The Ethics Committee of Jilin University First Hospital Clinical Research Institute in Changchun City (Jilin Province, China) approved our protocol. All clinical procedures were performed in our hospital (Phase I Clinical Trial Unit, The First Hospital of Jilin University). This study was conducted in agreement with the Declaration of Helsinki and the Guidelines for Good Clinical Practice. The registration number of this study is NCT04371185 (https://www.clinicaltrials.gov/). All participants provided written informed consent for this study.

BAT2206 injection (specification: 45 mg), a recombinant human IL-12 monoclonal antibody solution, was manufactured by Bio-Thera Solutions, Ltd. Reference drugs: EU-sourced ustekinumab and USA-sourced ustekinumab (Stelara®; specification: 45 mg) were purchased from Janssen-Cilag International NV (Schaffhausen, Switzerland) and Janssen Biotech, Inc. (Malvern, PA, USA), respectively. All subjects received BAT2206 or ustekinumab from a single lot.

The eligibility of participants was healthy male individuals aged from 18 to 55 years with a body weight between 55 and 85 kg and body mass index values between 18 and 28 kg/m2. Subjects with the following conditions were excluded from this study: diagnosis of cardiovascular disease and diseases of the central nervous system, liver, kidney, or other organs; abnormalities in vital signs, electrocardiograms, clinical laboratory tests (blood routine, biochemical test, thyroid function, antinuclear antibodies, C-reactive protein, rheumatoid factors), or chest X-ray image and ultrasonography; active infections including acute and chronic infections and local infections, within 2 months before screening; had used ustekinumab (or its biosimilar) or any anti-IL-12/23, anti-IL-12, or anti-IL-23 agents or had received treatment with tumor necrosis factor-targeted or IL-17-targeted agents within 6 months prior to screening, or use of any biological product or monoclonal antibody within 3 months prior to screening; has severe allergies caused by food or drugs; had taken alcohol or alcohol-containing drinks within 24 hours prior to assignment of a test drug; had positive results from the tuberculosis enzyme-linked immune SPOT test; or had hepatitis B virus, hepatitis C virus, or human immunodeficiency virus.

This was a randomized, double-blind, single-dose, parallel-group clinical trial for the evaluation of the pharmacokinetics, immunogenicity, and safety of BAT2206 injection compared to ustekinumab (EU) or ustekinumab (USA) in Chinese healthy male subjects. For ustekinumab, the inter-individual coefficient of variation of area under the plasma concentration–time curve from time zero to infinity (AUC0–inf) and Cmax did not exceed 36% [8, 9]. Given that the geometric mean ratio (GMR) of AUC0–inf and Cmax was equal to 0.95, we considered the power (1 – β) to be 90%, the individual coefficient of variation as 36%, and the one-sided test level α as 0.05. Thus, the 20% shedding rate was considered. Therefore, 270 subjects were enrolled (90 subjects for BAT2206, 90 subjects for ustekinumab [EU], and 90 for ustekinumab [USA]). The participants were randomly assigned at a 1:1:1 ratio to receive either BAT2206, ustekinumab (EU), or ustekinumab (USA) via a subcutaneous injection (45 mg/0.5 mL).

In a previous report on the population PK profiles using data from patients with psoriasis, body weight was found to be the most significant covariate factor that affects the clearance of ustekinumab. Additionally, in patients with Crohn’s disease and ulcerative colitis, body weight was the main covariate factor that affects the volume of distribution of ustekinumab. Therefore, body weight may have a significant influence on PK parameters, according to an analysis by the Summary of Product Characteristics of Stelara® [9]. Randomization was based on body weight stratification factors (weight ≥ 55 kg and < 65 kg, weight ≥ 65 kg and < 75 kg, weight ≥ 75 kg and < 85 kg), which were assigned by a computer-generated randomization schedule. In this way, the comparable distribution of body weight in the three arms can be ensured, allowing for distinguishing the differences in PK profiles between the proposed drug and the reference drug. All the enrolled subjects received subcutaneous administration on day 1 and were then allowed to leave the clinical study site after completing the required safety monitoring for at least 9 days. After that, subjects were required to return to the clinical site for safety evaluations, and blood tests of PK profiles on days 10, 11, 13, 15, 22, 29, 43, 57, 71, 85, 99, and 113 (end of the study period).

In this study, adverse events (AEs) were reported from initial drug administration to 113 days post-dose. The definition of AEs was according to the National Cancer Institute Common Terminology Criteria for the Classification of Adverse Events, version 5.0. Assessment of AEs included the following characteristics: the degree of severity (mild, moderate, and severe), duration of symptom severity, AE-related clinical outcomes, and association with the test drug. Additional parameters included physical examinations such as body temperature, sitting blood pressure, and heart rate, as well as electrocardiogram findings and clinical laboratory tests such as hematology, urinalysis, and biochemistry tests.

To study the PK profile, the blood samples were collected at the following timepoints: 0 hours (pre-dose), 4 hours, 12 hours, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 22, 29, 43, 57, 71, 85, 99, and 113 days post-dose. After 0.5 hour at room temperature, blood samples were centrifuged at 2000g at 4 °C for 15 minutes. The serum samples were stored at − 60 °C until subsequent measurement of the drug concentration by a validated enzyme-linked immunosorbent assay (Covance Inc., Shanghai, China). The lower limits of quantification of the assay for serum BAT2206 or ustekinumab were 50.0 ng/mL, and the calibration range was 50.0–1600 ng/mL. The accuracy was − 2.5 to 3.2%, with the precision within the 3.9% coefficient of variation.

Pharmacokinetic evaluations included the following parameters: Cmax, time to Cmax, area under the plasma concentration–time curve from time 0 to 672 hours (AUC0–672), AUC over the dosing interval (AUC0–t), AUC0–inf, elimination half-life, first-order rate constant of drug associated with the terminal portion of the curve, apparent clearance (CL/F), and apparent volume of distribution. The calculation of PK parameters was performed using WinNonlin software (version 8.0; Pharsight Corporation, St. Louis, MO, USA) with the non-compartment model.

Anti-drug antibody tests were performed at 0 hours (before drug administration) and on days 10, 15, 29, 57, 85, and 113. If the subjects had positive ADA results, a neutralizing antibody (Nab) was detected. An electrochemiluminescence assay was used for the quantification of ADAs and Nab that reacted to BAT2206 or ustekinumab [10, 11]. Anti-drug antibodies and Nab were analyzed by Covance Inc.

This electrochemiluminescence method was designed to detect the presence of anti- BAT2206, as well as the neutralizing antibodies of ustekinumab (USA) and ustekinumab (EU) in human serum. The control sample was prepared by adding an anti-BAT2206 positive control antibody to the mixed human serum. Samples were subjected to acid treatment and incubated with Sulfo tag-labeled BAT2206/ustekinumab (USA)/ustekinumab (EU) to form the complexes. After incubation, the antibody complex was added to the MSD plate coated with Pp40-Fc fusion protein. Pp40-Fc protein on the plate-bound to Sulfo Tag-labeled BAT2206/ustekinumab (USA)/ustekinumab (EU) would generate an electrochemiluminescence response. The plate was washed to remove any non-specific bound complexes; then a reading buffer (2X) was added to each well of the plate. The plate was read on the MSD Quickplex 120 or equivalent. The binding of free Sulfo tag-labeled BAT2206 to the Pp40-Fc protein could generate a maximal luminescent signal, meanwhile, the intensity of the signal will decrease in the presence of Nab.

The serum concentration versus time profile of three drugs (BAT2206, ustekinumab [EU], or ustekinumab [USA]) was determined by calculations using WinNonlin software with the non-compartment model. SAS Enterprise Guide 9.4 software was used for data analysis. Analysis of variance was used to compare the differences in PK parameters between the two drugs, including primary PK parameters (Cmax and AUC0–inf) and secondary PK parameters (AUC0–t, AUC0–672 h), after logarithmic transformation. In the model, the experimental groups were used as fixed effects, whereas body weight stratification (weight ≥ 55 kg and < 65 kg, weight ≥ 65 kg and < 75 kg, weight ≥ 75 kg and < 85 kg) was used as covariables. The standard bioequivalence margin was used for setting the criteria of PK similarity. The PK similarity of BAT2206 and its reference ustekinumab (EU or USA) could be determined by evaluating the mean log-transformed value of the 90% confidence interval (CI) for the GMR of test/reference. If the ratio of the values of two drugs was close, within the range of 0.8–1.25, the pairwise PK similarity of BAT2206 with ustekinumab (EU or USA) could be considered, according to the Guidelines for Bioequivalence Studies of Generic Products [12].