Introduction
Biosimilars are biopharmaceuticals that are approved by a regulatory agency and are highly similar to an approved marketed biologic product [
1‐
3]. There is strong interest throughout the world in developing biosimilars to many currently marketed biopharmaceuticals, with the hope of reduced costs for medical care coupled with increased accessibility to these medicines.
Many countries, including those in the European Union (EU) and the United States (US), have created abbreviated approval pathways for biosimilars (compared with the requirements for innovative molecules) that require a comparison of the proposed biosimilar with the approved product, generally termed the reference product, to demonstrate biosimilarity. The EU defines the reference product as the product that has been granted marketing authorization in the EU [
1]. In a similar manner, the US Biologics Price Competition and Innovation Act defines the reference product as the US Food and Drug Administration (FDA)-licensed product [
2]. Under certain circumstances, both the European Medicines Agency (EMA) and FDA allow the use of data derived from human or animal studies comparing a proposed biosimilar to a reference product licensed in another region that has scientific and regulatory standards compatible with the International Conference on Harmonization [
2,
4]. From a practical standpoint, this allows the possibility of conducting nonclinical in vivo studies with material from the EU or US to support global clinical trials. However, at the time of designing the nonclinical package in support of the development of PF-06438179, EMA guidance stipulated the use of the EU reference product, and hence, for the in vivo nonclinical studies, the EU reference product was employed as a comparator. Both regions suggest that biosimilar development should follow a stepwise approach, with a greater emphasis on in vitro assays, followed by generally limited or possibly no nonclinical in vivo studies prior to initiation of clinical trials in humans.
Remicade
® (Infliximab; Janssen Biotech, Inc. Horsham, PA, USA) is a chimeric human–mouse monoclonal antibody (mAb) approved throughout much of the world for the treatment of a variety of inflammatory conditions, including Crohn’s disease, pediatric Crohn’s disease, ulcerative colitis, pediatric ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis [
5]. The Fc region of infliximab is human, while the Fv region with the complementarity determining region (CDR) that binds human tumor necrosis factor alpha (TNF) is derived from the mouse. Infliximab binds soluble and transmembrane human TNF and blocks its interaction with p55 and p75 cell surface TNF receptors, thereby neutralizing the proinflammatory effects of TNF [
6]. A number of nonclinical in vivo studies were conducted during the development of infliximab. These included single- and repeat-dose (up to five doses) studies in chimpanzees, single- and repeat-dose (up to seven doses) studies in rats, and a local tolerance study in rabbits [
7]. Nonclinical toxicity studies were conducted primarily in chimpanzees because infliximab neutralizes human and chimpanzee TNF with similar potency. Infliximab does not have activity against mouse, rat, rabbit, pig, cotton top tamarin, marmoset (species not identified), pigtail macaque, rhesus monkey, cynomolgus monkey, or baboon TNF [
7]. Although weak activity against dog TNF was observed (0.01% of potency for human or chimpanzee TNF), nonclinical testing of infliximab in this species was discontinued because immediate dermal hypersensitivity (urticaria and angioedema) was observed with the initial intravenous (IV) infusion [
7]. In rats, minimal and reversible hepatic changes (Kupffer cell and hepatocellular hyperplasia) and slight reductions in erythrocyte counts, hemoglobin levels, and hematocrit values were observed after single or repeat daily doses at ≥10 mg/kg/day, but these findings were not considered relevant to humans, because they were considered to be related to the response of a normal rat reticuloendothelial system to large doses of chimeric (human–mouse) antibody, a foreign protein to this test animal [
8]. Infliximab was well tolerated in chimpanzees, rabbits, and rats. Additional studies were conducted in mice using a surrogate anti-mouse TNF mAb that was reported to have similar pharmacodynamic and pharmacokinetic properties as infliximab [
7]. No adverse infliximab surrogate-related findings were observed in 6-month repeat-dose or developmental and reproductive toxicity studies in mice using the surrogate anti-mouse TNF mAb up to 40 mg/kg [
7].
PF-06438179 is an anti-TNF mAb currently under development as a potential biosimilar to Remicade. Consistent with current regulatory authority guidelines [
1‐
3], a stepwise approach was used during development, whereby PF-06438179 was first evaluated for structural and functional similarity to the reference product in vitro. These analytical studies of PF-06438179 were performed side by side with Remicade sourced from the US (infliximab-US) [
5] and the EU (infliximab-EU) [
9]. Key attributes of primary structure, post-translational modifications, product purity, charge heterogeneity, and biological activity were shown to be similar [
10]. Based on the comparative structural and functional results providing strong support for analytical similarity, of which only a small subset is outlined in this nonclinical paper, PF-06438179 was then evaluated in limited in vivo nonclinical studies. This study reports results comparing the structural, functional, and in vivo nonclinical attributes of PF-06438179 with those of the reference product infliximab. Overall, the data demonstrated analytical and nonclinical similarity of PF-06438179 to infliximab, and have supported global clinical trials with PF-06438179.
Discussion
In vitro analytical characterization, of which only a small subset is presented in this study, demonstrated that the proposed biosimilar, PF-06438179, is structurally and functionally similar to its reference products, infliximab-US and infliximab-EU, and that the reference products are similar to each other. Peptide mapping and subunit analyses confirmed that PF-06438179 has the identical primary structure as that of the reference product infliximab. Peptide mapping, subunit analysis, and iCE focusing confirmed that PF-06438179 has post-translational modifications similar to those of the reference products, with the exception of the levels of C-terminal lysine. This is a minor difference that is not expected to be clinically significant because the C-terminal lysine is rapidly processed in vivo with a half-life of 62 min [
10,
11]. Size exclusion HPLC confirmed PF-06438179 has a product profile and a purity similar to those of the reference products. In the biological activity assay, PF-06438179 bound TNF, thus inhibiting TNF binding to its receptors on U937 cells and subsequently preventing TNF-related cell apoptosis. PF-06438179 had a similar dose response curve to that observed with infliximab-US and infliximab-EU. This side-by-side analytical characterization indicates that key structure/function attributes of reference product infliximab were successfully designed into PF-06438179 and provides strong support that PF-06438179 is analytically similar to infliximab-US and infliximab-EU. The analytical characterization also demonstrates that infliximab-EU is similar to infliximab-US.
Based on this similarity, only a limited in vivo nonclinical testing program was considered necessary. This strategy was consistent with global biosimilar guidance documents [
2,
3,
12,
13] and shaped by interactions with the EMA (EU), FDA (US), and Pharmaceuticals and Medical Devices Agency (Japan). The nonclinical in vivo program initially conducted consisted of a single-dose IV TK/tolerability study in male rats to compare the similarity of PF-06438179 versus infliximab-EU. The plan to conduct only this single-dose study in male rats was based on several factors:
1.
Infliximab is not pharmacologically active in typical nonclinical test species [
7]. The only animal species demonstrating significant pharmacologic activity is chimpanzees. However, the use of chimpanzees is not considered ethically acceptable [
14], and thus, a study in chimpanzees was not appropriate.
2.
Conducting toxicity studies in pharmacologically nonrelevant species is generally discouraged as they can be misleading [
15], and thus, repeat-dose toxicity studies in standard nonclinical species were not considered necessary.
3.
No significant infliximab-related findings were reported in a number of studies with various species conducted by the originator, including in chimpanzees as well as in a variety of pharmacologically-nonrelevant species. Thus, conducting toxicity studies with PF-06438179 (± infliximab-US or infliximab-EU) would not be expected to demonstrate any findings, and such studies were not considered necessary.
4.
Although the infliximab-CDR does not bind TNF from animal species other than chimpanzees, the neonatal Fc receptor (FcRn) from animal species including rats [
16] does recognize the Fc fragment of human immunoglobulin (Ig) G, and thus, it is possible to characterize FcRn-mediated/nontarget-related clearance and TK and assess Fc-related functionality in animals, including rodents.
5.
Infliximab clearance in humans decreases with increasing infliximab dose, likely related to saturation of target-mediated drug disposition (TMDD), and exposure appears to be linear at doses of 3–20 mg/kg [
5], which is within the range of the approved/therapeutic dose of 3–10 mg/kg. Thus, nontarget-mediated mechanisms (i.e., FcRn recycling) play a more significant role in the overall pharmacokinetics (PK) profile of infliximab in humans relative to TMDD, and therefore, PK/TK studies in animal species that lack TMDD are relevant with respect to the human PK of infliximab at these or higher doses.
6.
The use of rats (versus mice) enabled serial blood sampling for the evaluation of exposure and ADA response in individual animals over time.
7.
Safety and exposure information was publically available from studies conducted previously with infliximab in rats.
8.
Only one sex was considered necessary because no sex differences in TK were observed with the originator infliximab in rats [
17]. In addition, there were no major weight-, age-, or sex-related differences in clearance or volume of distribution of infliximab in patients [
5].
9.
The use of only the EU-approved product (infliximab-EU) as the comparator in the TK/tolerability study was considered reasonable because the in vitro assays demonstrated that infliximab-licensed products sourced from the US or EU were similar to each other.
10.
Overall, this scientifically based approach minimized animal use significantly, compared with a standard toxicology program.
Like the originator infliximab (Remicade) studies, there was no PF-06438179- or infliximab-EU-related toxicity observed during the single-dose study. The TK profile, systemic exposures, and ADA response of PF-06438179 were similar to those of infliximab-EU. Based on these results, the tolerability, TK, and ADA responses of PF-06438179 were considered similar to those of infliximab-EU when administered as a single IV bolus dose to male rats at doses of 10 or 50 mg/kg. It should be noted that the absence or presence of ADA nonclinically is not predictive of responses in humans, and because immunogenicity is a key attribute for establishing biosimilarity between the proposed biosimilar and the reference product, it has been/is being monitored in completed and ongoing clinical studies with PF 06438179.
The strategy of conducting a single-dose TK and tolerability study in male rats was successful in allowing initiation of comparative clinical (phase I) trials in humans in the US, and is consistent with recent publications, suggesting that the requirement for nonclinical in vivo testing of biosimilars shifts from being a default requirement to being necessary only under selected situations where there is residual doubt regarding similarity that remains following in vitro testing [
13,
18‐
20]. However, because pharmaceutical companies are seeking to develop biosimilar drug products globally, the overall program must meet the requirements of all countries where trials and registration are planned. During additional regulatory interactions, a 2-week toxicity study with PF-06438179 in both sexes was requested before clinical trials could proceed in one country/region. Based on this request, a 2-week IV toxicity study with PF-06438179 in male and female rats was conducted.
In the 2-week toxicity study in rats, once-weekly IV (bolus) administration of 10 or 50 mg/kg doses of PF-06438179 produced no adverse findings in any parameters evaluated. PF-06438179-related microscopic findings were limited to minimal to mild sinusoidal cell hyperplasia in the liver in both males and females administered 50 mg/kg. This finding was not adverse because of the minimal to mild severity and lack of other effects on the liver. The sinusoidal cell hyperplasia observed in this study was consistent with Kupffer cell hyperplasia observed in previous studies in rats following IV administration of Remicade at either single daily doses ≥10 mg/kg, or at seven daily doses ≥10 mg/kg/day [
8], or the EU-approved biosimilar of Remicade (Remsima
®, Celltrion Healthcare Co., Ltd.) at a dose of up to 40 mg/kg once weekly for 2 weeks [
21]. This finding could have potentially resulted from the response of a normal rat reticuloendothelial system to large doses of chimeric (human–mouse) antibody, a foreign protein to this test animal [
8], and therefore, it was not considered relevant to humans administered PF-06438179 or infliximab. Anti-drug antibodies were not evaluated in the 2-week study because there was no evidence of an ADA response in the single-dose study, and a lack of impact on serum concentrations of the study drug(s) in either study. Other test article-related differences in platelet counts, neutrophils, monocytes, large unstained cell counts, and fibrinogen were nonadverse, based on their small magnitudes and the lack of any correlative microscopic findings, such as inflammation in any organs. Minor hematologic findings (e.g., increases in reticulocytes and platelets at 50 mg/kg) have also been reported following administration of Remicade or Remsima to rats [
17].
Acknowledgments
This study was sponsored by Pfizer Inc. The article processing charges for this publication were funded by Pfizer Inc. Medical writing support was provided by Vardit Dror, Ph.D., of Engage Scientific Solutions and funded by Pfizer Inc. Dr. Zaher Radi conducted the peer review of the clinical and anatomic pathology data and reports from the 2-week toxicity study. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published. All authors had full access to all of the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis.