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Pharmacokinetic equivalence, comparable safety, and immunogenicity of an adalimumab biosimilar product (M923) to Humira in healthy subjects

Abstract The aims of this randomized, double‐blind, three‐arm, single‐dose study were to demonstrate pharmacokinetic (PK) equivalence of the adalimumab biosimilar M923 (hereafter referred to as “M923”) to each of 2 reference products, and to assess M923's safety and immunogenicity. Primary PK endpoints were maximum observed concentration (C max), area under the curve (AUC) from time 0 extrapolated to infinity (AUC0‐inf), and AUC from time 0 to 336 hours (AUC0‐336). Secondary endpoints included safety and immunogenicity assessments. Healthy subjects were randomized 1:1:1 to receive a 40‐mg dose of M923 (n = 107); adalimumab US Humira (n = 105), hereafter referred to as “US Humira”; or adalimumab EU Humira (n = 103), hereafter referred to as “EU Humira.” PK equivalence was demonstrated for all primary PK endpoints. Geometric least squares means ratios (GMRs) for C max, AUC0‐inf, and AUC0‐336 were 99.4, 100.9, and 100.5, respectively, between the M923 and EU Humira arms and 102.6, 104.2, and 102.9 between the M923 and US Humira arms. The 90% confidence intervals of the GMRs for all PK endpoints were within prespecified confidence bounds of 80%‐125%. Adverse event rates were similar across the M923 (47.7%), US Humira (50.9%), and EU Humira (53.3%) arms and were generally mild (73.7%) or moderate (22.0%). The proportion of subjects with a confirmed antidrug antibody (ADA) response was similar across study arms. This study demonstrated bioequivalent PK among M923, US Humira, and EU Humira and demonstrated that the PK parameters were consistent with similar safety and tolerability profile and ADA response rates.


| INTRODUCTION
Humira (adalimumab) is a fully human recombinant immunoglobulin (Ig) G1 monoclonal antibody that binds to the soluble form of human tumor necrosis factor (TNF)-a, thereby blocking interaction with its receptors, p55 (TNFR I) and p75 (TNFR II), and inhibiting the TNF-adependent proinflammatory cascade. 1 Humira also binds to the transmembrane form of TNF-a, which transmits strong inhibitory signals through transmembrane TNF-a. 2 The clinical efficacy and safety of Humira for the treatment of chronic inflammatory disorders, such as rheumatoid arthritis (RA), Crohn's disease, ankylosing spondylitis, and psoriasis, have been demonstrated in several large clinical studies. [3][4][5][6][7][8] The findings of these studies led to the approval of Humira by both the US Food and Drug Administration and the European Medicines Agency for these indications. 9,10 The high costs of biologic therapies (e.g., Humira) for the treatment of chronic inflammatory disorders, such as RA, can limit patient access to these treatments. 11 The development and incorporation of biosimilars into clinical practice has the potential to lower costs and increase access via affordability of these much-needed treatment options. 11 The availability of multiple biosimilars to a single agent has the potential to further increase options for access and lower costs by ensuring a more competitive marketplace. The marketing approval of biosimilar drug products requires assessment of risk and benefit based on equivalence studies, including clinical studies, to address any clinically significant differences in drug product that may arise due to a biological manufacturing process that differs from that used in the manufacture of the original drug product. 12 Adalimumab biosimilar M923 (hereafter referred to as "M923") is a proposed biosimilar to Humira (adalimumab [AbbVie Inc., North Chicago, IL]; chemical formula: C 6428 H 9912 N 1694 O 1987 S 46 ). This first-inhuman study, the first step in the clinical development process of M923, compared the pharmacokinetic (PK), safety, tolerability, and immunogenicity profiles of a single 40-mg subcutaneous injection of M923, adalimumab US Humira (hereafter referred to as "US Humira"), and adalimumab EU Humira (hereafter referred to as "EU Humira") in healthy subjects, with the objective of demonstrating PK equivalence and similarity with respect to short-term safety and immunogenicity.

| Eligibility criteria
Healthy men and women aged 18-55 years (inclusive) and with a body mass index (BMI) between 18.5 and 29.9 kg/m 2 (inclusive) were eligible to be included in this study as determined by medical history, physical examination, vital signs, and 12-lead electrocardiogram (ECG) at screening and admission. All subjects were to comply with the contraception requirements as specified in the study protocol or be of non-childbearing potential. Inclusion criteria are described in full in the Appendix S1.
Subjects were excluded from the study if they were previously treated with Humira or another recombinant human monoclonal antibody and/or had antidrug antibodies (ADAs) to Humira at screening. Exclusion criteria (described in full in Appendix S1) assisted selection of a homogeneous study population and minimized the risk of serious adverse drug reactions.

| Study design
The sample size (N = 324) was determined based on previously published estimates of variability in the primary PK parameters. 13,14 More specifically, Kaur et al. reported data from a study comparing ABP 501 to EU Humira, specifically a coefficient of variation (CV) of 41.7% for AUC 0-inf , 38.3% for AUC 0-last , and 30.5% for C max . Assuming the equivalence bounds of 80% to 125% for the relative bioavailability, a true ratio of 1.00, 90% joint power (Bonferroni adjustment for multiplicity) for the 3 comparisons (M923 vs EU Humira, M923 vs US Humira, EU Humira vs US Humira), and a population CV of 41.7% (for AUC 0-inf ), a sample size of 86 completers per arm (258 total) was required. Joint power was computed as 100% -(100%single comparison power) 9 3, using a Bonferroni correction for multiplicity. Assuming a loss of 20% of the data (15% for loss due to extrapolation to infinity, and 5% for dropouts), 108 subjects per arm (324 total) were needed to be randomized to achieve 90% power.
Subjects were screened between 45 days and 2 days prior to administration of study drug, and eligibility and baseline assessments were conducted 1 day before drug administration. The route of administration for M923 was chosen to be similar to those of approved reference products as recommended by regulators. 15 Regulators also recommend that the dose should be in the ascending part of the dose-response curve; this criterion was used to select the appropriate dose. Following confirmation of eligibility, subjects were randomized in an approximate 1:1:1 ratio to a single 40-mg subcutaneous injection via SC injection to the lower abdomen of

| Endpoints
The primary endpoints were maximum observed adalimumab concentration (C max ), area under the concentration vs time curve (AUC) from time 0 (predose) extrapolated to infinity (AUC 0-inf ), and AUC from time 0 (predose) to 336 hours (AUC 0-336 ). AUC 0-336 was selected as an additional primary parameter for this study, since it is within 24 hours of the sampling time (360 hours) used for total exposure comparisons in the original US and EU Humira submissions and reported to be free of ADA formation. Secondary PK endpoints included time to maximum concentration (t max ), AUC from predose to last dose (AUC 0-last ), AUC from predose to 1344 hours (day 57) (AUC 0-1344 ), apparent systemic clearance after extravascular dosing (CL/F), apparent volume of distribution (V z /F), and terminal half-life (t 1/2 ). Safety variables included incidence of adverse events (including injection site reaction), vital signs, 12-lead ECGs, routine laboratory tests, and immunogenicity assessments. was used as an isotype control. Colorimetric intensity was determined using a SpectraMax Plus 384 spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA) reading at 450 nm with a correction of 650 nm. The lower limit of quantification was 300 ng/mL and the upper limit of quantification was 4800 ng/mL. Interassay precision and accuracy were calculated from quality control samples. The CV for M923 and EU Humira was ≤10.2% and ≤8.8%, respectively.

| Immunogenicity
Analysis of ADAs was conducted using a screening assay based on M923 to identify samples with potentially positive binding ADA. Samples positive in the screening assay were confirmed in a tiered manner based on assays to EU Humira, US Humira, and M923. For samples that were confirmed positive, a third assay was used to determine the relative ADA titer; further assays were used to determine the presence of neutralizing ADAs (nADAs) and human IgE ADA.
ADAs were measured using an electrochemiluminescence assay validated to detect anti-adalimumab antibodies in human serum.

Screening
• Serial PK samples were collected on day 1, days 2-9, and days 11, 15, 22, 29, 36, 43, and 57 • Safety, tolerability, and immunogenicity were assessed periodically throughout the study Electrochemiluminescence was measured in relative light units using the MSD SECTOR Imager 2400 (Meso Scale Diagnostics, Rockville, MD, USA). The mean assay sensitivity was 1.96 ng/mL, and drug tolerances of 0.500 lg/mL, 2.00 lg/mL, and 0.417 lg/ mL were observed for M923, US Humira, and EU Humira, respectively, at the low-positive control (15.0 ng/mL). Inter-run assay precision for the quality control samples and negative control was 6.2% to 7.1%.
Serum neutralizing anti-adalimumab antibodies were measured using a validated fluorescence assay using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, LLC). The low-positive control was 920 ng/mL and the high-positive control was 3000 ng/ mL. The CV for high-and low-positive controls was 1.0% and 8.5%, respectively. Following a positive ADA result, samples were further evaluated for the presence of IgE (a specific isotype of ADA).

| Statistical analysis
The PK analysis set included all evaluable subjects in the safety analysis set with sufficient data to calculate at least 1 primary PK endpoint. Subjects with protocol deviations or events thought to affect PK were excluded. PK parameters were derived from the collected serum using noncompartmental methods with Phoenix â WinNonlin â version 6.4 (Certara, LP, Princeton, NJ, USA). The statistical analysis of the log-transformed primary endpoint and the select secondary PK endpoints (AUC 0-last and AUC 0-1344 ) was based on an analysis of covariance model with a fixed effect for administration arm, baseline age, and baseline body weight as continuous covariates.
PK equivalence across the 3 administration arms was established if the 90% confidence intervals (CIs) for the geometric least squares (LS) means of the primary PK parameters for each comparison (M923 vs US Humira, M923 vs EU Humira, and US Humira vs EU Humira) fell within the predefined equivalence boundaries of 80% to 125%.
The safety analysis set included all subjects who received study drug. Safety assessments were summarized using descriptive statistics, as appropriate. For immunogenicity, the number and percentage of subjects testing positive for ADAs or nADAs before the dose of M923, EU Humira, or US Humira (day 1) and at scheduled postdose assessments were summarized by administration arm and sampling time. Time to seroconversion was calculated for subjects with confirmed postdose ADA response only. All statistical programming was performed utilizing SAS â version 9.4 (SAS Institute Inc., Cary, NC, USA). The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. 16 3 | RESULTS

| Baseline characteristics and demographics
A total of 324 subjects were enrolled (109 in the M923 arm, 108 in the US Humira arm, and 107 in the EU Humira arm). The majority of subjects were male (315/324). Demographic and baseline characteristics, including age, sex, race, ethnicity, height, weight, and BMI, were similar across administration arms (Table 1). There were no premature withdrawals.

| Pharmacokinetics
Of the 324 subjects who received study drug, 9 subjects were excluded from the PK analysis set; 5 failed to receive a complete dose and 4 had quantifiable predose concentrations greater than 5% of C max . A total of 315 subjects were included in the PK analysis set (107 in the M923 arm, 105 in the US Humira arm, and 103 in the EU Humira arm). Twenty-seven of the 315 subjects in the PK analysis set had individual parameters which were excluded from the PK analysis set due to protocol deviations or failure to meet acceptability criteria. Half-life and related PK parameters could not be calculated in 6 subjects due to lack of defined terminal elimination phase (Table S1). Following administration of a single 40-mg subcutaneous dose, PK profiles of M923, US Humira, and EU Humira were similar ( Figure 2).
The geometric LS means ratio (GMR) of the primary endpoints C max , AUC 0-inf , and AUC 0-336 for the comparisons of M923 with either EU Humira or US Humira and between EU Humira and US Humira were fully contained within the 80% to 125% equivalence bounds ( Table 2). Individual and geometric mean results by study arm for the primary parameters (C max , AUC 0-336, and AUC 0-inf ) are illustrated in Figure 3. GMRs for the secondary endpoints AUC 0-last and AUC 0-1344 for all study arm comparisons were also close to unity and fully contained within the equivalence bounds. There were no relevant differences in median time to C max (144 hours for both M923 and EU Humira; 142 hours for US Humira) or geometric mean and range of the calculated t 1/2 , CL/F, and V z /F values (Table 3).

| Safety
All subjects dosed were included in the safety analysis. The percentage of subjects with at least 1 adverse event was comparable among the 3 administration arms, ranging from 47.7% to 53.3% (Table 4).
The majority (73.7%) of adverse events were considered by the investigators to be mild in severity and unrelated to administration of M923, US Humira, or EU Humira. There were no deaths. One subject from the EU Humira arm reported 3 serious adverse events: foot fracture, laceration, and wound infection; the first 2 events were considered severe while the third was moderate; all were considered unrelated to EU Humira. The types of adverse events were similar across study arms. The most common (≥2%) adverse event across all study arms was headache (11.4%) (see Table 4

| ADA assessment and analysis
The overall incidence of binding ADA response and nADA response is represented in Figure 4 and Table S2, and the mean time to first seroconversion was 42 to 45 days across the 3 administration arms.
Over time, the rates of ADA formation were similar across all study arms (

| DISCUSSION
As part of the evaluation of biosimilarity, comparative PK studies in a homogenous and sensitive population of healthy volunteers are recommended to detect potential differences in the PK of test and reference products. 17 This randomized, double-blind study was the first time that M923 was administered to healthy human subjects.
The primary aim of the study was to investigate and compare the PK profiles of M923, US Humira, and EU Humira after administra-  Although demographic and baseline characteristics were similar across administration arms, we cannot rule out potential gender differences that may exist or potential differences seen when a nonhealthy population is studied. It is noteworthy that no gender-related PK differences were observed after correction for a patient's body weight in RA patients and healthy volunteers. 18 Furthermore, these PK findings are in line with similarly designed phase 1 studies that also demonstrated PK similarity between single 40-mg subcutaneous injections of the biosimilar ABP 501, FKB327, LBAL, and MSB11022 and either US Humira or EU Humira in healthy subjects. 13,[19][20][21] Therefore, the PK profile for M923 observed in healthy subjects may be predictive of equivalent PK in patients with immune-mediated conditions for which Humira has been approved.
M923 was demonstrated to be well tolerated, with a safety profile that was comparable to that observed for EU Humira and US Humira. The incidence, severity, and type of adverse events Furthermore, there were no differences seen in adverse events between treatment groups in those subjects who developed an ADA response. Twenty-seven of the 315 subjects in the PK analysis set had individual parameters that were excluded from the PK analysis set due to protocol deviations or failure to meet acceptability criteria. Half-life and related PK parameters could not be calculated in 6 subjects due to lack of defined terminal elimination phase.  As previously reported, and as illustrated in this study, adalimumab is highly immunogenic in healthy volunteers. Multiple factors contribute to ADA formation, including composition of the biological agent and route of administration. 24 In patients with RA, the concomitant use of methotrexate has been implicated in the lower proportion of ADA-positive patients observed. 25 The presence of ADAs can limit drug effectiveness or induce hypersensitivity and other adverse reactions. 24,26 The generation of ADAs is increasingly recognized as a mechanism that contributes to primary and secondary failure of biological drugs in chronic inflammatory diseases, 24,25,27 although variability has been reported. 7 In this study, the percentages of subjects over time with binding ADA formation and nADA formation were similar among all study arms. The results were also consistent with other recent studies evaluating biosimilars of Humira. 13,19,21 In the M923 study, the proportion of subjects with a confirmed ADA response, the rate of ADA seroconversion over time, and the occurrence of nADAs were in line with that reported by Kaur et al. 13 A lower ADA response (44%) has been reported with LBAL, a Humira biosimilar that is currently in development. 20 The ADA responses with M923 and other Humira biosimilars 13,19,21 are higher (>70%) than historically reported for Humira registration trials 9,10 or in a real-world study in RA. 25 The lower ADA response may reflect the fact that in some of these studies, patients may have also been receiving agents that could suppress the immune system and thereby reduce the incidence of antibody formation. 25