In these two independent but similarly designed studies, a total of 68 volunteers (China n = 34; New Zealand n = 34) were included. In the study conducted in China, 100% of the volunteers were of Asian ethnicity and males accounted for 85.3%. In the New Zealand study, most of the volunteers were Caucasian (58.8%), and females accounted for 79.4% of all. With the exception of sex and ethnicity, all other baseline characteristics were similar between both studies (Table 2).

Of the total 68 volunteers across both studies, 52 received KJ103 and the remainder placebo. During the study, there were no DLTs or SAEs, and no treatment-emergent adverse events (TEAEs) or treatment-related adverse events (TRAEs) leading to study termination. All the predicted dose levels were escalated steadily and all volunteers received the proposed dose with one exception. One participant in New Zealand, receiving 0.40 mg/kg, experienced a self-limited infusion reaction leading to early termination of infusion, attributable to the protocol deviation of no pre-medication.

A total of 27 volunteers (79.4%) experienced 44 TEAEs in the Chinese study. 23 volunteers’ TEAEs (23/34, 67.6%) were grade 1, with fewer cases (4/34, 11.8%) being grade 2, and none grade 3 or above. A total of 9 volunteers (9/34, 26.5%) had 11 TRAEs in this study. Most of them (8/34, 23.5%) were grade 1, and only 1 volunteer (1/34, 2.9%) had a grade 2 TRAE (decreased lymphocyte count). No volunteers in the 0.40 mg/kg group experienced TRAE.

A total of 30 volunteers (88.2%) experienced 79 TEAEs in the New Zealand study. 26 (26/34, 76.5%) volunteers’ TEAEs were grade 1; 2 cases (2/34, 5.9%) were grade 2, and 2 volunteers (2/34, 5.9%) had a grade 3 AE (one volunteer at 0.04 mg/kg developed dental caries that was assessed as unlikely to be related to the test drug, and another participant at 0.40 mg/kg developed deranged liver blood tests that were assessed as probably related to the test drug, both of whom recovered). A total of 8 participants (8/34, 23.5%) reported 8 cases of TRAEs.

Combining the above results, 19 AEs observed in 17 of the 68 participants were classified as related (i.e. possible, probable or definite), as presented in Table 3. The most common AE related to KJ103 was elevated alanine aminotransferase (ALT). The primary concern with IgG-degrading enzymes is the risk of infection. In this regard, the following results affirm KJ103’s safety. One case of grade 1 infectious illness (suspected bacterial infection) occurred in the 0.25 mg/kg group in China, and was determined by the Investigator as probably related to KJ103. One case of grade 1 vulvovaginal candidiasis occurred in the 0.25 mg/kg group in New Zealand, and was determined possibly related to KJ103. There was no severe infection events related to KJ103 (Table 3).

Compared to the placebo, IgG showed mild degradation at dose of 0.01 mg/kg of KJ103. With increasing dose, a sharper “drop off” of IgG level was observed in a dose-dependent manner. A greater reduction in IgG after administration of KJ103 at a dose of 0.12 mg/kg to Asian volunteers in both studies. In both the 0.25 mg/kg and 0.40 mg/kg group, IgG levels reached their lowest point within 6 h of administration, falling by over 90%, and remained consistently below 70% of normal levels for the first week after dosing. After the initial fall ward, IgG levels gradually increased after one week of administration. IgG recovered to near or above baseline levels within 1 to 2 months post-dose in most volunteers. It is worth noting that dose levels of KJ103 did not affect the rate and extent of IgG recovery, only the initial “drop off”. These results indicate that the IgG changes related to dosing with KJ103 were consistent across different ethnicities and demonstrate a favorable pharmacological effect (Fig. 1).

A Mean IgG values for each dose group at each visit within 48 h after dosing in China. B Mean IgG values for each dose group at each visit within 48 h after dosing in New Zealand. C Mean IgG values for each dose at each visit within D63 after dosing in China. D Mean IgG values for each dose at each visit within D63 after dosing in New Zealand. E Mean IgG values for 0.25 mg/kg dose group at each visit within D63 after dosing. F Mean IgG values for 0.40 mg/kg dose group at each visit within D63 after dosing.

Based on the mechanism of action of KJ103, the digested fragments of scIgG, F (ab’)2 and Fc were also analyzed by SDS-PAGE. SDS-PAGE results were consistent with ELISA findings. Compared with the placebo group, in which there was no change, all the samples from KJ103 groups showed an increase in fragment concentration, rising in proportion to the corresponding decrease in intact IgG. All volunteers in the 0.04 mg/kg group and some volunteers in the 0.12 mg/kg group did not undergo complete IgG cleavage. Volunteers in the 0.25 mg/kg and 0.40 mg/kg groups experienced rapid, efficient, and complete cleavage of most IgG into F(ab’)2 and Fc fragments by KJ103 within 45 min - 6 h and 20 min - 6 h post-dosing, respectively. Combining the SDS-PAGE results, the IgG signal obtained 6 h post-dosing by ELISA primarily originates from scIgG. F(ab’)2 and Fc fragments decreased to the baseline levels within 7 days after dosing. Within one to two weeks, all volunteers demonstrated newly synthesized full-length IgG. Within 1 to 2 months post-dosing, IgG recovered to near or above baseline levels (Fig. 2).

A Subject R-405 in China received 0.25 mg/kg BW KJ103 and the exploratory indicators showed changes over time on SDS-PAGE. B Volunteer R-504 in China received 0.40 mg/kg BW KJ103 and the exploratory indicators showed changes over time on SDS-PAGE. C Volunteer R104-004 in New Zealand received 0.25 mg/kg BW KJ103 and the exploratory indicators showed changes over time on SDS-PAGE. D Volunteer R105-007 in New Zealand received 0.40 mg/kg BW KJ103 and the exploratory indicators showed changes over time on SDS-PAGE.

After a single i.v. infusion of KJ103, the overall blood drug concentrations of volunteers in the 0.01 mg/kg and 0.04 mg/kg dose groups were below the detection limit, as a result of low exposure and therefore low blood concentration of the administered drug. PK concentration results showed good reproducibility of PK curves in the dose range of 0.12 mg/kg to 0.40 mg/kg for all dose groups. The mean KJ103 concentration peaked immediately after administration, reaching the plateau phase during which the distribution and metabolism of the drug achieved equilibrium in the body, followed by slow elimination of the drug. Most of the KJ103 was eliminated within 24 h after administration (Fig. 3).

A The concentration of 0.12–0.40 mg/kg BW KJ103 in the blood of volunteers in China varies over time. B The concentration of 0.12–0.40 mg/kg BW KJ103 in the blood of volunteers in New Zealand varies over time.

After a single i.v. infusion of KJ103 in Chinese volunteers, the median Tmax of 0.12 mg/kg, 0.25 mg/kg and 0.40 mg/kg dose groups were 0.333 h, 0.333 h and 0.583 h, respectively. Cmax (Mean ± SD) were 2.208 ± 0.4985 mg/L, 5.142 ± 0.2922 mg/L and 9.200 ± 1.3049 mg/L, respectively. AUC0-t (Mean ± SD) were 28.405 ± 29.6554 h*mg/L, 43.114 ± 21.7201 h*mg/L and 170.261 ± 176.5502 h*mg/L, respectively. After excluding abnormal PK data, the average t1/2 of each dose group was 6.260 h, 4.862 h and 81.648 h, respectively. t1/2 showed a non-linear increase with the increase of dose.

After the New Zealand volunteers received a single i.v. infusion of KJ103, excluding the volunteer in the 0.40 mg/kg dose group who interrupted the dose due to an infusion reaction, the median Tmax of 0.12 mg/kg, 0.25 mg/kg and 0.40 mg/kg dose groups were 0.4583 h, 0.3333 h and 0.5833 h, respectively. Cmax (Mean ± SD) were 2.8135 ± 0.5216 mg/L, 6.5245 ± 0.9994 mg/L and 10.0124 ± 2.6712 mg/L, respectively. AUC0-t (Mean ± SD) were 45.2515 ± 47.9476 h*mg/L, 101.9423 ± 113.8636 h*mg/L and 61.9188 ± 29.7180 h*mg/L, respectively. After excluding abnormal PK data, the average t1/2 of each dose group was 11.3978 h, 2.2629 h and 8.4130 h, respectively (Table 4).

Based on the analysis of PK parameters using a non-compartmental model, KJ103 exhibited characteristics of rapid distribution and slow elimination in volunteers in China and New Zealand. The exposure of KJ103 in various dosage groups demonstrated a non-linear increase with dosage escalation.

Based on clinical trial data from both studies, we established a KJ103 population pharmacokinetic (PopPK) model to explore the covariates affecting PopPK parameters (Table S1). We found that only body weight affects the clearance of KJ103. For a participant with body weight at the 10th~90th percentile of the study population relative to the median body weight in the study population, the Cmax and AUC0~168 varied from -29% ~ 26% (Table 5). Other covariates such as region, ethnicity and gender did not impact PopPK parameters (P > 0.05). The results also indicate similar PK characteristics of populations between China and New Zealand.

The optimal PopPK model was a two-compartment model with first-order elimination. The population typical values (RSE%) of CL, Vc, Vp and Q were 0.162 L/h (13.6), 3.23 L/h (4.3), 14.2 L/h (18.5) and 0.591 L/h (5.0), respectively. The results of the prediction-corrected visual predictive check (pcVPC) are presented in Fig. 4. The median, upper and lower 5th percentiles of the observed values were mostly contained within the 95% confidence intervals of the predicted values. Additionally, the predicted interval encompassed most of the observed values, indicating a good predictive performance of the model.

Blue hollow point: measured value; Blue line: 5th and 95th quantiles of measured values; Red line: median measured value; Blue-shaded intervals: 95% prediction intervals of 5th and 95th quantiles predicted by the model; Red shaded interval: 95% prediction interval of the median predicted by the model. A Prediction corrected concentrations of KJ103 in the blood of healthy population (linear). B Prediction corrected concentrations of KJ103 in the blood of healthy population (logarithm).

A total of 48 volunteers were included in the PK/PD analyses, using IgG levels as the effect indicator. Individual volunteers’ KJ103 blood concentration data were obtained using the PopPK final model using Bayesian a posteriori estimation to explore the PK/PD model of KJ103 concentration and IgG level (Table S2). The relationship between KJ103 concentration and IgG level was described by an effector chamber model. The results revealed that IgG began to decline after the administration of KJ103 at a dose of 0.25 mg/kg, and was maintained near the nadir and close to the lower limit of detection after 5–19 h. The IgG recovered to more than 1 g/L after 36 h of administration, and more than 2 g/L after 96 h, and stayed lower than 4 g/L for 7 days. Analyses of covariates showed that gender influenced the IC50 (concentration of KJ103 when IgG level reaches half of its maximum inhibitory effect), whereas different ethnicities had no effect on the PK/PD model. The results of the prediction-corrected visual predictive check (pcVPC) are presented in Fig. 5. The median and upper and lower 5th percentiles of the observed values were mostly contained within the 95% confidence intervals of the predicted values. Additionally, the predicted interval encompassed most of the observed values, indicating a good predictive performance of the model.

Blue hollow point: measured value; Blue line: 5th and 95th quantiles of measured values; Red line: median measured value; Blue-shaded intervals: 95% prediction intervals of 5th and 95th quantiles predicted by the model; Red shaded interval: 95% prediction interval of the median predicted by the model. A Prediction corrected IgG in the blood of healthy population (linear). B Prediction corrected IgG in the blood of healthy population (logarithm).

Simulation of IgG change levels after a single administration of 0.25 mg/kg in male and female typical volunteers showed that the overall trend of IgG change in males and females was similar. IgG started to decline after administration, approaching near the trough value after 5–24 h, and then recovered slowly (Fig. 6). However, the IgG trough value was 0.66 g/L higher in females than in males, and females recovered faster than males, although both sexes were lower than 5 g/L for 7 days. Simulating IgG trough levels after a single dose in the range of 0.01 to 0.40 mg/kg in male volunteers shows that the IgG trough levels decrease with increasing dosage (Fig. 7). At 0.25 mg/kg of KJ103, enzymatic digestion of IgG is more than 90% effective and reaches a plateau. The IgG levels at 0.25 mg/kg dosage remained at a low level within a week under various conditions.

Simulation of 8.5–19.4 g/L baseline IgG range (10th-90th baseline IgG level of the included population), the change of IgG decline trough after a single dose of 0.25 mg/kg in typical male volunteers; Dots and solid lines represent predicted trough IgG values. The dashed line represents IgG=1 g/L.

The change of trough value of IgG in typical volunteers after a single dose of 0.01–0.40 mg/kg was simulated, and the baseline median IgG value of the included population was 12.8 g/L. Dots and solid lines represent predicted trough IgG values. The dashed line represents IgG=1 g/L.

The pre-existing ADA positivity rate for all enrolled volunteers was 33.82% (23/68), and the median value of pre-existing ADA titers was 0 (range: 0 to 1:429.61). There were no significant differences in the proportion and titer of pre-existing ADA among volunteers of different ethnicities enrolled in China and New Zealand (Fig. 8). The low proportion of pre-existing ADA in volunteers indicated that KJ103 has a low immunogenic background in the population. The relatively low levels of anti-KJ103 antibodies support the safety and efficacy of KJ103 administration. Only one volunteer from the 0.40 mg/kg group in New Zealand experienced an infusion reaction post-administration attributable to the failure to use prophylactic medication as per protocol. No infusion reactions occurred in the remaining participants from both studies.

Human serum samples were analyzed using a validated bridging ELISA method. Among the 34 healthy volunteers randomised in China, the pre-existing ADA positivity rate was 29.41% (10/34), and the median value of pre-existing ADA titer was 0 (range: 0 to 1:429.61). Among the 34 healthy volunteers randomised in New Zealand, the pre-existing ADA positivity rate was 38.2% (13/34), and the median value of pre-existing ADA titer was 0 (range: 0 to 1:242.58). No differences were found in pre-existing ADA positivity or titers between populations.

Among volunteers in the China cohort, the median baseline ADA value was 0 (titer range: 0 ~ 1: 429.61); on the 7th day after KJ103 administration, the median ADA titer was 0 (range: 0 ~ 1: 408.25); on day 14 post-dose, ADA levels were near peak, with a median ADA value of 1:1,041.97 (range: 0 to 1:144,433.38). Two months after administration, the median ADA titer for all KJ103 users was 1: 605.89 (range: 1: <10 ~ 1: 10,989.27). Six months after administration, the median ADA titer for all KJ103 users was 1:524.77 (range: 0 ~ 1: 4,921.42) (Table 6).

In the New Zealand cohort, the median baseline ADA titer was 0 (range: 0 ~ 1: 242.58); on the 7th day after KJ103 administration, the median ADA titer was 0 (range: 0 ~ 1: 246.78); on day 14 post-dose, ADA levels were near peak, with a median ADA value of 1:257.50 (range 0, 1: 20,855.78). Subsequently, ADA levels gradually declined, and after two months of administration, the median ADA titer for all KJ103 users was 1: 158.64 (range: 0 ~ 1: 7171.39), and the median value of ADA titer at 6 months after administration was 1:214.35 (range: 0 ~ 1: 5,782.43) (Table 6). After 2 weeks and 2 months of KJ103 administration, Chinese volunteers showed a wider range of ADA titer change in the 0.25 mg/kg and 0.40 mg/kg dose groups than New Zealand volunteers. There were no significant differences in the median values and ranges of change in ADA titers among participants in the two countries in each of the dose groups prior to KJ103 administration, and at 1 week and at 6 months after KJ103 administration.

The majority of volunteers in the two studies showed ADA changes beginning on Day 14 after KJ103 administration, peaking at approximately two weeks and then gradually declining. After 6 months of KJ103 administration, 56.86% (29/51) of volunteers’ ADA were back to baseline levels (Fig. 9). Although there are significant individual differences in the development of immunogenic responses, in general, immunogenic responses appear to be dose-related.

Comparison of ADA titers before and after administration of different doses of KJ103. A Changes in ADA titers in Chinese volunteers before and after receiving different doses of KJ103 administration (2 in 0.01 mg/kg group, 6 in other groups each). B Changes in ADA titers in New Zealand volunteers before and after receiving different doses of KJ103 administration (2 in 0.01 mg/kg group, 6 in other groups each).

It is reported that AAV2 exposure is high in Chinese population [21]. Therefore, AAV2 NAbs titers were examined in clinical samples from China to investigate the ability of KJ103 to remove pre-existing AAV NAbs. Another research indicates that NAb titers greater than 1:100 significantly increased innate immune responses in gene therapy [22]. Using 1:100 as the threshold, the percentage of pre-existing AAV2 NAbs before KJ103 administration was 73.5% (25/34), which was similar to that reported in the literature. Ten volunteers with pre-administration titers greater than 1:100 were selected from the 0.25 mg/kg and 0.40 mg/kg groups with fully cleavage of IgG. Their AAV2 NAb levels were estimated before and after administration of KJ103. Figure 10A–C displays the titration changes within 21 days for each individual. The re-grouping was based on the differences in pre-dose titers. Following one dose of 0.25 or 0.40 mg/kg KJ103, all pre-dose titers less than 1:1000 were reduced to below the 1:100 cutoff. Pre-dose titers ranging from 1:1000 to 1:2000 decreased close to the cutoff, whereas titers above 1:2000 did not reach it. The maximal degree of changes across individuals was similar, all achieving about 90% reduction when the lowest titers were observed at day 3 or 4. It indicates that one dose of KJ103 above 0.25 mg/kg can generally increase the threshold of NAb titer for AAV-based gene therapy by approximately 10-fold irrespective of the initial titer. For individuals with high titers, a 90% reduction still leaves the titers above the threshold. In such cases, a second dose of KJ103 may be required to further decrease the titers.

The titers of AAV2 Nab before and after administration were individually shown, grouped by their pre-dose titer as <1:1000 (A), 1:1000 ~ 1:2000 (B) and >1:2000 (C). The naming convention “R+number” represents the codes for different volunteers, with the corresponding dose indicated by the suffix. D The temporal variation of NAb, F(ab’)2 and IgG levels in the selected volunteers. Details regarding the selection and grouping referred to in the main text.

To estimate how KJ103 changed AAV2 NAbs titers and the possibility of a second dose, we then tested the changes of F(ab’)2 from these ten volunteers (Fig. 10D). Degradation of IgG by KJ103 resulted in rapid production of F(ab’)2. F(ab’)2 was gradually metabolized and almost cleared by day 7. During the first two days, AAV2 NAb titers declined more slowly than total IgG, probably because of neutralizing activity of newly produced and unmetabolized F(ab’)2. IgG levels gradually increased since day 2, whereas AAV2 NAb titers kept low from day 2 to day 4, leaving an ideal time window for gene therapy. Additionally, if the initial titer is too high, the lowest titer would still exceed the permissible threshold for gene therapy within this time window. A second dose of KJ103 could be considered to degrade remaining IgG and suppress IgG recovery. The strategy could help stabilize or extend the time window, as well as further expand the population eligible for gene therapy.