This multicenter, double-blinded, placebo-controlled phase 1 study evaluated the safety, tolerability, pharmacokinetics, and immunogenicity of a single injection of ALT-BB4, a recombinant hyaluronidase, in healthy adults. The participants were healthy men and women aged 18 years or older, with no prior history of significant dermatologic conditions. Additional inclusion and exclusion criteria are detailed on ClinicalTrials.gov (NCT05232175). The random allocation sequence was generated by an independent statistician using a computer-generated randomization plan [SAS® Version 9.4 64 bit (SAS Institute, Cary, NC, USA)]. Allocation concealment was maintained using sequentially numbered, sealed, opaque envelopes containing treatment assignments, which were provided to each study site. After confirming eligibility, investigators assigned participants their allocation numbers and prescribed the study medication accordingly. To ensure double-blinding, ALT-BB4 and placebo were prepared as clear, colorless solution in identical containers by unblinded pharmacy staff who were not involved in participant assessment or data collection. Investigators, participants, and all other study personnel remained blinded until study completion. During administration, measures were taken to prevent participants from viewing the injection procedure or site.

The study consisted of three parts: part I, designed for drug allergy assessment; part II-A, conducted for pharmacokinetics assessment; and part II-B, designed for safety and tolerability assessment (Fig. 1). In part I, participants were to receive a single intradermal injection (0.02 mL) of ALT-BB4 (recombinant hyaluronidase 1500 IU/mL) or placebo (0.9% NaCl), with the administration randomly assigned to their right or left forearm. Subsequent monitoring for allergic reactions was carried out at 30 min (immediate) and 48 h (delayed) post-injection. Participants who showed no signs of drug allergic reactions in part I proceeded to part II-A and part II-B assessments. In part II-A, participants received a single subcutaneous injection (1 mL) of ALT-BB4 into their right or left upper arm. Blood samples were collected at specific timepoints for subsequent pharmacokinetic analysis, and the detailed protocol is shown in the PK assessments of the material methods below. Part II-A was conducted as an open-label, single-arm study. In part II-B, participants were randomly assigned in a 2:1 ratio, with two-thirds receiving a single subcutaneous injection (1 mL) of ALT-BB4 and the remaining one-third receiving a placebo. Site and systemic adverse events were monitored from 30 min until 28 days after administration. No changes to eligibility criteria or other methodological modifications were made after trial commencement. The study was conducted according to the original protocol.

This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Council for Harmonisation (ICH) Good Clinical Practice guidelines. The study protocol was reviewed and approved by the Institutional Review Boards of the following institutions: Seoul National University Hospital (IRB number: H-2111-014-1268), Asan Medical Center (IRB number: 2021-1793), Konkuk University Medical Center (IRB number: KUMC 2021-10-034), and Kangbuk Samsung Hospital (IRB number: KBSMC 2021-10-049). Written informed consent was obtained from all participants prior to study enrollment.

The minimum target number of subjects, 231, for part I allergy evaluation was estimated by using the empirical cases of previous studies and statistical hypotheses. The statistical hypothesis was defined as follows: H0: pT ≥ p0 versus HA: pT < p0, where pT represents the allergy incidence rate in the ALT-BB4 group, and p0 represents the allergy incidence rate recommended by the FDA. On the basis of a prior study (NCT01689363 on ClinicalTrials.gov) involving a similar agent, p0 = 0.1 and pT = 0.05 were assumed. Using the “Numeric Results for Testing One Proportion using the Exact Test” feature in PASS 15, with a one-sided significance level of 2.5% and 80% power, the required sample size was calculated to be 231 subjects.

Of the 231 subjects completing part I, 23 (10%) were designated for enrollment in part II-A, while the remaining subjects were assigned to part II-B. The limited sample size for part II-A was based on preclinical monkey data showing minimal systemic exposure even at high doses, and was sufficient to confirm the expected PK profile while minimizing unnecessary burden to healthy volunteers. No interim analyses were planned or conducted during this trial. Given this was a single-dose phase 1 study primarily focused on safety assessment, no prespecified stopping guidelines were implemented.

All outcomes were prespecified in the study protocol and analyzed as planned. No additional outcome measures were added during the study period. Primary outcomes focused on assessing the safety, tolerability, and pharmacokinetics of a single injection of ALT-BB4 in comparison with placebo, administered to healthy adults. In part I, the incidence rate of drug allergy reactions was evaluated from 30 min to 48 h following intradermal administration. In part II-A, pharmacokinetic parameters were calculated on the basis of the plasma concentration of ALT-BB4. In part II-B, the incidence rate of treatment emergent adverse events (TEAEs), both at injection site and systemically, was assessed from 30 min until 28 days after subcutaneous administration.

Secondary outcomes encompassed the incidence of drug allergy reactions at specific timepoints (30 min and 48 h), and the incidence of TEAEs at specific timepoints (30 min, 2 days, 14 days, and 28 days). Additionally, the study explored immunogenicity by evaluating the presence of antidrug antibodies.

Blood samples for plasma pharmacokinetic analysis were collected at multiple timepoints: 2 h and 1 h before the start of the infusion, and at 0, 10, 20, 30, 40, and 50 min, as well as at 1, 1.25, 1.5, 2, 2.5, 3, and 24 h after the initiation of infusion. The plasma concentrations of ALT-BB4 were measured using a validated sandwich immunoassay. Plasma concentration versus time data were analyzed using noncompartmental pharmacokinetic analyses with Phoenix WinNonlin (Version 8.3 Certara, NJ, USA). Key descriptive pharmacokinetic parameters for ALT-BB4 were summarized, including Cmax, AUClast, t1/2, Tmax, CL/F, and VZ/F. Concentrations of immunoreactive ALT-BB4 in plasma were determined using a validated sandwich immunoassay on the basis of electrochemiluminescence (ECL) technology and MSD platform. High-binding-capacity 96-well plates were coated with an anti-ALT-B4 monoclonal antibody (Alteogen) at a concentration of 2.0 μg/mL. ALT-BB4 in diluted plasma samples, standards, and quality control samples were captured on the plates during a 2-h incubation period. Detection was performed using a biotinylated rabbit anti-ALT-B4 polyclonal antibody (Alteogen). After a series of washing with a solution containing 0.05% polysorbate 20, a sulfo-TAG-conjugated streptavidin reagent was applied for 30 min. Read buffer was added and the ECL signal was acquired using a MESO QuickPlex SQ120 instrument (MesoScale Discovery).

Blood samples for antidrug antibody (ADA) detection were collected at visit 1 of part I, visit 6 of part II-A, and visit 7 of part II-B. All plasma samples were stored in a deep freezer (designated at −80 to −60 °C) until assayed. The presence of ALT-BB4-reactive antibodies was assessed using a validated ECL immunoassay. In this assay, plasma samples were added to ALT-B4-coated plates and incubated with biotin-conjugated ALT-B4. The resulting immune complexes were captured using sulfo-TAG-conjugated streptavidin and detected with an ECL read buffer on the Meso Scale Discovery instrument. Samples with signal levels below the predefined cutoff point were classified as ADA-negative and not subjected to further evaluation. Samples exhibiting signal levels above the cutoff point were classified as ADA-positive and subsequently analyzed in a confirmatory assay.

The results are presented as counts (percentage) or averages (± standard deviation), unless stated otherwise. To compare ALT-BB4 and placebo groups, McNemar’s test was used for paired comparisons in part I, while the χ2 or Fisher’s exact test for numerical variables and paired t test or Wilcoxon signed-rank sum test for continuous variables were used in part II. For statistically significant binary outcomes in part II, both absolute risk differences with 95% confidence intervals and relative risks were calculated. No subgroup analyses were prespecified in the protocol or performed during the study. p-values less than 0.05 were considered statistically significant. All statistical tests were performed using SAS® Version 9.4.