This was a randomized, double-blinded, single-dose, parallel-group Phase I clinical trial conducted in Huashan Hospital of Fudan University and Wuxi People’s Hospital (China), which was designed to assess the PK bioequivalence of LZM008 to ACTEMRA^® as the primary endpoint, and the safety and immunogenicity similarity of two products as the secondary endpoints. The entire study was carried out in compliance with the Declaration of Helsinki principles and the Good Clinical Practices and Provisions for drug registration issued by the National Medical Products Administration. The ethics committee of the Huashan Hospital of Fudan University approved the study protocols and relative documents. All subjects who participated in the study signed informed consent documents before being screened. This trial was registered at www.chinadrugtrials.org.cn (Number: CTR20190889).

Subjects were randomly allocated in a 1:1 ratio to receive a single intravenous infusion of 4 mg/kg LZM008 or ACTEMRA^® in 60 min ± 5 min and were discharged from the Phase I clinical research center on day 4 and were required to return to assess safety, PK, and immunogenicity until the end of the study period (day 28). Blood samples for PK assessments were collected at pre-dose (within 2 h), and at 30 min, 1 h (immediately at the end of the infusion), 2 h, 4 h, 8 h, and 24 h (day 2); 48 h (day 3); 96 h (day 4); 168 h (day 7); 240 h (day 10); 336 h (day 14); 408 h (day 17); 504 h (day 21); 576 h (day 24); and 672 h (day 28). Anti-drug antibody (ADA) tests were performed at prespecified visits, including the screen visits on days 14 and 28. ADA-positive samples were further examined for the presence of neutralizing antibodies (NAbs).

Chinese healthy male subjects (age range of 18–45 years) having a body weight larger than 50 kg, with a body mass index (BMI) ranging from 18 to 26 kg/m² (inclusive), were enrolled into this study. Meanwhile, eligible subjects should use appropriate and effective contraceptive measures, such as abstinence, oral contraceptives, intrauterine devices, and condoms combined with a contraceptive diaphragm. In addition, the major exclusion criteria included 1) the history of clinically significant diseases based on their medical history, physical examinations, vital signs, electrocardiograms, laboratory findings, or chest X-ray; 2) subjects who participated in any other clinical trials and donated blood more than 200 mL in the past 3 months; and 3) positive results during an alcohol breath test, drug abuse test, or nicotine test.

An intravenous catheter was used for 8 h in each subject from the predose blood collection. Blood samples of 4 mL were collected in a serum separation tube with a coagulant each time and separation gel and kept at room temperature for 30 min ±15 min, and then centrifuged at 1,500 ∼ 2000 g for 10 ∼ 15 min at a temperature of 2 ∼ 8°C. The supernatant serum was separated and stored at −90 ∼ −60°C within 2 h.

Serum concentrations of LZM008 and ACTEMRA^® were determined using an enzyme-linked immunosorbent assay (ELISA), which was methodologically validated. The limit of quantification was 75.0 ∼ 2,400 ng/mL, and all serum concentrations lower than 75 ng/mL were recorded as 0 in the calculation of pharmacokinetic parameters. Meanwhile, if the sample concentration is higher than that in HOQ, the sample concentration would be diluted at a minimum of 30 times for detection. The precision values of an inter-batch were ≤ 20%, and the inter-assay accuracies ranged from 3.0% to ∼ 4.0%.

Pharmacokinetic parameters were calculated using a non-compartmental model (WinNonlin version 6.4), which included the maximum serum concentration (C_max), time to C_max (T_max), the area under the concentration–time curve (AUC) from time 0 to the last detectable drug concentration (AUC_0-t), the AUC from time 0 to infinity (AUC_0-∞), clearance (CL), and elimination half-life (T_1/2).

Blood samples for immunogenicity analysis were collected at the screening day and days 14 and 28 after infusion. The process of immunogenicity samples was the same as compared to PK samples, and the samples were analyzed for the presence of anti-drug antibodies (ADAs) using a bridging electrochemiluminescence immunoassay (bridging-ECLIA), which was methodologically validated. ADA-positive samples were further examined for the presence of neutralizing antibodies (NAbs). Based on the principle of specific combination of antibodies and drugs, the sample is mixed with a capture reagent (Biotin-Drag) and detection reagent (Drug-SULFO-TAG) to form an immune complex, which can be captured and recognized by the MSD Read Buffer Plate, and the signal (ECLU) is read by using an MSD instrument. The signal value is proportional to the content of the ADAs of the sample.

Safety was assessed by physical examinations, vital signs, laboratory tests, and electrocardiograms. The type, timing, seriousness, and relatedness of adverse events (AEs) were documented based on the results of vital signs, clinical laboratory data, and other medications used in this study. Laboratory tests were examined at screening and on days 3, 14, and 28. Vital sign examinations were performed at 3 h pre-dose; 2 h, 4 h, and 12 h after infusion on day 1; and on days 2, 3, 4, 7, 10, 14, 17, 21, 24, and 28 after infusion. Electrocardiographic (ECG) screening was performed 3 h pre-dose, 2 h after infusion on day 1, and on days 3 and 28 after infusion. The AEs were recorded and graded according to the National Cancer Institute Common Terminology Criteria for AEs (CTCAE; V 5.0) and were coded according to the Medical Dictionary for Regulatory Activities (MedDRA; V 22.0).

The sample size was determined according to previous studies on the bioavailability of ACTEMRA^®. The level of significance (α) has been set as 0.05, according to the Bonferroni adjustment method, and there should be 95% power for each endpoint at the same time. The coefficient of variation (CV) for AUC_0-∞ was 11%∼24%, and CV of C_max was 8%∼20% for ACTEMRA^® (ACTEMRA, 2016); the true ratio of AUC_0-t between LZM008 and ACTEMRA^® groups was 1 ± 0.05. A total of 84 subjects (42 per group) were needed in this trial. Moreover, considering that there may be about 15% of subject dropout rate, 96 healthy subjects should be enrolled for randomization.

After a logarithmic transformation of PK parameters, C_max, AUC_0–t, and AUC_0–∞, the ANOVA model with fixed effects (the group, the center, and the interaction between the center and the group) was used for analysis, and then, the t-test was used based on ANOVA analysis results to determine the pharmacokinetic bioequivalence of LZM008 and ACTEMRA^®. If the 90% CIs were observed to be within 80%–125% for all three PK parameters, bioequivalence was inferred. The safety analysis set included subjects who were administered the study drug. Descriptive statistics for PK parameters and demographic data were calculated. Between-group differences were assessed using the chi-squared test for categorical variables, the t-test for normally distributed continuous variables, and the Wilcoxon rank-sum test for non-normally distributed variables. All statistical analyses were performed using SAS version 9.4.

A total of 589 subjects were screened; 96 eligible male subjects were randomized (49 in the LZM008 group and 47 in the ACTEMRA^® group) and received a single intravenous infusion of 4 mg/kg LZM008 or ACTEMRA^® (Figure 1). In total, 49 subjects were randomized in the Huashan Hospital of Fudan University, and 47 subjects were randomized in Wuxi People’s Hospital. Although one subject in the LZM008 group withdrew from the clinical trial for personal reasons, with the last PK sample in 504 h and not being included in the bioequivalence set, the remaining 95 subjects completed the trial and entered the full analysis set, PK concentration analysis set, PK parameter analysis set, bioequivalence analysis set, safety analysis set, and immunogenicity analysis set. The two groups were well balanced and comparable in terms of demographics and baseline characteristics (Table 1).

The mean serum concentration–time profiles for LZM008 and ACTEMRA^® groups were almost overlapped (Figure 2). The AUC_0-t and AUC_0-∞ values of the LZM008 group were slightly higher than those of the ACTEMRA^® group, and the CL of the LZM008 group was slightly lower than that of the ACTEMRA^® group. The differences in other pharmacokinetic parameters between the LZM008 group and the ACTEMRA^® group were all within 10% (Table 2). Meanwhile, the last detectable timepoint of drug concentrations in the LZM008 group was 504 h (12 subjects), 576 h (28 subjects), and 672 h (8 subjects), while in the ACTEMRA^® group, the last detectable timepoint of drug concentrations was 408 h (2 subjects), 504 h (23 subjects), 576 h (15 subjects), and 672 h (7 subjects).

After a single intravenous infusion of 4 mg/kg of LZM008, the C_max and AUC_0-∞ values reached 87.99 μg/mL and 11,526.70 h*μg/mL, respectively, with T_max 1.98 h, and the half-life (t_1/2) was 83.45 h. The geometric mean ratios and 90% CI of AUC_0-t and C_max were 112.06% (105.46% and 119.08%) and 108.38% (102.50% and 114.60%), respectively. In addition, the geometric mean ratio and 90% CI of the secondary endpoint (AUC_0-∞) were 112.02% (105.44% and 119.02%). The 90% CIs of the GM ratio of all endpoints were still within the range of 80.00%–125.00%. Overall, PK profiles were similar in the two groups and can meet the bioequivalence criterion (Table 3).

In addition, the Wilcoxon rank-sum test was used for determining the T_max values of the two groups, and the p-value was greater than 0.05, and there was no statistical difference in the T_max values of LZM008 and ACTEMRA^®.

Meanwhile, after the natural logarithm transformation of AUC_0-t, AUC_0-∞, and C_max, the ANOVA model with fixed effects (the group, the center, and the interaction between the center and the group) was used for analysis, and then, the t-test was used based on ANOVA analysis results to determine the pharmacokinetic bioequivalence of LZM008 and ACTEMRA^®. The findings of the ANOVA test indicated that there were no significant differences, so the t-test was not required. However, in order to maintain consistency with the statistical analysis plan, t tests were still carried out. The p-values of all fixed effects were larger than 0.05. Therefore, there is no interaction between the center and the experimental grouping, and the effect of the experimental grouping between the two centers remains consistent.

The incidence of treatment emergent adverse events was similar in LZM008 and ACTEMRA^® groups (98.0% versus 100%) (Table 4). In the LZM008 group, 48 (98.0%) subjects had 189 cases of TEAEs, of which 160 cases were related to the investigated drug and 4 (8.2%) subjects had 10 cases of TEAEs of grade III or worse; 182 cases of TEAEs were observed in 47 (100%) subjects in the ACTEMRA^® group, of which 161 cases of TEAEs were related to the investigated drug and seven (12.8%) subjects had TEAEs of grade III or worse. There were no SAEs in this study and no TEAEs leading to discontinuation/withdrawal of treatment or death.

The incidence of TEAEs of grade III or worse in the trial was similar in LZM008 and ACTEMRA^® groups (8.2% vs. 12.8%) (Table 5). One subject in the LZM008 group had four cases of TEAEs of grade IV, which occurred on day 3; we observed a decrease in absolute neutrophils, agranulocytosis, and percentage of neutrophils, and an increase in heterozygous lymphocytes, and 300 μg of granulocyte colony-stimulating factor (G-CSF) was administered for treatment of TEAEs on day 7. All TEAEs of grade IV returned to baseline levels after day 36. Otherwise, five subjects (three in the LZM008 group and two in the ACTEMRA^® group) showed Mycobacterium tuberculosis complex test positive results at the last follow-up (day 28), while chest CT and symptom assessment results ruled out active tuberculosis infection of five subjects. A follow-up visit is conducted every 3 months for a total of 2 years, and anti-tuberculosis treatment (prophylactic) is considered if necessary.

All randomized subjects showed ADA-negative results at baseline. At the end of the trial (day 28), one (2.0%) subject in the LZM008 group and three (6.4%) subjects in the ACTEMRA^® group had ADA-positive test results, with the same titer value of 1:50. Meanwhile, one subject in the ACTEMRA^® group was observed to be NAb-positive on day 28, but there were no AEs attributable to the ADA finding for those subjects, and the PK profile of these subjects was similar to that of other subjects with ADA-negative results. Therefore, the incidence of ADA positivity in the LZM008 group was slightly lower than that in the ACTEMRA^® group, but it did not show clinically significant differences.