A randomized, single-blind, Phase I trial (INVICTAN-1) assessing the bioequivalence and safety of BI 695502, a bevacizumab biosimilar candidate, in healthy subjects

ABSTRACT

Objectives: This Phase I trial (INVICTAN®-1) evaluated three-way bioequivalence and safety of BI 695502 a bevacizumab biosimilar candidate, and reference product bevacizumab from two sources (US-approved Avastin®, Genentech; EU-approved Avastin, Roche).

Methods: Healthy male subjects (N = 91) were randomized 1:1:1 to receive a single intravenous infusion of 1 mg/kg of BI 695502 or US- or EU-approved Avastin. An interim analysis was planned when ~50% of subjects were evaluable for the primary end point to determine if the prespecified criteria for bioequivalence were achieved; if demonstrated, the study could be stopped early. The primary end point was area under the concentration–time curve (AUC) of the analyte in plasma from time zero extrapolated to infinity (AUC_0–∞). Other pharmacokinetic (PK) parameters, safety, and in vitro binding affinity were also evaluated.

Results: The interim analysis demonstrated three-way bioequivalence for all comparisons. The confidence intervals around the geometric mean ratios of the primary and secondary PK parameters were within the predefined acceptance ranges. Study drugs were well tolerated with no clinically relevant differences in safety.

Conclusion: BI 695502 and US- and EU-approved Avastin showed three-way bioequivalence with similar safety profile.

Clinical trial registration: NCT01608087.

KEYWORDS:

• Bevacizumab
• BI 695502
• bioequivalence
• biosimilar
• pharmacokinetics

1. Introduction

Bevacizumab is a recombinant humanized monoclonal immunoglobulin G1 antibody approved for the treatment of some solid tumors [1,2]. It binds to, and inhibits the activity of, human vascular endothelial growth factor (VEGF), also known as VEGF-A, preventing the development of new blood vessels (angiogenesis) [1]. VEGF-A is considered the most important VEGF family member with regards to regulation of angiogenesis in health and disease [3]. Neutralizing the biologic activity of VEGF leads to regression of tumor vascularization, normalizes remaining tumor vasculature, and prevents formation of new tumor vasculature, thereby inhibiting tumor growth [1].

Bevacizumab is approved for use in the US (Genentech, Avastin®) and the EU (Roche, Avastin) for the treatment of metastatic colorectal cancer (mCRC), non-squamous non-small cell lung cancer (nsNSCLC), renal cell cancer, epithelial ovarian, fallopian tube, or peritoneal cancer, and cervical cancer [1,2]. Bevacizumab is also approved for glioblastoma in the US [2] and breast cancer in the EU [1].

A biosimilar is a biologic that is highly similar to, and has no clinically meaningful differences from, the approved reference product [4]. While the use of biologics for the treatment of cancer has prolonged patient survival, the high cost of these agents has increased the financial burden on individual patients as well as healthcare systems [5]. Biosimilars have the potential to offer significant cost savings to both patients and healthcare systems.

Biologics are complex molecules that are derived from living cells using an intricate manufacturing process [6]. The focus of biosimilar development, and the regulatory approval process, is the extensive physicochemical and functional characterization of the biosimilar candidate to ensure similarity in terms of structure, mechanism of action, and pharmacology to the reference product [4,6,7]. Additionally, demonstration of bioequivalence is key to establishing biosimilarity [4,6,7].

BI 695502 is a bevacizumab biosimilar candidate being developed by Boehringer Ingelheim and is under investigation for use in the treatment of various cancers, including mCRC and nsNSCLC. The Phase I trial reported here (INVICTAN®-1) aimed to establish three-way bioequivalence and safety of a single dose of either BI 695502 or US- or EU-approved Avastin in healthy subjects.

2. Subjects and methods

2.1. Study objectives and end points

The objective of this study was to establish a three-way clinical pharmacokinetic (PK) equivalence bridge of BI 695502, US-, or EU-approved Avastin so that a global Phase III study could be conducted using only one of the reference products as a comparator drug, while satisfying global regulatory requirements for BI 695502. The primary end point for assessing bioequivalence was the area under the concentration–time curve (AUC) of the analyte in plasma over the time interval from zero extrapolated to infinity (AUC_0–∞). The secondary end points were maximum observed drug concentration in plasma (C_max) and AUC of the analyte in plasma over the time interval from time zero to the last quantifiable concentration (AUC_0–tz). Other PK parameters of interest and safety were exploratory end points.

2.2. Study design

This Phase I clinical trial was a randomized, single-blind, single-dose, two-stage, parallel-arm, active-comparator trial. A prespecified interim analysis was planned to be performed when approximately 50% of subjects were evaluable for the primary end point. The trial could be stopped if the interim analysis showed early significance for all comparisons.

Healthy subjects were enrolled at two sites in New Zealand and randomly assigned 1:1:1 to one of three treatment groups (Figure 1). Subjects received a single 30-min intravenous infusion (1 mg/kg) of either BI 695502, or US-, or EU-approved Avastin. Both US- and EU-approved Avastin were used in this Phase I clinical trial to comply with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines stating that comparisons must be made to the locally approved reference product [4,7]. As the PK profile of Avastin is linear for doses of 1–10 mg/kg [8], a dose of 1 mg/kg was selected in order to minimize exposure in this healthy population. A 30-min infusion time was considered safe for the 1 mg/kg body weight dose. The parallel design was chosen as bevacizumab has a long terminal half-life.

Figure 1. Patient flow.

PK: pharmacokinetic. ^aThe safety population consisted of subjects who received a single dose of study drug. ^bLeaking infusion set (subject may not have received the exact dose). ^cThe PK population consisted of all subjects who received a single dose of study drug and had at least one evaluable PK end point with no protocol violations relevant to the evaluation of bioequivalence.

The Independent Ethics Committees (IECs) reviewed and approved the trial prior to its initiation. The IECs met the requirements of the International Conference on Harmonization (ICH)/Good Clinical Practice (GCP) and local legislation. The trial was performed in compliance with the protocol, the principles of the Declaration of Helsinki, in accordance with the ICH Tripartite Guideline for GCP, and the applicable regulatory requirements.

2.3. Study population

Healthy adult males (≥21 to ≤50 years) with a body mass index (BMI) of ≤30 kg/m² were eligible to enroll in this study. Men of limited age range were enrolled into this study to control intersubject variability. Key exclusion criteria included hypertension or a relevant family history of hypertension; major injuries, surgery, or bone fracture within 4 weeks of the trial or planned surgical procedures during the trial; intended participation in collision sports during the trial; previous administration of a monoclonal antibody less than 15 weeks before the start of the trial; history of hemorrhagic or thromboembolic event; a known inherited predisposition to bleeding or thrombosis; and pre-existing proteinuria.

2.4. Safety evaluations

Safety evaluations including clinical laboratory parameters and vital signs were conducted by the investigators on all subjects who received the single dose of study drug (safety population). Adverse events (AEs) were classified using the most recent version of the Medical Dictionary for Regulatory Activities (MedDRA). AEs were assessed for severity (as defined by Common Terminology Criteria for Adverse Events [CTCAE] Version 4.0) and relationship to the study drug. All incidences of AEs that were observed during the trial were documented and reported as per the protocol. Any AEs occurring within 99 days of study drug administration were considered to be on-treatment.

2.5. Statistical analysis

The trial was performed as a two-stage design with Pocock boundaries. An interim analysis was planned to be conducted when approximately 50% of the subjects were evaluable for the primary end point. The predefined adjusted α-level for significance testing was 3.035% (one-sided), resulting in (two-sided) 93.93% confidence intervals (CIs) for the primary end point for both the interim and final analyses.

The sample size was calculated as 180 subjects, 90 subjects for Stage 1 of the trial and 90 subjects for Stage 2 if required (after interim analysis), so that the power to conclude three-way bioequivalence was approximately 90% assuming no treatment differences, and approximately 80% for a 5% treatment difference (ratio scale). The assumed variability was based on data in the literature for Avastin.

The ratios of the geometric means and their two-sided 90% CIs were provided for all secondary (90%) PK end points. The analysis was conducted using an analysis of covariance (ANCOVA) model on the logarithmic scale. Dependent variables were the logarithm of AUC_0–∞, AUC_0–tz, and C_max; independent variables were treatment and study site (categorical) as well as body weight at baseline (continuous). Bioequivalence was evaluated using the average bioequivalence method to test if the CIs of the ratio of the geometric means (BI 695502 vs. EU-approved Avastin, BI 695502 vs. US-approved Avastin, and US-approved vs. EU-approved Avastin) for the primary end points were within the prespecified acceptance range of 80–125%. These acceptance criteria needed to be reached for all three comparisons for bioequivalence to be declared; therefore, no adjustment of the type 1 error rate was required. All PK parameters were also analyzed descriptively. Graphics and statistical analyses were generated using SAS Version 9.2 (SAS, Cary, NC, USA).

2.6. Bioanalytic methods

Plasma, containing the K₃EDTA anticoagulant, was prepared by centrifugation (10 min at 2000–4000g) at 4–8°C. Plasma samples were frozen and stored at −20°C. Plasma concentrations of study drugs were measured using a validated, indirect enzyme-linked immunosorbent assay (ELISA). Goat anti-VEGF antibody (R&D Systems, Inc./Cat. No. AF-293-NA) was applied to the plate in order to immobilize VEGF (R&D Systems, Inc./Cat. No. 293-VE). Avastin and BI 695502 in plasma samples, after binding to the immobilized VEGF, were quantified by a secondary antihuman immunoglobulin G antibody (Millipore/Cat. No. AP504P) linked to horseradish peroxidase. The standard curve range was 30,000 ng/mL (upper limit of quantification) to 500 ng/mL (lower limit of quantification), prepared in neat plasma and diluted 1/100 in the assay. The resulting data were analyzed with a five-parameter logistic fit. All samples were tested using the same ELISA technique to ensure consistency in terms of methods used.

2.7. Pharmacokinetic analysis

The PK population included all subjects in the safety analysis set who provided at least one evaluable observation of any PK end point for bioequivalence evaluation. Blood samples for PK evaluation were collected on Day 1 (predose, just prior to the end of the infusion, and 2, 4, and 8 h after the start of the infusion) and on Days 2, 4, 5, 7, 14, 22, 35, 49, and 63.

PK parameters were determined using noncompartmental methods. C_max and the time of the maximum plasma concentrations (T_max) were measured directly from the observed plasma concentration–time data. The apparent terminal rate constant (λ_z) was determined using linear regression of the terminal phase of the log-transformed plasma concentration–time profiles; the terminal half-life was calculated as ln2/λ_z. AUC_0-tz, where tz is the time point of the last observed quantifiable plasma concentration, was calculated using linear up/log down trapezoidal summation. AUC_0–∞ was calculated as the sum of AUC_0–tz and C’_tz/λ_z, where C’_tz is the concentration at time tz, as predicted by the linear regression of the terminal phase of the log-transformed plasma concentration–time profiles. The total clearance of the analyte in plasma after intravenous administration (CL) was calculated by dividing the administered dose by AUC_0–∞, and the volume of distribution during the terminal phase (V_z) was calculated as CL/λ_z.

2.8. In vitro assays

The binding affinities of US- and EU-approved Avastin or BI 695502 to VEGF-A isoforms (VEGF-A₁₂₁, SinoBiological/Cat. No 10008-HNAH; VEGF-A₁₆₅, SinoBiological/Cat. No 11066-HNAH; and VEGF-A₁₈₉, Abcam/Cat. No ab106307) were measured by means of a surface plasmon resonance biosensor-based assay using Biacore T200 instrumentation. The assay involved immobilization of the recombinant VEGF-A proteins onto the sensor chip and subsequent measurement of antibody binding. Samples were diluted with running buffer (pH 7.4) to prepare a 1.23–100 nM concentration series before being injected onto the VEGF-A surface in single-cycle analysis format. The increase in response was measured over time. Association was determined by analysis of data obtained from multiple concentrations, whereas dissociation, which is independent of concentration, was determined over an extended time period (3600 s) following association of the highest concentration of sample. This method of analysis is widely used for high-affinity interactions in which dissociation is too slow to allow for a sufficient change in response units within the standard analysis time period.

To assess neutralization of VEGF function, proliferation of primary human umbilical vein endothelial cells (HUVECs; Promocell/Cat. No C-12203) was stimulated by incubating cells with VEGF-A₁₆₅ (10 ng/mL; PeproTech/Cat. No. 100–20) alone or pretreated with serial dilutions of BI 695502, US-, or EU-approved Avastin (0.03–150,000 ng/mL). After 72 h, cells were stained with the dye WST-1 and proliferation was quantified by measuring optical density (OD) at 440 nm. Proliferation was depicted as relative OD compared with HUVECs incubated with VEGF-A₁₆₅ alone. Mean curves for each test sample are presented from the results of four independent assays.

The assays described above are considered key for the development of this biosimilar. Extensive additional physicochemical characterization and in vitro functional assays were performed, but are not included in this manuscript describing the key PK outcomes of this clinical study.

3. Results

3.1. Subject demographics and baseline characteristics

Subject demographics and baseline characteristics were comparable between the three treatment groups. Overall, the mean age was 27 years with a range of 21–49 years. Mean BMI was 25.5 kg/m² and was comparable across the three treatment groups.

At the interim analysis, the trial met the prespecified criteria for bioequivalence and was stopped. Of the 91 randomized subjects, two who received US-approved Avastin discontinued prematurely and did not complete the planned observation period (Figure 1). One of these subjects was lost to follow-up and the other was unable to return to the trial site. These trial discontinuations did not impact on the PK evaluation and thus all primary and secondary PK parameters for these subjects were included in the analysis. One subject in the EU-approved Avastin group was excluded from the PK analysis due to a protocol deviation: the exact dose may not have been administered due to a leaking infusion set. Therefore, 30 subjects in each group were eligible for PK evaluation.

3.2. Pharmacokinetic evaluation

The median T_max was 2.0 h for all three treatment arms. Unadjusted geometric mean C_max levels were 23.7 µg/mL, 23.2 µg/mL, and 25.8 µg/mL for BI 695502, US-, and EU-approved Avastin, respectively (Table 1). Unadjusted geometric mean AUC_0–∞ levels were 7020 µg*h/mL, 7320 µg*h/mL, and 7580 µg*h/mL for BI 695502, US-, and EU-approved Avastin, respectively (Table 1). Mean plasma concentration–time profiles for all three treatment arms were comparable over the entire profiling period (Figure 2). An overview of the geometric mean and mean PK parameters is provided in Table 1.

Table 1. Summary of PK parameters in healthy subjects after a single dose of either BI 695502, US-, or EU-approved Avastin.

	BI 695502 (n = 30)		US-approved Avastin (n = 30)		EU-approved Avastin (n = 30)
	Geometric mean (gCV%)	Mean ± SD	Geometric mean (gCV%)	Mean ± SD	Geometric mean (gCV%)	Mean ± SD
AUC0–∞ (µg*h/mL)	7020 (20.0)	7160 ± 1490	7320 (16.1)	7400 ± 1140	7580 (17.7)	7700 ± 1490
AUC0–tz (µg*h/mL)	6580 (21.2)	6720 ± 1470	6680 (18.8)	6790 ± 1200	7110 (19.2)	7240 ± 1490
Cmax (µg/mL)	23.7 (15.5)	24 ± 3.58	23.2 (25.2)	23.8 ± 4.89	25.8 (16.4)	26.1 ± 4.44
T1/2 (h)	352 (17.7)	357 ± 67.1	374 (20.2)	381 ± 67.9	351 (25.3)	362 ± 94.6
CL (mL/h/kg)	0.142 (20.0)	0.145 ± 0.0276	0.137 (16.1)	0.138 ± 0.0231	0.132 (17.7)	0.134 ± 0.0217
Vz (mL/kg)	72.3 (22.9)	74.0 ± 15.0	73.7 (13.8)	74.4 ± 10.1	66.8 (23.7)	68.6 ± 15.6
	Median (minimum–maximum)
Tmax (h)	2.00 (0.483–8.0)		2.00 (0.467–168)		2.01 (0.483–24.0)

AUC: area under the concentration–time curve; AUC_0–∞: AUC of the analyte in plasma over the time interval from zero extrapolated to infinity; AUC_0–tz: AUC of the analyte in plasma over the time interval from time zero to the last quantifiable concentration; CL: total clearance of the analyte in plasma following intravenous administration; C_max: maximum observed drug concentration in plasma; gCV: geometric coefficient of variation; PK: pharmacokinetic; SD: standard deviation; T_1/2: terminal half-life of the analyte in plasma; T_max: time from administration to maximum observed concentration of the analyte in plasma; V_z: volume of distribution during the terminal phase λ_z (terminal rate constant in plasma) following intravenous administration.

Figure 2. Arithmetic mean plasma concentration–time profiles in healthy subjects after a single dose of study drug for BI 695502, US-, and EU-approved Avastin. The mean plasma concentration was not calculated for the BI 695502 and EU-approved Avastin arms after Day 49 as too many samples were below the lower limit of quantification.

i.v.: intravenous.

Results of the statistical comparison of primary and secondary PK end points corroborated the similarity of the mean plasma concentration–time curves (Figure 3). Bioequivalence was established by using the average bioequivalence method and hypothesis testing for the primary end point AUC_0–∞. Point estimates of the adjusted geometric mean ratio and corresponding CIs for all treatment comparisons of the primary and secondary PK end points are shown in Table 2.

Table 2. Pharmacokinetic bioequivalence results.

				Comparison
PK parameter	Comparison	Adjusted geometric mean	gCV	GMR (%)	CI^a
AUC0–∞ (µg*h/mL)	BI 695502 US-approved Avastin	7013 7261	19.52 15.57	96.58	88.54–105.35
	BI 695502 EU-approved Avastin^b	7013 7649	19.52 18.29	91.68	83.50–100.65
	US-approved Avastin EU-approved Avastin^b	7261 7649	15.57 18.29	94.92	87.14–103.40
AUC0–tz (µg*h/mL)	BI 695502 US-approved Avastin	6570 6639	20.68 18.46	98.96	90.97–107.64
	BI 695502 EU-approved Avastin^b	6570 7167	20.68 19.72	91.67	84.02–100.01
	US-approved Avastin EU-approved Avastin^b	6639 7167	18.46 19.72	92.63	85.18–100.74
Cmax (µg/mL)	BI 695502 US-approved Avastin	23.8 23.4	16.11 25.15	101.53	92.74–111.14
	BI 695502 EU-approved Avastin^b	23.8 25.5	16.11 15.18	93.25	87.12–99.81
	US-approved Avastin EU-approved Avastin^b	23.4 25.5	25.15 15.18	91.85	83.91–100.54

AUC: area under the concentration–time curve; AUC_0–∞: AUC of the analyte in plasma over time interval from zero extrapolated to infinity; AUC_0–tz: AUC of the analyte in plasma over the time interval from time zero to the last quantifiable concentration; CI: confidence interval; C_max: maximum observed drug concentration in plasma; gCV: geometric coefficient of variation; GMR: geometric mean ratio; PK: pharmacokinetic.

^a93.9% CI for AUC_0–∞ and 90% CI for AUC_0–tz and C_max.

^bOne subject was excluded.

Figure 3. Point estimates and confidence intervals of bioequivalence evaluation for primary and secondary PK parameters for BI 695502, US-, and EU-approved Avastin. Bioequivalence was declared if the confidence intervals (93.93% for primary and 90% for secondary end points) were within prespecified acceptance range of 80–125%.

AUC: area under the concentration–time curve; AUC_0–∞: AUC of the analyte in plasma over the time interval from zero extrapolated to infinity; AUC_0–tz: AUC of the analyte in plasma over the time interval from time zero to the last quantifiable concentration; C_max: maximum observed drug concentration in plasma; PK: pharmacokinetic.

Bioequivalence of BI 695502, US-, and EU-approved Avastin was concluded for all comparisons. Corresponding CIs for the adjusted geometric mean ratios of the primary (93.93% CI) and secondary (90% CI) end points were all within the prespecified acceptance range of 80–125%.

3.3. In vitro assays

Pharmacological in vitro assays analyzing the mode of action are part of the similarity assessment for comparing the biosimilar candidate BI 695502 with US- and EU-approved Avastin. These thorough evaluations are critical for proving initial similarity of the test compound before beginning clinical trials. VEGF-A₁₂₁, VEGF-A₁₆₅, and VEGF-A₁₈₉ are the main isoforms of VEGF-A which are neutralized by bevacizumab [3]. Surface plasmon resonance analysis serves as a state-of-the-art tool for measuring binding affinity and was used to compare the relative binding affinity of BI 695502 with the binding affinities of US- and EU-approved Avastin to each VEGF-A isoform. The analysis confirmed that binding affinity to VEGF-A isoforms −121, −165, and −189 was similar for BI 695502, US-, and EU-approved Avastin (Figure 4(a)). As shown in Figure 4(a), the maximal binding response of BI 695502 was comparable to the responses of US- and EU-approved Avastin. In addition, binding and dissociation (steepness of the curves) were similar between BI 695502, US-, and EU-approved Avastin.

Figure 4. In vitro VEGF-A binding assay (a) and HUVEC proliferation assay (b).

(a) Surface plasmon resonance analysis was used to assess the binding of all study drugs to VEGF-A₁₂₁, VEGF-A₁₆₅, or VEGF-A₁₈₉ (only relevant parts of the curves depicting the association and dissociation phases of the highest applied concentration are shown). Circles = US-approved Avastin Lot; Triangles = EU-approved Avastin Lot; Squares = BI 695502 Lot. Overlays of single representative Lots of each study drug are shown.(b) Proliferation of HUVECs was induced by recombinant VEGF-A₁₆₅ and the antiproliferative effects of BI 695502, US-, and EU-approved Avastin were compared. Circles = US-approved Avastin Lot; Triangles = EU-approved Avastin Lot; Squares = BI 695502 Lot. The mean dose–response curves of single representative Lots of each study drug tested in four independent assay runs are shown.HUVEC: human umbilical vein endothelial cell; VEGF: vascular endothelial growth factor.

The HUVEC proliferation assay demonstrated that BI 695502 inhibited VEGF-A₁₆₅-induced endothelial cell proliferation to the same extent as US- and EU-approved Avastin, providing indirect functional comparative data on the biologic neutralizing activity and similarity of BI 695502 (Figure 4(b)). A more comprehensive dataset for the in vitro assays and the pharmacologic approach for the development of BI 695502 will be published later elsewhere.

3.4. Safety

There were no clinically relevant findings for standard laboratory investigations or vital signs. Safety findings observed during and after intravenous infusion of bevacizumab (1 mg/kg) were comparable between the three treatment arms, with only mild and moderate AEs being observed (Table 3). No serious AEs, severe AEs (Grade 3 or higher as defined by CTCAE Version 4.0), or other significant AEs were reported and no subjects discontinued trial medication due to an AE. There were no deaths during the study period.

Table 3. Summary of AEs occurring in healthy subjects with one or more AEs, in any treatment group, after a single dose of BI 695502, US-, or EU-approved Avastin.

AE category n (%)	BI 695502 (n = 30)	US-approved Avastin (n = 30)	EU-approved Avastin (n = 31)
Subjects with ≥1 AE	26 (86.7)	21 (70.0)	26 (83.9)
Subjects with ≥1 severe AE^a	0 (0)	0 (0)	0 (0)
Subjects with serious AEs	0 (0)	0 (0)	0 (0)
Subjects with drug-related AEs	10 (33.3)	8 (26.7)	10 (32.3)
Upper respiratory tract infection	4 (13.3)	0	3 (9.7)
Headache	6 (20.0)	5 (16.7)	4 (12.9)
Epistaxis	1 (3.3)	0	1 (3.2)
Nausea	0	0	2 (6.5)
Abdominal pain	0	1 (3.3)	0
Diarrhea	1 (3.3)	1 (3.3)	1 (3.2)
Vomiting	1 (3.3)	0	0
Flatulence	0	0	1 (3.2)
Gastroesophageal reflux disease	0	0	1 (3.2)
Pain in jaw	0	1 (3.3)	0
AEs leading to discontinuation of study drug	0	0	0
Deaths	0	0	0

AE: adverse event.

^aGrade 3 or higher as defined by Common Terminology Criteria for Adverse Events Version 4.0.

The number of subjects with at least one drug-related AE was comparable between groups (10 in the BI 695502 group, eight in the US-approved Avastin group, and 10 in the EU-approved Avastin group). The most commonly reported drug-related AE was headache: six in the BI 695502 group, five in the US-approved Avastin group, and four in the EU-approved Avastin group (Table 3).

Immunogenicity data from this study will be published together with immunogenicity data from cancer patients in the BI 695502 Phase III studies to provide a comprehensive immunogenicity profile of BI 695502 compared with Avastin.

4. Discussion

BI 695502 is a bevacizumab biosimilar candidate being developed for the treatment of certain solid tumors, including mCRC and nsNSCLC. Current FDA and EMA guidelines require evaluating the bioequivalence of the biosimilar candidate to the reference product following extensive physicochemical and functional characterization [4,7]. This trial, INVICTAN-1, demonstrated three-way bioequivalence between the bevacizumab biosimilar candidate BI 695502, US-, and EU-approved Avastin based on all assessed PK end points. Together with the analytic data, the findings from this study justify the use of only one of the reference products as a comparator drug in a future Phase III study, which is the next step in the establishment of biosimilarity for BI 695502 to Avastin.

Safety results were similar between the study drugs, with no clinically meaningful differences observed across the three treatment groups. Few treatment-related AEs were reported, none of which were serious or severe in nature. Overall, no safety concerns were identified with BI 695502. Safety data in this trial are limited by the small sample size and the fact that a low subtherapeutic dose of BI 695502 was administered as a single infusion so that healthy subjects could be exposed to a drug that is normally used in cancer patients. Healthy subjects were chosen as a suitable population in which to detect differences in PK profiles of the compounds. In addition, the dose of 1 mg/kg was chosen as it is within the linear PK range of Avastin (1–10 mg/kg), allowing the PK results from this study to be extrapolated to the standard clinical dose. Ongoing Phase III studies will allow further assessment of the safety of BI 695502 at the appropriate dose and schedule for patients with NSCLC or mCRC.

In addition to bioequivalence, in vitro assays to measure binding of the antibody to the target antigen and neutralization of target bioactivity serve as an important part of the assessment of the molecular structure and function to demonstrate biosimilarity. Establishing similarity on a nonclinical level using analytic techniques is fundamental to the process of biosimilar development [4,6,7]. These data are important for the initiation of clinical trials, as Phase I and III trials cannot be conducted until sufficient evidence for structural and functional similarity has been demonstrated. In vitro assays showed that the three study drugs displayed similar binding affinities to three VEGF-A isoforms and inhibited the biologic effect of VEGF-A (HUVEC proliferation) to the same extent. A full and more rigorous analysis of the pharmacologic approach will be published elsewhere. Taken together, these data provide the first evidence of similarity of BI 695502 to both the US- and EU-approved Avastin in vitro.

The clinical development of BI 695502 continues with two ongoing pivotal Phase III clinical studies in patients with NSCLC (ClinicalTrials.gov NCT02272413) or with mCRC (ClinicalTrials.gov NCT02776683).

5. Conclusions

Three-way bioequivalence between BI 695502, US-, and EU-approved Avastin was demonstrated. Together with the safety observations and the physicochemical and functional results, these data provide the basis for the conduct of a larger Phase III study, which is the next step to establish full biosimilarity.

Declaration of interest

C. Wynne is an employee of, and holds shares in, an organization that received payment for carrying out the trial. B. Lang, M. Altendorfer, N. Czeloth, R. Lohmann, and D. Schliephake are employees of Boehringer Ingelheim. S. Athalye and W. Hettema were employees of Boehringer Ingelheim at the time this trial was performed and when this publication was initiated.

Acknowledgments

C. Wynne was an investigator in the trial and provided a critical review of the manuscript. W. Hettema, B. Lang, M. Altendorfer, N. Czeloth, R. Lohmann, S. Athalye, and D. Schliephake were responsible for the interpretation of the trial data and provided critical reviews throughout the development of this manuscript. All authors have approved the final manuscript for submission. Medical writing support was provided by SciMentum, UK, and GeoMed, an Ashfield company, part of UDG Healthcare plc, UK, and funded by Boehringer Ingelheim.

Trial registration number: NCT01608087.

Additional information

Funding

This study was funded by Boehringer Ingelheim.