2630 (52.4) 2920 ± 1270 2470 (51.2) 2760 ± 1290 2650 (38.5) 2830 ± 1040

AUC0–tz(µg*h/mL)

2440 (47.8) 2660 ± 1020 2300 (45.1) 2500 ± 1010 2480 (33.5) 2600 ± 798

Cmax (µg/mL) 3.9 (34.1) 4.1 ± 1.27 3.9 (34.5) 4.11 ± 1.26 4.1 (30.4) 4.31 ± 1.22λ/z (1/h) 0.003 (95.6) 0.005 ± 0.010 0.003 (83.4) 0.004 ± 0.003 0.003 (82.9) 0.004 ± 0.003T1/2 (h) 234 (95.7) 309 ± 228 243 (83.4) 313 ± 228 258 (82.9) 328 ± 234CL/F (mL/h) 15.2 (52.4) 17.8 ± 15.7 16.2 (51.2) 18.2 ± 9.7 15.1 (38.5) 16.2 ± 6.5Vz/F (mL) 5130 (51.8) 5770 ± 2860 5680 (46.1) 6250 ± 2870 5630 (56.6) 6420 ± 3400

Median (minimum–maximum)

Tmax (h) 132 (48–649) 109 (59–480) 120 (12–648)

AUC: Area under the concentration–time curve; AUC0–inf, pred: AUC of the analyte in plasma over the time interval from zero extrapolated to infinity; AUC0–tz: AUC ofthe analyte in plasma over the time interval from time zero to the last measurable concentration; CL/F: apparent clearance of the analyte in the plasma afterextravascular administration; Cmax: maximum observed drug concentration in plasma; gCV: geometric coefficient of variation; PK: pharmacokinetic; T1/2: terminalhalf-life of the analyte in plasma; Tmax: time from administration to maximum observed concentration of the analyte in plasma; Vz/F: apparent volume ofdistribution during the terminal phase following an extravascular dose; λ/z: terminal rate constant in plasma. an = 106 for all parameters except for Cmax and Tmax

which were n = 107; bn = 105 for AUC0–inf, pred, λ/z, T1/2, CL/F, Vz/F, n = 107 for AUC0–tz, and n = 108 for Cmax and Tmax;cn = 105 for all parameters except AUC0–tz,

which was n = 106 and Cmax and Tmax which were n = 107.

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Figure 3. Development of ADAs in healthy subjects after a single dose of study drug at Days 28 and 71 for BI 695501, US- and EU-approved Humira. (a) Frequency ofADA-positive responses, (b) median ADA titer and (c) end-of-study titers for healthy subjects with ADA-positive responses. Median values are depicted by a linewithin the 25% and 75% percentile boxes; arithmetic mean = diamond shape; individual outliers = individual points; minimum and maximum values or 1.5×interquartile range = vertical lines out of the box plot. ADAs: antidrug antibodies.

1366 C. WYNNE ET AL.

Titer values ≤16 appeared to have a minor effect on the PK,while a decrease in exposure (AUC0–inf, pred) was observed fortiter values >16. This effect was consistent across all treatmentgroups (Figure 5).

The time course of ADA development and ADA titers wassimilar for all three treatment groups. In addition, a similar impactof the ADAs on the PK profile of BI 695501, US- and EU-approvedHumira was observed.

One subject within the US-approved Humira group devel-oped a delayed hypersensitivity (erythema and itch at the injec-tion site that became a generalized urticaria over the next 24 h)11 days after administration of study drug, potentially linkedwithan immunogenicity response. This subject became ADA-positiveat the next sampling time point (Day 14) and had a relatively highend-of-study ADA titer (128); a positive nAb response wasobserved at Day 28 only for this subject.

3.4. Safety

Safety and tolerability results were comparable betweenBI 695501, US- and EU-approved Humira. Overall, 72%(n = 232) of subjects reported at least one AE (Table 2).The most commonly reported AE was headache (78 intotal; n = 25 in each of the BI 695501 and US-approvedgroups and n = 28 in the EU-approved group). There wereno deaths during the study period.

In total, 24.1% (n = 78) of subjects reported at least onedrug-related AE, 19.4% (n = 21) in the BI 695501 group, 26.9%(n = 29) in the US-approved Humira group, and 25.9% (n = 28)in the EU-approved Humira group. The most commonlyreported drug-related AE was headache (N = 27 in total;n = 6 in the BI 695501 group, n = 10 in the US-approvedgroup, and n = 11 in the EU approved group, Table 2).

2.8

11.2

59.8

0.9

17.8

63.9

1.98.4

58.3

0

20

40

60

80

Baseline Day 28 Day 71

BI 695501 US-approved Humira® EU-approved Humira®

nAb-

posi

tive

sub

ject

s (%

)

Figure 4. Neutralizing antibody development in healthy subjects after a single dose of study drug at baseline, Day 28 and Day 71 for BI 695501, US- and EU-approved Humira.nAb: neutralizing antibody.

Ada

limum

ab A

UC

0–in

f, p

red (

µg*h

/mL

)

High39BI 695501 US-approved

Humira®EU-approved

Humira®

Titern =

Low55

High36

Low53

High36

Low49

6,000

4,000

2,000

0

Figure 5. Impact of antidrug antibody titer (high [>16] vs. low [≤16]) on the exposure (AUC0–inf, pred) to the study drugs in healthy subjects after a single dose of BI 695501,US- or EU-approved Humira. Median values are depicted by a line within the 25% and 75% percentile boxes; arithmetic mean = diamond shape; individualoutliers = individual points; minimum and maximum values or 1.5× interquartile range = vertical lines out of the box plot.AUC0–inf, pred: AUC of the analyte in plasma over the time interval from zero extrapolated to infinity.

EXPERT OPINION ON INVESTIGATIONAL DRUGS 1367

Overall, 2.8% (n = 3) of subjects in each of the BI 695501and US-approved Humira groups, and 1.9% (n = 2) in the EU-approved Humira group reported serious AEs (Table 2). Ofthese, two serious AEs (abdominal pain in the BI 695501group and appendicitis in the US-approved Humira group)were considered related to the study drug. One subjectreported two serious AEs (hand and ankle fracture), whichwere not considered related to the study drug.

Five subjects reported one AESI each (Table 2), injectionsite hypersensitivity (1.9% [n = 2] in the BI 695501 group and0.9% [n = 1] in the EU-approved Humira group), hypersensi-tivity (0.9% [n = 1] in the US-approved Humira group), andurticaria (0.9% [n = 1] in the US-approved Humira group).

4. Discussion

BI 695501 is an Humira biosimilar candidate being developed forthe treatment of a variety of inflammatory diseases. Current guide-lines for biosimilarity require evaluation of the PK bioequivalenceof the proposed biosimilar to the reference product. The primaryobjective of this study was to demonstrate the three-way PKbioequivalence between BI 695501 and US- and EU-approvedHumira.

The 90% CIs for the geometric mean ratios of all primary PKparameters (for comparisons of BI 695501 to US- and EU-approved Humira, and for US- to EU-approved Humira) werewithin the prespecified acceptance ranges (80–125%), demon-strating three-way PK bioequivalence. Additionally, the statisticalcomparison of secondary and other PK parameters supportedsimilarity between BI 695501, US- and EU-approved Humira.

Data obtained in this study indicate comparable immuno-genicity results of BI 695501, US- and EU-approved Humira.Published data show variations in the incidence of ADA-posi-tive subjects with approved Humira, highlighting the differ-ence in data associated with individual assays and drug

tolerance [16–24]. Although it is not appropriate to indirectlycompare immunogenicity results across studies, it is evidentthat the results presented in this study are somewhat differentto those reported in the literature. The bridging electrochemi-luminescence method used in this study to detect ADAs wassensitive and highly drug tolerant and was able to detect verylow ADA levels under conditions where high circulating druglevels would normally impair the sensitivity of other methods.Of note, the methodology used in this study was able todetect early immunogenicity events and discriminate betweenADA levels that did or did not have an impact on PK para-meters. The time course of ADA development and ADA titersis important an consideration when evaluating immunogeni-city for biologics. In this study, these were similar for all threetreatment groups. In addition, a similar impact of the ADAs onthe PK profiles of BI 695501, US- and EU-approved Humira wasobserved. It can therefore be concluded that BI 695501 showssimilar immunogenicity results to that of US- and EU-approvedHumira in this study.

Likewise, the development of nAb was similar across allthree treatment groups and the ADCC method used in thisstudy appeared to be adequate to detect nAb responsesover the course of the study. Originally published immuno-genicity data relating to approved Humira results show thatthe rates of nAb development ranged from 3% to 9% in allapproved indications over a 6–12-month period [6,7]. Therates of nAb development presented in this study rangedfrom 58.3% to 63.9% and were quite different from thepublished data. These results are, however, similar to thehigher incidences recently observed in studies of other bio-similars, using state-of-the-art methods [25]. As with theADA results, differences could be attributed to study format,the type of assay used, and the fact that no concomitantimmunosuppressive drugs (e.g. methotrexate) were allowedin the study. In this study, analyses of BI 695501 compared

Table 2. Summary of adverse events occurring in healthy subjects with one or more AEs, in any treatment group, after a single dose of BI 695501,US- or EU-approved Humira.

n (%) BI 695501 (n = 108) US-approved Humira (n = 108) EU-approved Humira (n = 108)

Subjects with ≥1 AE 76 (70.4) 79 (73.1) 77 (71.3)Serious AEs (intensity) 3 (2.8) 3 (2.8) 2 (1.9)Concussion (severe) 1 (0.9) 0 0Fracture (moderate/severe) 2 (1.9) 0 0Abdominal pain (moderate/severe) 1 (0.9)ab 0 1 (0.9)a

Appendicitis (moderate) 0 2 (1.9)b 0Laceration (severe) 0 1 (0.9) 0Nephrolithiasis (moderate) 0 0 1 (0.9)

Drug-related AEs ≥2% in any of the treatment groups 21 (19.4) 29 (26.9) 28 (25.9)Headache 6 (5.6) 10 (9.3) 11 (10.2)Upper respiratory tract infection 4 (3.7) 3 (2.8) 5 (4.6)Rhinorrhea 1 (0.9) 2 (1.9) 4 (3.7)

AE of special interest (severity) 2 (1.9) 2 (1.9) 1 (0.9)Injection site reactivation/delayed hypersensitivity(mild)

2 (1.9) 0 1 (0.9)

Hypersensitivity reaction (mild) 0 1 (0.9) 0Urticaria (moderate) 0 1 (0.9) 0

AEs leading to discontinuation of study drug 0 0 0Death 0 0 0

Only the most severe occurrence of each subject was counted. Drug-related AEs are defined as any that are considered by the investigator to berelated to the study drug.

AEs: Adverse events. aThe incidence of abdominal pain in the BI 695501 group was severe, and the incidence in the EU-approved Humira group wasmoderate. bOne incidence each of abdominal pain and appendicitis was considered by the investigator to be related to the study drug.

1368 C. WYNNE ET AL.

with US- and EU-approved Humira conducted using thesame assay were similar. While assessment of immunogeni-city incidence and understanding its impact on drug expo-sure and safety are important, comparison of ADA incidencerates generated in different studies or with previously pub-lished data is not considered appropriate, as assay meth-odologies for immunogenicity evaluations are generally notconsistent between studies [12].

Safety and tolerability results were similar between BI 695501,US- and EU-approved Humira.

5. Conclusions

The three-way PK bioequivalence between BI 695501, US- and EU-approved Humira was demonstrated. Safety, tolerability, andimmunogenicity results across the three study treatments weresimilar.

Acknowledgments

Christopher Wynne, Lien Gheyle and Rod Ellis-Pegler were investigators inthe study and provided a critical review of the manuscript. MarioAltendorfer, Ivo Sonderegger, Susanne Buschke, Benjamin Lang, DeepakAssudani, Sandeep Athalye and Niklas Czeloth were responsible for theinterpretation of the study data and provided a critical review of themanuscript. The authors would like to thank the subjects who participatedin the study, and the staff of the clinical research units who carried out thestudy. The authors would also like to thank Niraj Chhaya who was respon-sible for safety monitoring in the study. Medical writing support wasprovided by Melissa Purves of SciMentum (Manchester, UK).

The data in this manuscript have been presented previously at AmericanAssociation of Pharmaceutical Scientists Annual Meeting and Exposition,Orlando, FL, USA, 25–29 October 2015 (poster number W4286) and AmericanCollege of Rheumatology/Association of Rheumatology Health Professionals(ACR/ARHP) Annual Meeting, San Francisco, CA, USA, 6–11 November 2015(poster number 2727).

Funding

This study was funded by Boehringer Ingelheim.

Declaration of interest

C. Wynne is an employee of, and holds shares in Christchurch ClinicalStudies Trust that received payment for carrying out the study. L.Gheyle is an employee of SGS, Clinical Pharmacology Unit, Antwerp,Belgium that received payment for carrying out the study. R. Ellis-Pegler is an employee of Auckland Clinical Studies Limited thatreceived payment for carrying out the study. Mario Altendorfer, IvoSonderegger, Susanne Buschke, Benjamin Lang, Deepak Assudani,Sandeep Athalye and Niklas Czeloth are employees of BoehringerIngelheim. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest inor financial conflict with the subject matter or materials discussed inthe manuscript apart from those disclosed.

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