FormalPara Key Summary Points

This phase 1 study established equivalence between BP02 (a trastuzumab biosimilar), EU-trastuzumab, and US-trastuzumab.

111 healthy male volunteers were randomized 1:1:1 to the three treatments.

Treatments were administered as a single 6-mg/kg intravenous infusion, and volunteers were evaluated for 78 days.

The 90% confidence intervals for the ratios of area under the serum concentration–time curve (AUC) from the time of dosing to infinity (AUC0-inf), AUC from the time of dosing until the time of the last quantifiable concentration (AUC0-t), and peak serum concentration of trastuzumab (Cmax) were within 80% to 125% for all three pairwise comparisons.

BP02, EU-trastuzumab, and US-trastuzumab were shown to have pharmacokinetic similarity, with no new safety and immunogenicity concerns for BP02.

Introduction

The overexpression or amplification of the human epidermal growth factor receptor 2 (HER2) gene that encodes the HER2 protein has been associated with numerous malignancies and accounts for around 20% of breast cancer and gastric cancer cases, 16% of ovarian cancer cases, and 1.3% of colorectal cancer cases [1]. In fact, HER2 is also an important biomarker in the management of several tumors, most prominently breast cancer [2]. Trastuzumab, marketed under the trade name Herceptin®, became the first monoclonal antibody targeting HER2 to receive United States Food and Drug Administration (USFDA) approval in 1998 and European Union (EU) approval in 2000 for the management of HER2-positive metastatic breast cancer, either alone or in combination with paclitaxel [3, 4]. Subsequently, trastuzumab was also approved for adjuvant therapy of HER2-positive early breast cancer and for HER2-positive gastric cancer [4, 5]. Trastuzumab works by binding to an extracellular domain of the HER2 receptor and inhibits AKT phosphorylation, leading to cell cycle arrest through AKT inhibition, although other mechanisms of action have been proposed [6].

Although the efficacy and safety of trastuzumab have been proven, and trastuzumab is also included in the WHO Model List of Essential Medicines, access to trastuzumab is a challenge, especially in low-to-middle-income countries and for patients with advanced or metastatic breast cancers, principally due to its cost [7]. According to a 2011 publication, trastuzumab was reported to have been used by only 8.6% of eligible patients, more than half of whom received it as part of clinical trials [8]. Improving patient access to trastuzumab can be achieved by reducing the cost of therapy; developing trastuzumab biosimilars and achieving marketing approval for them is an essential step in this direction [7]. The USFDA defines biosimilars as “a biological product that is highly similar to a US‐licensed reference product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of safety, purity, and potency of the product” [9]. Accordingly, prior to regulatory approval, it is important to prove that the potential biosimilar does not show clinically meaningful differences in safety, pharmacokinetics, immunogenicity, and efficacy when compared to the licenced biologic [9, 10].

BP02 is a recombinant IgG1 antibody against HER2 that is being developed by CuraTeQ Biologics, India, as a proposed biosimilar to trastuzumab. BP02 binds to the extracellular domain of HER2 receptors with high affinity and has an identical primary amino acid sequence to trastuzumab sourced from the European Union (EU-trastuzumab) and trastuzumab sourced from the United States (US-trastuzumab), where it is already marketed for approved use in several tumor types. Similarity has already been demonstrated between BP02 and both the reference trastuzumab products for physicochemical and functional quality attributes of the primary structure, protein composition, heterogeneity, higher-order structure, post-translation modifications, glycosylation, and biological functions [data on file]. The objective of this study was to establish equivalency in terms of the pharmacokinetics, safety, and immunogenicity of BP02 compared to EU-trastuzumab and US-trastuzumab in normal healthy adult male subjects after single-dose intravenous (IV) administration.

Methods

Study Design and Participants

This was a phase 1, randomized, double-blind, three-way parallel-group clinical trial conducted in two centers in New Zealand between July 2021 and May 2023. The drugs used in this trial were BP02 (CuraTeQ Biologics Private Limited, India), reference EU-trastuzumab (Herceptin®, Roche Pharma AG, Grenzach-Wyhlen, Germany), and reference US-trastuzumab (Herceptin®, Genentech, Inc., South San Francisco, CA, USA).

The trial was restricted to healthy male volunteers aged 18 to 65 years to control variability. Subjects were eligible for study inclusion if they had a BMI (body mass index) of 18–30 kg/m2, a body weight of 50–100 kg, and had no clinically relevant abnormalities of any body system based on history, physical examination, and laboratory tests, including thyroid function tests, 12-lead electrocardiogram (ECG), and echocardiogram. Subjects were required to refrain from donating sperm or fathering a child or they agreed to use two acceptable contraceptives for up to 9 months after the administration of the test drug. Subjects were also required to abstain from smoking for 48 h prior to recruitment.

Subjects with a known history of hypersensitivity to trastuzumab, those who had any clinically relevant condition of any organ system detected by a history check, physical examination, ECG, or laboratory investigation, or those who had a left ventricular ejection fraction (LVEF) < 55% were excluded from the study. Other major exclusion criteria were a history of cancer; testing positive for HIV, HBsAg, or HCV; the concurrent use of any drug or supplement that, in the opinion of the investigator, had the potential to interfere with the study results; the receipt of a COVID-19 vaccine within 14 days prior to or within 15 days after the completion of the study; the use of hematopoietic growth factors, monoclonal antibodies, or immunoglobulins within 6 months prior to screening (or five half-lives, whichever was longer); major surgery or trauma within the past 1 year; a history of or current alcohol abuse or drug abuse; and the receipt of any monoclonal antibody therapy as a part of a study within 90 days prior to dosing. The complete list of inclusion and exclusion criteria is available in Supplementary Table 1 in the Supplementary Information.

Eligible subjects were randomized 1:1:1 to receive a single 6-mg/kg dose of BP02, EU-trastuzumab, or US-trastuzumab via IV infusion over 90 min. Dosage selection was based on the approved maintenance dose of 6 mg/kg over 90 min for various indications [11] and similar doses used in previous studies [12, 13]. Randomization was carried out using a number list within coded envelopes and in a double-blind fashion, with treatment allocation concealed until the database was locked after study completion.

Outcomes and Assessments

Pharmacokinetic Evaluations

Blood samples were collected immediately pre-infusion (0 min); post-dose samples were taken 0.5, 1, 4, and 6 h after the end of infusion (±10 min window), at 24 h and 48 h post-dose relative to the start of infusion, and on days 3, 4, 8, 15, 22, 29, 36, 50, 64, and 78 (Fig. 1). A validated indirect sandwich enzyme-linked immunosorbent assay (ELISA) method was used to measure trastuzumab in human serum [14].

Fig. 1
figure 1

Study design. Healthy men were randomized to receive a single 90-min infusion of 6 mg/kg of BP02, US-trastuzumab, or EU-trastuzumab; blood samples were collected at pre-specified intervals. aOne subject who was randomized to EU-trastuzumab withdrew their consent prior to dosing. bReasons for discontinuation post-discharge: BP02, AEs (N = 1); US-trastuzumab, AEs (N = 3) and withdrawal of consent (N = 1). AEs adverse events

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The primary PK endpoints were area under the serum concentration–time curve (AUC) from the time of dosing to infinity (AUC0-inf), AUC from the time of dosing until the time of the last quantifiable concentration (AUC0-t), and peak serum concentration of trastuzumab (Cmax). Secondary PK endpoints were time to achieve Cmax (Tmax), half-life (T½), elimination rate constant (λz), and systemic clearance of trastuzumab (CL). The PK similarity of EU-trastuzumab and US-trastuzumab was also evaluated to provide bridging data that can justify the use of a single reference product in future comparative clinical trials in patient populations.

Safety Evaluations

Safety evaluations were performed based on physical examination, vital signs, ECG parameters, echocardiogram, clinical safety laboratory tests (hematology, clinical biochemistry, coagulation, urinalysis, and urine microscopy where clinically indicated), infusion-related reactions, adverse events (AEs), and concomitant medication.

All safety evaluations were conducted at screening, at subject discharge from the unit, and at study exit or early termination. Vitals were checked and a physical examination was conducted at every subject contact; a 12-lead ECG and an echocardiogram were conducted on days 8, 22, and 78, with two additional ECG recordings made on days 3 and 4; blood and urine analyses were performed on days 2, 4, 8, 15, 29, 50, and 78.

AEs were coded using a central coding dictionary, the Medical Dictionary for Regulatory Activities (MedDRA), version 25.1. An AE was defined as any clinically significant observation outside the normal range, and a treatment-emergent AE (TEAE) was defined as any AE that first occurred or worsened in severity after the administration of the study treatment.

Immunogenicity Evaluations

Blood samples were collected for the measurement of antidrug antibodies (ADA) and neutralizing antibodies (NAb) on days 1, 50, and 78. A validated electrochemiluminescence immunoassay (ECLIA) method was used for the determination of trastuzumab anti-drug antibodies and neutralizing anti-drug antibodies in human serum [15].

Subjects who were confirmed positive for anti-trastuzumab antibodies, including subjects who discontinued the study after receiving the drug, were followed up for up to 12 months. These subjects attended the clinical center at 6, 9, and 12 months post-dose for blood sampling to confirm their immunoglobulin status or until two consecutive samples had been confirmed negative for anti-trastuzumab antibodies, whichever occurred earlier.

Statistical Analysis

PK parameters were derived using non-compartmental methods with Phoenix® WinNonlin® version 8.3 (Certata, L.P., Princeton, NJ, USA). Cmax was obtained directly from the observed concentration versus time data, and Tmax was obtained from Cmax. AUC0-t was calculated by linear-up/log-down trapezoidal summation; AUC0-inf was calculated as the sum of AUC0-t plus the ratio of the last measurable plasma concentration to the elimination rate constant; and CL was calculated as dose divided by AUC0-inf. λz was calculated from a semi-log plot of the serum concentration–time curve by linear least-squares regression analysis using the maximum number of points in the terminal log-linear phase, and T½ was calculated as 0.693/λz.

The general linear models procedure (PROC GLM) of the SAS software (SAS Institute, Cary, NC, USA) was used for statistical analyses. If the 90% confidence intervals (CIs) for AUC0-inf, AUC0-t, and Cmax were within the 80–125% range (natural-log-transformed data, one-sided tests, α = 0.05), the comparison was considered to indicate bioequivalence.

Considering an expected treatment ratio of 95%, a maximum observed inter-subject coefficient of variation of approximately 20%, and a dropout rate of approximately 10%, a sample size of 37 subjects per arm was calculated as providing at least 90% power at a 5% significance level for all pairwise comparisons for the primary endpoints.

Ethical Conduct in the Study

This study was conducted in accordance with the ethical principles enshrined in the Declaration of Helsinki as well as all applicable local and regional laws. All subjects provided informed consent prior to study initiation. The study was approved by the Health and Disability Ethics Committee (HDEC; approval number 20/STH/190) and was registered at the Australian New Zealand Clinical Trials Registry (ANZCTR) prior to the initiation of subject screening (ACTRN12621000573853).

Results

Subject Disposition and Baseline Characteristics

Of the 241 male subjects who underwent screening, 112 subjects were randomized and 111 received the study drug (37 each received BP02, EU-trastuzumab, and US-trastuzumab), forming the safety and immunogenicity analysis set. One subject randomized to EU-trastuzumab withdrew consent before dosing. A total of 106 subjects completed the study; of the five subjects who discontinued the study, one subject belonged to the BP02 group (reason for discontinuation: AEs), and the remaining four belonged to the US-trastuzumab group (reasons for discontinuation: AEs (N = 3), withdrawal of consent (N = 1)). A total of 104 subjects were included in the final PK analysis set after excluding two subjects who received US-trastuzumab (reason for exclusion: major protocol deviations) (Fig. 1).

The baseline demographic characteristics in the three treatment groups were similar except for a small difference in mean age, which was slightly higher in the EU-trastuzumab group (38.6 years) compared with the US-trastuzumab (33.1 years) and the BP02 (30.6 years) groups (p = 0.021; Table 1).

Table 1 Baseline demographic characteristics
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Pharmacokinetic Analysis

Of the 104 subjects included in the PK analysis, 36 received BP02, 37 received EU-trastuzumab, and 31 received US-trastuzumab (Fig. 1). The trastuzumab serum concentration–time profile was similar for all three interventions (Fig. 2).

Fig. 2
figure 2

Linear (a) and semilogarithmic (b) serum concentration–time curves for BP02 (N = 36), EU-trastuzumab (N = 37), and US-trastuzumab (N = 31). SD standard deviation

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The evaluated primary PK parameters (AUC0-inf, AUC0-t, and Cmax) as well as the secondary PK parameters (median Tmax, T½, mean λz, and CL) were similar for the three treatment groups (i.e., BP02, EU-trastuzumab, and US-trastuzumab). For all primary and secondary PK parameters, the coefficients of variation for the three groups were comparable (Table 2).

Table 2 PK parameters (mean ± SD) of BP02, EU-trastuzumab, and US-trastuzumab
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The geometric mean ratios (GMRs) of AUC0-inf, AUC0-t, and Cmax are summarized in Table 3. The PK equivalence of BP02, EU-trastuzumab, and US-trastuzumab was established since the 90% CI of the GMR of each pair was observed to be within the 80–125% range for the three main PK outcomes (AUC0-inf, AUC0-t, and Cmax). Further, upon applying the same principles, EU-trastuzumab and US-trastuzumab were also found to have equivalent PK parameters. In summary, BP02, EU-trastuzumab, and US-trastuzumab were found to be biosimilar with respect to both primary and secondary PK parameters following IV infusion of a single 6-mg/kg dose in healthy adult male subjects.

Table 3 Comparative PK parameters of BP02, EU-trastuzumab, and US-trastuzumab
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Safety and Tolerability Analysis

Of the 111 subjects who received the intervention, a total of 281 TEAEs were reported in 87 subjects. Of these, 56 subjects had 106 TEAEs related to the study treatment. In general, TEAEs were either mild or moderate in severity, and no severe TEAE was reported in the study. The proportion of subjects with TEAEs was numerically higher in the US-trastuzumab group (89.2%) compared to the BP02 group (73.0%) and the EU-trastuzumab group (73.0%). The proportion of subjects with TEAEs that were found by the investigator to be related to the study treatment was numerically higher in the US-trastuzumab group (62.2%) compared with the BP02 group (48.6%) and the EU-trastuzumab group (40.5%). The US-trastuzumab group was also associated with a higher frequency of infusion-related reactions (43.2%) compared to the BP02 group (21.6%) and the EU-trastuzumab group (18.9%). The most frequently reported TEAE, with an incidence of ≥ 5% in all three groups, was headache (BP02 group: 27.0%, EU-trastuzumab group: 29.7%, and US-trastuzumab group: 37.8%) (Table 4).

Table 4 Summary of the most frequently reported TEAEs by treatment
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Although the proportion of subjects with a mild TEAE was similar in all the treatment groups (BP02: 62.2%, EU-trastuzumab: 59.5%, and US-trastuzumab: 59.5%), subjects in the US-trastuzumab group reported a numerically higher proportion of moderate TEAE (29.7%) compared with the EU-trastuzumab group (13.5%) and the BP02 group (10.8%). One subject treated with EU-trastuzumab suffered a lower-limb fracture, which was classified as a serious TEAE, but it was found not to be related to the study treatment. No deaths were reported during the study.

Four subjects (one in the BP02 group and three in the US-trastuzumab group) discontinued the study due to TEAEs. All of these subjects had infusion-related reactions during the study, which were probably related to the treatment administered. No clinically significant abnormalities in laboratory values, physical examinations, ECG, and echocardiogram values (including LVEF) were reported during the study. All subjects with TEAEs recovered completely with appropriate management.

A total of 31 subjects experienced 50 TEAEs of special interest in the study (Table 5). The proportion of subjects with TEAEs of special interest were numerically higher in the US-trastuzumab group (43.2%) compared with the BP02 group (21.6%) and the EU-trastuzumab group (18.9%).

Table 5 Summary of TEAEs of special interest by treatment
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Immunogenicity Analysis

The immunogenicity analysis set consisted of all 111 subjects who received the intervention (Fig. 1). Only two subjects (one subject each in the EU-trastuzumab and US-trastuzumab groups) tested positive for ADA in the pre-dosing sample on day 1; both subjects were negative for NAb. All subsequent samples collected from both subjects were negative for both ADA and NAb. Samples from other subjects were not positive for either ADA or NAb.

Discussion

The results of this phase 1, randomized, double-blind, three-way parallel-group study demonstrated equivalence in PK parameters and safety profiles between BP02, EU-trastuzumab, and US-trastuzumab among healthy men following a single 6-mg/kg IV infusion over 90 min.

For all three interventions, Cmax was achieved rapidly, and serum drug concentrations gradually declined, resulting in similar serum concentration–time profiles. The three products also had similar PK parameters, including AUC0-inf, AUC0-inf, Tmax, T½, λz, and CL. BP02 was found to have PK equivalence with both reference products (EU-trastuzumab and US-trastuzumab), as the 90% CIs of the estimated GMRs of the three primary PK endpoints (AUC0-inf, AUC0-t, and Cmax) were within the equivalence margin of 80–125%. PK equivalence between EU-trastuzumab and US-trastuzumab was also established in this study by the same approach.

BP02 was found to be safe and well tolerated, with safety and immunogenicity profiles similar to those of the two reference products. TEAEs were, on average, similar among the groups, although moderate TEAEs were observed at a numerically higher frequency in the US-trastuzumab group than in the other two groups. Further, well-powered studies are required to evaluate if there is indeed a difference in safety profile between US-trastuzumab and EU-trastuzumab and the potential reasons for this difference. No deaths were reported, no notable trends or clinically significant findings emerged, and no unexpected AEs were observed. Only one serious AE (a lower-limb fracture) was reported, and this was deemed to be unrelated to the treatment administered. Four subjects discontinued the study due to TEAEs (mainly infusion-related reactions), three of whom were from the US-trastuzumab group. The safety data reported in our study are consistent with the findings from previous PK studies that trastuzumab is well tolerated [12,13,14, 16, 17], and they also show that BP02 has a similar safety and tolerability profile to the reference products. Only two subjects tested positive for ADA on day 1; both were negative for ADA on subsequent days, and none of the other subjects tested positive for ADA throughout the study period. Also, those two subjects were not exposed to BP02 previously. This observation indicates the low immunogenicity of trastuzumab and is consistent with previous PK studies [12,13,14, 16, 17]; nevertheless, the impact of the development of ADAs and NAbs on the pharmacokinetics and safety remains to be studied in both healthy volunteers and in patients for whom trastuzumab is indicated.

A parallel-group design was the preferred design for this trial, considering that the average t½ of trastuzumab ranges from 1.7 to 12 days [18] and also considering the potential for immunogenicity development. Further, a crossover study design was impractical because the long washout period of trastuzumab (over 7 months) [11] would prolong the duration of a study with a crossover design. Both comparators were selected to meet the regulatory requirement of conducting  a head-to-head study of the biosimilar against reference products.

Our study has to be interpreted while considering the limitation that we recruited only healthy male volunteers. This was done to ensure a homogeneous population to reduce the potential variability in pharmacokinetics compared to patients with cancer. Exposing healthy female patients to trastuzumab may also potentially lead to the development of anti-trastuzumab antibodies, which may be harmful to the patients in the long term if they require trastuzumab treatment. This was another reason why our restriction of the study to male participants is justified. Despite the fact that this study population offered significant advantages with respect to recruitment and logistic aspects compared to a population of patients with cancer, we acknowledge that the results might not be generalizable to the wider population, especially to female patients. Nevertheless, in terms of the inclusion of only male participants and the selected study design and comparators, our study is similar to several previously published phase 1 studies for trastuzumab biosimilars [12,13,14, 16, 17].

Conclusions

BP02, EU-trastuzumab, and US-trastuzumab demonstrated bioequivalence in PK parameters following a single 6-mg/kg IV infusion in healthy adult male subjects. BP02 was also found to be safe and well tolerated and to have a similar immunogenicity profile to both EU-trastuzumab and US-trastuzumab. Finally, EU-trastuzumab and US-trastuzumab were also found to have equivalent PK, safety, and immunogenicity profiles. Boosted by these results, the establishment of the clinical safety and efficacy of BP02 in well-designed clinical trials could support the regulatory approval of BP02 as a biosimilar to trastuzumab, thereby improving patient access to this valuable biologic.